EACR24 EACR ad EJC.indd

European Journal of Cancer

Volume 61 Supplement 1, July 2016

Proceedings Book

th

24 Biennial Congress of the European Association for Cancer Research

Amsterdam • Boston • London • New York • Oxford • Paris • Philadelphia • San Diego • St Louis

European Journal of Cancer Editor-in-Chief:

Editors: Basic Science and Preclinical Research: Drug Development: Early Breast Cancer: Advanced Breast Cancer: Gastrointestinal Cancers: Genitourinary Cancers: Head and Neck Cancer: Hemato-Oncology: Lung Cancer: Gynaecological Cancers: Sarcomas: Melanoma: Neuro-oncology: Epidemiology and Prevention: Tumour Immunology and Immunotherapy: Paediatric Oncology: Founding Editor: Past Editors: Editorial Office:

Alexander M.M. Eggermont Institut Gustave Roussy Villejuif, France Richard Marais, Manchester, UK Ulrich Keilholz, Berlin, Germany Jordi Rodon, Barcelona, Spain Kathleen I. Pritchard, Toronto, Canada David Cameron, Edinburgh, UK Volker Heinemann, Munich, Germany Michel Ducreux, Villejuif, France Karim Fizazi, Villejuif, France J.P. Machiels, Brussels, Belgium Roch Houot, Rennes, France Mary O’Brien, London, UK Ignace Vergote, Leuven, Belgium Jean-Yves Blay, Lyon, France Dirk Schadendorf, Essen, Germany Roger Stupp, Zurich, Switzerland Jan Willem Coebergh, Rotterdam, The Netherlands Aure´lien Marabelle, Villejuif, France Rob Pieters, Utrecht, The Netherlands Henri Tagnon Michael Peckham, London, UK; Hans-Jo¨rg Senn, St Gallen, Switzerland; John Smyth, Edinburgh, UK Elsevier, The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Tel: +44 (0) 1865 843590, Email: [email protected]

EDITORIAL BOARD CLINICAL ONCOLOGY R. Baird (UK) N. Brünner (Denmark) R. Califano (UK) E. Calvo (Spain) F. Cardoso (Portugal) J. Cassidy (UK) H. Cody (USA) R. Coleman (UK) A. Costa (Italy) J. De Bono (UK) E. de Vries (The Netherlands) A. Dicker (USA) R. Dummer (Switzerland) F. Eisinger (France) S. Erridge (UK) G. Ferrandina (Italy)

H.Gabra (UK) H. Gelderblom (The Netherlands) B. Hasan (Belgium) J.C. Horiot (Switzerland) J. Jassem (Poland) A. Katz (Brazil) I. Kunkler (UK) C. Le Tourneau (France) C-C. Lin (Taiwan) P.E. Lønning (Norway) P. Lorigan (UK) C. Massard (France) K. McDonald (Australia) F. Meunier (Belgium) A. Miller (Canada) T. Mok (Hong Kong)

D. Nam (Korea) J. Overgaard (Denmark) J. Perry (Canada) J. Ringash (Canada) A. Rody (Germany) M. Schmidinger (Austria) S. Sleijfer (The Netherlands) S. Stacchiotti (Italy) M. van den Bent (The Netherlands) G. Velikova (UK) U. Veronesi (Italy) A. Voogd (The Netherlands) E. Winquist (Canada) T. Yap (UK)

BASIC SCIENCE, PRECLINICAL AND TRANSLATIONAL RESEARCH P. Allavena (Italy) J. Anderson (UK) M. Barbacid (Spain) M. Broggini (Italy) C. Catapano (Switzerland) M. Esteller (Spain) E. Garattini (Italy) A. Gescher (UK)

R. Giavazzi (Italy) J.M. Irish (USA) H.E.K. Kohrt (USA) J. Lunec (UK) A.G. Papavassiliou (Greece) V. Rotter (Israel) V. Sanz-Moreno (UK) S. Singh (Canada)

J. Stagg (Canada) C.G.J. Sweep (The Netherlands) P. Vineis (UK) A. Virós (UK) B. Weigelt (USA) N. Zaffaroni (Italy)

D. Forman (France) A. Green (Australia) K. Hemminki (Germany) C. Johansen (Denmark) L.A. Kiemeney (The Netherlands) E. Lynge (Denmark) M. Maynadie´ (France) H. Møller (UK) P. Peeters (The Netherlands)

S. Sanjose (Spain) M.K. Schmidt (The Netherlands) I. Soerjomataram (France) H. Storm (Denmark) L.V. van de Poll-Franse (The Netherlands) H.M. Verkooijen (The Netherlands) E. de Vries (The Netherlands) R. Zanetti (Italy)

G. Chantada (Argentina) F. Doz (France) A. Ferrari (Italy) M.A. Grootenhuis (The Netherlands) K. Pritchard-Jones (UK)

L. Sung (Canada) M. van den Heuvel-Eibrink (The Netherlands) M. van Noesel (The Netherlands)

EPIDEMIOLOGY AND PREVENTION B. Armstrong (Australia) P. Autier (France) V. Bataille (UK) J.M. Borras (Spain) C. Bosetti (Italy) H. Brenner (Germany) L.E.M. Duijm (The Netherlands) J. Faivre (France) S. Franceschi (France)

PAEDIATRIC ONCOLOGY C. Bergeron (France) A. Biondi (Italy) E. Bouffet (Canada) M. Cairo (USA) H. Caron (The Netherlands)

European Journal of Cancer Aims and Scope The European Journal of Cancer (EJC) is an international multidisciplinary oncology journal, which publishes original research, reviews, and editorial comments on basic and preclinical cancer research, translational oncology, clinical oncology – including medical oncology, paediatric oncology, radiation oncology, and surgical oncology, and cancer epidemiology and prevention. The EJC is the official journal of the European Organisation for Research and Treatment of Cancer (EORTC), the European CanCer Organisation (ECCO), European Association for Cancer Research (EACR) and the European Society of Breast Cancer Specialists (EUSOMA). For a full and complete Guide for Authors, please go to http://www.ejcancer.com Advertising information. 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Peer Review Policy for the European Journal of Cancer (EJC) The practice of peer review is to ensure that only good science is published. It is an objective process at the heart of good scholarly publishing and is carried out by all reputable scientific journals. Our reviewers therefore play a vital role in maintaining the high standards of the European Journal of Cancer (EJC) and all manuscripts are peer reviewed following the procedure outlined below. Initial manuscript evaluation The Editors first evaluate all manuscripts. In some circumstances it is entirely feasible for an exceptional manuscript to be accepted at this stage. Those rejected at this stage are insufficiently original, have serious scientific flaws, have poor grammar or English language, or are outside the aims and scope of the journal. Those that meet the minimum criteria are passed on to experts for review. Authors of manuscripts rejected at this stage will be informed within 2 weeks of receipt. Type of Peer Review The EJC employs single blind review, where the reviewer remains anonymous to the authors throughout the process. How the reviewer is selected Reviewers are matched to the paper according to their expertise. Our reviewer database contains reviewer contact details together with their subject areas of interest, and this is constantly being updated. Reviewer reports Reviewers are asked to evaluate whether the manuscript: – Is original – Is methodologically sound – Follows appropriate ethical guidelines – Has results which are clearly presented and support the conclusions – Correctly references previous relevant work Reviewers are not expected to correct or copyedit manuscripts. Language correction is not part of the peer review

process. Reviewers are requested to refrain from giving their personal opinion in the ‘‘Reviewer blind comments to Author’’ section of their review on whether or not the paper should be published. Personal opinions can be expressed in the ‘‘Reviewer confidential comments to Editor’’ section. How long does the peer review process take? Typically the manuscript will be reviewed within 2-8 weeks. Should the reviewers’ reports contradict one another or a report is unnecessarily delayed a further expert opinion will be sought. Revised manuscripts are usually returned to the Editors within 3 weeks and the Editors may request further advice from the reviewers at this time. The Editors may request more than one revision of a manuscript. Final report A final decision to accept or reject the manuscript will be sent to the author along with any recommendations made by the reviewers, and may include verbatim comments by the reviewers. Editor’s Decision is final Reviewers advise the Editors, who are responsible for the final decision to accept or reject the article. Special Issues / Conference Proceedings Special issues and/or conference proceedings may have different peer review procedures involving, for example, Guest Editors, conference organisers or scientific committees. Authors contributing to these projects may receive full details of the peer review process on request from the editorial office. Becoming a Reviewer for the EJC If you are not currently a reviewer for the EJC but would like to be considered as a reviewer for this Journal, please contact the editorial office by e-mail at [email protected], and provide your contact details. If your request is approved and you are added to the online reviewer database you will receive a confirmatory email, asking you to add details on your field of expertise, in the format of subject classifications.

Volume 61

EUROPEAN JOURNAL OF CANCER

Suppl. 1 2016

Contents Acknowledgements Official Media Partners Letter of Welcome Organising & Scientific Programme Committee Accreditation Information and Compliance General Information Awards About the EACR Floorplans List of Exhibitors Exhibitor Profiles Satellite Symposia QIAGEN Special Session Programme at a Glance Scientific Programme Saturday 9 July 2016 Sunday 10 July 2016 Monday 11 July 2016 Tuesday 12 July 2016 Poster Sessions, Sunday 10 July 2016 Poster Sessions, Monday 11 July 2016 Abstracts Proffered Papers (Sunday 10 July 2016) Proffered Papers (Monday 11 July 2016) Poster Sessions (Sunday 10 July and Monday 11 July 2016) Index of Presenting Authors

Indexed/Abstracted in: Current Contents; EMBASE/Excerpta Medica; Index Medicus; MEDLINE; CABS, BIOSIS Database; PASCAL-CNRS Database; CINAHLs®

VI VI VII VII IX XI XV XVIII XIX XXI XXIII XXXII XXXIV XXXV XXXVII XXXVII XXXVII XXXIX XLI XLII LV

S1 S1 S5 S9 S219

ISSN 0959-8049

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Acknowledgements The European Association for Cancer Research (EACR) would like to acknowledge the generous ongoing support of its Sustaining Members:

every step of the way.

And expresses sincere thanks for the generous support of the organisations sponsoring Symposia, Keynote and Award Lectures.

The EACR is also grateful for the support of the following sponsors: The EACR also wishes to thank the following companies and organisations for their support of the Congress by taking part in the exhibition: Acris Antibodies − OriGene EU Adaptive Biotechnologies Agilent Technologies LDA UK Ltd. American Association for Cancer Research ANGLE APTrans ArcherDX Aspect Imaging BD Life Sciences Best Theratronics Ltd. Bio-Techne Breast Cancer Now Cambridge Bioscience Cancer Research UK Cell Signaling Technology Charles River Chromatrap ECACC European Collection of Authenticated Cell Cultures

eLife OcellO Envigo Organisation of European Cancer Institutes ESMO − European Society for Medical Oxford Optronix & Baker Ruskinn Oncology Phase Holographic Imaging AB Fluidigm PlexBio Co., Ltd. GATC Biotech Polyplus-transfection GeneTex Promega UK Ltd. Greiner Bio-One Proteintech Europe Hybrigenics Services QIAGEN Indivumed Randox Biosciences LGC-ATCC Spandidos Publications LI-COR Biosciences Stratech Scientific Ltd Manchester Cancer Research Centre Taylor & Francis Group Medical and Biological Laboratories Co., Ltd. The Jackson Laboratory Menarini Silicon Biosystems Thermo Fisher Scientific Miltenyi Biotec Univadis Oncology NanoString Technologies VolitionRX New England Biolabs Worldwide Cancer Research

Official Media Partners On behalf of the EACR24 Congress Committees, we would like to acknowledge the collaboration and support of our official media partners: CRITICAL REVIEWS IN

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Letter of Welcome On behalf of the Scientific Committee, I am very pleased to welcome you to Manchester for the 24th Biennial Congress of the European Association for Cancer Research (EACR24) − Europe’s largest dedicated conference on basic and translational research. The theme of the 24th EACR Congress is ‘From Basic Research to Precision Medicine’. Precision medicine is an emerging important concept in cancer research and treatment today, and the Congress features world-class speakers covering all of the most innovative current research topics. EACR congresses are well known for providing a rich and diverse programme. During the next four days, EACR24 will provide a unique opportunity for participants to learn from and meet with leaders in the field of basic and translational research, apply new data to inform practice and ultimately improve patient outcomes. The programme has been designed to meet the needs of researchers at all stages of their careers, and to allow participants to benefit from chances to interact, discuss and reflect on the information presented. As well as highprofile plenary sessions, the programme offers parallel symposia, allowing participants to build their own scientific programme according to their interests. It will also include workshops for early-career researchers, as well as the opportunity for participants to share their work through oral and poster presentations. I am also pleased that this Congress will be held in my home city of Manchester − European City of Science 2016 − a modern, vibrant city with a great history of science and innovation, local culture and exceptional heritage. I hope that you will take the opportunity to experience the unique atmosphere of the city of Manchester. I am also delighted that the Congress is actively supported by the British Association for Cancer Research (BACR). I trust that you will return from the Congress inspired by colleagues from around the world and that you will have made new contacts and collaborations that will support you in your own important work. I am delighted to be welcoming you to what promises to be another highly educational, collaborative and successful EACR Congress. Kind regards, Professor Richard Marais Congress & Scientific Chair

Organising & Scientific Programme Committee Richard Marais (Congress & Scientific Chair)

Daniel Peeper

Anton Berns

Eli Pikarsky

Carlos Caldas

Joan Seoane

Donatella Del Bufalo

Emmy Verschuren

Clare Isacke

Christof von Kalle

Moshe Oren

Jane Smith

EACR 25 30 June - 3 July 2018

Amsterdam

SAVE THE DATE for this landmark congress celebrating 50 years of the EACR

25th Biennial Congress of the

European Association for Cancer Research From Fundamental Insight to Rational Cancer Treatment

ng

ati Celebr

50 years

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Accreditation Information The ‘24th Biennial Congress of the European Association for Cancer Research’ is accredited by the European Accreditation Council for Continuing Medical Education (EACCME) to provide the following CME activity for medical specialists. The EACCME is an institution of the European Union of Medical Specialists (UEMS), www.uems.net EACR24 is designated for a maximum of 18 hours of European external CME credits. Each medical specialist should claim only those hours of credit that he/she actually spent in the educational activity. The EACCME credit system is based on 1 ECMEC per hour with a maximum of 3 ECMECs for half a day and 6 ECMECs for a full-day event. European Accreditation is granted by the EACCME in order to allow participants who attend the above-mentioned activity to validate their credits in their own country. Through an agreement between the European Union of Medical Specialists and the American Medical Association, physicians may convert EACCME credits to an equivalent number of AMA PRA Category 1 Credits™. Information on the process to convert EACCME credit to AMA credit can be found at: www.ama-assn.org/go/internationalcme Live educational activities, occurring outside of Canada, recognised by the UEMS-EACCME for ECMEC credits are deemed to be Accredited Group Learning Activities (Section 1) as defined by the Maintenance of Certification Program of The Royal College of Physicians and Surgeons of Canada.

EACR24 is Compliant EFPIA The European Federation of Pharmaceutical Industries and Associations (EFPIA) ensures collaborations and partnerships between healthcare professionals and industry are subject to stringent legislation and requires that all parties respect high ethical standards. www.efpia.eu Ethical MedTech EthicalMedTech is a platform, supported by Eucomed, dedicated to ethics and compliance projects in the European MedTech industry. www.ethicalmedtech.eu

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General Information Congress Secretariat c/o ECCO − the European CanCer Organisation [email protected] www.ecco-org.eu/eacr24 During the congress, the Secretariat can be reached at +32 2 880 15 21

Congress Venue Manchester Central Convention Complex Petersfield, Manchester, M2 3GX United Kingdom Tel: +44 (0) 161 834 2700 www.manchestercentral.co.uk App All attendees can download the free ECCO App on iPhone, iPad, or Android supported devices. Features include EACR24 related information and news. The App contains the complete list of scientific sessions, session types, speakers, exhibitors, and satellite symposia. Users can save their selected sessions, notes, favourites, as well as exporting sessions to their smartphone calendar. To download the App, search for ECCO cancer on iTunes or Google Play. Learn more at www.ecco-org.eu/app or use the QR code below for direct download.

Badges For security reasons, delegates are requested to wear their badge at all times during the congress. Delegates having lost their badge can obtain a new badge at the registration desk. A replacement fee of 50 GBP per participant will be charged. Catering Coffee Breaks: Coffee breaks, courtesy of the organisers, have been scheduled as follows: Saturday 9 July: 15:00–15:30 Monday 11 July: 10:15−10:45 Sunday 10 July: 10:15−10:45 15:45−16:15 15:45−16:15 Tuesday 12 July: 10:15−10:45 Coffee breaks will take place in the catering areas of the exhibition, except on Tuesday, when refreshments will be served in the foyer areas on the ground floor. A variety of outlets will be open for the sale of refreshments at lunchtime and throughout the day. Free water dispensers are located throughout the venue. Certificate of Attendance Certificates of Attendance recording CME credits will be available online immediately after the Congress. You will receive an email link to a short questionnaire which also provides the link for you to print your Certificate of Attendance. We kindly ask you to keep your Congress badge as you will need the unique badge code to print your Certificate of Attendance. The Congress Secretariat will not mail Certificates of Attendance to participants after the Congress. For information on CME accreditation see page IX.

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EACR24, General Information / European Journal of Cancer 61, Suppl. 1 (2016) xi–xiv

Cloakroom A staffed cloakroom is located in the Entrance Hall and can be used at no cost. Cloakroom Opening Hours: Saturday 9 July: 10:30−20:00 Sunday 10 July: 08:00−21:00 Monday 11 July: 08:00−19:00 Tuesday 12 July: 08:00−14:00 Congress Dinner: Monday 11 July 20:00 The EACR24 Congress Dinner will take place at the Manchester Town Hall, located at Albert Square. A few last tickets may be available from the registration desk in the Central Foyer (price: 55 GBP). Ticket holders will be asked to present their ticket upon arrival at the dinner venue. Please kindly note that it will not be possible to buy tickets at the door of the dinner venue. EACR General Assembly and Awards Ceremony: Sunday 10 July 19:00−19:45 The General Assembly and Awards Ceremony of the European Association for Cancer Research will be held in room Charter 4 of the Congress Centre. This event is open to EACR members and award winners. Exhibition The EACR24 exhibition is held in Central Hall 2 on the ground floor of the Congress Centre. Entrance is free for registered delegates but limited to researchers, oncology professionals, press and exhibitors. Exhibition Opening Hours: Saturday 9 July: 14:45−19:30 Sunday 10 July: 10:00−17:15 Monday 11 July: 10:00−17:15 For the exhibition floorplan and list of exhibitors, please see the exhibition section (p. Book.

XX)

of this Proceedings

First Aid A first aid room is available for delegates. In case of emergency, please inform the concierge desk in the Central Foyer. Insurance The organisers of EACR24 do not accept liability for individual medical, travel or personal insurance. Participants are strongly recommended to obtain their own personal insurance policies. The organisers of EACR24 accept no responsibility for loss due to theft or negligence. Internet Wi-Fi Access General Wi-Fi access is available throughout the Congress Centre and gives 5 Mbps per device. Instructions to activate the Wi-Fi: (1) Connect to the wireless network ‘MCCC’. (2) You will automatically redirect to a portal page (if not, attempt to visit a web site in your browser e.g. www.bbc.co.uk). (3) Select ‘Free Wi-Fi’ (4) Enter your information and accept the terms and conditions. (5) Once successful you will be redirected to Manchester Central’s official website and will have access to the internet. Internet Zone The official EACR24 Internet Zone is available free of charge during the Congress. The terminals provide you with the following services: internet browsing, access to web-based mail, the congress searchable programme and exhibitor information. Language & Translation The official language of the congress is English. Translation is not provided.


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Lost & Found All enquiries should be directed to the information desk in the entrance hall. The organisers accept no responsibility for loss due to theft or negligence. Online Networking Community We have launched a secure, exclusive and interactive online community, allowing attendees to connect before, during and after EACR24. As a delegate, you can see who else is attending the Congress, start conversations, book private meetings with other attendees and much more. You will be able to view the profiles and interests of other attendees, discover which of your contacts from Facebook, Twitter and LinkedIn are also attending EACR24 and schedule oneto-one meetings with other attendees. Join the conversations on the topics that are critical to cancer research today! https://eacr24.pathable.com Opening Ceremony and Lecture: Saturday 9 July 17:00 The Opening Ceremony and Lecture will be held in the Exchange Hall. Access is free for all registered participants. Please refer to the Scientific Programme for further details. The Opening Ceremony and Lecture will be followed by an Exhibitors’ Reception taking place in Central Hall 2, where light refreshments will be served. Poster Sessions Posters will be on display for one day in the dedicated poster area (Sunday or Monday, across the various topics. For details please refer to the Scientific Programme). Poster presenters will be given access to the Exhibition Hall as of 08:00 to mount their poster on the day their poster is to be presented. Posters must be removed by 18:00 on the day the poster was presented. Any posters remaining after this time will be removed by the organisers and cannot be reclaimed. Posters in the Spotlight The Scientific Committee has made a special selection of posters which will be highlighted through short oral presentations followed by a moderated discussion. These sessions will facilitate dynamic exchange on some of the hottest topics at the Congress! Spotlight sessions will take place in the exhibition area from 16:15 until 17:05 on Sunday 10 and Monday 11 July. Registration EACR24 is open to all registered participants. Your official name badge is required for admission to the Congress Centre and all Congress events. For security reasons, participants are requested to wear their badge at all times. Registration Opening Hours: Saturday 9 July: 08:00−19:00 Sunday 10 July: 08:00−19:00 Monday 11 July: 08:00−17:30 Tuesday 12 July: 08:00−12:00 Registration Package The full congress registration package includes: − Entry to all scientific sessions, satellite symposia and to the Opening Ceremony and Lecture on Saturday 9 July; − Entry to the exhibition (restricted to researchers, oncology professionals and media); − EACR24 Proceedings Book; − EACR24 coffee breaks; − Internet access via the Internet Zone and Wi-Fi access in the Congress Centre; − Congress bag including a city map.

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The day registration package includes: − Access to all scientific sessions and satellite symposia on that day; − Entry to the exhibition (restricted to researchers, oncology professionals and media); − Proceedings Book (depending on availability); − EACR24 coffee breaks on that day; − Internet access via the Internet Zone and Wi-Fi access in the Congress Centre; − Congress bag including a city map (depending on availability). Satellite Symposia Several satellite symposia are taking place during EACR24. For the detailed programme, please see the satellite symposia section (page XXXII) of this Proceedings Book. Social Media Twitter is available during the Congress − tweet, network and follow updates using hashtag #EACR24. Follow the EACR on Twitter at @EACRnews, on Facebook at www.facebook.com/eacr.org or search ‘European Association for Cancer Research’ on LinkedIn. Speaker Preview Room The Speaker Preview Room is located in Central 3. Speakers are requested to bring their PowerPoint presentations to the Speaker Preview Room at least 4 hours before their session starts or one day in advance if the session starts early in the morning. Provision for presentations using laptops is not available in the session rooms. Speaker Preview Room Opening Hours: Saturday 9 July: 09:30−18:00 Sunday 10 July: 07:30−18:00 Monday 11 July: 07:30−17:30 Tuesday 12 July: 08:00−12:30

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Awards Mike Price Gold Medal Award The Mike Price Gold Medal is presented biennially to a senior researcher who has made an exceptional contribution to cancer research in Europe. The Award commemorates the life and work of perhaps the most significant figure in the EACR’s history, Mike Price, who served as Secretary General of the EACR for 21 years. He died in 2000 after fighting bravely for just over a year against an unusual form of cancer. We are delighted to announce that the 2016 award will be presented at EACR24 to Professor Sir Michael Stratton, Director of the Wellcome Trust Sanger Institute, UK. Professor Stratton will receive the Mike Price Gold Medal and deliver the closing lecture. Mike Stratton’s early research focused on inherited susceptibility. He mapped and identified the major high risk breast cancer susceptibility gene BRCA2 and subsequently a series of moderate risk breast cancer and other cancer susceptibility genes. In 2000 he initiated the Cancer Genome Project at the Wellcome Trust Sanger Institute which conducts systematic genome-wide searches for somatic mutations in human cancer. Through these studies he discovered somatic mutations of the BRAF gene in malignant melanoma and several other mutated cancer genes in lung, renal, breast and other cancers. He has described the basic patterns of somatic mutation in cancer genomes revealing underlying DNA mutational and repair processes. He is a Fellow of the Royal Society (FRS) and was knighted in 2013.

The Pezcoller Foundation − EACR Cancer Researcher Award The Pezcoller Foundation − EACR Cancer Researcher Award celebrates academic excellence and achievements in the field of cancer research. The award is presented biennially to a researcher of excellence with no more than 15 years post-doctoral experience. We are delighted to announce that the 2016 award goes to Professor Yardena Samuels (Department of Molecular Cell Biology and Director, Ekard Institute for Cancer Diagnosis Research, MICC, Israel). The Samuels laboratory uses various sequencing approaches to identify the genetic changes that underlie melanoma. Once these mutations are identified, her group focuses on characterising the biochemical, functional, and clinical aspects of the most highly mutated genes. Professor Samuels will give the Pezcoller Foundation − EACR Cancer Researcher Award Lecture and receive an unrestricted honorarium of €10 000.

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EACR24, Awards / European Journal of Cancer 61, Suppl. 1 (2016) xv–xvii

Anthony Dipple Carcinogenesis Award The winner of this senior award for major contributions to research in the field of carcinogenesis is Michael Karin (University of California, San Diego, USA). The Karin group has identified some of the fundamental mechanisms through which inflammation and obesity promote tumor development and progression and contribute to type II diabetes. They had established the mechanisms through which members of the IL-6 cytokine family contribute to the development of colorectal and liver cancer through activation of STAT3 and other transcription factors. They had also established the complex and cell type specific mechanisms through which NF-úB activation via IúB kinases (IKK) controls development and progression of colon, liver and prostate cancers. They were amongst the first to demonstrate that not only innate immune cells, such as macrophages, but also adaptive immune cells, including T regulatory cells and B lymphocytes, also contribute to tumorigenesis and its progression. Through this work, Dr Karin has contributed to the founding of the Inflammation and Cancer field. Dr Karin will lecture on his work at EACR24 and be presented with this award and a prize of $2500.

Carcinogenesis Young Investigator Award This award recognises a recent, significant contribution to carcinogenesis research by an investigator under the age of 40 on 1 July 2015. The winner is Dr Ludmil Alexandrov (Los Alamos National Laboratory, Los Alamos, USA). Ludmil Alexandrov is an Oppenheimer Fellow in the Theoretical Biology and Biophysics Group and the Center for Nonlinear Studies at Los Alamos National Laboratory. He earned his Bachelor of Science degree in Computer Science from Neumont University and received his Master’s of Philosophy in Computational Biology as well as his Ph.D. in Cancer Genetics from the University of Cambridge. During his PhD training, Ludmil developed the first mathematical model describing signatures of mutational processes in human cancer as well as the first computational framework for extracting these signatures from next-generation sequencing data from cancer genomes. He subsequently used this framework to map the mutational signatures in 7,042 cancer genomes providing the first comprehensive map of the mutational signatures in human cancer. More recently, he mapped the signatures of clock-like mutational processes operative in normal somatic cells and demonstrated that mutational signatures can be used for targeted cancer therapy. Dr Alexandrov will present the award lecture at EACR24 and be presented with the award and a prize of $2500.

EACR24, Awards / European Journal of Cancer 61, Suppl. 1 (2016) xv–xvii

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EACR Meeting Bursary Award Winners This year the EACR has provided 53 awards to support early-career researchers’ attendance at EACR24. Funds of £2000 to support the provision of additional EACR Meeting Bursaries have been generously provided by The Company of Biologists. Marie Mayrhofer, Germany; Lorea Valcarcel ´ Jimenez, ´ Spain; Marta Di Martile, Italy; Becky Bibby, UK; Andreana N. Holowatyj, USA; Anna-Lena Dittrich, UK; Joao Incio, USA; Manuel Pedro Jimenez-Garc´ ´ ıa, Spain; Marta Fernandez-Mercado, Spain; Marta Giussani, Italy; Oriol de Barrios Barri, Spain; Anh (Viet-Phuong) Le, Australia; Laura Escudero, UK; Brittany Wingham, UK; Nataliia Svergun, Canada; Lisa Dwane, Ireland; Marie Parsons, Australia; Mariia Inomistova, Ukraine; Sarah Argyle, UK; Alessandra Decio, Italy; Joana Vieira de Castro, Portugal; Lucilla D’Abundo, Italy; Francesca Ricci, Italy; Celine ´ Saraiva Gon¸calves, Portugal; Julie Nonnekens, Netherlands; Raghavendar Nagaraju, UK; Erika Larrea de Orte, Spain; Marina Bacci, Italy; Antonina Benfante, Italy; Felipe Maglietti, Argentina; Ka-Liong Tan, UK; Petar Ozretić, Croatia; Elisa Paolicchi, UK; Nauman Jadoon, Pakistan; Mei Fong Pan, USA; Kenan Izgi, Turkey; Sivan Gershanov, Israel; Nitin Shivappa, USA; Silva Giragosyan, Bulgaria; Oleg Tutanov, Russia; Gabor Pajor, Hungary; Rebecca Steele, Ireland; Roxana-Maria Petric, Romania; Catia ´ Alexandra Vicente Vaz, Portugal; Ana-Matea Mikecin, Croatia; Violetta Ritter, Germany; Katarzyna Jasinska, Poland; Katarina Zeljic, Serbia; Ivan Mamichev, Russia; Sylwia Katarzyna Krol, ´ Poland; Lajos-Zsolt Raduly, ¨ un, Romania; Fatma Ozg ¨ Turkey; Ana Oliveira, Portugal.

Mike Price and Lena Price Scholarship Award The Mike Price and Lena Price Scholarship Award is a commemorative bursary for one researcher from the University of Nottingham. It has been awarded in remembrance of Mike Price, who was EACR Secretary General for 21 years, and his mother, Lena. The winner of the Scholarship Award is Dr. Mohammed Aleskandarany.

Europe’s membership association for cancer researchers

WHY DO RESEARCHERS LIKE YOU JOIN THE EUROPEAN ASSOCIATION FOR CANCER RESEARCH? 98% of our members would recommend us to a colleague. Find out why: visit us at the EACR booth (A125) in the Exhibition Hall and join the EACR as a member today. Community of nearly 10,000 researchers with chances to meet new contacts & opportunities for collaborative research Communication about all the latest news, jobs and cancer research conferences *UHDWGLVFRXQWVRQUHJLVWUDWLRQUDWHVat EACR conferences & courses 7UDYHO)HOORZVKLSV of up to €2500 0HHWLQJ%XUVDULHV of up to €1200 $FFHVVWRVSRQVRUVKLS of up to €3000 for conferences you organise

-2,112: ZZZHDFURUJMRLQXV * Data from the EACR Members’ Survey, January 2016

$IIRUGDEOHPHPEHUVKLSIHHV 2SSRUWXQLWLHVWREHFRPHPRUHLQYROYHG through the EACR Ambassador Programme 5HSUHVHQWDWLRQ DGYRFDF\in European cancer policy discussion

98%

of members would recommend us to a colleague*

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Floorplans Venue Floorplan

EXCHANGE 9

EXCHANGE 10

EX. 8

UPPER LEVEL

GE

AN

CH

EX

1-7

EXCHANGE HALL

EXCHANGE AUDITORIUM

LOWER LEVEL

CHARTER 4

CHARTER 1

CLOAKROOM

CENTRAL FOYER

REGISTRATION

NETWORKING LOUNGE

CENTRAL HALL 2: EXHIBITION, POSTERS, CATERING

THE GALLERY

EXCHANGE 11

GROUND LEVEL

CENTRAL 3&4

EXCHANGE AUDITORIUM ENTRANCE

MAIN ENTRANCE

EACR24, Floorplans / European Journal of Cancer 61, Suppl. 1 (2016) xix–xx xx

Exhibition Floorplan

Posters

Catering

Posters

D495 D490

C385 C390

Posters

catering

B260

B255 B250

ACCESS TO SESSION ROOMS

Posters

catering

A160

A165 A170

A140

A135 A125

C375 C370

C380

catering

D485 D480

D475 D470

B235 B230

B240

Poster in the spotlight

Posters

A100

A115 A110

A120

catering

B225 B220

B205 B200

B210 C325 C320

B195 B190

Posters

MAIN ENTRANCE

C365 C360

C355 C340

D465 D460

D455 D440

C345

catering

D425 D420

C315 C310

B185 B180

C335 C330

D445

catering

D415 D410

C305 C300

D435 D430

D405 D400

Posters

CENTRAL FOYER

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List of Exhibitors Exhibitor name

Booth number

Acris Antibodies − OriGene EU

B205

Adaptive Biotechnologies

C310

Agilent Technologies LDA UK Ltd.

C340

American Association for Cancer Research

A110

ANGLE

D460

APTrans

D475

ArcherDX

D470

Aspect Imaging

D430

BD Life Sciences

A140

Best Theratronics Ltd.

A115

Bio-Techne

B230

Breast Cancer Now

D445

Cambridge Bioscience

B235

Cancer Research UK

C385

Cell Signaling Technology

A165

Charles River

B240

Chromatrap

B180

EACR: the European Association for Cancer Research

A125

ECACC European Collection of Authenticated Cell Cultures

C320

eLife

D490

Envigo

B220

ESMO − European Society for Medical Oncology

B190

Fluidigm

D420

GATC Biotech

C360

GeneTex

C315

Greiner Bio-One

D465

Hybrigenics Services

B185

Indivumed

B255

LGC-ATCC

B200

LI-COR Biosciences

B210

Manchester Cancer Research Centre

C325

Medical and Biological Laboratories Co., Ltd.

C305

Menarini Silicon Biosystems

D480

Miltenyi Biotec

A160

NanoString Technologies Europe Ltd

C355

New England Biolabs

C370

OcellO

A100

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EACR24, List of Exhibitors / European Journal of Cancer 61, Suppl. 1 (2016) xxi–xxii

Exhibitor name

Booth number

Organisation of European Cancer Institutes

A120

Oxford Optronix & Baker Ruskinn

C335

Phase Holographic Imaging AB

C390

PlexBio Co., Ltd.

C365

Polyplus-transfection

B225

Promega UK Ltd.

C330

QIAGEN

D450

Proteintech Europe

B250

Randox Biosciences

C380

Spandidos Publications

C300

Stratech Scientific Ltd

B195

Taylor & Francis Group

C375

The Jackson Laboratory

A170

Thermo Fisher Scientific

B260

Univadis Oncology

D440

VolitionRX

C345

Worldwide Cancer Research

A135

This list reflects confirmed exhibitors as of 1 June 2016.

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Exhibitor Profiles This list reflects confirmed exhibitors as of 1 June 2016

Acris Antibodies − OriGene EU Booth B205 www.acris-antibodies.com Acris Antibodies serves scientists since 1998 with an-easy-to-use internet platform to find the most useful antibodies. Recently acquired by OriGene, we now offer >1 million research products − everything you need for gene and protein based research: cDNA clones, siRNA, lysates, proteins, antibodies and many more. Acris Antibodies − OriGene EU is your “1 stop solution provider”. We support you as cancer research scientists in your gene as well as protein directed approaches! Adaptive Biotechnologies Booth C310 www.adaptivebiotech.com Adaptive Biotechnologies is the pioneer and leader in combining high-throughput sequencing and expert bioinformatics to profile T-cell and B-cell receptors. Adaptive is bringing the accuracy and sensitivity of its immunosequencing platform into laboratories around the world to drive groundbreaking research in cancer and other immune-mediated diseases. Adaptive also translates immunosequencing discoveries into clinical diagnostics and therapeutic development to improve patient care. Agilent Technologies LDA UK Ltd. Booth C340 www.agilent.com Agilent Technologies is a leading supplier of life science research systems that enable scientists to study complex biological processes and disease mechanisms. By integrating multiple comprehensive analyses—genomics, transcriptomics, proteomics, and metabolomics— Agilent is your partner to help you generate a better pathway-level understanding of your biological system and to be at the forefront of oncology research. American Association for Cancer Research (AACR) Booth A110 www.aacr.org The mission of the AACR is to prevent and cure cancer through research, education, communication, and collaboration. Through its programs and services, the AACR fosters research in cancer and related biomedical science; accelerates the dissemination of new research findings among scientists and others dedicated to the conquest of cancer; promotes science education and training; and advances the understanding of cancer etiology, prevention, diagnosis, and treatment throughout the world. ANGLE Booth D460 www.angleplc.com ANGLE is a UK and US based medical diagnostic company. ANGLE’s Parsortix technology enables the capture and harvest of circulating tumour cells from patient blood. The resulting liquid biopsy (simple blood test) provides a powerful tool to deliver personalised cancer care. ANGLE is currently developing its first diagnostic product in the area of ovarian cancer. The Parsortix system has CE Mark regulatory approval in Europe and an FDA approval process is underway in the US. APTrans Booth D475 www.apconix.com APTrans: Alderley Park Translational Science Consortium. A consortium that allows flexible and fully-integrated access to the core technical disciplines necessary for optimal project execution during drug development. Working with scientists, boards and investors, APTrans underpins successful drug selection and development by providing pre-eminent knowledge and experience in preclinical safety, pharmacokinetics, formulation and clinical study delivery, necessary to maximise product value.

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EACR24, Exhibitor Profiles / European Journal of Cancer 61, Suppl. 1 (2016) xxiii–xxxi

ArcherDX Booth D470 archerdx.com ArcherDX addresses the bottlenecks associated with using NGS in translational research by offering a robust platform for targeted sequencing applications. By combining proprietary Anchored Multiplexed PCR (AMP™) chemistry and easy-to-use, lyophilized reagents, Archer NGS assays generate highly enriched sequencing libraries to detect gene fusions, point mutations, CNVs and RNA abundance. Complemented by the Archer suite of bioinformatics software, ArcherDX technology dramatically enhances complex mutation identification and discovery. ArcherDX is headquartered in Boulder, Colorado.

Aspect Imaging Booth D430 www.aspectimaging.com Aspect Imaging’s M-Series one touch MRI systems are simple to operate, cost effective and high performance compact MRI scanners for rapid in vivo imaging of mice and rats. Ideal for longitudinal oncology studies, the M-Series can save hundreds of thousands of dollars when used for rapid drug efficacy studies complementing in vitro techniques. For rapid tumor screening, even behind the barrier, no MRI expertise or special works are required.

BD Life Sciences Booth A140 www.bdbiosciences.com BD Life Sciences, a segment of Becton, Dickinson and Company, is one of the world’s leading businesses focused on bringing innovative tools to life science researchers and clinicians. Its product lines include: flow cytometers and cell sorters, monoclonal antibodies, research reagents, diagnostic assays, and tools to support cell analysis.

Best Theratronics Ltd. Booth A115 www.theratronics.ca Best Theratronics Ltd., a TeamBest™ company, manufactures blood and research irradiators, and radiotherapy machines used to provide therapies to cancer patients worldwide. Best Cyclotron Systems manufactures cyclotrons ranging from energies of 15 to 70 MeV. TeamBest group of companies (including Best Medical and Best Nomos) also provides a variety of custom solutions for cancer diagnosis and treatment including brachytherapy sources and accessories, ultrasound imaging devices, treatment planning systems, QA/dosimetry instruments, and more. (www.teambest.com).

Bio-Techne Booth B230 www.bio-techne.com Bio-Techne brings together some of the most referenced brands in life science − R&D Systems, Novus Biologicals, Tocris Bioscience, and ProteinSimple providing innovative, high-quality research tools, including: • Bioactive proteins − R&D Systems premiere bioactive proteins • Application-qualified Antibodies − a diverse and extensive analyte selection from Novus and R&D Systems • Immunoassays − Legendary R&D Systems Quantikine ELISAs, our huge selection of Luminex Assays and cost effective Proteome Profiler Arrays • High quality small molecules − a unique collection of over 3,500 Tocris reagents

Breast Cancer Now Booth D445 breastcancernow.org We’re Breast Cancer Now, the UK’s largest breast cancer charity. We believe that if we all act now, by 2050, everyone who develops breast cancer will live. Our cutting-edge research is focused entirely on breast cancer. Come to our stand to find out about our grant funding opportunities and how to access high-quality breast tissues and derivatives through the Breast Cancer Now.


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Cambridge Bioscience Booth B235 www.bioscience.co.uk Cambridge Bioscience is a leading provider of life science products with a passion for bringing new and exciting technologies to researchers. We offer an innovative range of high quality products, services and instruments supporting cancer research. Our portfolio includes JuLI™ Stage, a real-time, fully-automated, fluorescence cell history recorder, Azure c600, a high performance gel and blot imaging system, and Avatar™, a ground-breaking cell culture incubator featuring accurate pressure, oxygen and CO2 control. Cancer Research UK Booth C385 www.cancerresearchuk.org Cancer Research UK is the world’s largest independent funder of cancer research, investing around £350 million every year across the entire research pipeline. We partner with a range of organisations, delivering progress in the treatment, diagnosis and prevention of all cancers. We support over 4,000 scientists, clinicians and nurses to meet our ambition of 3 in 4 people surviving cancer by 2034. Cell Signaling Technology Booth A165 www.cellsignal.com Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high quality research tools to the biomedical research community. Our employees operate worldwide from our U.S. headquarters in Massachusetts, and our offices in the Netherlands, China, and Japan. Charles River Booth B240 www.criver.com To help you identify the best fit for your oncology research, Charles River maintains a portfolio of animal models with varying levels of immunodeficiency and phenotypic characteristics. Charles River is also the exclusive distributor of JAX™ mice in Europe, including the highly immune deficient NSG mouse. every step of the way.

Chromatrap Booth B180 www.chromatrap.com Chromatrap products includes chromatin immuno-precipitation, enzymatic shearing and DNA clean-up kits with ChIP-validated antibodies and primers. Chromatrap solid-state ChIP assays are quicker, more sensitive and require less chromatin than others. The result is a quick, easy and sensitive protocol for producing high-purity DNA fragments which may be used for RT-PCR or sequencing. Chromatrap has research laboratories at Swansea University and R&D together with production facilities in the Porvair Sciences works at Wrexham, Wales. EACR Booth A125 www.eacr.org The European Association for Cancer Research (EACR) is Europe’s largest professional member society for cancer researchers with nearly 10,000 members worldwide. We provide a wide range of services to members, facilitate communication and collaboration, and organise a series of cancer research conferences where the latest research topics and interaction are the highest priorities. If you are not already an EACR member, we invite you to join us as a member at EACR24. ECACC European Collection of Authenticated Cell Cultures Booth C320 www.phe-culturecollections.org.uk ECACC currently holds over 40,000 cell lines representing 45 different species, 500 human cancer cell lines, and 50 tissue types and very recently 150 induced Pluripotent Cell Lines. ECACC is a member of EBiSC (European Bank of induced Pluripotent Cell Lines), a large European public-private partnership project. All the EBiSC cell lines are available to order direct from ECACC. ECACC has developed a comprehensive range of cell culture services including a new STR profiling service.

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eLife Booth D490 elifesciences.org eLife is a non-profit organisation inspired by research funders and led by scientists. Our mission is to help scientists accelerate discovery by operating a platform for research communication that encourages and recognises the most responsible behaviours in science. eLife publishes highly influential research in life sciences and biomedicine, including Cancer Biology, from advances in basic understanding to studies that demonstrate real-world outcomes. To explore this research, and learn more about eLife, please visit elifesciences.org

Envigo Booth B220 envigo.com Envigo provides essential research services, models and products for biopharmaceutical, crop protection, and chemical companies as well as universities, governments, and other research organizations. Our business is founded on a dedication to customer service and the expertise and experience of our 3,800 people.

ESMO − European Society for Medical Oncology Booth B190 www.esmo.org ESMO is the leading professional organisation for medical oncology. With more than 13,000 members representing oncology professionals from over 130 countries, ESMO is the society of reference for oncology education and information. ESMO’s educational resources support an integrated, multi-professional approach to cancer care. We have European roots and global reach: we welcome oncology professionals from around the world and we seek to erase boundaries in cancer care as we pursue our mission across oncology, worldwide. Fluidigm Booth D420 fluidigm.com We strive to partner with our customers to pursue truth in the complex biological world. Our contribution is the application of microfluidics and mass cytometry technologies to provide simplified and elegant workflows for life scientists making research breakthroughs and businesses providing quality services. Partner with us, whether the quest is to understand the profile and function of individual cells or to meet the high-throughput and data quality demands of a production-scale laboratory.

GATC Biotech Booth C360 www.gatc-biotech.com GATC Biotech is Europe’s leading sequencing services provider with more than 10,000 customers worldwide. Since 1990, the company has processed millions of samples from basic science to clinical diagnostics. Backed by ISO17025 accreditation and ISO13485 certification, the company handles every sample with scientific expertise and continuous innovation. GATC Biotech is particularly committed to advancing the human diagnostic field for personalized medicine by developing liquid biopsy solutions.

GeneTex Booth C315 www.genetex.com GeneTex is an internationally recognized antibody manufacturer. Our antibodies are supported by extensive research, development and validation, with products spanning cancer, cell biology, epigenetics, immunology, infectious disease, immunology and neuroscience. Founded in 1997 by three scientists with expertise in cancer research and infectious disease, GeneTex has evolved into a multinational organization, and one of the largest antibody manufacturers. We are dedicated to the highest standards of product performance, and the goal of accelerating your discovery.


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Greiner Bio-One Booth D465 www.greinerbioone.com Greiner Bio-One is a leading manufacturer and direct supplier of high quality laboratory products to the scientific research community. Focusing on the evolving needs of our customers in the development of innovative solutions our unsurpassed range includes products for cell and tissue culture, including 3D cell culture solutions, biobanking, high throughput screening, immunology, microbiology, liquid handling, molecular biology and clinical sample collection.

Hybrigenics Services Booth B185 www.hybrigenics-services.com Hybrigenics Services is a complete service provider of cutting-edge technologies dedicated to the study of proteins and protein interactions in every life science area. We help researchers understand protein functions by offering 4 lines of services: • DISCOVER novel protein interactions in various tissues and cell types • VALIDATE protein interactions and functions in cells • INHIBIT protein interactions with small molecules or natural compounds • HYBRIBODY, to select and validate highly affine single-domain antibodies targeting your protein

Indivumed Booth B255 indivumed.com Indivumed supports drug and biomarker development in the oncology field with a high quality human tumor tissue biobank and comprehensive clinical data. Key is a standardized biospecimen collection process to minimize post-operatively occurring molecular changes. Indivumed also provides services focused on target and biomarker validation for companion diagnostic, protein/phosphoprotein analytics and offers a drug testing platform for pathway analyses of therapeutic candidates. This makes Indivumed an ideal outsourcing partner for pre-clinical to clinical studies.

LGC-ATCC Booth B200 www.lgcstandards.com LGC supports cancer research throughout Europe by supplying researchers with ATCC’s extensive collection of biological resources including authenticated tumour cell lines, tumour cell panels, hTERT immortalized cells, primary cells, and human induced pluripotent stem cells (iPSCs). Moreover, LGC and ATCC have partnered to offer a complete human cell line authentication service which meets the requirements for funding, publication and quality control.

LI-COR Biosciences Booth B210 www.licor.com/bio LI-COR provides the complete solution for quantitative and chemiluminescent Western blot imaging, and a variety of other applications including in vivo imaging with its Odyssey® Infrared Imaging Systems, C-DiGit® Blot Scanner, Pearl® Trilogy Imaging System, Image Studio® analysis software and IRDye® Infrared Dye-based antibodies and reagents.

Manchester Cancer Research Centre Booth C325 www.mcrc.manchester.ac.uk The Manchester Cancer Research Centre (MCRC) is a unique collaboration that brings together the expertise, vision and resources of our partner organisations, all of which have formidable individual reputations in the field of cancer research. The Centre was formed in 2006 by The University of Manchester, Cancer Research UK and The Christie NHS Foundation Trust and unites researchers working in cancer biology, drug discovery and clinical trials across Manchester.

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Medical and Biological Laboratories Co., Ltd. Booth C305 www.mbl.co.jp/e Medical and Biological Laboratories Co., Ltd. provides MEBGEN RASKET KIT, a novel multiplex molecular tests for detection of extended RAS (KRAS and NRAS) gene mutations. MEBGEN RASKET KIT can simultaneously detect 48 RAS mutations in a single well. All the results will be obtained in 4.5 hours for up to 96 samples. MEBGEN RASKET KIT provides you a rapid batch analysis for molecular testing in all laboratories. For more information, visit our booth. Menarini Silicon Biosystems Booth D480 www.siliconbiosystems.com Menarini Silicon Biosystems has developed a platform for cell and molecular biology that sorts and collects individual or groups of rare cells. Using a proprietary electronic chip-based microfluidic cartridge and microscopic image analysis the DEPArray™ system recovers as little as one single cell from a suspension of tens of thousands of cells with 100% purity. The technology has application in the molecular characterization of Circulating Tumor Cells and analysis of FFPE samples. Miltenyi Biotec Booth A160 www.miltenyibiotec.com Miltenyi Biotec provides products that advance biomedical research and cellular therapy. Our innovative tools support research from basic research to translational research to clinical application. Our more than 25 years of expertise includes immunology, stem cell biology, neuroscience, and cancer. Miltenyi Biotec has more than 1,500 employees in 25 countries. NanoString Technologies Europe Ltd Booth C355 www.nanostring.com NanoString Technologies provides life science tools for translational research and molecular diagnostic products. The company’s nCounter® Analysis System, which has been employed in basic and translational research and cited in 500 peer review publications, has also now been applied to diagnostic use with the nCounter Dx Analysis System. The nCounter-based Prosigna™ Breast Cancer Prognostic Gene Signature Assay is marketed for use on the nCounter Dx Analysis System which is FDA 510(k) cleared. New England Biolabs Booth C370 www.neb.com New England Biolabs manufactures reagents for molecular biology and is a global market leader in the supply of restriction enzymes, polymerases, modifying enzymes and DNA ladders. The corner-stones of NEB’s reputation are service excellence and products you can trust. Cell Signaling Technology products are exclusively distributed in the UK by NEB. CST, active in systems biology research, produces, validates and supports all their antibodies in-house to help ensure high levels of specificity. OcellO Booth A100 ocello.nl OcellO is a CRO offering state-of-the-art phenotypic drug screening and profiling services using cultured human tissues and organoids. Culturing patient and PDX derived cells in 3D in natural extracellular matrix in a 384 well format enables high throughput testing while retaining optimum translation to in vivo models. Over 150 3D models available for oncology, immune-oncology, polycystic kidney disease and other indications. Organisation of European Cancer Institutes Booth A120 www.oeci.eu The Organisation of European Cancer Institutes, www.oeci.eu is a not-for-profit Organisation, formally registered as European Economic Interest Grouping and headquartered in Brussels. The OECI Members share a common mission of creating a critical mass of expertise to build and maintain a consensus on the best models of education, research, care, and organisation of a cancer centre. These objectives are supported by the OECI Accreditation and Designation Programme.


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Oxford Optronix & Baker Ruskinn Booth C335 www.oxford-optronix.com www.bakerco.com/baker-ruskinn-welcome Baker Ruskinn: The Ruskinn brand, your trusted source for precise environmental control technologies, has now evolved into Baker Ruskinn, developing solutions for cell biology, stem cell and regenerative medicine, including accurate and stable anaerobic chambers, hypoxia workstations, and precise oxygen regulation in culture media. Oxford Optronix: Pioneering instrumentation for the life scientist developed by Oxford Optronix Ltd. includes a physiological dissolved oxygen monitor and a novel, compact hypoxia workstation. Phase Holographic Imaging AB Booth C390 www.phiab.se Phase Holographic Imaging (PHI) develops time-lapse cytometry instrumentation and software. Cell proliferation, cell death and cell motility can be studied without toxic stains or labels on adherent cells. A multitude of quantifying data can be extracted from a single image or a time-lapse series of images. The instrument and software can be applied for among others cancer research, stem cell biology and toxicological studies. PHI trades through a network of international distributors. PlexBio Co., Ltd. Booth C365 www.plexbio.com PlexBio Co., Ltd., established in May 2010, designs, develops and manufactures CEIVD products and instrumentations in ISO-certified facilities. To this date, PlexBio has expanded from its headquarters in Taipei, Taiwan to Jiangsu Province, China, and has an Innovative Technology Center in San Francisco, USA. Polyplus-transfection Booth B225 www.polyplus-transfection.com Polyplus-transfection is a biotech company that has been developing transfection reagents for over 10 years. Its wide range of reagents can be used for the in vivo and in vitro delivery of nucleic acids (DNA, siRNA, miRNA, . . . ) for research, bioproduction and therapeutics. Promega UK Ltd. Booth C330 www.promega.co.uk Promega Corporation is a leader in providing innovative solutions and technical support to the life sciences industry. Promega Corporation’s 3,500 products enable scientists worldwide to advance their knowledge in life science research, particularly in genomics, proteomics, and cellular analysis. Our products and services support scientists asking fundamental questions about biological processes apply scientific knowledge to understand the mechanisms of disease, discover new therapeutics, and use genetics and DNA testing for human identification. Proteintech Europe Booth B250 www.ptglab.com Proteintech Group currently has over 10,000 antibodies in its catalog, all of which have been by a specialist in-house team in multiple species and multiple applications. Over 90% of our antibodies are raised against the whole recombinant protein which gives them superior protein recognition capabilities and versatility. QIAGEN Booth D450 www.qiagen.com QIAGEN is the leading global provider of Sample to Insight solutions to transform biological materials into valuable molecular insights. QIAGEN sample technologies isolate and process DNA, RNA and proteins from blood, tissue and other materials. Assay technologies make these biomolecules visible and ready for analysis. Bioinformatics software and knowledge bases interpret data to report relevant, actionable insights. Automation solutions tie these together in seamless and cost-effective molecular testing workflows.

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Randox Biosciences Booth C380 www.randox.com Randox Biosciences is dedicated to advancing scientific discovery, drug development and diagnostics. We provide custom solutions for clinical and research use along with an extensive product range including over 85 oncology biomarkers that can be assayed on our award winning Biochip Array Technology. We produce the majority of our products in-house and are therefore much more flexible in terms of adapting to the methods of your research project, making it cost effective, efficient and safe.

Spandidos Publications Booth C300 www.spandidos-publications.com Spandidos Publications was founded in 1992 and has developed into a leading publishing group in the biomedical sciences field. We currently publish eight journals: International Journal of Molecular Medicine, International Journal of Oncology, Molecular Medicine Reports, Oncology Reports, Experimental and Therapeutic Medicine, Oncology Letters, Biomedical Reports and Molecular and Clinical Oncology. All journals published by Spandidos Publications maintain the highest standards of quality and the members of their Editorial Boards are world-renowned scientists.

Stratech Scientific Ltd Booth B195 www.stratech.co.uk Stratech Scientific Ltd supplies over 1.8 million life science products for researchers who need consistent, reproducible results. We have built an excellent reputation over 34 years for supplying high quality, competitively priced and reliable products to our end users. We are a family run business dedicated to delivering exceptional product quality with unbeatable technical support. We are so confident you will love our products, we guarantee them all with a full money back promise.

Taylor & Francis Group Booth C375 www.tandfonline.com Find the right journal for your research with Taylor & Francis. Taylor & Francis publish more than 250 peer-reviewed journals spanning the complete spectrum of medicine − from bench to bedside and beyond. If you are looking to submit research, then we have a publication to match. Explore our titles in Oncology: bit.ly/hemtandf Visit www.tandfonline.com for more information about our titles, or drop by the Taylor & Francis stand at any time during the conference.

The Jackson Laboratory Booth A170 www.jax.org The Jackson Laboratory is a leading provider of mouse models and in vivo oncology services. Drawing on decades of mouse-based research experience, we provide oncology study protocols ranging from traditional xenograft services to patient-derived xenograft cancer models, model generation services and advanced humanized mouse models for immuno-oncology drug development.

Thermo Fisher Scientific Booth B260 www.thermofisher.com Thermo Fisher Scientific is the world leader in serving science. Our mission is to enable our customers to make the world healthier, cleaner and safer. We support cancer researchers with innovative technologies and service through our Thermo Scientific, Applied Biosystems, Invitrogen, Gibco, Ion Torrent and Affymetrix brands.


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Univadis Oncology Booth D440 www.univadis.com Univadis® Oncology offers a range of evidence-based oncology information and decision-support tools to help Oncology Clinicians: − Stay up to date with practice-changing clinical research − Easily find relevant clinical trials for their patients − Keep up with the latest clinical breakthroughs in oncology − Access a comprehensive source of authoritative guidelines & decision-support tools Univadis® is a free service from Aptus Health, exclusively for healthcare professionals. VolitionRX Booth C345 www.volitionrx.com VolitionRx is developing blood-based diagnostic tests for the early detection of cancer using our proprietary Nucleosomics® immunoassay platform to profile global levels of epigenetic features of circulating cell-free nucleosomes. Our goal is to provide affordable, accessible and easily implemented tests for a range of cancers. VolitionRx’s R&D and manufacturing activities are centred in Belgium with initial focus on bringing CE marked NuQ® diagnostic products to market in Europe, followed by the U.S. and worldwide. Worldwide Cancer Research Booth A135 www.worldwidecancerresearch.org Worldwide Cancer Research funds some of the most ambitious early-stage cancer research around the world and in 35 years has awarded almost £200 million in research grants to support some of the world’s best scientists across 34 different countries, supporting over 1,750 research projects. Our horizons are broad and our vision bold but we believe we will, with the right research, see no life cut short by cancer.

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Satellite Symposia Sunday 10 July 2016

Satellite Symposium: Thermo Fisher Scientific Pioneering Precision Medicine: innovative approaches for solid tumour research

Charter 4, 12:30−14:00

12:30−12:40 Personalised Medicine Solutions Andy Felton. Product Management, Thermo Fisher Scientific 12:40−13:00 MicroRNAs in Nipple Aspiration Fluid (NAF): A novel, non-invasive way to study breast cancer Cathy Moelans, PhD. Department of Pathology, University Medical Center Utrecht (UMCU), The Netherlands 13:00−13:20 NGS analysis of Cell-free DNA and CTCs in breast cancer Professor Jacqui Shaw, Professor of Translational Cancer Genetics, Cancer Diagnostics Facility, Department of Cancer Studies, University of Leicester, UK 13:20−13:40 What sample: from tissue to liquid biopsy Prof. Aldo Scarpa, Director of the ARC-Net Research Centre for Applied Research on Cancer, Verona, Italy 13:40−13:50 A role for proteogenomics in precision oncology? Martin Hornshaw, Marketing Manager, Thermo Fisher Scientific

EACR24, Satellite Symposia / European Journal of Cancer 61, Suppl. 1 (2016) xxxii–xxxiii

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Monday 11 July 2016

Satellite Symposium: Affymetrix Clinical Application of Microarrays in Cancer

Charter 4, 12:30−14:00

12:30−13:00 Identification/Discovery of biomarkers for Bladder Cancer Rik Bryan, Chief Investigator Institute Cancer & Genomic Sciences, University of Birmingham, UK 13:00−13:30 Development of the Decipher Bladder cancer diagnostic product Hugh Wellman, Director, Licensing and IP, GenomeDX, Canada 13:30−14:00 CytoScan HD array analysis of childhood leukemia Karin Nebral, St. Anna Kinderkrebsforschung, CCRI, Children’s Cancer Research Institute, Vienna, Austria

Satellite Symposium: BD Life Sciences Innovative flow cytometry and genomic solutions to embrace the heterogeneity in tumours and accelerate your cancer research with confidence

Exchange 9, 12:30−14:00

12:30−13:15 Deciphering tumour heterogeneity using cell surface markers Ievgenia Pastushenko, IRIBHM, Universite´ Libre de Bruxelles, Belgium 13:15−14:00 From Tissue to Cells to Molecular Analyses: Innovative Tools for Cancer Studies Rainer Blaesius PhD. BD Life Sciences

xxxiv

QIAGEN Special Session Sunday 10 July 2016

QIAGEN’s new NGS solutions for Cancer Research − a sample to insight approach

Exchange 9, 19:00−20:30

19:00−19:30 The GeneReader NGS System: experience of a complete NGS workflow solution ¨ PD. Dr. Margarethe Odenthal, Pathology Institute, Universitatsklinikum Cologne, Germany 19:30−20:00 Digital sequencing with molecular barcodes: a sample-to-insight solution for DNA and RNA analysis Raed Samara, PhD, Global Product Manager BRC, QIAGEN, USA 20:00−20:30 Evaluating the potential of blood borne biomarkers through NGS in a clinical setting Dr Dominic Graham Rothwell, CEP Staff Scientist, Clinical and Experimental Pharmacology Group, Cancer Research UK Manchester Institute, The University of Manchester, UK

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Programme at a Glance Saturday 09 July 2016 Exchange Hall

Sunday 10 July 2016 Exchange Hall

Symposium 08:30 - 10:15 Tumour Heterogeneity and Evoluon Chair: Carlos Caldas Benjamin Izar Andrea So oriva Proffered Paper: V. Chigancas Carlos Caldas

Exchange Auditorium

Charter 1

Symposium 08:30 - 10:15 DNA Damage Chair: Stephen Jackson Fabrizio D'Adda di Fagagna Don W. Cleveland Proffered Paper: N. Cordes Stephen Jackson

Symposium 08:30 - 10:15 Tumour Metabolism Chair: Karen Vousden Ma Vander Heiden Eyal Go lieb Proffered Paper: J. Pelleer Celeste Simon

Opening Address 12:30 - 13:15 Chair: Richard Marais Kumar Harpal

Mühlbock Lecture 13:15 - 14:15 Serine Metabolism and Cancer Therapy Chair: Moshe Oren Karen Vousden

Poster Viewing / Exhibion 12:30 - 14:00

Satellite Symposium Charter 4 12:30-14:00

Worldwide Cancer Research Lecture 14:15 - 15:00 Chair: Anton Berns Mariano Barbacid

Symposium 14:00 - 15:45 The Tumour Microenvironment Chair: Clare Isacke Krisan Pietras Alan Serrels Proffered Paper: E. Farge Peter S. Nelson

Opening Ceremony / Opening Lecture supported by Susan G. Komen 17:00-17:50 Chair: Richard Marais David Livingston

Followed by Exhibitors' Welcome Recepon 18:00 - 19:30

Exhibion 14:45 - 19:30

Coffee Break (Exhibion Hall) 15:00 - 15:30 OECI supported Plenary Symposium 15:30 - 17:00 Cancer Immunology Chair: Christof von Kalle James P. Allison Adrian Hayday Paul Tumeh

Symposium 10:45 - 12:30 Mechanisms of Drug Resistance Chair: Daniel Peeper Ravid Straussmann Susan Galbraith Proffered Paper: M. Smith Daniel Peeper

Symposium 14:00 - 15:45 Liquid Biopsies in Cancer Chair: Caroline Dive Alberto Bardelli Klaus Pantel Proffered Paper: C. Massie Caroline Dive

Coffee Break 15:45-16:15 Poster Defence / Poster Spotlight Session / Exhibion 15:45-17:15 Anthony Dipple Carcinogenesis Award 17:15 - 18:00 Chair: Curs C. Harris Michael Karin Carcinogenesis Young Invesgator's Award 18:00 - 18:45 Chair: Curs C. Harris Ludmil B. Alexandrov

General Assembly Charter 4 19:00 - 19:45

Special Session Exchange Room 9 19:00 - 20:30

Symposium 10:45 - 12:30 Epigenecs Chair: Geneviève Almouzni Rolf Ohlsson Maarten van Lohuizen Proffered Paper: V. Marcel Geneviève Almouzni

Young Cancer Researcher Workshop: Scienfic Speed Networking Foyer 13:00 - 14:00 Coordinators: Eli Pikarsky Emmy Verschuren

Symposium 14:00 - 15:45 Senescence and Ageing Chair: Clemens Schmi Valery Krizhanovsky William Keyes Proffered Paper: P. Morancho Clemens Schmi

Exhibion 10:00 - 17:15

Symposium 10:45 - 12:30 Cancer Immunotherapy Chair: To be announced Zelig Eshhar Sanago Zelenay Proffered Paper: C. Herold-Mende To be announced

Poster Viewing 10:15 - 17:15

Coffee Break / Poster Viewing / Exhibion 10:15-10:45

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EACR24, Programme at a Glance / European Journal of Cancer 61, Suppl. 1 (2016) xxxv–xxxvi

Monday 11 July 2016 Exchange Hall

Exchange Auditorium

ESMO Symposium 08:30 - 10:15 Clinical Developments in the Diagnosis and Treatment of Solid Tumors Chairs: Richard Marais & Fortunato Ciardiello Fabrice André Fortunato Ciardiello Proffered Paper: V. Jendrossek Rolf Stahel

Symposium 08:30 - 10:15 Stem Cells Chair: Sean Morrison Cedric Blanpain Connie J. Eaves Proffered Paper: JD. Barros-Silva Sean Morrison

Tuesday 12 July 2016 Charter 1

Exchange Hall Plenary Lecture 08:45 - 09:30 Mouse Models of Lung Cancer Chair: Donatella Del Bufalo Anton Berns

Symposium 08:30 - 10:15 Breakthrough Tools in Cancer Genecs Chair: Emmy Verschuren Joakim Lundeberg Zoltan Szallasi Proffered Paper: S. Mamlouk Serena Nik-Zainal

The Pezcoller Foundaon - EACR Cancer Researcher Award Lecture 09:30 - 10:15 Chair: Moshe Oren Yardena Samuels

Coffee Break 10:15-10:45

BACR supported Symposium 10:45 - 12:30 Invasion and Metastasis Chairs: Michelle Garre & Ingunn Holen Erik Sahai Gerhard Christofori Proffered Paper: H. Hosseini Margaret Frame

Satellite Symposium Exchange Room 9 12:30-14:00

Satellite Symposium Charter 4 12:30-14:00

Young Cancer Researcher Workshop: Women in Science Foyer 13:00 - 14:00 Coordinators: Françoise Meunier Anne-Lise Børresen-Dale

Symposium 14:00 - 15:45 Inflammaon and Cancer Chair: Eli Pikarsky Karin de Visser Alberto Mantovani Proffered Paper: J. Incio Eli Pikarsky

Symposium 14:00 - 15:45 Breakthrough Cancer Models Chair: Ultan McDermo Richard A. Flavell Chris Bakal Proffered Paper: F. Kuehnel Ultan McDermo

Poster Viewing / Exhibion 12:30 - 14:00

Symposium 14:00 - 15:45 Novel Targeted Therapies Chair: Joan Seoane Tim Somervaille Stefan Pfister Proffered Paper: J. Koach Joan Seoane

Symposium 10:45 - 12:30 Tumour Suppressors Chair: Moshe Oren Xin Lu To be announced Proffered Paper: T. Hieken Moshe Oren

Coffee Break 15:45-16:15 Poster Defence / Poster Spotlight Session / Exhibion 15:45-17:15

The Company of Biologists supported Plenary Symposium 17:15 - 18:45 Signalling Networks in Cancer Chair: Joan Seoane Michael Yaffe Douglas A. Lauffenburger

Congress Dinner 20:00

Plenary Symposium 10:45 - 12:15 Precision Medicine Chair: Carlos Caldas Jean-Charles Soria Christophe Le Tourneau Rodrigo Dienstmann

Exhibion 10:00 - 17:15

Symposium 10:45 - 12:30 Diagnosing Cancer Earlier Chair: Paul Pharoah William C. Hahn Yoryos Lyratzopoulos Proffered Paper: E. Harkness Paul Pharoah

Poster Viewing 10:15 - 17:15

Coffee Break / Poster Viewing / Exhibion 10:15-10:45

Closing Remarks & EACR24 Highlights 12:30 Mike Price Gold Medal Award Lecture 12:50 - 13:30 Chair: Anton Berns Mike Stra on

xxxvii

Scientific Programme Abstract Nr.

Abstract Nr.

Saturday 9 July 2016

Sunday 10 July 2016

12:30–13:15

08:30–10:15

Opening Address (Exchange Hall)

12:30

Welcome Message R. Marais (United Kingdom)

12:40

K. Harpal (United Kingdom)

13:15–14:15 13:15

Muhlbock ¨ Lecture (Exchange Hall) Chair: M. Oren (Israel) Serine metabolism and cancer therapy K. Vousden (United Kingdom)

14:15–15:00

Worldwide Cancer Research Lecture (Exchange Hall) Chair: A. Berns (Netherlands)

14:15

Deciphering the RAS/MAPK signaling pathway in cancer M. Barbacid (Spain)

15:30–17:00

OECI supported Plenary Symposium: Cancer Immunology (Exchange Hall) Chair: C. Von Kalle (Germany)

15:30

Immune checkpoint blockade in cancer therapy: New insights, opportunities, and prospects for a cure J.P. Allison (USA)

16:00

Novel candidates for check point regulation of T cells in tissues and tumours A. Hayday (United Kingdom)

16:30

Defining the clinical value of mapping cancer organization P. Tumeh (USA)

17:00–17:50

17:00

18:00–19:30

Opening Ceremony: Opening Lecture supported by Susan G. Komen (Exchange Hall) Chair: R. Marais (United Kingdom) New insights into BRCA1 function D.M. Livingston (USA) Exhibitors’ Welcome Reception (Exhibition Hall)

Symposium: Tumour Heterogeneity and Evolution (Exchange Hall) Chair: C. Caldas (United Kingdom)

08:30

Dissecting malignant and non-malignant heterogeneity by single-cell rna-seq B. Izar (USA) 09:00 Functional and non-functional intra-tumour heterogeneity A. Sottoriva (United Kingdom) 09:30 Proffered Paper: Functional study of glioblastoma (GBM) intratumour genomic heterogeneity and evolution using patientderived xenografts (PDXs) V. Chigancas, A. Gonzalez-Junc ` a, ` J.L. Parra, R. Mayor, J. Seoane (Spain) 09:45 Tumour heterogeneity in breast cancer − what do we know? C. Caldas (United Kingdom) 08:30–10:15

1

Symposium: DNA Damage (Exchange Auditorium) Chair: S.P. Jackson (United Kingdom)

08:30

The role of non coding RNA in DNA damage response activation F. D’Adda di Fagagna (Italy) 09:00 Boveri revisited: Aneuploidy and tumorigenesis D.W. Cleveland (USA) 09:30 Proffered Paper: Targeting of beta1 integrins compromises DNA damage repair for radiosensitization of head and neck cancer cells E. Dickreuter, I. Eke, M. Krause, K. Borgmann, M. Van Vugt, N. Cordes (Germany) 09:45 Cellular responses to DNA damage: From molecular insights to new cancer therapies S.P. Jackson (United Kingdom) 08:30–10:15

2

Symposium: Tumour Metabolism (Charter 1) Chair: K. Vousden (United Kingdom)

08:30

Metabolic regulation in tumours M. Vander Heiden (USA)

09:00

Adaptable metabolic traits of cancer cells under environmental and genetic stress E. Gottlieb (United Kingdom)

09:30

Proffered Paper: Targeting GMP synthesis reveals a hierarchy of p53-cell cycle checkpoints in c-Myc driven CRCs J. Pelletier , F. Riano-Canalias, ˜ R. Salazar, A. Villanueva, O. Yanes, S. Kozma, G. Thomas (Spain)

09:45

Hypoxia, metabolism, and tumour progression C.M. Simon (USA)

3

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EACR24, Scientific Programme / European Journal of Cancer 61, Suppl. 1 (2016) xxxvii–lxviii

Abstract Nr.

Abstract Nr. 10:45–12:30

13:00–14:00

Symposium: Cancer Immunotherapy (Exchange Hall) Chair: To be announced

10:45

The CAR approach; From the mouse cage to the patient’s health Z. Eshhar (Israel)

11:15

The mechanistic basis of cancer immunotherapy S. Zelenay (United Kingdom)

11:45

Proffered Paper: Immunological changes during tumor progression from primary to recurrent glioblastoma C. Herold-Mende, C. Rapp, R. Warta, A. Unterberg, P. Beckhove (Germany)

12:00

Melanoma immunotherapy: Where are we now? To be announced

4

Experts to meet: R. Marais (United Kingdom), J. Seoane (Spain), C. Dive (United Kingdom), C. Caldas (United Kingdom), M. Oren (Israel), D. Del Bufalo (Italy), D. Peeper (Netherlands), C. von Kalle (Germany), C. Isacke (United Kingdom), E. Sahai (United Kingdom), K. de Visser (Netherlands), A. Berns (Netherlands) This is an informal one-hour session to enable participants to make lots of new connections in a short space of time. Workshop participants are divided in six groups based on their research interests; each group will include two experts/board members. Every six minutes participants will be asked to rotate and speak with a next person in their group. This is a series of one-on-one speed networking meetings, aiming for a concrete action post EACR24. 14:00–15:45

10:45–12:30

Young Cancer Researcher Workshop: Scientific Speed Networking (Foyer) Coordinators: E. Pikarsky (Israel), E. Verschuren (Finland)

Symposium: The Tumour Microenvironment (Exchange Hall) Chair: C. Isacke (United Kingdom)

Symposium: Mechanisms of Drug Resistance (Exchange Auditorium) Chair: D. Peeper (Netherlands)

14:00

10:45

The tumour microbiome and its effects on drug resistance R. Straussman (Israel)

Microenvironmental control of breast cancer subtype K. Pietras (Sweden)

14:30

11:15

Developing combinations to overcome resistance and pathway feedback S. Galbraith (United Kingdom)

New connections in cancer cell–host immune signalling A. Serrels (United Kingdom)

15:00

11:45

Proffered Paper: Utilising non-mutational drug-tolerance prolongs and improves targeted melanoma therapy M.P. Smith, H. Brunton, E.J. Rowling, M.R. Girotti, R. Marais, R. Dummer, K.T. Flaherty, Z.A. Cooper, J.A. Wargo, C. Wellbrock (United Kingdom)

12:00

Systematic genetic perturbations to reveal cancer cell dependencies D. Peeper (Netherlands)

Proffered Paper: Mechanical induction of the tumourogenic beta-catenin pathway by tumour growth pressure M.E. Fernandez-S ´ anchez, ´ S. Barbier, J. Whitehead, G. Bealle, ´ A. Michel, H. Latorre-Ossa, C. Rey, L. Fouassier, A. Claperon, L. Brulle, ´ E. Girard, N. Servant, T. Rio-Frio, H. Marie, S. Lesieur, C. Housset, J.-L. Gennisson, M. Tanter, C. Menager, ´ S. Fre, S. Robine, E. Farge (France)

15:15

Targeting microenvironment damage responses to improve treatment outcomes P.S. Nelson (USA)

14:00–15:45

Symposium: Liquid Biopsies in Cancer (Exchange Auditorium) Chair: C. Dive (United Kingdom)

10:45–12:30

Symposium: Epigenetics (Charter 1) Chair: G. Almouzni (France)

10:45

Epimutations and cancer: The nuclear architecture connection R. Ohlsson (Sweden)

11:15

Role of Polycomb repressors in cell fate control and cancer: Consequences for epigenetic therapy M. van Lohuizen (Netherlands)

11:45

Proffered Paper: The rRNA epigenetic hypothesis: role of ribosome heterogeneity in tumorigenesis V. Marcel, F. Nguyen Van Long, N. Pion, J. Erales, J.C. Bourdon, A. Puisieux, I. Motorine, P. Bouvet, F. Catez, J.J. Diaz (France)

12:00

Chromatin determinants of centromeric identity and their deregulation in cancer G. Almouzni (France)

5

14:00

Clonal evolution and drug resistance: From cancer avatars to liquid biopsies A. Bardelli (Italy)

14:30

Molecular and functional characterization of circulating tumour cells K. Pantel (Germany)

15:00

Proffered Paper: Enhancing sensitivity for analysis of circulating tumour DNA C. Massie, N. Rosenfeld (United Kingdom)

15:15

Lung cancer circulating tumour cells − What are they good for? C. Dive (United Kingdom)

6

7

8


Abstract Nr.


Symposium: Senescence and Ageing (Charter 1) Chair: C.A. Schmitt (Germany)

14:00

Directed elimination of senescent cells − mechanisms and consequences V. Krizhanovsky (Israel)

14:30

Cellular senescence in cancer and ageing W. Keyes (Spain)

15:00

Proffered Paper: Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence B. Morancho (Spain)

15:15

16:15–17:05

Monday 11 July 2016 08:30–10:15

9

Senescence and cancer therapy C.A. Schmitt (Germany)

917

16:25

Comprehensive sequencing-based characterisation of the DNA methylation landscape of 1300 breast tumours R.N. Batra (United Kingdom)

102

16:35

Phosphopeptides as novel tumour antigens in colorectal cancer S. Penny (United Kingdom)

915

16:45

Modelling the tumour microenvironment and employing functional genomics identifies CREBBP as a novel tumour suppressor in triple negative breast cancers B. Peck (United Kingdom)

362

Genomic evolution and heterogeneity in 2,800 cancers D. Wedge (United Kingdom)

361

16:55

17:15–18:00

17:15

18:00–18:45

18:00

Award Lecture: Anthony Dipple Carcinogenesis Award (Exchange Hall) Chair: C.C. Harris (USA) p62-dependent NRF2 activation and CD44 signaling: New insights to liver cancer development and treatment M. Karin (USA) Award Lecture: Carcinogenesis Young Investigator Award (Exchange Hall) Chair: C.C. Harris (USA)

ESMO Symposium: Clinical Developments in the Diagnosis and Treatment of Solid Tumors (Exchange Hall) Chairs: R. Marais (United Kingdom), F. Ciardiello (Italy)

08:30

Breast cancer F. Andre´ (France)

09:00

GI malignancies F. Ciardiello (Italy)

09:30

Proffered Paper: Radiation-induced lung 10 fibrosis: from molecular mechanisms to novel treatment concepts F. Wirsdorfer, ¨ S. De Leve, F. Cappuccini, A.V. Meyer, L.F. Thompson, H. Karmouty-Quintana, D. Klein, M.R. Blackburn, M. Stuschke, V. Jendrossek (Germany)

09:45

Big leaps in the treatment of lung cancer R.A. Stahel (Switzerland)

Poster in the Spotlight Session I (Exhibition Hall) IKKe/IKBKE integrates LPS and IL-17A signaling cascades to establish an inflammatory microenvironment in intestinal tumors S. Goktuna (Turkey)

16:15

xxxix

08:30–10:15

Symposium: Stem Cells (Exchange Auditorium) Chair: S.J. Morrison (USA)

08:30

Cancer cell of origin and tumour heterogeneity C. Blanpain (Belgium)

09:00

Clonal dynamics of malignant stem cells C.J. Eaves (Canada)

09:30

Proffered Paper: Exploring prostate progenitor compartment complexity as a route of targeting prostate cancer heterogeneity J.D. Barros-Silva, D. Linn, I. Steiner, G. Guo, G.C. Yuan, S. Orkin, Z. Li, E. Baena (United Kingdom)

09:45

Recent progress in in vivo reprogramming S.J. Morrison (USA)

08:30–10:15

Symposium: Breakthrough Tools in Cancer Genetics (Charter 1) Chair: E. Verschuren (Finland)

08:30

In situ sequencing for mutation and expression profiling within tumour tissue sections J. Lundeberg (Sweden)

09:00

Detecting and quantifying DNA repair pathway aberrations and mutational processes in human tumor biopsies in order to enable personalized cancer therapy Z. Szallasi (USA)

09:30

Proffered Paper: NGS-based 3D reconstruction reveals copy number changes as the major source of genomic heterogeneity in colorectal cancer S. Mamlouk, L. Childs, C. Oliveira, H. Daniel, F. Klauschen, D. Aust, M. Morkel, T. Wolf, R. Schafer, ¨ C. Sers (Germany)

09:45

A compendium of 560 breast cancer genomes S. Nik-Zainal (United Kingdom)

Understanding mutational processes in human cancer L.B. Alexandrov (USA)

11

12

xl


Abstract Nr.


Symposium: Diagnosing Cancer Earlier (Exchange Hall) Chair: P. Pharoah (United Kingdom)

10:45

Systematic functional interrogation of cancer W.C. Hahn (USA)

11:15

Markers and measures of timeliness of cancer diagnosis in symptomatic patients, and their predictors and outcomes: Lessons from the past and challenges ahead Y. Lyratzopoulos (United Kingdom)

11:45

12:00

10:45–12:30

Proffered Paper: A comparison of four 13 methods of mammographic density measurement in the UK Predicting Risk Of Cancer At Screening (PROCAS) study − on behalf of the PROCAS Study team S. Astley, E. Harkness, J. Sergeant, P. Stavrinos, R. Warren, M. Wilson, A. Brentnall, J. Cuzick, A. Howell, G. Evans (United Kingdom) Polygenic risk and the targeted prevention of cancer P. Pharoah (United Kingdom) BACR supported Symposium: Invasion and Metastasis (Exchange Auditorium) Chair: M. Garrett (United Kingdom) Co-Chair: I. Holen (United Kingdom)

13:00–14:00

Young Cancer Researcher Workshop: Women in Science (Foyer) Coordinators: F. Meunier (Belgium), A. Børresen-Dale (Norway)

14:00–15:45

Symposium: Novel Targeted Therapies (Exchange Hall) Chair: J. Seoane (Spain)

14:00

Epigenetic approaches to therapy in acute myeloid leukaemia T. Somervaille (United Kingdom)

14:30

Novel targeted therapies in relapsed childhood malignancies S. Pfister (Germany)

15:00

Proffered Paper: Targeting PA2G4, a novel MYCN co-factor, for the treatment of neuroblastoma J. Koach, J.E. Murray, J. McCarroll, G. Milazzo, G. Perini, M. Haber, M.D. Norris, J.I. Fletcher, B.B. Cheung, G.M. Marshall (Australia)

15:15

New therapeutic targets in glioblastoma J. Seoane (Spain)

14:00–15:45

Symposium: Inflammation and Cancer (Exchange Auditorium) Chair: E. Pikarsky (Israel)

10:45

Invasion and beyond − the role of cancer-associated fibroblasts E. Sahai (United Kingdom)

14:00

Cancer-induced systemic inflammation promotes breast cancer metastasis K. de Visser (Netherlands)

11:15

Transcriptional regulation of EMT and cancer metastasis G. Christofori (Switzerland)

14:30

11:45

Proffered Paper: Cell density, Her2 and progesterone signaling regulate dissemination of breast cancer cells H. Hosseini, K. Harper, M. Obradovic, M. Soledad Sosa, L.K. Nanduri, M. Hoffmann, C. Werno, J.A. Aguirre-Ghiso, C.A. Klein (Germany)

A complement/macrophage dependent oncosuppressor pathway in mouse and human tumours A. Mantovani (Italy)

15:00

Proffered Paper: Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy J. Incio, P. Suboj, S. Chin, H. Liu, R. Soares, Y. Boucher, D. Fukumura, R. Jain (USA)

15:15

Immune microniches promoting tumor progenitor cells E. Pikarsky (Israel)

12:00

10:45–12:30 10:45

Targeting and imaging invasion and metastasis M. Frame (United Kingdom) Symposium: Tumour Suppressors (Charter 1) Chair: M. Oren (Israel)

14:00–15:45

Cellular plasticity, tumour heterogeneity and single cell biology X. Lu (United Kingdom)

11:15

To be announced To be announced

11:45

Proffered Paper: In young women with atypical hyperplasia, high ERb expression in background breast lobules correlates with decreased risk of future breast cancer T. Hieken, J. Carter, J. Hawse, T. Hoskin, M. Bois, M. Frost, L. Hartmann, D. Radisky, D. Visscher, A. Degnim (USA)

12:00

14

RNF20: Linking chromatin, ubiquitin and tumour suppression M. Oren (Israel)

15

16

17

Symposium: Breakthrough Cancer Models (Charter 1) Chair: U. McDermott (United Kingdom)

14:00

Inflammasomes in health, dysbiosis and disease R.A. Flavell (USA)

14:30

Describing how phenotypic heterogeneity emerges in cancer cell populations C. Bakal (United Kingdom)

15:00

Proffered Paper: Establishment of resectable transgenic mouse models of pancreatic ductal adenocarcinoma for investigations on adjuvant therapies E. Guerlevik, B. Fleischmann-Mundt, J. Brooks, N. Woller, M. Manns, S. Kubicka, F. Kuehnel (Germany)

15:15

Modelling drug resistance in cancer using genome-wide CRIPSR and ENU genetic screens U. McDermott (United Kingdom)

18


Abstract Nr.

Abstract Nr. 16:15–17:05 16:15

16:25

16:35

16:45

16:55

17:15–18:45

17:15

18:00

Poster in the Spotlight Session II (Exhibition Hall)

Tuesday 12 July 2016

Mimicking disease progression features by modulation of the tumour microenvironment in stirred-tank culture systems M.F. Estrada (Portugal)

363

Genome-wide AFB1-induced mutational signature in cells, mice and human tumors − implications for molecular epidemiology J. Zavadil (France)

676

RNAi and CRISPR/Cas9 based in vivo models for drug discovery P. Premsrirut (USA)

898

Transcriptional regulation of prostate cancer metabolism L. Valcarcel (Spain)

756

Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing J. Demeulemeester (United Kingdom)

145

08:45–09:30 08:45

09:30–10:15

The Company of Biologists supported Plenary Symposium: Signalling Networks in Cancer (Exchange Hall) Chair: J. Seoane (Spain) Modulating growth factor and stressresponse signaling to improve cytotoxic cancer therapy M.B. Yaffe (USA)

xli

Plenary Lecture (Exchange Hall) Chair: D. Del Bufalo (Italy) Mouse models of lung cancer A. Berns (Netherlands) The Pezcoller Foundation − EACR Cancer Researcher Award Lecture (Exchange Hall) Chair: M. Oren (Israel)

09:30

Introduction of the Award Lecturer D. Bassi (Italy)

09:35

Towards deciphering the functional genetics and neo-antigenic landscape in melanoma Y. Samuels (Israel)

10:45–12:15

Plenary Symposium: Precision Medicine (Exchange Hall) Chair: C. Caldas (United Kingdom)

10:45

Precision medicine trials in oncology J.C. Soria (France)

11:15

The SHIVA Trial: Results, lessons and perspectives C. Le Tourneau (France)

11:45

Precision medicine in colorectal cancer using gene expression classifiers: ongoing efforts, future directions R. Dienstmann (Spain)

Systems analysis of resistance to targeted kinase inhibitors D.A. Lauffenburger (USA) 12:30–12:50

Closing Ceremony (Exchange Hall) Closing Remarks & EACR24 Highlights Chair: R. Marais (United Kingdom)

12:50–13:30

12:50

Mike Price Gold Medal Award Lecture (Exchange Hall) Chair: A. Berns (Netherlands) Insights from cancer genomes into the causes of cancers M. Stratton (United Kingdom)

xlii


Abstract Nr.

Abstract Nr.

Expression of hypoxia related miRNAs in laryngeal 114 squamous cell carcinoma S. Giragosyan, G. Stancheva, T. Popov, D. Konov, J. Rangachev, O. Stoyanov, V. Mitev, S. Todorov, R. Kaneva (Bulgaria)

Poster Sessions, Sunday 10 July 2016 10:15–17:15

115

100

Antitumor effect of pharmacological and genetic ablation of selected histone modifying enzymes in human glioma cells ´ M. Maleszewska, B. Wojta´s, B. Kaminska S.K. Krol, ´ (Poland)

116

A distinct epigenetic state sensitizes enzalutamide-resistant prostate cancer cells to BET bromodomain inhibition N. Shah, V.K. Arora, W. Karthaus, J. Wongvipat, D. Zheng, C. Sawyers (USA)

101

Identification of chemotherapeutic resistant mutations in castration-resistant prostate cancer F. Ozgun, ¨ H. Sarac, N. Lack (Turkey)

117

Comprehensive sequencing-based characterisation of the DNA methylation landscape of 1300 breast tumours R.N. Batra, A.T. Vidakovic, S.F. Chin, H. Clifford, M. Callari, A. Bruna, A.S. Batra, S.J. Sammut, O.M. Rueda, C. Carlos (United Kingdom)

102

Somatic mutation of the ATRX and gene in the ERBB4Akt/mTOR pathway are frequent in cervical small cell neuroendocrine tumors M. Choi, S. Cho, H.J. Ban, C.H. Lee, H.K. Kim, C. Kim, J.Y. Lee (Korea)

118

Functional genomic screening identifies USP11 as a novel therapeutic target in breast cancer L. Dwane, A. O’Connor, L. Mulrane, R. Klinger, A. Dirac, K. Jirstrom, J. Crown, R. Bernards, W. Gallagher, D. O’Connor (Ireland)

103

Evolutionary trajectory of Asian EGFR mutation positive lung adenocarcinomas leads to “high intratumor heterogeneity” R. Nahar , W. Zhai, T. Zhang, A. Takano, A.J. Khng, Y.Y. Lee, X. Liu, C.H. Lim, T.K.H. Lim, T.P.T. Koh, Z.W. Aung, A.S.M. Teo, C.X. Chan, C.K. Toh, W.T. Lim, B. Lim, W.L. Tam, E.H. Tan, D.S.W. Tan, A.M. Hillmer (Singapore)

119

Frameshift mutations of rapamycin pathway genes and their regional heterogeneity in sporadic colorectal cancers C. An, J.I. Lee, N.J. Yoo, S.H. Lee (Korea)

104

Overexpression of TMPRSS2:ERG variants activates TGF-b signaling and promotes invasion of prostate cancer cells L. Ratz, M. Laible, P. Altevogt, S.M. Klauck, H. Sultmann ¨ (Germany)

106

p53-associated microRNAs and MDM2 expression in the biology and prognosis of neuroblastoma M. Inomistova, N. Khranovska, O. Skachkova, G. Klymnyuk, N. Svergun, N. Adamchuk (Ukraine)

107

TMPRSS4 protein overexpression and its promoter hypomethylation predict poor prognosis in squamous lung cancer patients M. Villalba, A. Diaz-Lagares, M. Redrado, A.L. de Aberasturi, M.J. Pajares, R. Pio, L.M. Montuenga, M. Esteller, J. Sandoval, A. Calvo (Spain)

120

High expressed miR-183 as a potential biomarker of poor survival in tongue cancer patients K. Zeljic, G. Supic, A. Divac Rankov, A. Nikolic, R. Kozomara, N. Jovic, D. Radojkovic, Z. Magic (Serbia)

121

Endogenous Ago2 PAR-CLIP reveals novel target genes of deregulated miRNAs in DLBCL M. Fernandez-Mercado, E. Larrea, I. Goicoechea, I. Ceberio, M. Landthaler, M. Arauzo-Bravo, C.H. Lawrie (Spain)

108

Upregulated lncRNA-HNGA1, a target of miR-375, contributes to aerobic glycolysis of head and neck squamous cell carcinoma through increasing levels of the glucose transporter protein SCL2A1 Y. Wang (China)

122

Investigating the impact of telomere dysfunction on the cancer genome L. Escudero, C. Fegan, C. Pepper, K. Ashelford, K. Norris, K. Cleal, K. Liddiard, D.M. Baird (United Kingdom)

109

Exploiting loss of heterozygosity for allele-selective cancer chemotherapy V. Rendo, I. Stoimenov, R. Svensson, T. Sjoblom ¨ (Sweden) Histone H2B monoubiquitylation − the Ying and Yang of breast cancer O. Tarcic, R. Granit, I. Ben-Porath, M. Oren (Israel)

123

Follicular lymphoma cases harbour recurrent mutations in micro-RNA binding sites of genes associated with lymphomagenesis E. Larrea, M. Fernandez-Mercado, I. Ceberio, J.A. GuerraAssun¸cao, ˜ J. Okosun, J. Fitzgibbon, C. Lawrie (Spain)

110

TP53 variations in human cancers: new lessons from the IARC TP53 Database and genomic studies M. Olivier , L. Bouaoun, D. Sonkin, M. Ardin, M. Hollstein, G. Byrnes, J. Zavadil (France)

124

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Genomic analysis of multi-site fresh prostate samples M. Parry, A. Cannistraci, V. Ramani, M. Lau, J. Shanks, D. Nonaka, N. Clarke, N. Dhomen, E. Baena, R. Marais (United Kingdom)

125

Tumour promoting HER2 splice variant D16HER2: Regulation and implication in breast cancer A.L. Dittrich, H. Gautrey, A. Tyson-Capper (United Kingdom)

New software for detecting somatic mutations at low level in Sanger sequencing traces E. Schreiber, H. Leong, S. Berosik, S. Schneider, J. Marks, W. George, Y.P. Lim, E. Chan, A. Gerstner (USA)

126

Direct input of biological samples into highly multiplexed target amplification reactions for next-generation sequencing (NGS) C. Van Loy, T. Biorac, M. Allen, D. Topacio, M. Carvallo, J. Kilzer, K. Rhodes, M. Andersen (USA)

127

Cancer Genomics, Epigenetics and Genome Instability I Understanding estrogen receptor transcription in breast cancer J. Carroll (United Kingdom)

Whole-genome sequencing of circulating tumor DNA reveals 112 relevance of focal amplifications for the management of metastatic prostate cancer J. Belic, P. Ulz, R. Graf, M. Auer, K. Fischereder, G. Hoefler, T. Bauernhofer, J.B. Geigl, E. Heitzer, M.R. Speicher (Austria) Identification of candidate predisposing factors in familial polycythemia vera with exome sequencing E. Hirvonen, E. Pitkanen, ¨ K. Hemminki, L.A. Aaltonen, O. Kilpivaara (Finland)

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Abstract Nr.

Abstract Nr. Genetic profiling of mutations in pheochromocytoma and paraganglioma by targeted next generation sequencing − A pilot study S. Pillai, V. Gopalan, A. King Y. Lam (Australia)

128

High sensitivity Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples in precision oncology A. Gerstner , E. Schreiber, S. Jackson, K. Varma (USA)

129

Functional assessment of low-frequency mutations in breast cancer A. Leonidou, S.L. Maguire, P.T. Wai, P. Huang, B. Peck, R.C. Natrajan (United Kingdom)

130

Assessing the interplay of RNA-fusion events with cancer driver mutations in melanoma patients A. Mandal, M.R. Girotti, N. Dhomen, A. Viros, G. Gremel, E. Galvani, F. Baenke, R. Lee, K.H.J. Lim, P. Lorigan, R. Marais (United Kingdom)

131

Circulating tumour DNA analysis for monitoring and 132 stratification of patients with small cell lung cancer S. Mohan, M. Ayub, H.S. Leong, S. Sahoo, N. Smith, C. Miller, F. Blackhall, D. Rothwell, C. Dive, G. Brady (United Kingdom) CD44 mRNA expression in hormonally treated and non-treated prostate cancer cases A.S. Navazio, P. Rizzolo, V. Zelli, V. Silvestri, V. Valentini, R. Santi, G. Nesi, L. Ottini (Italy)

133

Overexpression of PBK/TOPK relates to tumor malignant 134 potential and poor outcome of gastric carcinoma T. Ohashi, S. Komatsu, D. Ichikawa, M. Miyamae, W. Okajima, T. Imamura, J. Kiuchi, K. Okamoto, H. Tsuda, E. Otsuji (Japan) Methylome profiling of BRCA-positive and BRCA-negative MBCs P. Rizzolo, V. Silvestri, V. Licursi, A.S. Navazio, V. Valentini, V. Zelli, S. Bianchi, D. Palli, S. Fox, L. Ottini (Italy)

135

Confirming variants discovered by Next Generation Sequencing (NGS) with Sanger sequencing using innovative bioinformatics tools E. Schreiber, S. Berosik, M. Wenz, S. Chang, S. Jackson, J. Zhai, S. Schneider, P. Brzoska (USA)

136

Effects of histone acetyltransferases GCN5/PCAF knockdown on urothelial carcinoma cells E. Koutsogiannouli, C. Hader, M. Pinkerneil, M.J. Hoffmann, W.A. Schulz (Germany)

137

Combined circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer G. Brady, M. Ayub, D. Rothwell, S. Mohan, J. Chudziak, C. Miller, S. Gulati, A. Lamarca, J.W. Valle, C. Dive (United Kingdom)

138

Genomic characterization of the role of chromatin 139 remodelling genes in human cancer T. Moreno, C. Revilla, S. Montes, L. Cereceda, E. Salido, A. Astudillo, P. Isidro, I. Esposito, M. Ramirez, I. Varela (Spain) Detection and molecular analysis of circulating tumour cells 140 isolated from SCLC patients following short and long term storage B. Mesquita, D. Burt, M. Carter, C. Smowton, L. Priest, F. Blackhall, D.G. Rothwell, C. Dive, G. Brady (United Kingdom) A novel ESR2 frameshift mutation predisposes to medullary 141 thyroid carcinoma and causes inappropriate RET expression M. Read, J. Smith, V. Smith, N. Morgan, N. Wake, J. Watkinson, Y. Wallis, E. Maher, C. McCabe, E. Woodward (United Kingdom)

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Pan-cancer analysis of mutation hotspots in protein domains 142 M. Miller , N. Gauthier, E. Reznik, C. Sander (United Kingdom) Characterization of the genomic profile of pseudomyxoma peritonei using amplicon sequencing combined with exome sequencing P. Nummela, L. Saarinen, A. Thiel, R. Lehtonen, P. Jarvinen, ¨ H. Jarvinen, ¨ L.A. Aaltonen, A. Lepisto, ¨ S. Hautaniemi, A. Ristimaki ¨ (Finland)

143

DNA damage responses and chemosensitivity of breast cancer tissue slices ex vivo D. Van Gent, K. Naipal, N. Verkaik, T. Meijer, J. Hoeijmakers, C. Van Deurzen, R. Kanaar, A. Jager (Netherlands)

144

Carcinogenesis I miR-15a expression and deregulation in colorectal cancer biology and its impact on clinicopathological features F. Ebrahimi, V. Gopalan, A. Lam (Australia)

191

Conditional transgenic expression of PIM1/PIM2 kinases in hormone-dependent tissues induces mammary gland and female reproductive system tumours ´ M.P. Jimenez-Garc´ ıa, A. Lucena-Cacace, B. Felipe-Abrio, E.M. Verdugo-Sivianes, D. Otero-Albiol, M. Perez, S. Munoz-Galvan, ´ J. Peinado-Serrano, J.M. Garc´ıa-Heredia, M. Narlik-Grassow, C. Blanco-Aparicio, A. Carnero (Spain)

192

Determination of the proto-oncogenic role of PIM1/PIM2 kinases in male reproductive system pre-neoplastic lesions by using conditional transgenic murine models ´ M.P. Jimenez-Garc´ ıa, M.J. Robles-Frias, B. Felipe-Abrio, A. Lucena-Cacace, D. Otero-Albiol, E.M. Verdugo-Sivianes, J. Peinado-Serrano, M. Perez, S. Munoz-Galvan, J.M. Garc´ıa-Heredia, M. Narlik-Grassow, C. Blanco-Aparicio, A. Carnero (Spain)

193

Expression and localization of GAEC1 oncogene in peripheral blood from patients with colorectal adenocarcinoma V. Gopalan, M. Orr, S. Pillai, A. Lam (Australia)

194

YAP signalling promotes aggressive tumour development over heterotopia in the brain M. Mayrhofer , V. Gourain, M. Reischl, P. Affaticati, A. Jenett, J.S. Joly, D. Sieger, M.C. Mione (Germany)

195

Reprogramming bladder cancer cells for studying cancer initiation and progression B. Iskender Izgi, K. Izgi, H. Canatan (Turkey)

196

COX-2 gene variations and risk of developing breast cancer G. Ozhan, Z. Kara, H. Kara (Turkey)

198

The role of Fanconi Anaemia pathway in sporadic non-FA associated head and neck squamous cell carcinoma F. Alsobahi, S. Collis, K. Hunter (United Kingdom)

199

HER2 cooperates with YWHAZ to promote to invasive esophago-gastric cancer by inducing epithelial– mesenchymal transition S. Komatsu, D. Ichikawa, M. Miyamae, T. Kosuga, H. Konishi, A. Shiozaki, H. Fujiwara, K. Okamoto, H. Tsuda, E. Otsuji (Japan)

200

Plasma microRNA profiles; down-regulation of tumor suppressive miR-X level in plasma relates to poor outcomes and is a novel treatment target in gastric cancer S. Komatsu, D. Ichikawa, T. Imamura, W. Okajima, T. Ohashi, J. Kiuchi, T. Arita, H. Konishi, A. Shiozaki, E. Otsuji (Japan)

201

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Abstract Nr.

Abstract Nr. Cell and Tumour Biology I Frequent and functional derepression of the Iroquois homeobox gene IRX3 in human acute leukemia T. Somervaille, T. Somerville (United Kingdom)

215

Absence of MYBBP1A translocation from nucleolus to nucleoplasm under glucose starvation in renal carcinoma cell lines B. Felipe Abrio, M.P. Jimenez ´ Garc´ıa, E.M. Verdugo Sivianes, A. Lucena Cacace, D. Otero Albiol, M. Perez, J. Peinado Serrano, S. Munoz Galvan, ´ J.M. Garc´ıa Heredia, A. Carnero Moya (Spain)

234

Tumour suppressor genes of common fragile sites: Active players in DNA damage response?! R. Aqeilan (Israel)

216

Study of the biomarker potential of circulating non-coding PRNCR1 & CCAT2 RNAs in plasma of breast cancer patients E. Babaei, V. Montazeri (Iran)

218

Role of bcl-2 in cancer–stroma interplay V. Farini, D. Trisciuoglio, M. Desideri, M. Di Martile, M.G. Tupone, S. D’Aguanno, D. Del Bufalo (Italy)

236

237

Determination of pyruvate metabolic fate regulates head and neck tumourigenesis W.C. Li, T.Y. Chen, J.M. Huang, C.W. Chang, C.Y. Chen, W.J. Chang, C.J. Liu, H.M. Chen, J.F. Lo (Taiwan)

219

Investigating epithelial–mesenchymal plasticity in circulating tumour cells from breast cancer xenograft models A.V.P. Le, A. Tachtsidis, T. Blick, D. Gunasinghe, M. Waltham, A. Dobrovic, E. Thompson (Australia)

238

Multiple myeloma cells reprogram bone marrow mesenchymal stem cells’ translation initiation thereby promoting their migration M. Dabbah, O. Attar-Schneider, S. Tartakover Matalon, V. Zismanov, L. Drucker, M. Lishner (Israel)

220

The induction of metformin-inhibited breast cancer cell line proliferation in different conditions Z. Safari, S. Reza (Iran) MDM2 antagonists − a therapeutic approach for patients with hepatocellular carcinoma? A. Mahdi, J. Lunec, H. Reeves (United Kingdom)

240

ILK as a signaling nexus for induction of breast cancer stem cells in response to tissue stiffness and hypoxia M.F. Pang, M. Stallings-Mann, M.J. Siedlik, S. Han, D.C. Radisky, C.M. Nelson (USA)

222

241

Modulation of microRNA-1288 in oesophageal squamous cell carcinoma via targeting tumour suppressor FOXO1 F. Islam, S. Pillai, V. Gopalan, A. King-Yin Lam (Australia)

225

Caveolin-1-negative head and neck squamous cell carcinoma primary tumors display increased epithelial to mesenchymal transition and prometastatic properties S. Martin, A. Jung, A.M. Ray, L. Ramolu, C. Macabre, J. Abecassis, M. Donten (France) Biochemical study on the effect of quercetin along with Adriamycin on DEN induced experimental hepatocellular carcinoma R. Venkateswari, D. Sakthisekaran (India)

243

244

ZEB1 induces the Wnt antagonist DKK1 to jointly determine 226 poorer survival in colorectal carcinomas O. De Barrios, B. Gyorffy, ˝ M.J. Fernandez-Ace ´ nero, ˜ E. Sanchez´ Tillo, ´ J.I. Casal, D.S. Darling, A. Castells, A. Postigo (Spain) The progression of K-ras induced early cancerous lesions is driven by a COPD-like inflammation C. Jungnickel, P. Schnabel, R. Bohle, R. Bals, C. Beisswenger (Germany)

227

Crosstalk between glial and glioblastoma cells triggers the “go-or-grow” phenotype of tumor cells A.I. Oliveira, S.I. Anjo, S. Serra, A.J. Salgado, B. Manadas, B.M. Costa (Portugal)

245

GLUT1/GLUT4 balance is a marker of androgen-insensitivity in prostate cancer P. Gonzalez-Menendez, D. Hevia, R. Alonso, I. Gonzalez-Pola, J.C. Mayo, R.M. Sainz (Spain)

228

Ultra-deep sequencing reveals the subclonal structure and genomic evolution of oral squamous cell carcinoma S. Tabatabaeifar , M. Thomassen, M. Larsen, S. Rosenkilde Larsen, T.A. Kruse, J. Ahm Sørensen (Denmark)

246

The effects of aspirin on growth and COX-2 expression in prostate cancer cells R. Wang, U.K. Shah, G. Jenkins, S. Doak (United Kingdom)

229

Regucalcin in hormone-dependent cancers: towards a candidate tumour suppressor gene? C.V. Vaz, R. Marques, C.J. Maia, S. Socorro (Portugal)

247

MiR-302b improves chemotherapy efficacy in human breast cancer and affects tumor microenvironment A. Cataldo, I. Plantamura, E. D’Ippolito, S. Baroni, D. Palmieri, M.V. Iorio (Italy)

230

Integration of gene expression and miRNAs reveals amino acid metabolism as key metabolic hub of adaptation to long term oestrogen deprivation in ER+ breast cancer cells M. Bacci, M. Ferracin, M. Ramazzotti, L.A. Martin, G. Pintus, P. Chiarugi, A. Morandi (Italy)

231

Regulation of WNT6 by HOXA9 in glioblastoma: functional and clinical relevance C. Gon¸calves, M. Pojo, A. Xavier-Magalhaes, ˜ J. Vieira de Castro, A.A. Pinto, R. Taipa, F. Pardal, R.M. Reis, N. Sousa, B.M. Costa (Portugal)

248

Extracellular matrix proteins derived from the tumor microenvironment as circulating breast cancer diagnostic markers M. Giussani, E. Landoni, G. Merlino, F. Turdo, S. Veneroni, V. Cappelletti, M.G. Daidone, R. Miceli, R. Orlandi, T. Triulzi, E. Tagliabue (Italy)

249

Using lentivirally encoded miRnome library as a functional screen to identify miRNAs associated with invasion and metastasis in breast cancer I. Goicoechea, E. Larrea, L. Manterola, R. Gomis, I. Schultz, R. Schaapveld, C.H. Lawrie (Spain)

232

Loss of Popdc protein expression promotes a more malignant phenotype in glioblastoma and breast cancer cells J.N. Amunjela, S.J. Tucker (United Kingdom) Autofluorescence as a new biomarker to identify glioblastoma stem cells J. Vieira de Castro, I. Miranda-Lorenzo, M.T. Cerqueira, A.A. Pinto, C. Heeschen, B.M. Costa (Portugal)

250

Role of d16HER2 in HER2-positive breast cancer stem cells L. Castagnoli, G.C. Ghedini, A. Koschorke, C. Chiodoni, P. Nanni, E. Tagliabue, S.M. Pupa (Italy)

233

Ovarian cancer patient-derived xenografts resistant to cisplatin exhibit metabolic changes F. Ricci, L. Brunelli, F. Spriano, R. Pastorelli, G. Damia (Italy)

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Abstract Nr.

Abstract Nr. The role of profilin I and II in glioma progression N. Svergun, U. Shahzad, C. Figueiredo, S. Agnihotri, B. Golbourn, A. Luck, C. Smith, J. Rutka (Canada)

252

Radium-223 in the treatment of metastatic prostate cancer I.A. Marques, A.M. Abrantes, A.S. Pires, G. Costa, E. Tavares-Silva, M.F. Botelho (Portugal)

253

A new targeted combination therapy overcomes the acquired resistance of colorectal cancer stem cells A. Benfante, L.R. Mangiapane, M.L. Colorito, M. Gaggianesi, A. Nicotra, A. Giammona, E. Scavo, A. Chinnici, M. Todaro, G. Stassi (Italy)

255

Increasing mechanistic understanding of the role of p53 mutations in triple negative breast cancer and identifying potential roles in chemoresistance R. Steele, S. Eddie, S. McDade, P. Mullan (United Kingdom)

256

17b-Estradiol stimulates generation of reactive oxygen species and nitric oxide in ovarian adenocarcinoma cells M.H. Moghadasi (Iran)

257

Regulation mechanism of Notoginsenoside R1 on human colorectal cancer metastasis C.C. Wu, S. Hsieh, L.C. Hsieh, Y.H. Kuo, S.L. Hsieh (Taiwan)

259

Next generation sequencing in laryngeal cancer specimens reveals alterations of the DIAPH2 gene that can putatively contribute to the metastatic potential of squamous carcinoma M. Giefing, E. Byzia, N. Zemke, K. Kiwerska, M. Kostrzewska-Poczekaj, M. Jarmuz-Szymczak, M. Wierzbicka, G. Greczka, R. Grenman, K. Szyfter (Poland)

261

The expression of cyclooxygenase-2 is increased by nerve 262 growth factor in epithelial ovarian cancer C. Romero, I. Hurtado, M. Garrido, A. Selman, M. Vega (Chile)

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TMPRSS4 expression enhances cancer stem cell-like properties in lung cancer cells and correlates with a CSC phenotype in NSCLC patients M. Redrado, A.L. de Aberasturi, M. Villalba, L. Larzabal, J. Garcia, S.R. Evans, E. Jantus Lewintre, C. Camps, L. Montuenga, A. Calvo (Spain)

272

Targeting endothelial secreted factors to impair glioblastoma initiating cells E. Harford-Wright, J. Gavard (France)

273

A highly sensitive method for circulating tumour cell antigen quantification in liquid biopsy K. Mumford (United Kingdom)

274

Cellular and molecular regulations of hexokinase 2 mediated head and neck tumourigenesis C.H. Huang, T.Y. Chen, C.Y. Chen, C.W. Chang, L.H. Cheng, C.J. Liu, J.F. Lo, H.M. Chen, W.C. Li (Taiwan)

275

Ly6C+inflammatory monocytes regulate neutrophils inflammation response induced by lung injury L. Liu, X. Wei, Y. Wei (China)

276

Hypoxia-induced treatment resistance correlates with EMT markers in head and neck squamous cell carcinoma cell lines K. Roberg, E. Wiechec, K. Tiefenbock, ¨ L. Alexandersson (Sweden)

277

Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumourigenesis in murine mammary epithelium K. Reed, C. Daly, P. Shaw, L. Ordonez, G. Williams, J. Quist, A. Grigoriadias, J. Van Es, H. Clevers, A. Clarke (United Kingdom)

278

Target-independent suppression of angiogenesis by human IgG1 antibodies and IVIg via FcgRI S. De Falco, V. Cicatiello, L. Tudisco, V. Tarallo, I. Apicella, J. Ambati (Italy)

279

Unexpected role of Alu RNAs in cancer progression V. Tarallo, F. Di Ruocco, S. De Falco (Italy)

280

The role of monocarboxylate transporters in human ovarian cancer cell lines A. Boyers, L. Hutchinson, I. Statford (United Kingdom)

281

Molecular-genetic analysis of BRCA1 and BRCA2 mutations in Bulgarian patients with hereditary breast ovarian cancer (HBOC) syndrome R. Dodova, A. Mitkova, D. Pencheva, S. Valev, M. Taushanova, A. Nachev, K. Timcheva, R. Dimitrov, V. Mitev, R. Kaneva (Bulgaria)

282

Stromelysin-3 as a marker of metastasis and predictor of poor survival in oral squamous cell carcinoma S.H. Lin, S.F. Yang, K.T. Yeh, C.W. Lin (Taiwan)

283

285

MicroRNAs mediate regulation of chemoresistance of hepatocellular carcinoma cells with involvement of p53 tumour suppressor N. Wang, Y. Feng (Hong Kong)

263

Autophagy mediated activation of RelB/p52 responsible for the reprogramming of tumour associated macrophages H.Y. Tan, N. Wang, Y. Feng (Hong Kong)

264

Downregulation of AKT/mTOR signaling pathway are critical for Salmonella medical suppression of MMP-2/MMP-9 and metastasis ability in mouse tumor models K. Chun-Yu, C.H. Lee (Taiwan)

265

Tumor suppressor crosstalk: Hippo and p53 N. Furth, N. Bossel, Y. Pozniak, T. Geiger, E. Domany, Y. Aylon, M. Oren (Israel)

266

Tracing the path of cancer initiation A. Adjiri (Algeria)

267

Melatonin regulates oral cancer cell migration by suppressing histone acetylation on the CBP/P300 gene S.F. Yang, C.W. Lin, C.M. Yeh (Taiwan)

268

Neutrophil gelatinase-associated lipocalin suppress metastases of oral cancer through miRNA-4505-regulated carbonic anhydrase IX C.W. Lin, W.E. Yang, S.F. Yang (Taiwan)

269

Eribulin differentiates cetuximab resistant oral squamous cell carcinoma cells to sensitive by inducing mesenchymal– epithelial transition (MET) H. Kitahara, M. Hirai, H. Nakamura, S. Kawashiri (Japan)

286

Nobiletin inhibits human osteosarcoma cells metastasis by NF-úB-, and CREB-dependent down-regulation of MMPs via ERK and JNK pathway J.S. Yang, S.F. Yang (Taiwan)

270

The role of TEX19 in growth and maintenance of cancer stem-like cells V. Planells, L. Parry, J. Wakeman, R. McFarlane (United Kingdom) Control of epithelial splicing regulatory proteins in epithelial– mesenchymal transitions L. Li, S. Oltean (United Kingdom)

287

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Abstract Nr.

Abstract Nr. New insights of mutant calreticulin in myeloproliferative neoplasms M. Morlan Mairal, A. Aziz (United Kingdom)

288

Gene-specific interactions between ultraviolet radiation and 289 melanoma ´ M. Pedersen, S.J. Furney, M.R. Girotti, K. Hogan, A. Viros, G. Saturno, E. Galvani, B. Sanchez-Laorden, C. Ng, J.S. Reis-Filho, P. Lorigan, M. Cook, R. Marais (United Kingdom)

Deregulation of peripheral circadian clock in murine colorectal tumor deregulates the genes of cell cycle and proliferation M. Sotak, K. Balounova, P. Ergang, A. Sumova, J. Pacha (Czech Republic)

302

Crosstalk of CCL7 and CCR3 promotes metastasis of colon cancer cells via MAPK signaling pathways Y.S. Lee, Y.R. Lee, S.J. Song, B.Y. Oh, W.Y. Lee, Y.B. Cho (Korea)

304

305

PDGFRb inhibition by Gint4.T aptamer prevents recruitment of bone marrow-derived mesenchymal stem cells into breast cancer microenvironment R. Fontanella, S. Camorani, L. Cerchia, A. Zannetti (Italy)

290

MALDI-MS and [11 C] acetate PET imaging of lipid heterogeneity in non-small-cell lung cancer F. Henderson, D. Lewis, D. Soloviev, K. Brindle, A. McMahon, K.J. Williams (United Kingdom)

291

Establishment of three-dimensional culture of cholangiocarcinoma cells S. Sukphokkit, P. Kiatwuthinon, S. Kumkate, T. Janvilisri (Thailand)

306

Metabolic rewiring in melanoma cell lines that acquired resistance to BRAF inhibitors F. Baenke, B. Chaneton, M. Smith, N. Van Den Broek, K. Hogan, H. Tang, A. Viros, N. Dhomen, E. Gottlieb, R. Marais (United Kingdom)

292

Study of Moesin regulation and effects in breast cancer cells W.M. Abdel-Rahman, F. Alam, F. Mezhal, M.S. Ayad, A. El-Serafi, H. El Hassasna, J. Thiery, Z. Noman, S. Chouaib (U.A.E.) Assessment of WT1 expression as a marker of treatment outcome in karyotype normal acute myeloid leukemia patients in Pakistan Z. Ahmed (Pakistan)

307

The role of vasculogenic mimicry in small cell lung cancer S. Williamson, F. Trapani, B. Abbott, M. Galvin, R. Metcalf, M. Hendrix, F. Blackhall, K. Frese, K. Simpson, C. Dive (United Kingdom)

293

Influence of autophagy inhibitors on chemotherapeutic efficacy in oral squamous cell carcinoma A. Anderson, J. O’Sullivan (Ireland)

308

New models for investigating integrin antagonists A. Elsharif , H. Sheldrake, L. Patterson, S. Shnyder (United Kingdom)

294

Analysis of murine stromal components in patient derived xenograft (PDX) models of pancreatic cancer D. Behrens, U. Pfohl, C. Hallas, B. Buttner, ¨ J. Hoffmann (Germany)

309

Identification of new combination therapies for NSCLC tumours harbouring KRAS mutations M. Molina-Arcas, D.C. Hancock, C. Moore, S. Horswell, N. Matthews, J. Downward (United Kingdom)

295

310

Modes of cell death induced by novel tetrahydroisoquinolinone-based analogues in breast- and lung cancer cell lines M. Verwey, A.M. Joubert, W. Dohle, B.V.L. Potter, A.E. Theron (South Africa)

296

Human papillomavirus status and the microenvironment in oropharyngeal carcinoma; determinants of invasion and potential therapeutics R. Bolt, D. Lambert, C. Murdoch, S. Thomas, B. Foran, K. Hunter (United Kingdom)

311

Lysyl oxidase regulates EGFR signalling through the extracellular matrix H. Tang, L. Leung, G. Saturno, A. Viros, G. Di Leva, E. Morrison, D. Smith, L. Johnson, N. Dhomen, C. Springer, R. Marais (United Kingdom)

297

Down-regulation of TFPI-2 in the progression of ovarian cancer J. Fry, S. Kato, L. Abarzua, P. Gonzalez, C. Ramirez, E. Cumsille, J.C. Roa, C. Ibanez, ˜ M. Cuello, G. Owen, M.L. Bravo (Chile) The role of BAP1 loss of function in uveal melanoma K. Brooks, N. Dhomen, R. Marais (United Kingdom)

312

299

SIRT1 in bladder cancer E. Chapman, E. Baxter, J.A. Roulson, M. Sanchez-Carbayo (United Kingdom)

313

Apoptosis and proliferation in micropapillary structures of colorectal polyps and carcinomas M. Patankar , S. Vayrynen, A. Tuomisto, M. Makinen, T. Karttunen (Finland)

314

AICAR, an AMPK activator, sensitizes TRAIL-mediated apoptosis through p38 MAPK-dependent and AMPKindependent signaling pathways in human bladder cancer T24 cells Y.H. Choi, M.H. Han, S.H. Hong, J.W. Jeong, E.O. Choi, K.Y. Jeon, C. Park (Korea)

300

Activation of ER stress and autophagy induced by pterostilbene via Akt/mTOR pathway in human hepatocellular carcinoma cells H.L. Chiou, C.L. Yu, Y.H. Hsieh (Taiwan)

315

Regulation of AMPK-dependent MDR1 expression by resveratrol in cisplatin-resistant human bladder cancer T24 cells via attenuating NF-úB and CRE transcriptional activity S.H. Hong, M.H. Han, C. Park, S.Y. Ji, J.W. Jeong, E.O. Choi, Y.H. Choi (Korea)

301

Exploiting cross-talk between lipid metabolism and oncogenic signaling for treatment of ovarian cancer R. Wagner, G. Stubiger, ¨ P. Lanzerstorfer, J. Weghuber, E. Karteris, K. Nowikovsky, N. Wilfinger, R. Colomer, M.L. Lopez-Rodr´ ´ ıguez, T.W. Grunt (Austria) Platycodon grandiflorum-induced autophagy and growth inhibition in human lung cancer cells C. Park, J.W. Jeong, E.O. Choi, M.H. Han, Y.H. Choi, S.H. Hong (Korea)

316

Overexpression of CTEN relates to malignant outcome in adenocarcinoma of the esophagogastric junction J. Kiuchi, S. Komatsu, D. Ichikawa, K. Aratani, H. Konishi, A. Shiozaki, H. Fujiwara, K. Okamoto, H. Tsuda, E. Otsuji (Japan)

317


xlvii

Abstract Nr.

Abstract Nr. Functional characterization of novel tumour suppressor galectin-2 in hepatocellular carcinoma F.C.F. Ko, K.W. Lee, W.C.S. Tai, Z. Leung, J.W.P. Yam (Hong Kong)

318

New tools for the study of intratumour heterogeneity in cancer L. Gonzalez-Silva, L. Quevedo Palacio, T. Moreno Rodriguez, C. Revilla Gomez, R. Rad, D. Saur, I. Varela (Spain)

333

Bcl-xL modulates cell survival and drug resistance in edelfosine-treated glioblastoma cells S. Melo-Lima, M.C. Lopes, F. Mollinedo (Portugal)

319

Combination therapy to target ERBB receptors in HER2 low breast cancer M. Berdiel-Acer , E. Sofyali, S. Burmester, R. Will, U. Korf, S. Wiemann (Germany)

334

Molecular mechanisms involved in the immunomodulation induced by LIF in cancer A. Sala Hojman, A. Soto, I. Huber-Ruano, V. Chigan¸cas, C. Raventos, ´ M. Vidal-Jorge, E. Mart´ınez-Saez, ´ J. Sahuquillo, C. Espejo, J. Seoane (Spain)

336

A relay of IL-1R and CXCR2 signals in the tumour microenvironment confer tolerance to MAPK antagonism in melanoma H. Young, E. Rowling, M. Smith, M. Bugatti, W. Vermi, N. Luheshi, C. Wellbrock, A. Hurlstone (United Kingdom)

337

Widespread epigenetic activation of DNp73 in small cell lung cancer causes vulnerability to Tip60−p400 inhibition A. Nist, A.M. Krampitz, K. Schlereth, M. Mernberger, M.C. Moßner, T. Muley, R. Dammann, T. Stiewe (Germany)

338

Role of the HIV matrix protein p17 in EBV-driven lymphomagenesis K. Mastorci, D.A. Fae, ` C. Giagulli, A. Carbone, P. De Paoli, A. Caruso, R. Dolcetti (Italy)

340

Leukemic cell death induced by the Notch-1 fragmentderived peptide evokes the immunogenic inflammation A. Kuniyasu, M. Makise (Japan)

341

The AMPK-related kinase NUAK1 is a target for treatment of colorectal cancer J. Port, N. Muthalagu, M. Raja, T. Monteverde, M. Mezna, F. Ceteci, G. Murray, O. Sansom, S. Zanivan, D. Murphy (United Kingdom)

342

Metabolic profile of osteolysis in bone metastases S. Avnet, S. Lemma, M. Sboarina, P. Porporato, N. Zini, P. Sonveaux, N. Baldini (Italy)

343

344

Mutant p53 and tumour cell engulfment activity in cancer 320 H. Mackay, J. Le Quesne, D. Moore, P. Muller (United Kingdom) Role of nuclear Met-derived exosomes in hepatocellular carcinoma metastasis and lung premetastatic niche formation S.K. Tey, X. Mao, J.W.P. Yam (Hong Kong)

321

Overexpression of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) in a subset of hepatocellular carcinoma (HCC) promotes cancer stemness properties and cell migration of HCC cells Y.M. Tsui, K.M.F. Sze, E.K.K. Tung, T.K.W. Lee, I.O.L. Ng (Hong Kong)

322

S100A8 inhalation prolongs survival in murine orthotopic 323 lung cancer S.W. Wong, K. Hsu, J. Mccarroll, C. Geczy, N. Tedla (Australia) Tumour matrix stiffness regulates metastasis by promoting cancer cell interactions with the endothelium S. Reid, A.T. Henze, J. Serneels, L. Neilson, A. Ramon-Fernandez, R. Adams, K. Blyth, D. Bryant, M. Mazzone, S. Zanivan (United Kingdom)

324

The epigenetic repressor EZH2 controls tumour escape mechanisms during immunotherapy D. Zingg, N. Arenas-Ramirez, R.A. Rosalia, A.T. Antunes, J. Haeusel, O. Boyman, L. Sommer (Switzerland)

325

miR-29c modulates chemoresistance in esophageal cancer cells by suppressing FBXO31 A.L. Cheung, B. Li, W.W. Xu, J. Liu, K.W. Chan (China)

326

Acidosis meets the “hallmarks of cancer”: transcriptome analysis to uncover its role in melanoma S. De Summa, A. Ferretta, P. Rosamaria, P. Orazio, M. Carella, G. Guida, A. Azzariti, S. Tommasi (Italy)

327

The bone metastatic niche promotes breast cancer stem cell activity via IL-1b−Wnt signalling R. Eyre, K. Spence, D. Alferez, A. Santiago-Gomez, C. Hart, B. Simoes, M. Brown, A. Gurney, G. Farnie, R. Clarke (United Kingdom)

328

Coordinated erasure and adding up epigenetic marks define transcriptional programs during microglia reprogramming B. Kaminska, M. Maleszewska, A. Steranka, M. Smiech, B. Kaza, M. Dabrowski (Poland)

345

Clinical relevance and functional role of galectin-1 in hepatocellular carcinoma Z. Leung, F.C.F. Ko, J.W.P. Yam (Hong Kong)

329

Targeting Ewing Sarcoma cells and the tumor microenvironment with OMTX003 anti-endoglin monoclonal antibodies P. Puerto Camacho, A.T. Amaral, J.L. Ordonez, ˜ M.J. Robles, S. Dom´ınguez, C. Jordan ´ Perez, ´ ´ M. Biscuola, M. Lopez ´ Alvarez, M. Fabre, E. Alava (Spain)

Mutant p53 promotes tumor progression by the endoplasmic reticulum UDPase ENTPD5 F. Vogiatzi, D.T. Brandt, J. Fuchs, K. Grikscheit, O. Timofeev, A. Nist, M. Mernberger, R. Grosse, T. Stiewe (Germany)

330

346

TGFb-induced JMJD3 controls tumor cell microenvironment and myeloid cell polarization I. Huber Ruano, C. Raventos, ´ A. Arias, A. Cascante, I. Cuartas, J. Seoane (Spain)

331

N-acetylglucosaminyltransferase 5 (Mgat5) mediated N-glycosylation of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) enhances EGFR signaling of cell invasion and metastasis in head and neck cancer M.H. Wu, W.F. Chiang, T.M. Cheng (Taiwan)

347

Specific EMT-inducers signature associates with oncogenic events in breast tumour progression C. Moyret-Lalle, E. Ruiz, C. Bardel, I. Treilleux, S. Courtois-Cox, A. Puisieux (France)

332

YAP, Cdc42EP3 and septins modulate mechanotransduction and the emergence of cancer-associated fibroblasts F. Calvo, R. Ranftl, S. Hooper, E. Sahai (United Kingdom) The contribution of the cancer initiating cell markers EpCAM and claudin7 to metastatic progression S. Heiler , M. Zoller ¨ (Germany)

348

xlviii


Abstract Nr.

Abstract Nr.

Dual roles of MMP2 activity on brain tumor progression and invasion C.S. Chiang, C.F. Yu (Taiwan)

516

517

350

Enhancement of doxorubicin cytotoxicity by histone deacetylase inhibition in human sarcoma cells M.G. Tupone, D. Trisciuoglio, M. Desideri, M. Di Martile, S. Buglioni, R. Loria, V. Ferraresi, N. Baldini, R. Biagini, D. Del Bufalo (Italy) Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells M. Di Martile, M. Desideri, T. De Luca, S. Buglioni, D. Del Bufalo, D. Trisciuoglio (Italy)

518

351

Delivery of microRNA-26a by CD38-conjugated nanoparticles into chronic lymphocytic leukemia cells of the Em-TCL1 mouse model L. D’Abundo, E. Callegari, A. Bresin, C. Bassi, P. Guerriero, G. Russo, S. Sabbioni, F. Malavasi, M. Negrini (Italy)

519

Development of novel tirapazamine-loaded liposomes for hypoxia targeted therapy V.L. Silva, W. Al-Jamal (United Kingdom)

520

Cediranib affects tumor dissemination and prolongs the survival of mice bearing patient-derived ovarian cancer xenografts (EOC-PDX) A. Decio, M. Cesca, F. Bizzaro, M.R. Bani, D. Belotti, R. Giavazzi (Italy)

522

The effects and molecular mechanisms of crown ethers on drug efflux pumps I. Guberovic, M. Marjanovic, K. Ester, A.M. Mikecin, I. Martin-Kleiner, T. Sumanovac-Ramljak, K. Mlinaric-Majerski, M. Kralj (Croatia)

523

Effects of telomerase blockage on the advanced triple negative breast cancer cells culture by activated lymphocytes A. Tavartkiladze, R. Khutsishvili, A. Gogiberidze (Georgia)

524

Salinomycin affects Golgi apparatus function in cancer stem-like cells A.M. Mikecin, M. Marjanovic, I. Guberovic, M. Kralj (Croatia)

525

1-Methylhistidine and high dose methotrexate influence on triple negative breast cancer in experiment A. Tavartkiladze, R. Khutsishvili, A. Gogiberidze (Georgia)

526

The role of reactive oxygen species in photodynamic therapy combined with acetylsalicylic acid in colon and esophagus cancer cells M. Laranjo, N. Almeida, A. Serra, A.M. Abrantes, M. Pineiro, A.C. Gon¸calves, J. Casalta-Lopes, A.B. Sarmento-Ribeiro, M.F. Botelho (Portugal)

528

miRNA pattern modifications in bladder cancer R.M. Cojocneanu Petric, C. Braicu, L. Budisan, C. Ivan, S. Chira, B. Petrut, L. Raduly, A. Jurj, G.A. Calin, I. Berindan-Neagoe (Romania)

529

The hypoxia marker CA-IX is prognostic in soft tissue sarcoma patients treated in the UK phase III VORTEX trial L. Forker , S. Sioletic, P. Shenjere, J. Irlam, H. Valentine, D. Hughes, A. Hughes, P. Gaunt, M. Robinson, C. West (United Kingdom)

349

Monitoring the dynamics of clonal tumor evolution in vivo using secreted luciferases J.P. Charles, J. Fuchs, M. Hefter, J.B. Vischedyk, M. Kleint, F. Vogiatzi, A. Nist, O. Timofeev, M. Wanzel, T. Stiewe (Germany) Cancer stem cell markers and exosomes S. Heiler , M. Zoller, ¨ U. Erb (Germany)

PKCtheta controls invasion of triple-negative breast cancer 352 by direct phosphorylation of FAK L. Chadelle, V. Cadamuro, J. Liu, X. Wang, K. Belguise (France) Quantitative analysis of estrogen receptors alpha and beta expression in ovarian cancer patients by flow cytometry E. Dudko, T. Bogush, S. Kolomiytsev, O. Rjabinina, A. Tjulandina, V. Kirsanov, E. Bogush, S. Tjulandin, I. Mamichev (Russian Federation)

353

Colorectal adenocarcinoma stem cells show distinct growth patterns depending on the culture environment A. Borys, P. Wołkow (Poland)

354

Approaches to elucidate cancer metabolism using zebrafish models M.C. Mione, A. Kalkbrenner, V. Gourain, M. Mayrhofer, S. Burkart, C. Muhle-Goll, B. Luy (Germany)

355

Non-canonical Hedgehog/AMPK-mediated control of polyamine metabolism is required for medulloblastoma growth G. Canettieri, S. Coni, D.M. Laura, G. Sdruscia (Italy)

356

The impact of Mycobacterium obuense on innate and adaptive immunity J. Crooks, S. Brown, C. Forss, A. Phythian-Adams, P. Cook, L. Rosa Brunet, A. MacDonald (United Kingdom)

357

Novel methods for the isolation of tumor cells from human, 358 mouse, and xenografted tumors D. Agorku, A. Langhammer, L. Willnow, K. Klingner, S. Tomiuk, J. Kollet, S. Ruberg, ¨ J. Schuler, ¨ A. Bosio, O. Hardt (Germany) Dissecting the pro-tumoural role of the essential amino-acid transporter complex CD98/LAT1 Y. Cormerais, S. Giuliano, P.A. Massard, J. Durivault, E. Hitoshi, W. Michael, S. Parks, J. Pouyssegur (Monaco)

359

The role of E3 ubiquitin ligase Smurf2 in cancer M. Blank (Israel)

360

Genomic evolution and heterogeneity in 2,800 cancers D. Wedge, P. Spellman, P. Van Loo (United Kingdom)

361

Modelling the tumour microenvironment and employing functional genomics identifies CREBBP as a novel tumour suppressor in triple negative breast cancers B. Peck, S. Maguire, E. Morrison, P. Wai, R. Natrajan (United Kingdom)

362

Experimental/Molecular Therapeutics, Pharmacogenesis I Thermally responsive ELP-dnMAML protein and its effect 514 on U251 cells in vitro T. Opacak-Bernardi, J.S. Ryu, D. Raucher (Croatia) Cinacalcet inhibits neuroblastoma tumor growth and upregulates cancer-testis antigens ´ C. De Torres, C.J. Rodr´ıguez-Hernandez, S. Mateo-Lozano, M. Garcia, C. Casala, ` N. Castrejon, ´ E. Rodr´ıguez, M. Sunol, C. Lavarino, J. Mora (Spain)

515

Dysregulations of circulating microRNAs are possible 530 biomarkers for colorectal cancer L.Z. Raduly, C. Braicu, M.A. Jurj, O. Zanoaga, R. Cojocneanu Petric, M.S. Muresan, I. Berindan-Neagoe (Romania) Ascorbic acid and conventional chemotherapeutic agents synergistically inhibit colorectal cancer cells proliferation and tumor growth A.S. Pires, C.R. Marques, J. Encarna¸cao, ˜ J. Casalta-Lopes, A.C. Gon¸calves, A.M. Abrantes, A.B. Sarmento-Ribeiro, M.F. Botelho (Portugal)

531


Abstract Nr.

Abstract Nr. Photodynamic therapy combined with acetylsalicylic acid: the mechanisms involved in colon and esophagus cancer cell death N. Almeida, M. Laranjo, A. Serra, M. Abrantes, M. Pineiro, A.C. Gon¸calves, J. Casalta-Lopes, A. Sarmento-Ribeiro, M.F. Botelho (Portugal)

532

Drugs driven synthetic lethality: getting rid of tumor genetics with combination of Dbait and PARP inhibitors M. Dutreix, W. Jdey, S. Thierry, F. Devun, I. Kuperstein, A. Zinovyev, E. Barillot (France)

534

COX-2 inhibitor derivatives reduce MDA-MB-231 535 invasiveness caused by mesenchymal stem cells M. Salimi, K. Moradi, F. Barneh, S. Irian, A. Amanzadeh (Iran)

xlix

DNA nanosphere as a drug delivery system for cancer cells S. Chaithongyot, T. Kangsamaksin, A. Udomprasert, N. Chomanee, K. Charngkaew (Thailand)

547

Vitamin E phosphate (VEP) nucleoside prodrugs: a platform for intracellular delivery of monophosphorylated (MP) nucleosides R. Daifuku (USA)

549

In vitro and in vivo testing of a novel DNA methyl transferase inhibitor (DNMTI) with activity against hematogenous and solid tumors whether p53 null or wild type (WT) R. Daifuku (USA)

550

551

Targeting miR-155 to resensitize lung cancer to chemotherapy K. Van Roosbroeck, F. Fanini, T. Setoyama, C. Ivan, C. Rodriguez-Aguayo, X. Zhang, I. Wistuba, G. Lopez-Berestein, M. Fabbri, G. Calin (USA)

536

Melanoma-directed activation of apoptosis using a novel bispecific antibody directed against MCSP and Death Receptor 5 Y. He, D. Hendriks, R. Van Ginkel, D. Samplonius, E. Bremer, W. Helfrich (Netherlands)

552

Increased uptake of a novel doxorubicin-containing formulation in liver macrophages might indicate better hepatocellular cancer therapy V. Vshyukova, S. Swedmark, V. Nasek, I. Rebeko, E. Sanko-Schyslionak (Belarus)

537

Ex vivo culture of circulating tumour cell derived explants to facilitate rapid therapy testing in small cell lung cancer A. Lallo, U. Warpman Berglund, K. Frese, D. Potter, T. Helleday, C. Dive (United Kingdom)

553

Characterization of dabrafenib resistance mechanisms in multiple myeloma V. Figueroa Vazquez, S.S. Mughal, B. Brors, M.S. Raab (Germany)

538

Flubendazole as potential anti-neuroblastoma therapy option M. Michaelis, B. Agha, R. Florian, N. Loschmann, ¨ Y. Voges, F. Westermann, M. Wass, J. Cinatl Jr (United Kingdom)

554

Oligonucleotide aptamers as innovative therapeutic tools for triple-negative breast cancers S. Camorani, E. Crescenzi, M. Passariello, R. Fontanella, M. Fedele, A. Zannetti, L. Cerchia (Italy)

539

Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport M. Michaelis, F. Rothweiler, M. Wurglics, N. Aniceto, M. Wiese, M. Wass, T. Ghafourian, M. Schubert-Zsilavecz, J. Cinatl Jr (United Kingdom) The Resistant Cancer Cell Line (RCCL) collection M. Michaelis, M. Wass, J. Cinatl Jr (United Kingdom)

555

Carnosol inhibits tumor growth and metastasis of breast cancer through inhibition of the histone acetytransferase activity of p300 and proteasome-dependent degradation of STAT3 R. Iratni, E.H. Hussain, A.D. Yusra, V. Ranjit (U.A.E.)

540

Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status M. Michaelis, F. Rothweiler, N. Loschmann, ¨ M. Sharifi, T. Ghafourian, J. Cinatl Jr (United Kingdom)

556

Plasma microRNA profiles; down-regulation of plasma miR-107 level contributes to poor outcomes in pancreatic cancer T. Imamura, S. Komatsu, D. Ichikawa, M. Miyamae, R. Morimura, H. Ikoma, H. Konishi, A. Shiozaki, H. Taniguchi, E. Otsuji (Japan)

541

Systems level analysis of T-DM1 resistance in in vitro and in vivo models of HER-2 overexpressing breast cancer O. Sahin, O. Saatci, S. Durmus, E. Eyupoglu, S. Wiemann (Turkey)

557

558

mTOR inhibition overcomes docetaxel chemoresistance in prostate cancer cell and mouse models through downregulation of sphingosine kinase 1 H. Alshaker , Q. Wang, C. Cooper, M. Winkler, D. Pchejetski (Jordan)

543

Development of tumour spheroid models for cancer research and drug discovery R. Polanski, M. Vazquez-Chantada, S. Peel, P. Hopcroft, F. Ghari, C. Roberts, K. Roberts (United Kingdom)

559

CDK6 as a predictive marker of response to fulvestrant treatment and a potential indicator of benefit from CDK4/6 inhibitor therapy in estrogen receptor-positive metastatic breast cancer C. Alves, D. Elias, M. Lyng, M. Bak, A. Lykkesfeldt, H. Ditzel (Denmark)

544

Quambalarine B halts proliferation and reprograms metabolism of leukemic cells K. Valis, V. Grobarova, E. Stodulkova, M. Kuzma, D. Kavan, M. Kolarik, S. Bartova, P. Talacko, M. Flieger, J. Cerny, P. Novak (Czech Republic) Novel anion transporters as cytotoxic agents in lung cancer P. Manuel-Manresa, L. Korrodi-Gregorio, ´ A.M. Rodilla, R. Quesada, R. Perez-Tom ´ as, ´ V. Soto-Cerrato (Spain)

560

Identifying high quality, potent and selective inhibitors of ATM kinase: Discovery of AZD0156 B. Barlaam, K. Pike (United Kingdom)

545

Anionophores induce cell death and dysregulation of cancer-related miRNAs A.M. Rodilla, L. Korrodi-Gregorio, ´ P. Manuel-Manresa, R. Quesada, R. Perez-Tom ´ as, ´ V. Soto-Cerrato (Spain)

561

CRISPR-Cas9-based target validation for p53-reactivating model compounds M. Wanzel, J. Vischedyk, M. Gittler, N. Gremke, M. Mernberger, J. Charles, J. Schneikert, A.C. Bretz, A. Nist, T. Stiewe (Germany)

562

Identification of anti-angiogenic and anti-metabolic 546 compounds in-vitro and in-vivo in zebrafish to determine if novel dual action drugs can enhance radiosensitivity in oesophageal adenocarcinoma A. Buckley, N. Lynam Lennon, A. Cannon, R. Byrne, A. Reynolds, J.V. Reynolds, B. Kennedy, J. O’Sullivan (Ireland)

l


Abstract Nr.

Abstract Nr. Combined CDK and HDAC inhibition induces apoptosis in both uveal and cutaneous melanoma R. Heijkants, K. Willekens, A. Teunisse, M. Nieveen, J.C. Marine, A. Jochemsen (Netherlands)

563

Ritonavir interacts with ixazomib synergistically to cause ubiquitinated protein accumulation and endoplasmic reticulum stress in bladder cancer cells A. Sato, T. Asano, M. Isono, K. Okubo, T. Asano (Japan)

564

PI3K blockade reverses primary resistance and adaptation to eribulin in PI3K-pathway activated breast cancer tumors A. Gris-Oliver , C. Saura, M. Oliveira, A. Piris, P. Nuciforo, J. Perez-Garc´ ´ ıa, J. Arribas, J. Baselga, J. Cortes, ´ V. Serra (Spain)

565

Retargeting of anti-viral immune responses to solid tumors using bispecific adapters J. Niemann, N. Woller, M.P. Manns, S. Kubicka, F. Kuhnel ¨ (Germany)

566

Development of a drug discovery platform focused on SENPs as therapeutic targets for oncology Y. Ofir-Rosenfeld, N. Arnaudo, F. Enjalbert, J. Harrigan, M. Woodrow, A. Jones, M. Kemp, H. Robinson, X. Jacq (United Kingdom)

567

In vitro antitumor cytotoxicity of some macrofungi extracts 568 Z. Zizak, A. Klaus, M. Kozarski, J. Vunduk, M. Niksic (Serbia) The co-administration of anticancer and pro-apoptotic agents as a novel approach in liver cancer therapy W. Al-Shakarchi (United Kingdom)

569

RAD51 and BRCA2 enhance oncolytic adenovirus type 5 activity in ovarian cancer L. Tookman, B. Ashley, C. Connell, G. Bridge, C. Ingemarsdotter, S. Dowson, A. Shibata, L. Michelle, S. Martin, I. McNeish (United Kingdom)

570

Identification of molecular mechanisms of acquired resistance to trastuzumab in gastric cancer A. Sampera, M. Gelabert-Baldrich, F.J. Sanchez-Mart´ ´ ın, A. Dalmases, O. Arpi, M. Iglesias, A. Mart´ınez, A. Rovira, J. Albanell, C. Montagut (Spain)

571

sVEGFR1, the VEGFR1 splice variant: A dual function in the response of squamous cell lung carcinoma to anti-angiogenic therapies C. Abou Faycal, E. Brambilla, J. Agorreta, A. Lucas, P. Lacal, L. Montuenga, R. Pio, S. Gazzeri, B. Eymin (France)

572

Nuclear translocation of IGF-1R by amphiregulin: a regulator of the response of lung adenocarcinoma to EGFR-TKI? M. Guerard, T. Robin, P. Perron, C. Barial, J.L. Coll, B. Eymin, A. Hurbin, S. Gazzeri (France)

573

Design and characterization of novel polyurea/polyurethane 574 nanocapsules developed to improve cancer chemotherapy delivery ´ ´ (Spain) as C. Cusco, V. Soto-Cerrato, J. Rocas, R. Perez-Tom Design, synthesis and evaluation of novel and clinically used anti-cancer agents targeted to mitochondria O. Mohammed, S. Hussain, D. Mincher, A. Turnbull (United Kingdom)

575

Characterization of RNA aptamers as specific ligands of integrin alpha5 beta1 L. Choulier , M. Fenat, P. Fechter, A.F. Blandin, G. Renner, N. Etienne-Selloum, I. Lelong-Rebel, S. Martin, M. Lehmann, M. Dontenwill (France)

576

Novel cell-permeable PARG inhibitors are selective and sensitize cells to alkylating DNA damage D. James, E. Fairweather, L. Griffiths, G. Hopkins, A. Jordan, A. McGonagle, K. Smith, A. Stowell, I. Waddell, D. Ogilvie (United Kingdom)

577

Photodynamic therapy in combination with doxorubicin and methotrexate as an option in osteosarcoma G. Brites, B. Serambeque, M. Laranjo, G. Chohfi de Miguel, A. Serra, M. Pineiro, A.M. Abrantes, J. Casalta-Lopes, A. Rocha-Gonsalves, D. Priolli, M.F. Botelho (Portugal)

578

Development and synthesis of a potential versatile theranostic nanocarrier for cancer X. Siwe Noundou, C. Rambanapasi, R.W.M. Krause (South Africa)

579

Mechanistic differentiation of PI3K/mTOR, Aurora kinase and EZH2 inhibitor classes by comparative cancer cell line profiling J.C.M. Uitdehaag, J.A.D.M. De Roos, M.B.W. Prinsen, J.R.F. De Vetter, J. Dylus, A.M. Van Doornmalen, S.J.C. Van Gerwen, J. De Man, R.C. Buijsman, G.J.R. Zaman (Netherlands)

580

Evaluation of pre-analytical procedures for the detection of BRAF V600 mutations in melanoma patients: comparison between Sanger sequencing and Competitive allele-specific TaqMan PCR (Cast-PCR) R. Barbano, B. Pasculli, M. Coco, A. Fontana, M. Copetti, M. Rendina, V.M. Valori, P. Graziano, E. Maiello, V.M. Fazio, P. Parrella (Italy)

581

Molecular determinants of sensitivity and resistance to FGFR inhibition in FGFR2-amplified gastric cancer I. Babina, R. Cutts, J. Ning, E. McKnight, A. Pearson, A. Swain, N. Turner (United Kingdom)

582

Effects of doxorubicin-loaded dextran coated magnetic nanoparticles: on gene and protein expression profile of p53, survivin and bcl-2 in doxorubicin sensitive/resistant MCF-7 cell lines S. Yalcın, O. Onguru, U. Gunduz (Turkey)

583

miR-625-3p regulates oxaliplatin resistance by 584 directly targeting MAP2K6/MKK6 in human colorectal adenocarcinoma I. Lyskjær , M. Heilskov Rasmussen, R. Rakownikow JersieChristensen, L. Schmidt Tarpgaard, M. Muhlig Nielsen, J. Skou Pedersen, T. Falck Ørntoft, C. Lindbjerg Andersen (Denmark) Gli1/DNA interaction is a druggable target for Hedgehogdependent tumors P. Infante, M. Mori, R. Alfonsi, C. Ingallina, B. Botta, L. Di Marcotullio (Italy)

585

The unique binding mode of NTRC 0066-0, a novel inhibitor of the spindle assembly checkpoint kinase TTK (Mps1), leads to long target residence time and potent anti-tumor activity G. Zaman, J. Uitdehaag, J. De Man, N. WillemsenSeegers, J.G. Sterrenburg, J. De Wit, J. De Roos, M. Prinsen, R. Buijsman (Netherlands)

586

Thermally triggered theranostics for pancreatic cancer M. Malekigorji, P. Kong Thoo Lin, M. Lees, M. Gueorguieva, A. Curtis, C. Hoskins (United Kingdom)

587

Molecular and Genetic Epidemiology I Cancer of unknown primary is associated with diabetes X. Li (Sweden)

666


li

Abstract Nr.

Abstract Nr. Effects of WW domain-containing oxidoreductase (WWOX) gene polymorphisms in the Kozak sequence on the risk and progression of oral cancer C. Ying-Erh, S.F. Yang, C.W. Lin (Taiwan)

669

The role of MET in tumor resistance to radiation therapy: a phosphoproteomic approach E. Orlando, A. Bensimon, M. Medova, ` D.M. Aebersold, R. Aebersold, Y. Zimmer (Switzerland)

702

The effect of the CYP1A1*2A variant on colorectal cancer susceptibility in a British population E. Ozta¸s, G. Ozhan, A.K. Daly (Turkey)

670

703

Carcinogenic and non-carcinogenic risk assessment for children exposed to DDTs residues in pasteurized cow milk from Iran market N. Rastkari, M. Zare Jeddi, R. Ahmadkhaniha, M. Yunesian (Iran)

671

Single- and double strand breaks induced by proton beam radiation in human uveal melanoma cells ´ K. Jasinska, K. Berniak, P. Olko, B. Romanowska-Dixon, K. Urbanska, ´ J. Dobrucki, M. Elas (Poland)

Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms exacerbate bladder cancer risk associated with alcohol drinking: Gene−environment interaction H. Masaoka, H. Ito, N. Soga, A. Yokomizo, M. Eto, K. Matsuo (Japan)

672

Association between night shift work and methylation status of circadian rhythm genes among Polish nurses and midwives − preliminary results A. Bukowska, E. Wieczorek, B. Peplonska, M. Przybek, E. Jablonska, E. Reszka (Poland)

673

Prevention and Early Detection I A novel detection method of metastatic cells in the cerebrospinal fluid of pediatric population with medulloblastoma using fluorescence lifetime imaging microscopy S. Gershanov, H. Toledano, S. Michowiz, G. Yahav, O. Barinfeld, A. Hirshberg, M. Salmon-Divon, D. Fixler, N. Goldenberg-Cohen (Israel)

Cellular motility inhibition by proton beam irradiation 704 ´ K. Jasinska, K. Pochylczuk, E. Czajka, M. Michalik, M. Sarna, P. Olko, B. Romanowska-Dixon, K. Urbanska, ´ M. Elas (Poland)

685

Awareness and understanding of disease among hospitalized cancer patients in Pakistan N.A. Jadoon, F.U. Sulehri, N.A. Shair, M. Hussain (Pakistan)

686

Inflammatory potential of diet and risk of laryngeal cancer in a case–control study from Italy N. Shivappa, J. Hebert, V. Rosato, D. Serraino, C. La Vecchia (USA)

687

Blood-circulating nucleoprotein complexes: DNA and protein content in healthy donors and breast cancer patients O. Tutanov, S. Tamkovich, T. Duzhak, Y. Tsentalovich, P. Laktionov (Russian Federation)

689

Transcriptional variations associated with time to breast cancer development among African American women with benign breast disease A.N. Holowatyj, J.J. Ruterbusch, R. Ali-Fehmi, S. Bandyopadhyay, G. Dyson, D. Radisky, M.L. Cote (USA)

690

Circulating microRNA profiles in plasma: identification of 691 miR-224 as a novel diagnostic biomarker in hepatocellular carcinoma independent of hepatic function W. Okajima, S. Komatsu, D. Ichikawa, M. Miyamae, T. Ohashi, T. Imamura, J. Kiuchi, H. Taniguchi, O. Eigo (Japan)

Radiobiology/Radiation Oncology I Exploiting the DNA damage response to improve peptide receptor radionuclide therapy outcome J. Nonnekens, M. Van Kranenburg, M. De Jong, D. Van Gent (Netherlands)

701

Inhibition of anti-apoptotic Bcl-2 proteins by ABT-263 sensitizes hypoxic cancer cells to ionizing radiation V. Ritter , J. Rudner, J. Matschke, V. Jendrossek (Germany)

705

MicroRNA-330-5p downregulation in oesophageal adenocarcinoma is a potential therapeutic target for enhancing chemoradiation sensitivity in patients B. Bibby, C. Cawthorne, J. Reynolds, S. Maher (United Kingdom)

706

Investigating the role of microRNA-187 as a modulator of chemoradiation sensitivity in oesophageal adenocarcinoma S.D. Argyle, B.A.S. Bibby, N. Lynam-Lennon, J.V. Reynolds, C.J. Cawthorne, S.G. Maher (United Kingdom)

707

Protein kinase Ba (AKT1) plays a role in the cellular response to ionizing radiation K. Al-Refae, S. Oeck, H. Riffkin, G. Iliakis, V. Jendrossek (Germany)

708

Could radiotherapy produce a systemic synergistic effect when combined with abiraterone in patients with prostate cancer resistant to castration? F. Casas, K. Holub, B. Mellado, G. Oses, T. Herreros (Spain)

709

Characterization of biological effects in radiotherapy 710 applications of ultra-high dose rate pulses from a plasma focus device F. Buontempo, L. Isolan, I. Zironi, G. Castellani, R. Nano, F. Pasi, A. Tartari, D. Mostacci, M. Sumini, A.M. Martelli (Italy) Radiosensitization by interfering with arginine metabolism: New insights using 2-D and 3-D HNSCC models F. Manig, O. Chen, L. Lehmann, M. Huther, ¨ O. Stasyk, M. Baumann, L. Kunz-Schughart (Germany) Signalling Pathways I Wnt secretion is not required to sustain Wnt/b-catenin signaling in b-catenin mutant hepatocellular carcinoma cells W. Wang (Netherlands)

711

724

Targeting GSK3b downregulates the Hedgehog–Gli signalling pathway and reduces cell proliferation in human colon cancer D. Trnski, M. Sabol, A. Gojevic, M. Martinic, P. Ozretic, V. Musani, S. Ramic, S. Levanat (Croatia)

725

Expression of PTCH1b tumor suppressor gene is controlled by different 5 -untranslated region cis-regulatory elements P. Ozretic, A. Bisio, V. Musani, D. Trnski, M. Sabol, A. Inga, S. Levanat (Croatia)

727

USP15 regulates SMURF2 kinetics through C-lobe mediated deubiquitination P. Eichhorn, P. Iyengar, P. Jaynes, D. Lama, C. Verma (Singapore)

728

lii


Abstract Nr.

Abstract Nr. The LATS2 tumor suppressor inhibits SREBP and suppresses hepatic cholesterol accumulation Y. Aylon, A. Gershoni, R. Rotkopf, A. Koh, X. Sun, M.I. Fiel, Y. Hoshida, S. Friedman, R. Johnson, M. Oren (Israel)

729

Expression of mTOR pathway components in gastric neuroendocrine neoplasms I. Peregorodiev, V. Delektorskaya, V. Bokhian, I. Stilidi (Russian Federation)

744

Role of the estradiol in the development of papillary thyroid carcinoma in women E. Paolicchi, G. Figlioli, M. Cipollini, K. Hemminki, A. Forsti, ¨ S. Landi, F. Gemignani (Italy)

730

745

Messenger-RNA characteristics in triple negative breast cancer A. Tavartkiladze, R. Khutsishvili, A. Gogiberidze (Georgia)

731

Targeting the E3-ubiquitin ligase RNF144B to inhibit proliferation in oestrogen receptor negative endometrial cancer cells Q. Zhou, T. Jackson, S. Elhakdakhny, P. Townsend, E. Crosbie, B. Sayan (United Kingdom)

746

Cancer heterogeneity is incompatible with a unique cell metabolic map J. Dumont (Belgium)

732

Restricting growth and spreading of paediatric medulloblastoma by blocking kinase signalling-dependent brain infiltration D. Tripolitsioti, K. Santhana Kumar, A. Neve, M. Pillong, J. Kunze, G. Schneider, T. Shalaby, M. Grotzer, M. Baumgartner (Switzerland)

Detection of dual role of reactive oxygen species in experimental RS-1 hepatocellular carcinoma M.I. Gruia, A. Busca, E.M. Panait, V. Negoita, M. Dumitru, I. Gruia (Romania)

733

miRNA in cancer physiopathology and diagnosis: A complex tool J.E. Dumont (Belgium)

747

734

From proteomic to therapeutic analysis: functional profile of two lung cancer cell lines widely studied in pre-clinical research L. Korrodi-Gregorio, ´ V. Soto-Cerrato, R. Vitorino, ´ ´ (Spain) as M. Fardilha, R. Perez-Tom

748

The roles of AMPK and TAK1/NF-kB signaling cascade in governing cancer cell aggressiveness in omental microenvironment R. Chen, H.Y. Ngan, D.W. Chan (Hong Kong) XBP1 modulates luminal breast cancer by transcriptional regulation of SRC-3 during estrogen signalling S. Gupta, A. Gupta, M.M. Hossain, G. Callagy, K. Michael (Ireland)

735

Protein phosphatase 4 regulates cell survival and responses 749 to PI3K/AKT/mTOR pathway inhibitors in breast cancer cell lines H.N. Mohammed, M. Mourtada-Maarabouni (United Kingdom)

Mammaglobin-A in human breast cancer pathology and survival L. Baker , D. Wilson, N. Whiteoak, L. Hall, J. France, P. Bhaskar (United Kingdom)

736

E2F7 regulates transcription and maturation of multiple microRNAs to restrain cell proliferation J. Vallejo-Rodr´ıguez, J. Mitxelena, A. Apraiz, M. Malumbres, A.M. Zubiaga (Spain)

750

FGFR1 upregulation causes resistance to erlotinib in non-small cell lung cancer cell lines K. Jacobsen, H.C. Beck, H.J. Ditzel (Denmark)

737

751

Heregulin-1b/HER3 system in hepatocellular carcinoma: status and regulation by insulin C. Buta, E. Benabou, M. Lequoy, H. Regnault, ´ D. Wendum, O. Scatton, O. Rosmorduc, F. Praz, L. Fartoux, C. Desbois-Mouthon (France)

738

Loss of viability correlates with altered organ homeostasis in the GI tract of adult, Sos1/2-depleted mice ´ C. Gomez, R. Garc´ıa-Navas, P. Liceras-Boillos, B. Anta, F.C. Baltanas, ´ D. Jimeno, A. Fernandez-Medarde, N. Calzada, M. Jimenez, ´ E. Santos (Spain) Human adipose tissue macrophages display activation of cancer-related pathways T. Mayi, D. Mehdi, B. Gael, W. Wouters, P. Fran¸cois (France)

753

BRAF and Src/FAK are required for TGFb-induced EMT of thyroid cancer cells P. Baquero, E. Jimenez-Mora, ´ A. Santos, B. Gallego, M. Lasa, A. Chiloeches (Spain)

739

‘Notch’ wings to target cancer G. Monticone, H. Shimizu, M. Baron (United Kingdom)

754 755

Sos1 disruption increases mitochondrial oxidative stress in primary MEFs R. Garc´ıa-Navas, P. Liceras-Boillos, B. Anta, C. Lillo, C. Gomez, ´ D. Jimeno, N. Calzada, M.S. Jimenez, F.C. Baltanas, ´ E. Santos (Spain)

740

HER2 positivity is high in breast cancer of young women: Experience of a single institute in Turkey E. Celikmakas, Y. Karslioglu, I. Hacibey, S. Kilic, M. Ozturk, M. Safali, O. Onguru (Turkey)

The dual inhibition of RNA Pol I transcription and PIM kinase 741 as a new therapeutic approach to treat advanced prostate cancer R. Rebello, E. Kusnadi, D. Cameron, H. Pearson, A. Lesmana, J. Devlin, D. Drygin, A. Clark, L. Porter, J. Pedersen, S. Sandhu, G. Risbridger, R. Pearson, R. Hannan, L. Furic (Australia) ETV5 as a mediator of the oncogenic effects of mutant FGFR3 in bladder cancer E. Di Martino, M. Knowles (United Kingdom)

742

The prognostic significance of eukaryotic initiation factors 4E and 5 in male breast cancer M. Humphries, S.R. Sreekumar, A. Hanby, V. Speirs (United Kingdom)

743

Translational Research I The molecular rationale for steroid based therapy of leukemia: diagnostic and therapeutic implications E. Yefenof , P. Stepensky, K. Shlomit (Israel)

788

Development of a deregulating MicroRNA panel for the detection of early relapse in postoperative colorectal cancer patients J.Y. Wang, I.P. Yang (Taiwan)

789

A new somatic mutation in the coding region of KRAS gene (G48A) found in a NSCLC patient E. Caiola, M. Marabese, M.C. Garassino, G. Rastelli, G. Settanni, S. Brugnara, M. Broggini, M. Ganzinelli (Italy)

790

Genomics study to identify novel cancer genes predictive for prognosis and 5-FU benefit in stage II/III colorectal cancer M. Parsons, O. Sieber, D. Mouradov (Australia)

791


Abstract Nr.

Abstract Nr. BAP1-deficient mesothelioma: identifying mechanisms for response to HDAC inhibitors and screening for novel drug sensitivities Z. Butt, J. Kenyani, J. Sacco, J. Coulson (United Kingdom)

792

Horses for courses − analytical study on the feasibility of ALK dual colour break-apart FISH assay for the detection of inv2(p21p23) G. Pajor , G. Smuk, A. Boka, L. Pajor, T. Tornoczky, I. Chudoba (Hungary)

793

liii

Discovery of high-Gleason prostate cancer biomarker 807 candidates from exosomes in urine after massage by proteomic analysis and subsequent verification using selected reaction monitoring K. Fujita, H. Kume, K. Matsuzaki, A. Kawashima, T. Ujike, A. Nagahara, M. Uemura, T. Tomonaga, N. Nonomura (Japan) Prognostic potential of EphA2 expression in prostate cancer patients A. Bromby, C. Hart, M. Brown, N. Clarke (United Kingdom)

808

Presence of TUBB3-positive cells in morphologically normal 794 lung tissue: the cancerization field or cancer cells spreading outwards the primary site? I. Mamichev, T. Bogush, E. Dudko, S. Kaliuzhny, O. Ryabinina, A. Grishanina, B. Polotsky, M. Davydov (Russian Federation)

Prostate cancer: A personalised approach through the development of patient-derived xenografts A. Cannistraci, M. Parry, M. Smith, V. Ramani, M. Lau, J. Shanks, N. Daisuke, N. Clarke, N. Dhomen, E. Baena, R. Marais (United Kingdom)

809

The EGFR synonymous polymorphism rs1050171 predicts responsiveness to anti-EGFR therapy in metastatic colorectal cancer patients S. Bonin, M. Donada, G. Bussolati, E. Nardon, M. Pichler, A.M. Chiaravalli, C. Capella, L. Annaratone, G. Hoefler, G. Stanta (Italy)

795

An open source R package for Droplet Digital PCR analysis 810 A. Chiu, G. Brady, M. Ayub, C. Dive, C. Miller (United Kingdom)

Cathepsin B overexpression regulates the migration and progression in oral squamous cell carcinoma W.E. Yang, S.F. Yang, M.K. Chen (Taiwan)

797

Combination of CDK4/6 inhibitor PD-0332991 with standard chemotherapy is synergistic in RB high pancreatic cancer A. Chou, A. Steinmann, D. Froio, A. Drury, D. Wohl, V. Chin, A. Nagrial, A. Gill, M. Pajic (Australia)

798

Humanizing the NOD/SCID/IL-2Rgnull (NSG) mice using busulfan and retro-orbital injection of umbilical cord blood-derived CD34+ cells Y. Ko, J.H. Seo, M. Lee, J.A. Lee (South Korea)

799

Hsa-miR-375 and local control in early stage breast cancer B. Zellinger , F. Zehentmayr, C. Hauser-Kronberger, P. Strasser, U. Bodenhofer, G. Fastner, R. Reitsamer, T. Fischer, F. Sedlmayer (Austria)

800

DNp63 is critical for progression of high grade non801 muscle invasive bladder cancer through deregulation of specific genetic pathways M. Castillo-Martin, N. Gladoun, A. Collazo Lorduy, J.M. Gaya, F. Algaba, J. Palou Redorta, C. Cordon-Cardo (Portugal) Quantitative estimation of BRCA1 protein expression in 802 breast cancer tissue using the method of flow cytometry E. Shestakova, E. Dudko, A. Grishanina, V. Kirsanov, N. Vichljantzeva, S. Kolomiytsev, T. Bogush (Russian Federation)

PATZ1 is a new prognostic marker of diffuse large B cell 811 lymphomas R. Franco, G. Scognamiglio, E. Valentino, M. Vitiello, L. Panico, A. Pinto, G. Botti, A. De Chiara, L. Cerchia, M. Fedele (Italy) Molecular profiling of circulating tumour cells (CTCs) in non-small cell lung cancer within the TRACERx study of intratumoural heterogeneity and evolution S. Gulati, D.G. Rothwell, D. Burt, B. Mesquita, C. Wirth, G. Wilson, J. Pierce, G. Brady, C. Swanton, C. Dive (United Kingdom)

812

The prognostic potential of the long non-coding RNA MALAT1 in prostate cancer K. Hiew, S.M. Bokobza, C.A. Hart, T. Elliott, N.R. Smith, M. Brown, N.W. Clarke (United Kingdom)

813

A novel PCR error correction algorithm for cell-free DNA next generation sequencing data using high performance computing C.S. Kim, S. Gulati, M. Ayub, D.G. Rothwell, S. Mohan, C. Dive, G. Brady, C. Miller (United Kingdom)

814

CHL1 methylation may act as a tumour suppressor gene and predicts poor prognosis in breast cancer ´ E. Mart´ın-Sanchez, S. Mendaza, A. UlaziaGarmendia, I. Monreal-Santesteban, A. Cordoba, ´ F. Vicente-Garc´ıa, J.J. Illarramendi, I. Blanco-Luquin, E. Pernaut-Leza, D. Guerrero-Setas (Spain)

815

Rab Coupling Protein dependent recycling of P-glycoprotein promotes drug/chemo-resistance V. Phatak, P. Muller (United Kingdom)

816

Platelet derived growth factor receptors regulated microRNAs in non-small cell lung cancer S. Naidu, M. Garofalo (United Kingdom)

817

Identifying novel DDR targets; the Cancer Research UK Manchester Institute approach M. Watson, D. James, H. Begum, S. Durant, L. Goodwin, L. Griffiths, A. Jordan, H. Small, I. Waddell, D. Ogilvie (United Kingdom)

803

804

Investigating blood cells’ chromosomal conformation patterns as new diagnostic markers for prostate cancer D. Pchejetski, H. Alshaker, M. Winkler, A. Akoulitchev (United Kingdom)

818

Targeting LRG1 potentiates vascular normalisation and reduces growth in subcutaneous tumours D. Kallenberg, M. O’Connor, L. Dowsett, J. George, M. Gourlaouen, N. Jeffs, S. Moss, J. Greenwood (United Kingdom)

820

Systematic longitudinal analysis of circulating tumour DNA in melanoma patients undergoing systemic therapy G. Gremel, R.J. Lee, M.R. Girotti, G. Garner, A.K. Mandal, S. Valpione, P. Serra-Bellver, S. Wood, A. Fusi, N. Dhomen, P. Lorigan, R. Marais (United Kingdom)

805

CDH22 is downregulated by methylation and acts as an independent prognostic factor in breast cancer S. Mendaza, E. Mart´ın-Sanchez, ´ A. Ulazia-Garmendia, I. Monreal-Santesteban, A. Cordoba, ´ F. Vicente-Garc´ıa, S. De la Cruz, I. Blanco-Luquin, D. Guerrero-Setas (Spain) Dichloroacetate-induced metabolic reprogramming into oral squamous cell carcinomas deeply impacts on mitochondrial morphology and dynamics V. Ruggieri, F. Agriesti, T. Tataranni, C. Mazzoccoli, C. Piccoli (Italy)

821

liv


Abstract Nr.

Abstract Nr. Tumour–microenvironment mediates resistance to 822 immuno and targeted therapies in acral melanoma R. Lee, M.R. Girotti, G. Khandelwal, F. Baenke, A. Viros, A. Mandal, J. Bridgeman, E. Galvani, G. Gremel, M. Kalaitsidou, G. Ashton, I. Peset, M. Smith, R. Hawkins, A. Fusi, C. Miller, D. Gilham, N. Dhomen, P. Lorigan, R. Marais (United Kingdom)

Application of sequencing, liquid biopsies and patientderived xenografts for personalized medicine in melanoma M.R. Girotti, G. Gremel, R. Lee, E. Galvani, D. Rothwell, A.K. Mandal, K.H.J. Lim, G. Saturno, S.J. Furney, F. Baenke, M. Pedersen, M. Smith, A. Fusi, N. Dhomen, G. Brady, P. Lorigan, C. Dive, R. Marais (United Kingdom)

836

New insights in the molecular pathogenesis of Epstein–Barr virus-positive and -negative post-transplant diffuse large B-cell lymphoma J. Morscio, J. Finalet Ferreiro, D. Dierickx, G. Verhoef, I. Wlodarska, T. Tousseyn (Belgium)

823

Modelling anti-PD-1 treatment in a transgenic murine model of BRAFV600E-driven melanoma E. Galvani, K. Hogan, L. Boon, G. Gremel, A. Viros, A. Mandal, M. Smith, J. Swan, A. Banyard, G. Ashton, N. Dhomen, R. Marais (United Kingdom)

837

Bcl-XL antagonism is essential for sensitisation of ovarian cancer cell lines to carboplatin M. Najim Abed, R. Alan (United Kingdom)

824

839

Combined inhibition of PD1 and CD96 checkpoints improves survival in a resectable murine model of pancreatic cancer J. Brooks, B. Fleischmann-Mundt, N. Woller, M.P. Manns, S. Kubicka, E. Gurlevik, ¨ F. Kuhnel ¨ (Germany)

825

Clinical significance of non-coding RNAs expression in human hepatocellular carcinoma K. Hur , S.Y. Jang, S.Y. Park, G. Kim, Y.H. Choi, Y.R. Lee, S.H. Lee, S.K. Jang, W.Y. Tak, Y.O. Kweon (South Korea) Dickkopf-1 (DKK-1) expression is a novel lymph node metastasis predictor in early gastric cancer Y.J. Huh, H.M. Lee, M.S. Cho, J.H. Lee (Korea)

840

Phenotypic characterisation of a panel of SCLC circulating tumour cell derived explant models (CDX) F. Trapani, D. Nonaka, N. Simms, K. Frese, I. Peset, K. Simpson, M. Carter, L. Franklin, F. Blackhall, C. Dive (United Kingdom)

826

Selective inhibition of notch signalling and cancer stem cells by an antibody targeting active ADAM10 L. Atapattu, N. Saha, C. Chheang, M. Vail, M. Eissman, M. Ernst, A. Scott, D. Nikolov, P. Janes (Australia)

841

NUB1 protein regulates cullins and E3 ligases in estrogen receptor negative breast cancers K.L. Tan, S. Haider, C. Zois, H. Turley, R. Leek, A. Harris, F. Buffa, O. Acuto, F. Pezzella (United Kingdom)

827

Hypermethylation and downregulation of glutathione peroxidase 3 are related to pathogenesis of melanoma J.Y. Bae, X. Zhang (Korea)

828

Bisphosphonates potentiate the activity of pitavastatin against ovarian cancer cells M. Abdullah, A. Richardson (United Kingdom)

829

Whole exome analysis of HER-2 positive human breast cancers: molecular mechanisms underlying response to neoadjuvant therapy with trastuzumab M. La Ferla, P. Aretini, C. Scatena, M. Menicagli, F. Lessi, S. Franceschi, L. Cantini, G. Bevilacqua, A.G. Naccarato, A. Fontana, C.M. Mazzanti (Italy)

830

Similarities between the Marfan syndrome and cancer: Implications of the Fibrillin−TGFb axis on cancer biology and treatment J.L. Parra, R. Mayor, I. Huber, I. Cuartas, A. Arias, C. Raventos, ´ J. Seoane (Spain) NSCLC − multiplex immunohistochemical staining for diagnosis in small biopsies B.S. Nielsen, K. Holmstrøm, T. Møller, H. Stender, M. Kristensson, E. Santoni-Rugiu (Denmark)

Tumour Immunology I A combination of T regulatory cell-specific markers to determine the expanded subsets in cancer patients E. Elkord, M. Abd Al Samid, B. Chaudhary, K. Yazan, B. Ammori (U.A.E.)

900

Development of an autoimmune-mediated strategy for bladder cancer vaccination in mice K. Izgi, B. Iskender, H.B. Ulusoy, H. Canatan (Turkey)

901

IL-17C promotes tumor-associated inflammation and lung tumor growth C. Jungnickel, L. Bittigkoffer, A. Kamyschnikow, C. Herr, R. Bals, C. Beisswenger (Germany)

902

Rewiring the cytokine network in melanoma R. Nagaraju, C. Dee, J. Barriuso, H. Young, M. Abdelmouti, A. Hurlstone (United Kingdom)

903

831

Programmed Death Ligand 1 (PD-L1)-targeted TRAIL combines PD-L1-mediated checkpoint inhibition with TRAIL-mediated apoptosis induction D. Hendriks, Y. He, I. Koopmans, V. Wiersma, R. Van Ginkel, D. Samplonius, W. Helfrich, E. Bremer (Netherlands)

904

832

Electrochemotherapy immune response enhancement by gene electrotransfer using IL-2 and IL-12 genes in canine patients F. Maglietti, S. Michinski, S. Emanuela, M. Tellado, G. Marshall (Argentina)

905

Population analysis of patients diagnosed with renal 833 carcinoma − A retrospective single center study in Argentina M.N. Gandur Quiroga, M.D.N. Juarez Rusjan, M.A. Krasnapolski, C. Pulero, L.B. Todaro, P.J. Azurmendi (Argentina) Monitoring circulating tumour DNA in patients receiving selective internal radiation therapy for liver metastases and intrahepatic cholangiocarcinoma H. Winter , P. Kaisaki, A. Cutts, R. Carter, T. Greenhalgh, A. Schuh, J. Taylor, R. Sharma (United Kingdom)

834

Pitavastatin is a potential treatment for drug-resistant ovarian cancer E. Robinson, S. Jones, K. Menezes, M. Abdullah, E. Stronach, C. Hoskins, A. Richardson (United Kingdom)

835

Nanoplex activated DCs sparking T cells as tumour 906 cytotoxic mediators B. Wingham, J. Greenman, C. Dyer, E. Rosca (United Kingdom) Increased serum levels of IL-8 and TGF-b accompanied with CD26 alterations in patients with metastatic colorectal cancer I. Matic, I. Besu Zizak, M. Djordjic Crnogorac, A. Damjanovic Velickovic, B. Kolundzija, J. Spasic, D. Radosavljevic, J. Milovanovic, N. Todorovic-Rakovic, Z. Juranic (Serbia)

907

Irradiated autologous multicellular vaccine isolated from fresh carcinoma induces multiple-target antitumour immunity F. Chunju, M. Xiyan, W. Yuquan (China)

908


lv

Abstract Nr.

Abstract Nr. Natural killer cells from patients with malignant or inflammatory pleural effusions display decreased cytotoxicity and a decidual NK-like phenotype S. Zanellato, A. Musco, A. Bruno, B. Bassani, C. Sampietro, M. Cattoni, A. Imperatori, A. Albini, D.M. Noonan, L. Mortara (Italy)

909

A TLR7 agonist enhances the anti-tumour efficacy of obinutuzumab through an NK cell/CD4 dependent mechanism in a murine lymphoma model E. Cheadle, G. Lipowska-Bhalla, E. Fagnano, C. Klein, S. Dovedi, J. Honeychurch, T. Illidge (United Kingdom)

910

Combined aerosol immunotherapy in the treatment of lung metastases V.M. Le Noci, M. Sommariva, C. Storti, E. Tagliabue, A. Balsari, L. Sfondrini (Italy)

911

The expression of MNT, a MYC antagonist, is autoregulated at the mRNA and protein level ˜ F. Ourique, M.C. Lafita, J. Liano, J. Aresti, P.J. Hurlin, J. Leon (Spain)

146

A novel peptide-specific monoclonal antibody with a potential use for immunotherapy recognizes AMHR2 expressed by ovarian cancer cells C. Sakalar , H. Aksu, A. Turan, S. Kaya, C. Mustafa, K. Busra, S. Sedat, C. Halit (Turkey)

912

147

Early functional activation of natural killer cell and dendritic cell during the antitumor therapeutic response induced by TNFa tumor vessel delivery and melphalan S. Zanellato, E. Balza, A. Poggi, D. Reverberi, A. Rubartelli, L. Mortara (Italy)

913

Molecular analysis of circulating tumour cells identifies distinct profiles in treatment na¨ıve chemosensitive and chemorefractory small cell lung cancer patients D.G. Rothwell, B. Mesquita, L. Carter, C. Smowton, H.S. Leong, D. Burt, C. Miller, F. Blackhall, C. Dive, G. Brady (United Kingdom) The binding profile of proteins is maintained in snap-frozen tissue A. Uwineza, C. Kenny, M. O’Sullivan (Ireland)

148

The expressions of genes causing histone modifications in colorectal carcinomas with signet ring cells S. Yalcin, Y. Karslioglu, E. Celikmakas, B. Kurt, O. Onguru (Turkey)

149

Deregulation of IGF2, FZD10, MAPK3, SMAD4 and SRF expression in colorectal cancer M. Ibarrola-Villava, N. Tarazona, V. Gambardella, C. Mongort, S. Navarro, S. Garcia-Botello, S. Rosello, A. Cervantes, G. Ribas (Spain)

150

Methylation profile of candidate genes in gastric cancer with microsatellite instability using high-throughput MALDI-TOF mass array technology: The role of RUNX3 in cancer progression ˜ M.J. Llorca Cardenosa, T. Fleitas, M. Ibarrola-Villava, M.C. Pena-Chilet, ˜ C. Mongort, L. Navarro, S. Navarro, G. Ribas, A. Cervantes (Spain)

151

The Shiga Toxin receptor Gb3/CD77, a promising therapeutical target in gastrointestinal cancer, is epigenetically regulated M. Perl, M. Gehrmann, U. Nitsche, G. Multhoff, L. Johannes, K.P. Janssen (Germany)

152

Poster Sessions, Monday 11 July 2016 10:15–17:15

TGF-b and immunity to gliadin and cow’s milk proteins in 914 patients with metastatic colorectal cancer I. Besu, I. Matic, D. Radosavljevic, J. Spasic, Z. Juranic (Serbia) Phosphopeptides as novel tumour antigens in colorectal 915 cancer S. Penny, J. Abelin, A. Saeed, S. Malaker, P. Trantham, J. Shabanowitz, S. Ward, D. Hunt, M. Cobbold (United Kingdom) Abolition of solid tumors and their metastases by intratumoral alpha radiation in combination with CpG and inhibitors of immune suppressor cells Y. Keisari, H. Confino, M. Scmidt, M. Efrati, V. Umansky, I. Kelson (Israel)

916

IKKe/IKBKE integrates LPS and IL-17A signaling cascades to establish an inflammatory microenvironment in intestinal tumors S. Goktuna, K. Shostak, T.L. Chau, L. Heukamp, P. Close, S. Rahmouni, G. Van Loo, R. Buttner, F. Greten, A. Chariot (Turkey)

917

Modeling the immunological response to clinically relevant radiotherapy in a murine prostate tumour model D. Mukherjee, E. Ani, J. Honeychurch, T. Illidge (United Kingdom)

917A

Cancer Genomics, Epigenetics and Genome Instability II Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing J. Demeulemeester , E.K. Møller, P. Kumar, N. Silje, D.C. Wedge, K.P. White, B. Naume, V.N. Kristensen, P. Van Loo, T. Voet (United Kingdom)

145

Exploiting DNA damage repair defects for effective targeting 153 of acute myeloid leukaemia by PARP inhibitors M.T. Esposito, L. Zhao, T.K. Fung, J. Rane, A. Wilson, N. Martin, J. Gil, A.Y. Leung, A. Ashworth, C.W.E. So (United Kingdom) Targeted next generation sequencing of Bulgarian prostate 154 cancer patients finds new somatic mutations and reflects disease heterogeneity D. Kachakova, A. Vlahova, K. Mihova, A. Mitkova, I. Popov, E. Popov, S. Christova, C. Slavov, V. Mitev, R. Kaneva (Bulgaria) CRISPR/Cas-9-mediated targeting of TP53 and MYC to investigate antimitotic mode of action S. Littler , H. Whalley, S. Sousa, S. Taylor (United Kingdom)

155

Search for predisposing alleles in Hungarian non-BRCA breast and ovarian cancer families ´ T. Pocza, A. Bozsik, J. Papp, T. Vaszko, ´ E. Olah ´ (Hungary)

156

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Abstract Nr. Genetic and epigenetic evaluation of potentially important subtypes of clear cell sarcoma of kidney (CCSK) C. Kenny, S. Bausenwein, A. Lazaro, R. Furtwangler, ¨ S.L.M. Gooskens, M. Van den Heuvel Eibrink, C. Vokuhl, I. Leuschner, N. Graf, M. Gessler, M.J. O’Sullivan (Ireland)

157

Deep resequencing of the regions assigned by GWAS reveals new candidate breast cancer susceptibility variants ´ A. Bozsik, J. Papp, T. Vaszko, ´ T. Pocza, T. Gyuris, B.L. Balint, ´ E. Olah ´ (Hungary)

158

In vivo overexpression of EMI1 promotes chromosome instability and tumorigenesis P. Duijf (Australia)

159

Understanding loss of SDF1 expression in colon tumors R. Benbrika Nehmar , B. Romain, L. Marisa, C. Bour, I. Duluc, E. Pencreach, D. Guenot (France)

160

Functional evaluation of breast cancer case-associated non-coding variants in BRCA1/2 J. Sevcik, M. Janatova, Z. Kleibl, P. Kleiblova, M. Borecka, P. Zemankova, K. Zdarilova, L. Stolarova, M. Brown (Czech Republic)

161

Identification of pancreatic cancer susceptibility genes in the Czech Republic M. Borecka, M. Janatova, P. Zemankov ´ a, ´ L. Stolarova, K. Zdarilova, J. Soukupova, J. Sevcik, P. Kleiblova, Z. Kleibl (Czech Republic)

162

CHEK2 gene analysis in 1020 high-risk breast and ovarian cancer patients in the Czech Republic L. Stolarova, P. Kleiblova, P. Zemankova, M. Janatova, M. Borecka, K. Zdarilova, J. Sevcik, J. Soukupova, Z. Kleibl (Czech Republic)

Genetic variant in miR-618 in the risk of chronic lymphocytic leukemia I. Mart´ın-Guerrero, A. D´ıaz-Navarro, A. Gutierrez-Camino, A. Garc´ıa-Orad (Spain)

173

Involvement of miR-3117 in pediatric acute lymphoblastic 174 leukemia susceptibility A. Gutierrez-Camino, S. Varona-Fernandez, ´ N. Garc´ıa de Andoin, A. Navajas, A. Sastre, A. Carbone´ Baneres, ˜ V. Dolzan, I. Astigarraga, I. Mart´ın-Guerrero, A. Garc´ıa-Orad (Spain) The KDM5B demethylase in the normal and malignant mammary gland F. Kogera, C. Steven, B. Spencer-Dene, G. Picco, V. Tajadura-Ortega, Y.H. Chen, E. Bennett, J. Quist, J. Taylor-Papadimitriou, J. Burchell (United Kingdom)

175

Comprehensive characterization of matched pre-treatment biopsies and residual disease of chemotherapy treated breast cancer M. Hoogstraat, E. Lips, L. Mulder, P. Nederlof, G. Sonke, S. Rodenhuis, J. Wesseling, L.F.A. Wessels (Netherlands)

177

Epigenetic control of CD24 expression in colorectal cancer M. Ayub, W. Bodmer (United Kingdom)

178 179

163

Digital sorting enables whole-exome and low-pass wholegenome sequencing from low tumor-content formalin-fixed paraffin embedded (FFPE) biopsies C. Forcato, J. Laliberte, C. Bolognesi, C. Schumacher, G. Buson, C. Mangano, P. Tononi, G. Medoro, T. Harkins, N. Manaresi (Italy)

180

Epigenetic and genetic characterization of pancreatic acinar cell carcinoma ¨ F. Bergmann, C. Plass, C. Siebenkas, O. Popanda, P. Schmezer (Germany)

164

An oncogenomics-based in vivo screen identifies novel melanoma tumor-suppressors M. Olvedy, J.C. Tisserand, F. Luciani, B. Boeckx, F. Rambow, J. Wouters, J. Van den Oord, D. Lambrechts, P. De Sepulveda, J.C. Marine (Belgium) Novel regulators of ESR1 activity T. Campbell, B. Ponder, K. Meyer (United Kingdom)

181

Integrative analysis of methylation and copy number alteration reveals molecular subgroups of cholangiocarcinoma R. Toth, B. Goeppert, D.B. Lipka, D. Brocks, M. Baehr, S. Roessler, P. Schirmacher, C. Plass, D. Weichenhan (Germany)

166

The characterisation of potential fusion genes in breast cancer A. Mohd Noor , S. Maguire, J. Watkins, J. Quist, H. Mirza, K. Ougham, A. Tutt, C. Gillett, R. Natrajan, A. Grigoriadis (United Kingdom)

182

183

Single nucleotide variants at the MAP3K1/SETD9 locus on chromosome 5q11.2 are associated with somatic PIK3CA mutations in breast cancer U. Pfeffer , R. Puzone (Italy)

167

Genetic analyses of early-onset gastric cancer P. Kapusta, J. Machlowska, A. Bogdali, P. Radkowski, F. Morsink, W. Polkowski, J. Offerhaus, P. Wołkow, R. Maciejewski, R. Sitarz (Poland)

The somatic mutation profiles of 173 genes in over 2400 168 primary breast cancers B. Pereira, S.F. Chin, O. Rueda, H. Northen, M. Ross, J. Peden, D. Bentley, S. Aparicio, C. Caldas (United Kingdom) The emerging role of ATRX and chromatin remodeling in pleomorphic sarcomas oncogenesis M. Michaud, G. Perot, ´ T. Lesluyes, N. Desplat, L. Delespaul, C. Lucchesi, A. Neuville, J.Y. Blay, J.M. Coindre, F. Chibon (France)

169

MicroRNA profiling of pediatric low-grade gliomas (pLGGs) 171 G. Catanzaro, Z.M. Besharat, A. Mastronuzzi, A. Carai, E. Miele, A. Po, V. Alfano, F. Giangaspero, F. Locatelli, E. Ferretti (Italy) 14q32 miRNA cluster: A hot spot for osteosarcoma risk in a Spanish population I. Mart´ın-Guerrero, N. Bilbao-Aldaiturriaga, J. Uriz, A. Garc´ıa-Orad (Spain)

172

Deregulated microRNAs in breast ductal carcinoma in situ 184 (DCIS) with invasive propensity S. Volinia, V. Bertagnolo, F. Brugnoli, S. Grassilli, M. Galasso, C. Scatena, F. Lessi, C.M. Croce, S. Capitani (Italy) Targeted enrichment method using molecular barcodes to improve low frequency allele detection K. Zobeck, L. Forsmark, C. LeCocq, H. Tao, B. Arezi, H. Johansson (USA)

185

“Melanomics”: analysis and integration of whole genomes, 186 transcriptomes and miRNomes of primary melanoma patients S. Reinsbach, A. Wienecke-Baldacchino, A. Ginolhac, L. Vallar, A. Muller, A. Krishna, P. Nazarov, P. May, S. Kreis (Luxembourg) Investigation on Korean gastric tumorigenesis by performing whole transcriptome and miRNA sequence analysis Y.H. Koh, H.J. Seul, J.B. Seo, H.M. Kim, K. Ahn, H.S. Kang, B. Han, H.S. Kim, G. Jang, J. Seo, K.C. Kim, H.S. Na, S.E. Choi, J.W. Cho, D.Y. Zang (Korea)

187


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Abstract Nr.

Three-dimensional ECM-based cell culture models for cancer research K. Storch, E. Dickreuter, A. Vehlow, N. Cordes (Germany)

364

The bone microenvironment as a master regulator of tumour cell dormancy in breast cancer − evidence from novel in vivo models P. Ottewell, H. Brown, G. Allocca, M.T. Haider, N. Brown, I. Holen (United Kingdom)

365

PI3KC2a, a new spindle associated protein involved in genomic instability and tumorigenesis M.C. De Santis, M. Martini, G. Federico, A. Ghigo, C.C. Campa, R. Chiarle, A. Sapino, E. Hirsch (Italy)

366

Angiogenic markers in hepatocellular carcinoma R. Albulescu, L.G. Necula, A.I. Neagu, V. Herlea, S.O. Dima, C. Tanase, I. Popescu (Romania)

367

New data on molecular phenotype of ascitic tumor cells in ovarian cancer patients T. Bogush, S. Kaliuzhny, E. Dudko, O. Rjabinina, N. Vichljantzeva, A. Tjulandina, E. Bogush, S. Tjulandin, M. Davydov, I. Mamichev (Russian Federation)

369

UNG deficient BALB/c mice do not develop hyperplasia or 205 B cell lymphomas L. Alsøe, A.B. Wennerstrom, ¨ T. SenGupta, H. Nilsen (Norway)

NEAT1, a long non-coding RNA, controls cell survival and is up-regulated in breast cancer Z.A. Almnaseer , M. Mourtada-Maarabouni (United Kingdom)

370

Mutation screening of Bulgarian hereditary breast and ovarian cancer patients with multi-gene cancer panel A. Mitkova, R. Dodova, D. Pencheva, A. Vlahova, M. Taushanova-Hadjieva, S. Valev, K. Timcheva, S. Christova, V. Mitev, R. Kaneva (Bulgaria)

206

A novel role for p21-activated kinase 4 (PAK4) in endocrine 371 resistance and disease progression ´ A. Santiago-Gomez, I. Dragoni, R. NicAmhlaoibh, E. Trivier, J.M. Gee, A.H. Sims, S.J. Howell, R.B. Clarke (United Kingdom)

Protective effects of Sanicula europaea extracts on nuclear DNA damage E. Onay U¸car , M. Pekmez, E. Mertoglu, ˘ L. Dalyan, N. Arda (Turkey)

207

Prognostic significance of TERT promoter mutations and association with other common genomic aberrations in Bulgarian glioblastoma patients G. Stancheva, T. Goranova, M. Laleva, A. Mitkova, G. Poptodorov, N. Velinov, M. Kamenova, V. Mitev, N. Gabrovsky, R. Kaneva (Bulgaria)

208

Genome-wide sequencing identifies genetic relationship between first and late-onset second cancers in aristolochic acid nephropathy patients X. Castells, M. Ardin, S. Rorive, N. Broeders, A. Heguy, P.P. Bringuier, T. Quackels, T. Roumeguere, J. Nortier, J. Zavadil (France)

209

Reactivation of thyroid hormone receptors in fully developed rat hepatocellular carcinomas causes their regression and prevents lung metastasis A. Perra, M.A. Kowalik, L. Cabras, E. Puliga, G.M. Ledda-Columbano, A. Columbano (Italy)

210

Breast cancer and Epstein–Barr virus infection Y. Shlyakhtunou, A. Savchenko (Belarus)

211

New approach to analyzing gene expression in neuroendocrine tumors M.V. Comanescu, M. Dobre, F. Vasilescu (Romania)

212

A pre-operative, diagnostic gene panel for guiding primary treatment choices in endometrial cancer: Advancing beyond the decades-old technology of dilation and curettage (D&C) J. Martignetti, B. Reva, P. Elena, O. Camacho-Vanegas, D. Rykunov, S. Kendall, H. Shah, N. Nair, M. Strahl, W. Hamou, T. Kalir, E. Schadt, R. Sebra, P. Dottino (USA)

188

Identifying effective single-cell sequencing strategies for intra-tumor heterogeneity estimation J. Alves (Spain)

190

Carcinogenesis II Comparative analysis of human and mouse expression data identifies distinct proto-oncogene PTTG- and PBF-associated genes in thyroid cancer M. Read, J. Fong, W. Imruetaicharoenchoke, B. Modasia, H. Nieto, J. Watkinson, K. Boelaert, V. Smith, A. Turnell, C. McCabe (United Kingdom) Rab20 and Rab30 regulate hepatocellular carcinoma (HCC) for tumour suppression H.M. Liu, S.K. Tey, J.W.P. Yam (Hong Kong)

Cell and Tumour Biology II Mimicking disease progression features by modulation of the tumour microenvironment in stirred-tank culture systems M.F. Estrada, S.P. Rebelo, V.E. Santo, E.J. Davies, S. Abreu, M.T. Pinto, W. Sommergruber, P.M. Alves, E. Anderson, C. Brito (Portugal)

202

204

363

Neuropilin 2 regulates lung disseminated tumor cells escape from dormancy and progression into metastasis P. Bragado, R. Alonso, M. Mancino, P. Fernandez-Nogueira, G. Fuster, P. Gascon (Spain)

372

Targeting mismatch repair deficiency in endometrial cancer R. Begum, S. Martin (United Kingdom)

373

Apobec3B expression in drug resistant MCF-7 breast cancer cell lines O. Onguru, S. Yalcin, C. Rosemblit, P. Zhang, S. Kilic, U. Gunduz (Turkey)

374

Sequential treatment of HCC cells with PI3K/Akt/mTOR pathway inhibitors prior to sorafenib attenuates cancer stem cell population D.C. Kahraman, T. Kahraman, R.C. Atalay (Turkey)

375

Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumours ˜ B.M. Simoes, A. Denis, R. Eyre, K. Spence, A. Santiago-Gomez, ´ A. Sarmiento-Castro, I. Tanaka, D. Howat, S. Howell, R. Clarke (United Kingdom)

376

Myc-dependent cell cycle progression through the activation of CDK1 and phosphorylation of p27 L. Garcia-Gutierrez, G. Bretones, I. Arechaga, D. Santamaria, M. Barbacid, J. Leon (Spain)

377

The role of High-Mobility Group Box 1 protein in mesenchymal–epithelial signalling in the tumour microenvironment A. Evans, S. Sharma, E. Hemers (United Kingdom)

378

Glioblastoma cancer stem cells: Adaptive or stable phenotype? A. Dirkse, A. Golebiewska, N.H.C. Brons, T. Buder, A. Deutsch, S. Leite, N. Sauvageot, S. Senn, C. Herold-Mende, S. Niclou (Luxembourg)

379

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Abstract Nr.

Relevance of TERT promoter mutations in poorly differentiated and anaplastic thyroid carcinomas. Association with RAS, BRAF or PIK3CA activating mutations ´ N. Feas-Rodr´ ıguez, R. Munoz, ˜ T. Alvarez Gago, J.J. Mateos Otero, J.M. Cameselle Tejeiro, G. Garcia-Rostan (Spain)

380

Loss of c-KIT in thyroid cancer cells: A functional study to investigate its role in tumor differentiation and progression F. Lessi, S. Franceschi, E. Tantillo, F. Panebianco, M. Menicagli, M. La Ferla, P. Aretini, G. Bevilacqua, I. Marchetti, C.M. Mazzanti (Italy)

397

Role of AXL in invasion and drug resistance of breast and colon cancer cells W.M. Abdel-Rahman, N.A. Al-khayyal, V.A. Nair, M.S. Ayad (U.A.E.)

381

Characterization of the role of ASB6 in head and neck cancer initiating cells C.K. Chen, P.F. Su, J.F. Lo (Taiwan)

398

Leptin sustains hypoxic phenotype through modulation of mitochondrial homeostasis in prostate cancer cells M. Bologna, A. Calgani, C. Vicentini, D. Biondi, A. Ciafarone, P. Muzi, A. Angelucci (Italy)

382

The impact of monocarboxylate transporter expression on metabolic function in prostate cancer cells L. Hutchinson, A. Boyers, A. Chadwick, I. Stratford (United Kingdom)

383

The heterogeneity of circulating lung tumor cells from non-small cell lung cancer patients S.L. Kong, S.J. Tan, T.K.H. Lim, H.M. Poh, T.Z.X. Yeo, X. Liu, Y.W. Chua, A.A. Bhagat, W.T. Lim, A.M. Hillmer (Singapore)

385

Induction of senescence in primary glioblastoma cells by serum and TGFb R. Kumar , A. Gont, T. Perkins, I. Lorimer (Canada) Yeast as a model system to screen purine derivatives against human CDK1 and CDK2 kinases T. Mayi, M.E. Huang, L. Vernis, M. Legraverend, G. Faye (France)

ATRX expression and ALT phenotype in uterine leiomyomas 399 T. Ahvenainen, N. Makinen, ¨ R. Butzow, ¨ P. Vahteristo (Finland) Activation of CXCR4 by aberrant promoter demethylation in lung cancer N. Kang, S.J. Kim, Y.K. Kim (South Korea)

400

Neuroendocrine differentiation of prostate cancer cells grown in nude mice in presence of bone marrow stromal cells in androgen deprivation conditions G.L. Gravina, A. Mancini, A. Colapietro, A. Vetuschi, S. Pompili, R. Sferra, S. Delle Monache, C. Festuccia (Italy)

401

K-Ras modulated microRNAs induce tumorigenesis and drug resistance in NSCLC L. Shi, M. Garofalo (United Kingdom)

402

386

TGF-b induced stemness acquisition using next-generation sequencing in lung cancer S.J. Kim, N. Kang, Y.K. Kim (South Korea)

403

387

Acriflavine inhibits the epithelial-to-mesenchymal transition in vitro in liver and pancreatic cancer cells: restoring the phenotype and drug sensitivity and reducing ATF4 mediated stress response A. Bulle, J. Dekervel, P. Windmolders, L. Lambrechts, E. Van Cutsem, C. Verslype, J. Van Pelt (Belgium)

404

Depletion of g-glutamylcyclotransferase inhibits cancer cell growth via cellular senescence caused by CDK inhibitor induction K. Matsumura, N. Susumu, I. Hiromi, A. Eishi, K. Susumu, K. Akihiro, Y. Tatsuhiro (Japan)

405

The vacuolar ATPase subunit E controls apoptosis and is 388 up-regulated in breast cancer S.N. Mohammed, M. Mourtada-Maarabouni (United Kingdom) Distinct HER2 distribution and homo-dimerization patterns on subpopulations of breast cancer cells − correlative light- and electron microscopy in liquid for cancer stem cell characterization D. Peckys, N. De Jonge (Germany)

390

Isochaihulactone-induced NAG-1 overexpression mediated by endoplasmic reticulum stress-independent DDIT3 leads to glioblastoma multiforme cell apoptosis S.F. Tsai, H.J. Harn (Taiwan)

391

Grb2 facilitates EGF-dependent GEP100-Arf6 pathway activation leading to lung cancer invasion and metastasis T. Menju, K. Hijiya, H. Motoyama, A. Aoyama, F. Chen, T. Sato, M. Sonobe, S. Feller, H. Sabe, H. Date (Japan)

392

N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents C. Mazzoccoli, V. Ruggieri, T. Tataranni, F. Agriesti, I. Laurenzana, A. Fratello, N. Capitanio, C. Piccoli (Italy)

393

Glucose deprivation as new therapeutic approach to target pancreatic cancer cell metabolism T. Tataranni, F. Agriesti, V. Ruggieri, C. Mazzoccoli, I. Laurenzana, R. Scrima, N. Capitanio, C. Piccoli (Italy)

394

Hypoxia-induced angiogenesis, EMT and stem cell characteristics using next-generation sequencing in lung cancer N. Kang, S.J. Kim, Y.K. Kim (South Korea)

395

Induction of cellular senescence and hair follicle stem cell dysfunction upon p16INK4a expression in the skin N. Azazmeh, R. Amiel-Tokarsky, A. Helman, I. Ben-Porath (Israel)

396

Targeting autophagy in pancreatic neuroendocrine tumors 406 T. Wiedmer , M.P. Tschan, A. Perren, I. Marinoni (Switzerland) Histone deacetylases-1 promotes urothelial cell migration and invasion by modulating p63/Rho-kinase-1/pMLC2 signalling K. Srivastava, V. Smith, C. Breen, K. McCloskey (United Kingdom)

407

Development of ex vivo phenotypic assays to enable precision medicine in the context of high grade serous ovarian cancer L. Nelson, A. Tighe, A. Clamp, R. Edmondson, G. Jayson, S. Taylor (United Kingdom)

408

Neural crest derived progenitors give rise to tumor stroma and contribute to aggressiveness in high-risk neuroblastoma P. Linares-Clemente, D. Aguilar-Morante, R. Pardal (Spain)

409

ALDH1 expression and activity increase during tumor evolution in sarcoma cancer stem cell populations L. Martinez-Cruzado, J. Tornin, L. Santos, A. Rodriguez, R. Rodriguez (Spain)

410

Notch3–EGFR crosstalk in triple negative breast cancers (TNBC): new therapeutic possibilities G. Diluvio, F. Del Gaudio, G. Franciosa, M.V. Giuli, M.P. Felli, D. Bellavia, I. Screpanti, S. Checquolo (Italy)

411

Phenotypic and miRNA transcriptomic evaluation of cellular tumour spheroid as model of breast cancer stem cell studies W.Y. Ho, S.K. Yeap (Malaysia)

412


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Abstract Nr. The human L-asparaginase ASPG is cytotoxic for leukemic cells S. Belviso, M. Menniti, N. Perrotti, R. Iuliano (Italy)

413

Tumour suppression by a protein hydroxylase 429 A. Zayer, H. Smith, M. Pillai, R. Sekirnik, J. Zak, X. Lu, C. Schofield, M. Bix, P. Ratcliffe, M. Coleman (United Kingdom)

Runt-related transcription factor 2 (Runx2) is responsible for galectin-3 overexpression in human thyroid carcinoma E. Kaptan, S. Sancar Bas, A. Sancakli, H. Gumushan Aktas, S. Bolkent (Turkey)

414

Biomimetic microfluidic platform for 2D3D co-culture cancer 430 models M. Virumbrales-Munoz, J.M. Ayuso, R. Monge, G. Llamazares, M. Olave, M. Doblare, L. Fernandez, I. Ochoa (Spain)

Notch3 sustains CXCR4 expression in acute T cell lymphoblastic leukemia progression F. Ferrandino, G. Bernardini, P. Grazioli, A.F. Campese, D. Bellavia, S. Checquolo, I. Screpanti, M.P. Felli (Italy)

415

CD133+ subpopulation was identified in miR-135a induced 431 early transformation process of HPV-infected cells to cervical cancer W. Zhu, D.C.K. Yuen, R.T.K. Pang, W.S.B. Yeung (Hong Kong)

Identification of novel cancer biomarkers of prognostic value using specific gene regulatory networks D. Chudasama, V. Bo, M. Hall, V. Anikin, G. Pados, A. Tucker, E. Karteris (United Kingdom)

416

DigiWest multiplex protein profiling of extracellular vesicles to decipher cancer metastasis and to identify biomarkers G. Erdmann, C. Sachse, M. Templin, J. Gross (Germany)

432

417

Fasting in combination with curcumin induces a fatal energy “black out” in tumor cells L. Raffaghello, G. Bianchi, N. Bertola, A. Amaro, G. Angelini, L. Emionite, S. Ravera, U. Pfeffer (Italy)

433

Somatic MED12 exon 1 nonsense mutation in T-cell acute lymphoblastic leukemia escapes nonsense-mediated mRNA decay and prevents protein nuclear localization ¨ ¨ K. Kampj arvi, T. Heikkinen, S. Keskitalo, P. Von Nandelstadh, V. Rantanen, E. Pitkanen, ¨ K. Lehti, S. Mustjoki, M. Varjosalo, P. Vahteristo (Finland)

Crizotinib overcomes microenvironment mediated drug resistance in Ph positive leukemia O. Regev, N. Kidan, H. Khamisie, J. Mahajna (Israel)

434

Impact of Aspirin on factors associated with breast cancer lymph node metastasis S. Khan, K. Gilligan, K. O’Brien, B. Moloney, I.S. Miller, E. Ramphul, T.I. Barron, K. Bennett, A.T. Byrne, M.J. Kerin, R.M. Dwyer (Ireland)

435

RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation E.J. Arenas Lahuerta, R. Gomis Cabre, ´ M. Morales (Spain)

436

Role of integrated TGF-b superfamily signaling in melanoma progression E. Tuncer , D. Zingg, P. Cheng, A. Antunes, J. Haeusel, C.X. Deng, I. Kleiter, L. Sommer (Switzerland)

437

Expression profiling of tumour budding cells in colorectal 419 cancer suggests an EMT-like phenotype and molecular subtype switching L. De Smedt, S. Palmans, O. Govaere, J. Wouters, D. Barras, J. Dekervel, S. Tejpar, D. Lambrechts, X. Sagaert (Belgium) The role of CD109 in glioma progression P. Filppu, V. Le Joncour, M. Hyvonen, ¨ H. Sihto, H. Joensuu, K. Lehti, P. Laakkonen (Finland)

420

S100A14 increases tumorigenesis in triple-negative breast 421 cancer S. Ehmsen, K. Nørgaard, H.J. Ditzel, R. Leth-Larsen (Denmark) Cell-of-origin links histotype spectrum to immune microenvironment diversity in non-small cell lung cancer driven by mutant Kras and loss of Lkb1 A. Nagaraj, J. Lahtela, A. Hemmes, M. Mayr ¨ anp ¨ a¨ a, ¨ E.W. Verschuren (Finland) K. Salmenkivi, K. Narhi, ¨

422

Visualizing ZO-1-mediated coupled migration between cancer and stromal cells in lung cancer metastasis via a 5-dimensional cell imaging system H.H. Lu, B.C. Chen, C.C. Ho, H.W. Chen, C.J. Yu (Taiwan)

423

Glioma cell-of-origin and tumor cell plasticity L. Rousso-Noori, O. Pilo Kerman, S. Talmor, P. Magod, D. Friedmann-Morvinski (Israel)

424

Mechanisms of p53-mediated chemosensitivity of VMY on medulloblastoma cells A. Naeem, V. Yenugonda, O. Rodriguez, M.L. Avantaggiati, B. Rood, S. Karam, C. Albanese (USA)

425

LOX and LOXL2 in pancreatic cancer microenvironment K. Gardian, M. Durlik (Poland)

426

Tissue transglutaminase correlates with disease progression 438 and the acquisition of epithelial–mesenchymal transition in colorectal cancer cells O. Ayinde, Z. Wang, C. Tselepsis, M. Griffin (United Kingdom)

Endometrial cancer stem cells: tumorospheres 427 characterization and cancer stem cells markers M.J. Carvalho, M. Laranjo, T. Costa, N. Almeida, M. Abrantes, J. Casalta-Lopes, A. Paiva, M.F. Botelho, C. Oliveira (Portugal) Engineering glioblastoma microenvironment in a chip to study cell response J.M. Ayuso, R. Monge, G. Llamazares, M. Virumbrales-Munoz, A. Lacueva, M. Olave, M. Doblare, I. Ochoa, L. Fernandez (Spain)

428

Targeting COPZ1 in thyroid tumour cells M.C. Anania, E. Cetti, D. Lecis, L. Cleris, M. Mazzoni, S. Pagliardini, G. Manenti, A. Greco (Italy)

439

miR-214 in stroma cells and tumor progression D. Dettori, F. Orso, E. Penna, L. Salmena, P.P. Pandolfi, M. Caselle, D. Taverna (Italy)

440

Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein S. D’Aguanno, D. Trisciuoglio, M. Desideri, V. Farini, M. Di Martile, T. De Luca, M.G. Tupone, A. Urbani, D. Del Bufalo (Italy)

442

Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage S. Alsafadi, A. Houy, A. Battistella, T. Popova, M. Wassef, E. Henry, F. Tirode, A. Constantinou, S. Piperno-Neumann, S. Roman-Roman, M. Dutertre, M.H. Stern (France)

444

The pseudokinases KSR link metabolism to the MAPK/ERK pathway A. Verlande, D. Poteˇ ˇ sil, M. Krafˇc´ıkova, ´ Z. Zdrahal, ´ L. Trant´ırek, S. Uldrijan (Czech Republic)

446

Targeting breast cancer stem cells by inhibiting Wnt pathway: Combination of Pd (II) complex and niclosamide E. Ulukaya, M. Erkisa, S. Aydinlik, B. Cevatemre, F. Ari, V. Yilmaz (Turkey)

447

lx


Abstract Nr.

Abstract Nr. Dysregulated cell signalling as an oncogenic basis for the development of clear cell sarcoma of kidney (CCSK) N. McDonagh, E. O’Meara, M. Alsulami, E. Dillon, G. Cagney, M.J. O’Sullivan (Ireland)

448

Adhesion signalling in melanoma biology Z. Miskolczi, M. Smith, C. Wellbrock (United Kingdom)

449

The ERBB network facilitates KRAS-driven lung tumorigenesis S. Neidler, A. Hedley, B. Kruspig, T. Monteverde, K. Hewit, B. Neiswandt, A. Rosenwald, E. Shanks, C. Dick, D. Murphy (United Kingdom)

450

Hypoxic conditions regulate the molecular content, release and uptake rates of extracellular vesicles produced by colorectal cancer cells ¯ E. Zandberga, A. Abols, C. Bajo A. Line, Santos, R. Toleikiene, P. Zayakin, D. Pupola, ¯ K. Shvirksts, M. Grube, U. Riekstina (Latvia)

451

Chloroquine enhances the antiangiogenic effect of 452 doxorubicin on the chorioallantoic membrane (CAM) in vivo E. Armutak-Ikitimur, E. Gurel-Gurevin, H.T. Kiyan, O.B.B. Esener, S. Aydinlik, A. Uvez, K. Dimas, E. Ulukaya (Turkey) Protein kinase CK2 regulates prostate cancer cell migration and proliferation through phosphorylation of PRH/HHEX E. Marcolino de Assis Junior , Y. Hasan Siddiqui, R.M. Kershaw, E.H. Humphreys, S. Chaudhri, P.S. Jayaraman, K. Gaston (United Kingdom)

453

Tumor heterogeneity testing in gastrointestinal stromal tumors using droplet digital PCR ´ K. Jaˇsek, M. Grendar, Z. Lasabova, ´ P. Szepe, ´ L. Plank (Slovak Republic)

454

Chemotherapy resistance in breast cancer: A role for senescent fibroblasts D.W. Perkins, L. O’Leary, C.M. Isacke (United Kingdom)

455

Chemotherapy and inflammation show a NFúB- and STAT3-dependent pro-tumorigenic potential ` F. Alessandrini, Y. Ciribilli (Italy) L. Pezze,

456

The role of tumour heterogeneity in melanoma progression E. Rowling, A. Chapman, B. Telfer, A. Hurlstone, C. Wellbrock (United Kingdom)

457

Low primary intra-tumoural heterogeneity versus high genomic disparity between primary and distant sites in NSCLC B. Polzer , N. Wendler, R. FahriogluYamaci, F. Elsner, B. Cucuruz, M. Lindner, H.S. Hofmann, B. Passlick, C.A. Klein (Germany)

458

The activities of cell surface NADH-oxidases, pyruvate kinase and lactate dehydrogenase: Signatures for cancer cells in precarious nutrient conditions A.M. Otto, P. Biechl, H. Fernandes, M. Gkiouli, J. Hintermair, J. Hutterer (Germany)

459

Investigation of the possible relationship of cancer stem cell subpopulation and metastatic potential of an oral squamous cell carcinoma cell line N. Martins Lopes, R. Alves da Silva Alavarce, R. Carneiro Ortiz, T.J. Dion´ısio, G. Pompermaier Garlet, E. Graner, V. Soares Lara, C. Oliveira Rodini (Brazil)

460

Investigating the molecular significance of aberrant HER2, HER3 and downstream partner signalling in castrate resistant prostate cancer M. Aalsamraae (United Kingdom)

461

Spheroids versus monolayers: are the cells metabolically the same? M. Gkiouli, A. Otto, J. Hintermair (Germany)

463

Development and characterisation of a novel syngeneic, 465 spontaneous breast cancer metastasis model U. Jungwirth, G. Qiong, S. Wantuch, C. Isacke (United Kingdom) A kinome-scale synthetic lethality screen reveals an essentiality of CDK13 for 19q12 amplified cancer cells R. Stark, W. Krek (Switzerland)

466

Mcl-1 dynamics influence mitotic slippage and death in mitosis O. Sloss, C. Topham, S. Taylor (United Kingdom)

467

Functional characterization of the interaction between the stem cell factor SOX2 and the cell cycle regulator P27 in normal and cancerous stem cells V. Moncho-Amor , L. Garros, C. Galichet, K. Rizzoti, M. Matheu, R. Lovell-Badge (United Kingdom)

468

Gadd45: A new player in liver tumorigenesis U. Rosato, J.M. Salvador (Spain)

469

Nandrolone affects cell growth and differentiation in 470 hepatoma cells C. Piccoli, F. Agriesti, T. Tataranni, C. Mazzoccoli, V. Ruggieri, R. Scrima, O. Cela, C. Pomara, N. Capitanio (Italy) ZEB1 induce chemoresistance through epithelial– mesenchymal transition in prostate cancer cells H.R. Contreras, O. Orellana-Serradell, D. Herrera, E.A. Castellon ´ (Chile)

471

Exploring the role of inflammation in ETV1-expressing prostate tumours V. Ubertini, P. Wang, H.S. Leong, K. Glass, E. Baena (United Kingdom)

473

Extracellular matrix anisotropy in breast cancer invasion and metastasis D. Park, R. Jenkins, A. Labernadie, E. Wershof, X. Trepat, P. Bates, L. Jones, E. Sahai (United Kingdom)

474

Functional dissection of the role of Myc in pancreatic tumourigenesis N. Muthalagu, J. Morton, W. Clark, A. Hedley, O. Sansom, D. Murphy (United Kingdom)

475

Multi-omics studies of intra-tumour heterogeneity in colorectal cancer G. Wa˛tor , M. Suski, A. Borys, J. Swirta, E. Chronowska, M. Barczynski, ´ P. Wołkow (Poland)

476

Human aldehyde dehydrogenase 7A1 (ALDH7A1) expression affects cancer cell proliferation, migration and cell protection against oxidative stress L. Elsalem, A. Fotopoulos, A. Papathanasiou, S. Allison, J. Moreb, Z. Cournia, K. Pors (United Kingdom)

477

Progesterone receptor antagonists are potential inhibitors of breast cancer stem cell activity D. Alferez, R. Eyre, K. Spence, W. Hayhurst, C. Chresta, H. Sacha, R. Clarke (United Kingdom)

478

The dual role of ALCAM in endometrial cancer L. Devis, N. Masia, ´ E. Mart´ınez, F. Brochard-Wyart, A. Gil-Moreno, S. Dufour, A. Santamaria, F. Alameda, J. Reventos, ´ E. Colas ´ (Spain)

479

BRAF inhibition promotes BRAF mutant human melanoma cell survival under nutrient-deprived conditions through activation of mitochondrial metabolism T. Delgado-Goni, S. Wantuch, P. Workman, R. Marais, M.O. Leach, M. Beloueche-Babari (United Kingdom)

480


Abstract Nr. Single cell analysis of intratumour heterogeneity in childhood acute lymphoblastic leukaemia V. Turati, H. Mike, J. Herrero, A. John, S. Richardson, B. Gaal, L. Mark, S.E. Jacobsen, T. Enver (United Kingdom)

481

sept4/Arts regulates stem cell apoptosis and skin regeneration Y. Fuchs, S. Brown, T. Gorenc, J. Rodriguez, E. Fuchs, H. Steller (Israel)

483

Tumor progression marker for breast cancer − survivin gene (BIRC5) Y. Shlyakhtunou (Belarus)

484

WWP2 controls endothelial permeability by regulating the RAP1–RADIL signaling pathway F. Renzi, M.F. Baietti, L. Abbasi, K. Gevaert, A. Sablina (Belgium)

485

Expression of survivin gene (BIRC5) and ErbB-2 (Her-2/neu) in lymphocytes in breast cancer Y. Shlyakhtunou, V. Semenov (Belarus)

486

Identification and exploitation of collateral vulnerabilities 487 to transcription and RNA processing inhibitors in tumor subtypes L. Frischknecht, Y. Christinat, C. Britschgi, W. Krek (Switzerland)

lxi

Abstract Nr. Comparison of antitumour effects of tributyltin chloride and triphenyltin chloride in different human breast cancer cell lines, MCF-7 and MDA-MB-231 L. Hunakova, L. Toporova, D. Macejova, J. Brtko (Slovak Republic)

503

Ceramide is a key factor that regulates the crosstalk between TGF-b and sonic hedgehog signaling at the basal cilia to control cell migration and tumor metastasis S. Gencer , B. Ogretmen (Turkey)

505

Development of an organotypic system for the study of cancer dormancy M. Montagner , E. Sahai (United Kingdom)

506

MicroRNA induced autophagy confers chemoresistance in glioblastomas U. Baumgartner , N. Wirth, S. Haemmig, A. Zulliger, M.P. Tschan, E. Vassella (Switzerland)

507

Pluripotent genes role in normal melanocyte lineage commitment and malignant transformation D. Sheinboim, I. Maza, I. Dror, J. Hanna, C. Levy (Israel)

510

Metabolic targeting and antagonism of the EGFRvIII/PDK1 axis in temozolomide resistant glioblastoma models K.K. Velpula, M.R. Guda, K. Sahu, J. Tuszynski, S. Asuthkar, S.E. Martin, J.D. Lathia, A.J. Tsung (USA)

511

512

513

ERK5 is required for pro-tumour macrophage activation E. Giurisato, W. Vermi, C. Tournier (United Kingdom)

488

A preliminary characterisation of de novo lipogenesis in pancreatic (Capan-1) and liver (HepG2) cancer cell lines N. Lambri, A. Snabaitis, A. Le Gresley, H. Modjtahedi, M. Stolinski (United Kingdom)

489

Influence of stemness genes on metastatic capacity of cancer stem cells in prostate cancer ´ R. Valenzuela, F. Cifuentes, E.A. Castellon, T. Thomson, H.R. Contreras (Chile)

490

Pro-survival p53 target genes have evolved clusters of interacting polymorphic response elements that can affect cancer risk P. Zhang, G. Stracquadanio, X. Wang, M. Pybus, J. ZeronMedina, S. Nornes, S. Moore, Y. Bi, M. Wallace, E. Bond, B. Davies, N. Sacilotto, S. De Val, B. Schuster-Boeckler, D. Bell, G. Bond (Nuffield Department of Clinical Medicine)

Functional characterization of a tumor suppressor micro-RNA in ovarian cancer B. Majem, A. Parrilla, A. Soriano, A. Gil-Moreno, M.F. Segura, A. Santamaria (Spain)

492

A central role for the syntenin exosomal pathway on the SRC metastatic function in colorectal cancer C. Lecointre, N.S. Imjeti, F. Lembo, V. Simon, P. Zimmermann, S. Roche (France)

Interrogating tumour–stroma interactions mediating escape from breast cancer metastatic dormancy L. O’Leary, A. Van Weverwijk, C. Isacke (United Kingdom)

493

Anticancer effects of lipid encapsulated Bilberry J. Thornthwaite, S. Thibado, T. Ballard, B. Goodman (USA)

495

A novel pre-clinical model for imaging cancer-associated inflammation K. Finegan, M. Babur, D. Forster, J. O’Connor, C. Tournier, K. Williams (United Kingdom)

496

The role of lysyl oxidase activity in ccRCC cell adhesion and mobility in vitro Q. Wang, A. Jeylani, A.K. Fallah, L. Lee-Jones, S. Kumar, M. Slevin (United Kingdom)

497

GRP94 protein and prosurvival autophagy, the Achilles heel on brain metastasis progression A. Sierra, N. Santana-Codina, R. Sanz-Pamplona, L. Muix´ı (Spain)

498

DNAJB1 renders cancer stem cell-like and metastatic traits to colorectal cancer cells dependent on b-catenin Y. Guo, T. Zhang, Y. Liu, C. Zhao (China)

499

Benzyl butyl phthalate (BBP) induces the expression of CD44+/CD24− leading to enhanced colony formation in MCF-7 mammosphere cell T.H. Hsieh, T. Eing-Mei (Taiwan)

500

Experimental/Molecular Therapeutics, Pharmacogenesis II Stratification of breast cancer patients using Tip60 staining 589 paired with evaluation of a designed Tip60 specific histone acetyltransferase inhibitor for targeted treatment A. Shalaby, H. Emma, E. Bourke, C. Gao, S. Martin, K. Michael, L. Eriksson, H. Thomas, J. Brown (Ireland) Trastuzumab-based photoimmunotherapy integrated with viral HER2 transduction inhibits HER2-negative gastric cancer S. Kagawa, M. Ishida, T. Fujiwara (Japan)

590

Lichen compound protolichesterinic acid inhibits 591 DNA synthesis and DNA repair in cultured cancer cells H. Ogmundsdottir , H. Hauksdottir, J. Thorsteinsdottir (Iceland) A 3D image-based phenotypic screen of bi-specific antibodies targeting stem cells in a panel of patient derived colon carcinoma organoids B. Herpers, R. Roovers, B. Ebbink, M. Van de Wetering, K. Yan, L. Salinaro, H. Clevers, W. De Lau, R. Vries, M. Throsby, L. Price (Netherlands)

592

Targeting HER3 by interfering with its Sec61-mediated cotranslational insertion into the endoplasmic reticulum A. Ruiz-Saenz, M. Sandhu, Y. Carrasco, R. Maglathlin, J. Taunton, M. Moasser (USA)

593

lxii


Abstract Nr.

Abstract Nr. Urokinase kringle-derived peptide UP7 suppresses tumor angiogenesis and breast cancer metastasis Y.A. Joe, H.K. Kim, P. Naidansuren, S.W. Lee (South Korea)

608

Inhibition of neuroblastoma growth by the thiadiazolidinone compound TDZD-8 D. Aguilar-Morante, R. Pardal (Spain)

609

595

The MRN complex: A potential target for MYCN amplified neuroblastoma M. Petroni, F. Sardina, M. Sahun ` Roncero, C. Heil, P. Infante, B. Ricci, E. Petricci, E. Locatelli, M. Comes-Franchini, G. Giannini (Italy)

610

Docosahexaenoic acid induces cell death through downregulation of hedgehog signaling via the activation of SIRT6 in human non-small cell lung carcinomas harboring EGFR mutations S. Jeong, K. Jing, S. Shin, Y.J. Jeon, J.Y. Heo, G.R. Kweon, S.K. Park, J.I. Park, K. Lim (Korea)

596

The challenges of an autologous cell therapy product in clinical trials R. Smith, M. Bryan, K. Campbell, L. Cooney, F. Gilbert, T. Hamill, A. Scott, K.J. Williams (United Kingdom)

611

612

Docosahexaenoic acid induces ROS-mediated apoptosis through the Nrf2–Klf9–Txnrd2 pathway in HeLa cells K. Jing, S. Shin, S. Jeong, S. Kim, Y.J. Jeon, J.Y. Heo, G.R. Kweon, S.K. Park, J.I. Park, K. Lim (Korea)

597

Idelalisib sensitivity and mechanisms of disease progression in relapsed TCF3-PBX1 acute lymphoblastic leukemia S. Eldfors, H. Kuusanmaki, ¨ M. Kontro, M.M. Majumder, A. Parsons, H. Edgren, T. Pemovska, O. Kallioniemi, K. Wennerberg, N. Gokbuget, ¨ T. Burmeister, K. Porkka, C. Heckman (Finland)

Rational design of combination therapies and blockage of acquired targeted drug resistance H. Reuveni, L. Kupershmidt, S. Carmi, N. Moskovitz, M. Rozen, M. Shlapoberski, E. Golomb, M. Lotem, S.M. Stemmer, I. Haviv (Israel)

598

Digoxin is a modifier increasing platinum drug anticancer activity E. Dudko, V. Chernov, T. Bogush, Y. Dyakova, V. Kirsanov, Z. Shprakh, A. Kamensky, B. Polotsky, S. Tjulandin, E. Shestakova (Russian Federation)

613

Esculentin-2PLa, a frog skin antimicrobial peptide, causes necrotic cell death in breast cancer cell lines S. Sancar-Bas, S. Bolkent (Turkey)

600

Bioactivities of novel boehmeriasin derivatives in liver cancer cells E. Akhan Guzelcan, M. Baumann, I. Baxendale, R. Cetin Atalay (Turkey)

601

Induction of EMT is WNT-independent in cisplatin resistant 615 urothelial carcinoma cell lines M.A. Skowron, G. Niegisch, G. Fritz, J.G.H. Van Roermund, A. Romano, P. Albers, W.A. Schulz, M.J. Hoffmann (Germany)

Improving nelarabine efficacy in refractory/relapsed T-cell acute lymphoblastic leukemia (T-ALL) by targeting aberrant PI3K/mTOR signaling F. Chiarini, A. Cappellini, A. Lonetti, A. Bertaina, F. Locatelli, F. Melchionda, A. Pession, A.M. Martelli (Italy)

602

S-phase arrest independent activation of the DNA-damage response under hypoxic conditions M. Likhatcheva, R. Gieling, C. Demonacos, K. Williams (United Kingdom)

603

Mutant p53 modulates the signal of hepatocyte growth factor (HGF) to endow cancer cells with drug resistance Y. Stein, A. Jacob, N. Goldfinger, R. Straussman, V. Rotter (Israel)

Insulin/insulin-like growth factor-1 receptors mediate acquired resistance to anti-EGFR therapy in human cholangiocarcinoma cells by regulating an epithelial to mesenchymal transition/cancer stem cell axis J. Vaquero, C. Lobe, A. Claperon, ´ M. Mergey, C. Desbois-Mouthon, F. Praz, L. Fouassier (France)

594

Trefoil Factor 3 (TFF3) is HER2-regulated and substitutes for HER2 signalling in acquired trastuzumab resistance in HER2+/ER+ breast cancer Q.Y. Chong, V.K. Pandey, A. Banerjee, Y.J. Chen, M.L. You, P.E. Lobie (Singapore)

Elucidation of molecular action mechanisms of cytotoxic palladium derivatives on various cancer cells O. Kacar, I. Hatipoglu, V. Yilmaz, N. Arda, E. Ulukaya, C.A. Acilan (Turkey)

616

Synthesis, biological characterization and evaluation of 618 molecular mechanisms of novel copper complexes as anticancer agents C. Acilan, B. Cevatemre, Z. Adıguzel, D. Karakas, E. Ulukaya, N. Arda, N. Ribeiro, I. Correiae, J.C. Pessoa (Turkey) Biological and biophysical characteristics of a new polymer platform for drug delivery systems ´ a, ´ A. Galisov M. Jiratov ´ a, ´ M. Paˇr´ızek, A. Posp´ısˇ ilova, ´ M. Rabyk, D. Jirak, ´ M. Hrubý (Czech Republic)

619

604

Investigating a novel binuclear palladacycle complex for anti-cancer activity in rhabdomyosarcoma cells J. Bleloch, R. Ballim, S. Aliwaini, S. Mapolie, S. Prince (South Africa)

620

Identification of CDK4/6-response biomarkers using estrogen receptor-positive breast cancer patient-derived xenografts (PDX) M. Palafox, M.T. Herrera, M. Bellet, J. Arribas, C. Saura, E. Di Tomaso, N.C. Turner, J. Cortes, ´ J. Baselga, V. Serra (Spain)

605

Resistance to sunitinib in clear cell renal cell carcinoma results from drug sequestration in lysosomes and inhibition of autophagic flux S. Giuliano, Y. Cormerais, M. Dufies, R. Grepin, ´ P. Colosetti, A. Belaid, J. Parola, P. Auberger, B. Mograbi, G. Pages ` (Monaco)

621

A novel role for Top2a as a mediator of resistance to ribosomal DNA transcription inhibition D. Cameron, E. Sanij, M. Bywater, N. Hein, J. Lim, G. McArthur, G. Poortinga, R. Hannan (Australia)

606

622

Induction of apoptosis in cancer cells by p-coumarate ester derivatives O. Firuzi, F. Borges, J.C. Menezes, N. Edraki, S. Kamat, M. Khoshneviszadeh, H.H. Mirzaei, R. Miri, J.A. Cavaleiro, L. Saso (Iran)

607

Thymoquinone confers higher cytotoxicity against triple negative breast cancer compared to estrogen receptor positive breast cancer through modulation of TNF receptor superfamily genes and P65 activity M. Cakir, C. Sakalar , S. Sezen, H. Aksu, B. Kurt, H. Canatan (Turkey)


Abstract Nr.

lxiii

Abstract Nr.

Characterisation of a novel and potent STAT3 inhibitor, VS-43 H. Valentine, V. Satam, P. Patil, R. Sjoholm, M. Lee, M. Lee, J.A. Hartley, K. Kiakos (United Kingdom)

623

Speeding up the S5 XL sequencing system: Sequencing in an hour enables sample to answer in a 8 hr workday R. Qi, C. Parikh, A. Luchetti, D. Mandelman, H. Latif, A. Harris, S. Ghosh (USAUSA)

638

A platinum-blue complex exerts its cytotoxic activity via DNA damage and induces apoptosis in cancer cells Z. Adiguzel, S. Ozalp-Yaman, G. Celik, S. Salem, T. Bagci-Onder, Y.C. Cetin, N. Arda, C. Acilan (Turkey)

624

Cold atmoshperic plasma as an approach to retinoblastoma R. Silva-Teixeira, M. Laranjo, G. Brites, A.M. Abrantes, T. Rodrigues, A.C. Gon¸calves, S. Raquel, A.B. SarmentoRibeiro, P. Matafome, F. Caramelo, M.F. Botelho (Portugal)

639

Statin-induced metabolic modulation in cancer: a biomarker for targeting monocarboxylate transporters M. Mehibel, F. Ortiz-Martinez, A. Boyers, B. Telfer, K. Williams, I. Stratford (United Kingdom)

625

640

MicroRNA-31 regulates chemosensitivity in malignant pleural mesothelioma via altered intracellular drug localisation H. Moody, M. Lind, S. Maher (United Kingdom)

626

Selective inhibition of HDAC1 and HDAC2 counteracts medulloblastoma cell growth in mouse models through Gli acetylation S. Coni, A.B. Mancuso, L. Di Magno, G. Sdruscia, D. Rotili, A. Mai, G. Canettieri (Italy)

641

Oxidized products of a-linolenic acid negatively affect cell survival and motility of breast cancer cells J. Gutierrez-Pajares, C. Oger, J.M. Galano, T. Durand, S. Chevalier, P. Frank (France)

627

Hyperbaric oxygen therapy combined with photodynamic therapy as a new therapeutic approach against retinoblastoma A. Neves, A. Abrantes, A. Pires, R. Teixo, M.F. Botelho (Portugal)

Identification of a pyrrolo-pyrimidin derivative to overcome the resistance to apoptosis in non-small cell lung cancer cells P. Gilson, F. Mahuteau, C. Beauvineau, J.L. Coll, A. Hurbin, B. Busser (France)

628

Targeting the PI3K/AKT/mTOR pathway with a dual mTORC1/2 inhibitor in bladder cancer. Rationale for its testing in clinical trials ´ A. Hernandez-Prat, A. Martinez, O. Arp´ı, S. Menendez, ´ N. Iarchoukc, F. Rojo, J. Albanell, R. Brake, A. Rovira, J. Bellmunt (Spain)

629

Resistance to photodynamic therapy in glioblastoma cancer cells S. Shahmoradi Ghahe, B. Majchrzak, K. Kopania, A. Ciuba, B. Tudek (Poland)

630

Combination of photodynamic therapy with doxorubicin in osteosarcoma: Cell death and the role of oxidative stress B. Serambeque, G. Brites, M. Laranjo, G. Chohfi de Miguel, A. Serra, M. Pineiro, A.M. Abrantes, J. Casalta-Lopes, A. Rocha-Gonsalves, A.C. Gon¸calves, A.B. Sarmento-Ribeiro, D. Priolli, M.F. Botelho (Portugal)

631

MicroRNA SNPs as novel markers of methotrexate toxicity in pediatric acute lymphoblastic leukemia L. Iparraguirre, M. Umerez, A. Gutierrez-Camino, I. Mart´ın-Guerrero, N. Garc´ıa de Andoin, A. Sastre, A. Navajas, I. Astigarraga, A. Garc´ıa-Orad (Spain)

Targeting cell metabolism to improve prostate cancer 642 therapeutics M. Bedaj, K. Rao, C. Robson, S. McCracken (United Kingdom) Photodynamic therapy in EGFR targeted nanoparticles for lung cancer M. Laranjo, R. Teixo, A.M. Abrantes, A.C. Gon¸calves, A.B. Sarmento Ribeiro, M. Pineiro, A. Serra, S. Oliveira, M.F. Botelho (Portugal)

644

Investigation of the anti-cancer potential of natural products and their synthetic analogues B. Adegbesan, C. Demonacos (United Kingdom)

645

AAV-shRNA vectors as an alternative therapy for human 646 basal-like breast cancer C. Pinto, A.S. Ribeiro, G. Silva, M. Oliveira, A.S. Coroadinha, C. Peixoto, A. Barbas, C. Brito, J. Paredes, P.M. Alves (Portugal) In-vitro and in vivo preclinical evaluation of a 5T4 specific antibody–drug conjugate for treating ovarian cancer Y.L. Wan, P. Sapra, D. Gilham, H.C. Kitchener, P.L. Stern (United Kingdom)

647

648

632

Membrane Hsp70 as a biomarker for aggressive prostate cancer and therapeutic target G.A. Foulds, N. Dunning-Foreman, S. Stangl, M. Gehrmann, J. Vadakekolathu, D. Boocock, R. Rees, G. Multhoff, A.G. Pockley (United Kingdom)

649

Identification and development of novel Smoothened antagonists for the treatment of Hh-dependent tumors R. Alfonsi, M. Mori, P. Infante, F. Ghirga, B. Botta, L. Di Marcotullio (Italy)

633

Vascular endothelial growth factor (VEGF) isoform mediated tumour response to combination treatment with a blocking antibody to VEGFR-2 and radiotherapy D. Mukherjee, S.J. Lunt, M. Fisher, W. English, C. Kanthou, G. Tozer (United Kingdom)

650

Design and evaluation of novel theranostic fluorogenic dual probe-prodrug in cancer S. Mathur , D. Mincher, A. Turnbull, C. Stevens, A. Poole (United Kingdom)

634

Photodynamic therapy in osteosarcoma. Is there involvement of the apoptosis pathway mediated by p53, or the proliferative pathway mediated by beta-catenin? What is the action mechanism of PDT? G. De Miguel, B. Camporeze, A.Y.K. Grizotto, A.M. Abrantes, M.F. Botelho, J.A. Pereira, D.G. Priolli (Brazil)

635

Antiangiogenic potential of flavonoids in an animal model of human colon adenocarcinoma D. Priolli, G.D.C. Orfali, I.R.O. Assun¸cao, ˜ I.M. Marchesi, N.P. Martinez, D.D.C. Silva, J.A. Pereira, P.D.O. Carvalho, A.Y.K. Grizotto (Brazil)

651

Unveiling the mechanisms of exemestane-acquired resistance: The role of autophagy and PI3K pathway C. Amaral, T. Augusto, E. Tavares-da-Silva, F.M.F. Roleira, G. Correia-da-Siva, N. Teixeira (Portugal) Performance evaluation of a new integrated blood collection/ sample preparation system for the stabilization and extraction of circulating cell-free DNA (ccfDNA) A. Ullius, T. Voss, D. Grolz ¨ (Germany)

636

Anti-cancer properties of secondary metabolites derived from marine bacteria L. Lee-Jones, Q. Wang, S. Bohan, O. Martin, P.E. Linton (United Kingdom)

653

lxiv


Abstract Nr. Flavonoids Q3G and hydrolyzed rutin demonstrated antitumor activity in colon adenocarcinoma − in vivo study D. Priolli, D.D.C. Silva, A.C. Duarte, G.D.C. Orfali, N.P. Martinez, P.D.O. Carvalho, A.Y.K. Grizotto (Brazil)

654

New biocompound for the treatment of glioma: Is rutin 655 hydrolyzed by hesperinase an alternative? C. Pacheco, P.M. Moro, R. Colenci, A.C. Duarte, M.C.F. Messias, G. Mamprim, C.T. Parisi, D.G. Priolli, T.W. Santos (Brazil) Hydrolyzed rutin in an animal model of human glioma P. Prado, C. Pacheco, C.T.P. De Oliveira, P.D.O. Carvalho, G. Mamprim, D. Priolli, A.Y.K. Grizotto (Brazil)

656

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK positive neuroblastoma cells, Drosophila and mice J. Siaw (Sweden)

657

Clearing mutant p53 with natural compounds: implication for anticancer therapeutical approaches G. D’Orazi, A. Garufi, M. Cirone (Italy)

658

Combination of BGJ398 with either a pan-PI3K inhibitor or a specific PIK3CA inhibitor shows synergy in FGFR2 mutant endometrial cancer cell lines L. Packer , X. Geng, V. Bonazzi, C. Mahon, R. Ju, S. Stephenson, P. Pollock (Australia)

661

The anti-leukemic drug nilotinib inhibits metastatic properties of colorectal cancer cells by targeting the receptor tyrosine kinase DDR1 P. Tosti, C. Leroy, M. Jeitany, V. Simon, D. Bonenfant, L. Thouennon, S. Elmessaoudi, C. Mollevi, A. Thierry, P. Martineau, B. Robert, A. Sirvent, S. Roche (France)

662

Antiproliferative and anti-inflammatory activities of a dichloromethane-methanol extract from Pterocarpus mildbraedii leaves C. Otuechere, G. Santosh, O. Farombi (Nigeria)

663

Molecular and Genetic Epidemiology II IKZF1 gene deletions are strongly associated with Ikaros dominant-negative isoforms expression in acute lymphoblastic leukemia V. Vshyukova, A. Meleshko (Belarus)

674

Development of a prediction model and estimation of cumulative risk for upper aerodigestive tract cancer based on aldehyde dehydrogenase 2 (ALDH2) genotype and alcohol consumption in a Japanese population K. Matsuo, Y. Koyanagi, H. Ito (Japan)

675

Genome-wide AFB1-induced mutational signature in cells, mice and human tumors − implications for molecular epidemiology W. Yu, M. Huang, W.W. Teoh, M. Ardin, S. Villar, A. Jusakul, R. Othman, K. Sabapathy, J. Zavadil, S. Rozen (France)

676

Implementation of liquid biopsies for detection of activating EGFR mutations in Bulgarian patients with NSCLC S. Bichev, A. Savov (Bulgaria)

677

Diagnostic and prognostic value of long non-coding RNA expression in urothelial carcinoma J. Droop, T. Szarvas, W.A. Schulz, C. Niedworok, G. Niegisch, K. Scheckenbach, M.J. Hoffmann (Germany)

678

Searching for circulating miRNA markers in gastric cancer ˙ ´ J. Toton-Zura nska, M. Sier˙ze˛ga, K.W. Maria, P. Radkowski, J. Kulig, P. Wołkow (Poland)

680

Abstract Nr. Correlation of genetic polymorphism of TNFa and TGFb genes with protein level in patients with pancreatic and colorectal cancer in Polish population M. Zagozda, A. Sarnecka, Z. Staszczak, M. Nowak-Niezgoda, W.L. Olszewski, M. Durlik (Poland)

Prevention and Early Detection II Dose–response relationship between inorganic arsenic exposure and lung cancer among arseniasis area residents with low methylation capacity: A population-based cohort study P.J. Liao, K.H. Hsu (Taiwan)

682

692

Novel insights into Notum and glypicans regulation in colorectal cancer M. De Robertis, M. Arigoni, L. Loiacono, F. Riccardo, R.A. Calogero, Y. Feodorova, D. Tashkova, V. Belovejdov, V. Sarafian, F. Cavallo, E. Signori (Italy)

693

Screening and secondary prevention of colorectal cancer among limited contingent as a first step of implementing screening in Belarus I. Rebeko, A. Petkevich, S. Krasniy, I. Abelskaya, A. Gerasimovich, E. Lobachevskaya (Belarus)

694

Circulating cell free DNA (cfDNA) as a novel tool to monitor Barrett’s esophagus neoplastic progression E. Boldrin, E. Rumiato, M. Fassan, L. Balsamo, S. Realdon, G. Battaglia, M. Rugge, A. Amadori, D. Saggioro (Italy)

695

Prospective validation of a blood-based 9-miRNA profile for early detection of breast cancer in a cohort of women examined by clinical mammography M. Lyng, A. Kodahl, H. Binder, H. Dtizel (Denmark)

696

Breast stromal collagen micro-organisation, mammographic density, and cancer C. Streuli, M. Sherratt, S. Astley, A. Gandhi, C. Kirwan, J. McConnell (United Kingdom)

697

Risk factors for breast cancer: a case–control study among post-menopausal women in Pakistan N.A. Jadoon, M. Hussain, F.U. Sulehri, N.A. Shair (Pakistan)

698

Transcriptome profiling of the colorectal adenoma– adenocarcinoma sequence J. Reumers, L. Van Wesenbeeck, G. Chu, S. Gaj, I. Van den Wyngaert, K. Smans, G. Borzillo, J. Arts (Belgium)

699

A capillary electrophoresis-sequencing based screening solution for identifying and quantifying hotspot mutations in solid tumors S. Jackson, A. Gerstner, K. Varma (USA)

700

Radiobiology/Radiation Oncology II Secretome signature identifies ADAM17 as novel target for radiosensitization of non-small cell lung cancer S. Bender , A. Sharma, A. BrogginiTenzer, M. Pruschy (Switzerland)

712

Arginine-auxotrophic pancreatic cancer cells are radiosensitized by enzymotherapeutic arginine-deprivation therapy N. Sorour, M. Huether, A. Azad, E. Fokas, O. Stasyk, C. Pilarsky, L. Kunz-Schughart, C. Von Neubeck (Germany)

713

Discoidin domain receptor 1 targeting impairs GBM cell invasion and mediates radiochemosensitization by induction of autophagy E. Klapproth, A. Vehlow, N. Cordes (Germany)

714


lxv

Abstract Nr.

Abstract Nr.

Thromboxane-mediated protein kinase C-related kinase (PRK) signalling: implications for thromboxane- and androgen-dependent neoplastic responses in prostate cancer A. O’Sullivan, E. Mulvaney, T. Kinsella (Ireland)

763

Pathway deregulation networks in breast cancer young patients: Own data with METABRIC and TCGA databases ˜ M. Pena-Chilet, S.S. Oltra, M.T. Martinez, J. Fores, G. Ayala, G. Ribas (Spain)

764

KMT5A regulated gene expression signature as a potential biomarker panel for advanced prostate cancer Z.A.H. Alebady (United Kingdom)

765

Investigating the p53 dependency of synergy of HDAC inhibition with chemotherapy in colorectal cancer F. Falcone, A. McIntyre, J. Patrick, D. Longley, S. McDade (United Kingdom)

766

Combinatorial targeting of PI3K and MAPK signaling pathways using microRNAs to inhibit cell proliferation and invasion in breast cancer O. Sahin, M. Mutlu, O. Saatci, S. Ansari, U. Raza (Turkey)

767

721

MEK5-ERK5 inhibitors target chronic myeloid leukemia stem cells and in combination with imatinib reduce the expression of stem cell genes I. Tusa, G. Cheloni, N.S. Gray, A. Gozzini, P. Dello Sbarba, E. Rovida (Italy)

768

Monitoring pharmacodynamic response to hypoxiamodifying therapy using non-invasive positron emission tomography and magnetic resonance imaging V. Tessyman, F. Duncan, K. Finegan, R. Little, S. Cheung, Y. Watson, M. Babur, M.C. Asselin, J. O’Connor, K. Williams (United Kingdom)

722

Predicting cancer cell line sensitivity to metabolic targets: A network approach F. Ortega (United Kingdom)

769

Systems biology of immunogenic cell death in melanoma N. Rufo, T. Sauter, J.C. Marine, A.D. Garg, P. Agostinis (Belgium)

770

Radio-sensitizing effects of all trans retinoic acid (ATRA) on human chronic lymphocytic leukemia and osteosarcoma cell lines M. Russo, F. Messano, M. Basile, S. Carmela, I. Tedesco, S. Moccia, G.L. Russo (Italy)

723

Pan-omic profiling of RAS oncogene-mediated transformation and therapy resistance K. Kasack, R. Schafer, ¨ H. Koch, B. Kuster ¨ (Germany)

771

The molecular mechanisms underlying the ERa-36mediated signaling in breast cancer S. Omarjee, J. Jacquemetton, C. Poulard, N. Rochel, A. Dejaegere, Y. Chebaro, I. Treilleux, E. Marangoni, L. Corbo, M. Le Romancer (France)

772

Integrin a5b1 regulates both survival and migration of highgrade glioma cells through divergent AKT-driven pathways G. Renner , M.C. Mercier, F. Noulet, M. Dontenwill (France)

773

Intestinal bacterial indicator phylotypes convey radiationinduced genotoxicity I. Maier , J. Liu, J. Borneman, R. Schiestl (Austria)

716

Cordycepin increases radiosensitivity by modulating radiation-induced G2/M arrest in cervical cancer cells W.Y. Park, D.B. Seong, S. Hong, J.R. Yu (South Korea)

717

Role of c-MET inhibition in radiosensitization of hepatocellular carcinoma cell lines M. Gothwal, T. Brunner, A. Thomsen (Germany)

718

Depletion of FOXM1 via MET targeting underlies establishment of a DNA damage-induced senescence program in gastric cancer P. Francica, N. Llu´ıs, D.M. Aebersold, R. Langer, F. Bladt, A. Blaukat, D.M. Stroka, M. Rodr´ıguez Mart´ınez, Y. Zimmer, M. Medova´ (Switzerland)

719

Investigating a novel potential ATM/ATR/DNA-PK phosphorylation site on the MET RTK as a link between MET addiction and radioresistance J.P. Koch, S.M. Roth, A. Quintin, J. Gavini, D.M. Stroka, D.M. Aebersold, Y. Zimmer, M. Medova´ (Switzerland)

720

Treatment with the novel Akt inhibitor AZD5363 following radiotherapy improves tumour control in mouse models of head and neck cancer E. Searle, B. Telfer, D. Forster, K. Williams, B. Davies, T. Illidge, I. Stratford (United Kingdom)

Signalling Pathways II Transcriptional regulation of prostate cancer metabolism L. Valcarcel, V. Torrano, A.R. Cortazar, X. Liu, J. Urosevic, M. Castillo, G. Morciano, M. Graupera, P. Pandya, M. Unda-Urzaiz, N. Schultz, A. Aransay, V. Sanz-Moreno, R. Barrio, G. Velasco, P. Pinton, C. Cordon-Cardo, R.R. Gomis, J. Locasale, A. Carracedo (Spain)

756

758

Tumor suppressor role of miR-551a by targeting focal adhesion kinase in breast cancer A. Kaushik, G. Venkatraman, S.K. Rayala (India)

774

PREX1 integrates G protein-coupled receptor and phosphoinositide 3-kinase signaling to promote glioblastoma invasion A. Gont, M. Daneshmand, J. Woulfe, I. Lorimer (Canada)

775

Oncogenic reciprocal signalling C. Tape (United Kingdom)

759

A single cell approach to understanding cell cycle entry in cancer A. Barr , F.S. Heldt, S. Cooper, B. Novak, C. Bakal (United Kingdom)

760

Altered NFATc1 activity blocks T cell development and facilitates the pathogenesis of T-acute lymphoblastic leukemia A. Patra, S. Klein-Hessling, K.P. Knobeloch, J.C. Andrau, V. Ellenrieder, I. Screpanti, E. Serfling (United Kingdom)

776

The role of intracellular Ca2+ concentration for macromolecular drug carrier uptake into tumor cells in an acidotic tumor environment D. Gundel, ¨ O. Thews (Germany)

761

Yes-associated protein signalling in pancreatic cancer microenvironment B. Xie, S. Ward, M. Furutani-Seiki, A. Mackenzie, R. Mrsny (United Kingdom) Proteome and phosphoproteome analysis of stratified meningiomas S. Ferluga, J. Dunn, E. Lasonder, V. Sharma, D.A. Hilton, C. Adams, C.O. Hanemann (United Kingdom)

777

Investigation of the functional significance of YAP1 in prostate cancer cells and tissues F. Kisaayak Collak, F. Sagir, ˘ U. Demir, S. Ozkanlı (Turkey)

778

ADNP is a repressor of WNT signaling in colon cancer that 762 can be therapeutically induced C. Blaj, A. Bringmann, M. Urbischek, S. Krebs, H. Blum, T. Frohlich, ¨ G. Arnold, A. Jung, T. Kirchner, D. Horst (Germany)

lxvi


Abstract Nr. The role of the HER2 and HER3 in prostate cancer and their potential as therapeutic targets K. Rao, L. Gaughan, C. Robson, S. McCracken (United Kingdom)

779

Biomarker detection in pancreatic ductal adenocarcinoma H. Klett, M. Klose, S. Kowar, P. Bronsert, S. Kusters, ¨ M. Werner, P. Christoph, H. Busch, M. Boerries (Germany)

780

Role of microRNAs in signal transduction pathways of the inflammatory cytokine interleukin-6 in hepatocellular carcinoma cell lines and primary hepatocytes F. Servais, M. Kirchmeyer, M. Casper, M. Hamdorf, C. Haan, P. Nazarov, L. Vallar, C. Rubie, M. Glanemann, F. Lammert, S. Kreis, I. Behrmann (Luxembourg)

781

Indirect control of the p53-induced apoptosis based on RNA interference R. Jaksik, A. Lalik, K. Puszynski (Poland)

783

Regulation of the p53 pathway by siRNA and antisense oligonucleotides A. Lalik, R. Jaksik, K. Puszynski (Poland)

784

MEF2C is an ALK target gene that regulates cell invasion and migration in neuroblastoma G. Umapathy, R. Palmer, B. Hallberg (Sweden)

785

Polymorphisms in the p53 pathway are enriched in cancer 786 susceptibility loci and share characteristics with somatic pathway mutations G. Stracquadanio, M. Wallace, A.M. Grawenda, P. Zhang, J. Hewitt, J. Zeron-Medina, F. Castro-Giner, I.P. Tomlinson, C.R. Goding, K.J. Cygan, W.G. Fairbrother, L.F. Thomas, P. Sætrom, F. Gemignani, S. Landi, B. Schuster-Boeckler, D.A. Bell, G.L. Bond (Nuffield Department of Clinical Medicine) Controlling the intercellular processes K. Puszynski, A. Lalik, R. Jaksik (Poland)

Translational Research II Treating malignant glioma and brain metastasis with nanoparticles: Challenges of a peptide-based targeting and passage through the blood–brain-barrier V. Le Joncour , M. Hyvonen, ¨ E. Casals, P. Filppu, J. Tanjore Ramanathan, A. Ayo, J. Westermarck, J. Rosenholm, P. Laakkonen (Finland)

787

844

Development of a new workflow to identify novel marker in normal and adenoma tissue of CRC patients N. Lange, M. Schoeppler, A. Tiedemann, M. Spyra, F.T. Unger, H. Juhl, K.A. David (Germany)

845

The utilisation of analytical chemistry techniques in detecting novel biomarkers in oral cancer N. Sinevici, O. Jeffrey (Ireland)

846

Validation of a model of drug-induced aggressive liver cancer: gene expression and tumor recurrence J. Van Pelt, J. Dekervel, A. Bulle, H. Van Malenstein, P. Windmolders, E. Van Cutsem, C. Verslype (Belgium)

847

A computational approach to discriminate between human and mouse reads in sequencing data from Patient Derived Tumour Xenografts M. Callari, A. Sati Batra, R.N. Batra, H. Clifford, W. Greenwood, S.J. Sammut, S.F. Chin, A. Bruna, O.M. Rueda, C. Caldas (United Kingdom)

848

High throughput capture and profiling of CTCs using innovative technologies for gene expression C. Jarman, A. Lejeune-Dodge, S. Peter, K. Hull, D. Donnelly (USA)

849

Abstract Nr. Association of angiopoietin-2 and Ki-67 expression with vascular density and sunitinib response in metastatic renal cell carcinoma A. Lampinen, J. Rautiola, T. Mirtti, A. Ristimaki, ¨ H. Joensuu, P. Bono, P. Saharinen (Finland)

851

Combined anti-MET/EGFR treatment results in complete tumor regression and prevents resistance onset in a MET-amplified gastroesophageal xenopatient cohort S. Giordano, M. Apicella, C. Migliore, T. Capeloa, S. Menegon, M. Cargnelutti, A. Sapino, P. Cassoni, S. Marsoni, S. Corso (Italy)

852

Monitoring metastatic melanoma treatment resistance using 853 circulating tumour DNA S. Murphy, J. Wan, D. Gale, J. Morris, F. Mouliere, G. Bignell, C. Alifrangis, C. Parkinson, A. Durrani, U. McDermott, C. Massie, P. Corrie, N. Rosenfeld (United Kingdom) Development of a tool inhibitor of GCN5 towards the treatment of ccRCC S. Walker , K. Duffell, J. Downs, S. Ward (United Kingdom)

854

Evaluating the efficiency of hyaluronic acid for specific tumour targeting A. Spadea, J.M. Rios de la Rosa, M. Mehibel, A. Marianne, N. Tirelli, I.J. Stratford (United Kingdom)

855

High HMGN5 expression confers resistance to tamoxifen and its expression is predictive of response to tamoxifen in ER+ breast cancer patients D. Elias, L. Suntharalingam, H. Vever, A. Lykkesfeldt, M. Bak, H. Ditzel (Denmark)

856

Identifying changes in mutational dynamics in patients with early breast cancer undergoing neoadjuvant chemotherapy S.J. Sammut, S.F. Chin, O.M. Rueda, S.J. Dawson, M. Callari, E. Provenzano, J. Abraham, L. Hughes-Davies, H. Earl, C. Caldas (United Kingdom)

857

Plasma miRNAs as prognostic markers in colorectal cancer M. Maierthaler , A. Benner, L. Jansen, P. Knebel, J. ChangClaude, M. Hoffmeister, H. Brenner, B. Burwinkel (Germany)

858

SEARCHBreast; making surplus material from in vivo models of breast cancer available for research B. Morrissey, K. Blyth, P. Carter, C. Chelala, L. Jones, I. Holen, V. Speirs (United Kingdom)

859

Phosphorylated YB-1 (Y-box binding protein 1) as a biomarker of poor prognosis in patients with breast cancer S. Casimiro, M. Bettencourt, A. Fernandes, T.R. Pacheco, M. Maia-Matos, A.L. Costa, C. Abreu, A. Alves, L. Correia, L. Costa (Portugal)

860

A gemcitabine based peptide conjugate with improved metabolic properties, dual mode of action and efficacy in animal models T. Karampelas, E. Skavatsou, O. Argyros, C. Tamvakopoulos (Greece)

861

Therapeutic efficacy of the paradox-breaking panRAF and SRC drug CCT3833/BAL3833 in KRAS-driven cancer models G. Saturno, F. Lopes, M.R. Girotti, I. Niculescu-Duvaz, D. Niculescu-Duvaz, A. Zambon, L. Davies, L. Johnson, N. Preece, A. Viros, M. Pedersen, R. McLeary, R. Knight, R. Lee, D. Holovanchuk, A. Fusi, P. Lorigan, N. Dhomen, R. Marais, C. Springer (United Kingdom)

862


lxvii

Abstract Nr.

Abstract Nr. Proteomic analysis and interactions network for penile cancer characterization according to HPV status E. De Jesus Silva, A. Bulgarelli, F. Marchi, Z. Stenio, ˆ I. Werneck, G. Guimaraes, ˜ N. Antonio ˆ Assun¸cao, ˜ F. Augusto Soares, J. Vassallo (Brazil)

863

Patient-derived xenografts effectively capture patient clinical 877 responses to oncology therapy A. Davies, J. Stebbing, S. Zacharoulis, A. Gaya, W. McGuire, W. Harris, R. Maki, M. Hidalgo, D. Vasquez-Dunddel, D. Ciznadija, A. Katz, D. Sidransky (USA)

RNA sequencing reveals a distinct transcriptomic profile predictive of clinical outcome in stage III colorectal cancer patients treated with adjuvant chemotherapy E. Mini, R. D’Aurizio, G. Perrone, A. Magi, A. Lapucci, R. Tassi, C. Napoli, L. Picariello, I. Landini, S. Nobili (Italy)

864

879

Pathway based adaptive therapy in head and neck cancer R.B. Reddy, B. Ramachandran, S. Rajendran, V. Dhavale, H. Vardhan, N. Hedne, V. Kekatpure, A. Jayaprakash, A. Suresh, M.A. Kuriakose (India)

865

Detection of colorectal cancer and adenomas by epigenetic profiles of circulating nucleosomes: A pilot study with 58 subjects L. D’Hondt, M. Herzog, J.F. Rahier, L. Faugeras, A. Druez, E. Josseaux, K. Scoubeau, F. George, T. De Ronde, J. Micallef (Belgium)

880

Differential methylation of a CpG site in the CD95-ligand promoter predicts the response to therapy with APG101 in glioblastoma C. Gieffers, C. Merz, J. Sykora, C. Kunz, M. Thiemann, B. Wiestler, W. Wick, H. Fricke (Germany)

866

Patient-derived tumor xenografts in humanized NSG-SGM3 mice: A new immuno-oncology platform L.C. Yao, M. Cheng, M. Wang, B. Soper , K. Snow, J. Banchereau, K. Palucka, J. Keck (USA)

Dynamics of relapse in breast cancer O.M. Rueda, S.J. Sammut, S.F. Chin, B. Pereira, R.N. Batra, M. Callari, E. Provenzano, C. Curtis, C. Caldas (United Kingdom)

867

DigiWest multiplex protein profiling for identification of chemotherapy biomarkers Y. Beiter, F. Treindl, J. Naskuo, H. Neubauer, G. Erdmann, C. Sachse, M. Templin (Germany)

868

Ribosome biogenesis factors: novel clinical markers of breast cancer outcome F. Nguyen Van Long, N. Pion, A. Lardy-Cleaud, E. Lavergne, J.C. Bourdon, S. Chabaud, I. Treilleux, F. Catez, J.J. Diaz, V. Marcel (France)

869

Caspase modelling is a viable innovative tool in the precision medicine arsenal for stage III colorectal cancer (CRC) M. Salvucci, M. Cremona, A. Lindner, A. Resler, E. Kay, B. Hennessy, P. Laurent-Puig, S. Van Schaeybroeck, M. Rehm, J. Prehn (Ireland)

870

Proteomic profile in prostate cancer − From translational research to precision medicine C. Tanase, I.D. Popescu, E. Codrici, S. Mihai, A.M. Enciu, L. Necula, A. Preda, R. Albulescu (Romania)

871

Effects of drugs active on tumor metabolism in head and neck paraganglioma cell lines R. Florio, L. De Lellis, V. Di Giacomo, M.L. Gallorini, A. Natale, M.C. Di Marcantonio, F. Verginelli, D. Verzilli, R. Mariani-Costantini, A. Cama (Italy)

872

Changes in ctDNA levels established with the untargeted 886 mFAST-SeqS method reflect response to therapy in metastasized breast cancer patients E. Heitzer , M. Auer, C. Suppan, N. Dandachi, M. Balic (Austria)

Examining the tumour suppressive capabilities of microRNA-379 and elucidating a clinically relevant means of delivery K. O’Brien, S. Khan, M. Mannion, D. Joyce, P. Dockery, P. Lalor, H. Ingoldsby, M. Kerin, R. Dwyer (Ireland)

874

Inhibition of oncogenic MAPK signaling in melanoma triggers SOX2-dependent adaptive drug resistance programs S. Flueckiger , A. Aloia, L. Frischknecht, A. Tuozzo, D. Croci, O. Shakhova, C. Britschgi, M.P. Levesque, R. Dummer, W. Krek (Switzerland)

RATHER: High resolution molecular profiling of invasive lobular breast cancers S.F. Chin, C. Caldas (United Kingdom)

875

Urine biomarker profiling contains structure and predicts 881 prostate cancer hormone therapy relapse H. Curley, M. Yazbek-Hanna, R. Hurst, R. Kumar, I. Mills, J. Schalken, J. Clark, D. Brewer, C. Cooper (United Kingdom)

Micro-fluidic enrichment of circulating or disseminated 876 cancer cells from blood or lymph nodes independent of surface marker expression N. Stojanovic, K. Weidele, S. Scheitler, B. Alberter, M. Raab, A. Markiewicz, S. Haferkamp, C.A. Klein, B. Polzer (Germany)

Expression patterns and prognostic value of Cyclindependent kinase 5 (Cdk5) in colorectal tumours V. Ruiz de Porras, S. Cabrero-De las Heras, S. Bystrup, J.L. Subirats, E. Musulen, ´ J.L. Manzano, V. Moreno, L. Layos, C. Buges, ´ E. Martinez-Balibrea (Spain)

882

UM Cure 2020 − A consortium of European experts in uveal melanoma to identify new therapies for patients with metastatic disease N. Dhomen, R. Marais, M.J. Jager, B.E. Snaar-Jagalska, S. Coupland, B. Romanowska-Dixon, M.C. Mione, A. Valente, B. Ryll, R. Ruijtenbeek, A. Prestat, E. Vinolo, S. Roman-Roman (United Kingdom)

883

PIK3CA mutant allele differential expression (MADE) 884 associates with breast cancer clinical features J. Xavier, B. Almeida, C. Sun, J. Silva, A. Marreiros, M. Eldridge, R. Bernards, C. Carlos, S.F. Chin, A.T. Maia (Portugal) NF-úB2 genetic variants (rs7897947 and rs12769316) are strongly correlated with the survival of NSCLC patients F.I. Dimitrakopoulos, A. Antonacopoulou, A. Kottorou, S. Maroussi, N. Panagopoulos, C. Scopa, A. Koutras, T. Makatsoris, D. Dougenis, H. Kalofonos (Greece)

885

887

Loss of LIM-domain proteins LIMD1, Ajuba and WTIP 888 as a novel molecular mechanism for disease aetiology in clear cell renal cell carcinoma (ccRCC) K. Smith, M. Sheaff, G. Elia, A. Clear, G. Trevisan, D. Foxler, K. Bridge, S. Shepherd, T. Powles, T. Sharp (United Kingdom) Imaging tumour vasculature using optoacoustics: Breast cancer xenograft models differ in tumour vascularity and oxygenation I. Quiros Gonzalez, M. Tomaszewski, J. Joseph, S. Bohndiek (United Kingdom)

889

lxviii


Abstract Nr.

Abstract Nr.

Tumour infiltrating (TINKs) and tumour associated (TANKs) natural killer cells: a new paradigm in colorectal cancer angiogenesis A. Bruno, B. Bassani, D. D’Urso, L. Boni, L. Mortara, D. Noonan, A. Albini (Italy)

926

Control of immune tolerance by the SIRPa−CD47 pathway and myeloid-derived suppressor cells N. Poirier , S. Pengam, N. Dilek, U. Claire, M. Bernard, V. Daguin, G. Vanessa, B. Gilles, V. Bernard (France)

928

Dual targeting of adaptive and innate immune checkpoints induce potent memory anti-tumor response V. Gauttier, N. Poirier , S. Pengam, B. Vanhove, S. Conchon (France)

929

Selective targeting of the SIRPa immune checkpoint, but not its counter-receptor CD47, controls the polarization of human macrophages V. Gauttier, S. Pengam, C. Mary, V. Thep ´ enier, ´ B. Vanhove, N. Poirier (France)

930

931

898

Glycosylated and methylated peptides as neoantigens in leukaemia S. Penny, S. Malaker, L. Steadman, P. Trantham, D. Bai, J. Shabanowitz, D. Hunt, M. Cobbold (United Kingdom)

899

Influence of adjuvants in vaccine formulations for 933 inducing enhanced HAGE-specific cytotoxic-T-lymphocytes responses against triple-negative breast cancer cell lines D. Nagarajan, G. Pockley, R. Rees, S. McArdle (United Kingdom)

Characterization of a novel dormant, drug resistant, stem cell subpopulation in acute lymphoblastic leukemia S. Ebinger , E. Ozdemir, S. Tiedt, C. Ziegenhain, C. Castro-Alves, W. Enard, I. Jeremias (Germany)

890

Clinicopathologic significance of androgen receptor expression in invasive breast cancer: A comprehensive analysis in large cohorts M.A. Aleskandarany, R. Abduljabbar, S. Sonbul, C.F. Lai, S. Ali, M. Diez-Rodriguez, C. Nolan, A.R. Green, I.O. Ellis, E.A. Rakha (United Kingdom)

892

Photo-acoustic molecular imaging advancement by unmixing and optical flow method with unfocused ultrasound sonoporation method as improvement in tumor treatment P. Giustetto, D. Fuchs, P. Trochet (Italy)

893

Mathematical biology framework for exploring heterogeneous drug responses within a patient H. Mistry (United Kingdom)

894

Targeting cancer cells with mutated KRAS with krasin, a novel synthetic cycloheptapeptide R. Sheikhnejad, F. Tahoori, B. Shokri (Iran)

895

RNAi and CRISPR/Cas9 based in vivo models for drug discovery P. Premsrirut, G. Martin, L. Dow, S.Y. Kim, J. Zuber, G. Hannon, S. Lowe (USA) The palliative treatment efficacy of glucose inhibition combined with chemotherapy on a case of non-small cell lung cancer (NSCLC) patient with widespread bone metastases Y. Liu, T.C. Tsang, Y. Zhang, X. Mao, Q. Qi, M. Zhu, C. Zhang, X. Pan, M.E. Pennington, Y. Ling (China)

Tumour Immunology II Nanotechnology-based immunotherapeutic approach for tumour eradication E. Zupanˇciˇc , C. Curato, L. Eisenbach, S. Jung, H. Florindo (Portugal)

918

IL-2 and IL-15 cytokine treatments increase NKG2D receptor expression on T, NKT-like and NK cell lymphocyte subsets from regional lymph nodes of melanoma patients regardless of lymph node involvement A. Vuletić , I. Jovanić, Z. Milovanović, S. Nikolić, V. Juriˇsić, G. Konjević (Serbia)

919

Knockdown of osteopontin (SPP1) in glioma cells blocks protumorigenic activation of infiltrating myeloid cells and restores activation of cytotoxic T cells A. Gieryng, D. Pszczolkowska, M. Dabrowski, B. Kaminska (Poland)

921

Differential dendritic cell inhibition by conditioned media from oesophageal and colorectal cancer M. Morrissey, N. McCabe, M. Dunne, J.V. Reynolds, J. O’Sullivan (Ireland)

922

APG101 blocks pro-apoptotic CD95/CD95L signaling and protects immune cells from activation-induced cell death C. Merz, J. Sykora, B. Thamara, D. Richards, H. Fricke, C. Gieffers (Germany)

924

APG1233 is a novel hexavalent CD40 agonist acting on monocyte differentiation and macrophage-polarization and promotes increased activation of CD4+ T cells by shifting M1/M2 macrophage balance C. Merz, J. Sykora, T. Beyer, D. Richards, H. Fricke, C. Gieffers, O. Hill (Germany)

925

Determining a mechanism of synergistic immunochemotherapy in ovarian cancer J. Wahba, M. Natoli, L. Whilding, A. Parente-Pereira, J. Maher, R. Smith, S. Ghaem-Maghami (UK)

934

PD-1 blockade enhances synergistic killing of ovarian tumour cells by combination chemotherapy and T cell immunotherapy J. Wahba, M. Natoli, L. Whilding, A. Parente-Pereira, J. Maher, R. Smith, S. Ghaem-Maghami (UK)

935

European Journal of Cancer (2016) 61(S1), S1–S4

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.ejcancer.com

Sunday 10 July 2016 Sunday 10 July 2016

08:30−10:15

Sunday 10 July 2016

08:30−10:15

Symposium

Symposium

Tumour Heterogeneity and Evolution

DNA Damage

1 Proffered Paper: Functional study of glioblastoma (GBM) intratumour genomic heterogeneity and evolution using patient-derived xenografts (PDXs)

2 Proffered Paper: Targeting of beta1 integrins compromises DNA damage repair for radiosensitization of head and neck cancer cells

V. Chigancas1 , A. Gonzalez-Junc ` a` 1 , J.L. Parra1 , R. Mayor1 , J. Seoane1 . 1 ´ Institut of Oncology VHIO, Translational Research, Barcelona, Vall D’Hebron Spain Background: Glioblastoma (GBM) is a dismal disease with an extremely short overall survival. Almost invariably tumors relapse after surgery and standard of care (SOC) therapies. The intratumor subclonal landscape of GBM changes and evolves in response to treatment leading to a substantial divergence between the primary and the secondary tumors. We hypothesized that subclones that are enriched in tumor relapse might present a selective advantage over the rest of subclones and, hence, might contain genomic alterations that could confer resistance to SOC. In order to identify subclones enriched in secondary tumors, we performed exome sequencing of tumor specimens from primary and secondary GBM. Two cases presented an enrichment in clones carrying mutations in the neurofibromatosis 1 (NF1) gene after tumor relapse, indicating that these mutations could confer resistance to SOC. We decided to generate patient-derived xenograft (PDX) models of the primary tumors of both cases and selectively generate the identified NF1 mutations using the CRISPR technology to assess whether NF1 alterations conferred resistance to treatment. Materials and Methods: We generated patient-derived neurospheres cultures and PDX models of two primary GBM tumors. NF1 genetic alterations were generated through two strategies: (i) small heterogeneous RNA (shRNAs) to decrease NF1 expression and (ii) CRISPR, generating exactly the same mutations observed in a patient after tumor recurrence. Parental and NF1deficient models were engineered to express dsRed and green fluorescent protein (GFP), respectively. Parental dsRed and NF1 deficient cells GFP were ortothopically concomitantly inoculated in the brain of Balb/c nude mice in the proportion 3:1 respectively. The animals received SOC treatment, and after detection of tumor growth by luciferase expression in in vivo imaging system (IVIS), the PDXs were sorted and analyzed by FACS. Results: When analyzing PDXs-derived tumor cells after SOC treatment in mice, we observed an enrichment of cells with decreased NF1 mRNA expression (shNF1) or NF1 CRISPR cells (carrying the same mutation observed in the patient) over time in comparison to non-treated animals. These results indicate that cells deficient in NF1 exhibit an advantage in response to SOC and are enriched in tumor relapse. Conclusion: Our results show that the NF1 mutations observed to be enriched in tumor recurrence confer a selective advantage and resistance to SOC. Early detection of alterations in NF1 in GBM patients can therefore be decisive for prognosis and treatment choice. In addition, our data highlight the need to consider intratumor genomic heterogeneity and evolution to improve the treatment of GBM. No conflict of interest.

E. Dickreuter1 , I. Eke1 , M. Krause2 , K. Borgmann3 , M. Van Vugt4 , N. Cordes1 . Oncoray − National Center for Radiation Research in Oncology TU Dresden, Medical Faculty Carl Gustav Carus, Dresden, Germany, 2 German Cancer Consortium DKTK, Medical Faculty Carl Gustav Carus, Dresden, Germany, 3 Laboratory of Radiobiology and Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4 Department of Medical Oncology, University Medical Centre Groningen, Groningen, Netherlands 1

Introduction: Resistance to cancer therapies is a major unsolved challenge. Responsible is, among others, integrin-mediated adhesion to extracellular matrix. Studies of us and others identified targeting of beta1 integrin receptors as promising approach for radio- and chemosensitization of tumor cells. Although different prosurvival beta1 integrin-mediated signaling pathways are known, it remains unclear whether they are critically involved in the repair of radiation-induced DNA double strand breaks (DSB). Therefore, we examined the impact of beta1 integrin targeting on DSB repair and describe a regulatory function of beta1 integrins for DNA-PK-dependent but not PARP-dependent non-homologous end-joining (NHEJ). Material and Methods: Experiments were performed by employing a physiological 3D cell culture model based on laminin-rich extracellular matrix and tumor xenografts of human head and neck squamous cell carcinoma (HNSCC) cell lines. beta1 integrin inhibition was accomplished using the monoclonal antibody AIIB2. AIIB2, X-ray irradiation, siRNA-mediated knockdown and inhibitor treatment were performed and residual DSB number, NHEJ activity, expression and phosphorylation of various DNA repair proteins as well as clonogenic survival were determined. Results: beta1 integrin targeting impaired the repair of radiogenic DSB (gammaH2AX/p53BP1, pDNA-PKcs T2609 foci) in vitro and in vivo, decreased NHEJ activity and reduced the expression and phosphorylation of Ku70, Rad50, Nbs1 and pDNA-PKcs T2609. Further, we identified Ku70, Ku80 and DNA-PKcs but not PARP-1 to reside in the beta1 integrin signaling pathway. Intriguingly, combined inhibition of beta1 integrin and PARP using Olaparib was significantly more effective than either treatment alone in non-irradiated and irradiated HNSCC cells. Moreover, FAK and JNK1 were identified to mediate beta1 integrin-dependent DNA-repair. Conclusions: Here, we support beta1 integrins as promising cancer targets and highlight a regulatory role for beta1 integrins in the DNA-PK-dependent repair of radiation-induced DSB. Further, our findings show combined targeting of beta1 integrins and PARP-dependent NHEJ using Olaparib as promising strategy for radiosensitization of HNSCC cells. No conflict of interest.

0959-8049/$ – see front matter © 2016 Elsevier Ltd. All rights reserved.

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EACR24 Oral Presentations, Sunday 10 July 2016 / European Journal of Cancer 61, Suppl. 1 (2016) S1–S4

Sunday 10 July 2016

08:30−10:15

Symposium

Tumour Metabolism 3 Proffered Paper: Targeting GMP synthesis reveals a hierarchy of p53-cell cycle checkpoints in c-Myc driven CRCs 1 J. Pelletier1 , F. Riano-Canalias ˜ , R. Salazar1,2 , A. Villanueva1,2 , O. Yanes3 , S. Kozma1,2 , G. Thomas1,2 . 1 Laboratory of Cancer Metabolism, IDIBELL, Hospital Duran i Reynals, Spain, 2 Institut Catala` d’Oncologia, 08908 L’Hospitalet de Llobregat, Barcelona, 3 Centre for Omic Sciences, 43204 Reus, Tarragona, Spain

Introduction: Sporadic colorectal cancer (sCRC) is the most frequent cancer shared by men and women in Europe and represents ~80% of CRC cases. Almost all sCRCs display deregulated c-Myc signaling, which drives hyperactivation of ribosome biogenesis. Ribosome biogenesis, a complex multi-step molecular process, is highly energy consuming and a key determinant in c-Myc driven tumorigenesis, serving as a potential ‘Achilles heel’ for the treatment of sCRCs. Thus, it is important to elucidate the underlying mechanisms of crosstalk between ribosome biogenesis and oncogene triggered cell cycle checkpoints, and to clinically exploit these findings. Mycophenolic acid (MPA), an FDA approved drug, is an inhibitor of inosine monophosphate dehydrogenase (IMPDH), which carries out the rate limiting step in the de novo synthesis of guanosine monophosphate (GMP). MPA has been shown to impair ribosome biogenesis and induce p53. Clinically MPA is used as an immunosuppressant through its ability to activate p53, induce cell cycle arrest and provoke apoptosis in blood cells. Material and Methods: In this study, we set out to elucidate the underlying mechanisms that sense purine nucleotide levels following MPA mediated IMPDH inhibition in c-Myc driven CRCs cell lines. Results and Discussion: The impaired ribosome biogenesis checkpoint (IRBC) is executed by a preribosomal complex made-up of two ribosomal proteins (RP)L5, RPL11, and noncoding 5S rRNA, which upon ribosome impairment is re-directed to the binding and inhibition of E3-ubiquitin ligase human double minute 2 (HDM2). We have found that exponentially proliferating cells first sense a reduction in GMP levels by activating the IRBC. However, if the lesion in ribosome biogenesis is not resolved, cells exit G1, and engage a second checkpoint involving replicative stress, and the activation of ATR-CHK1 checkpoint, leading to S-phase arrest. Both checkpoints can be rescued by the supplementation of guanosine, which can be converted into GMP by the purine ‘salvage pathway’. But, if GMP depletion persists and activation of the ATRCHK1 checkpoint is not resolved DNA double strand breaks ensue provoking the activation of the ATM-CHK2 checkpoint. Conclusion: Taken together, our results suggest that reduction of purine pools is first sensed by the IRBC, but if the checkpoint is not resolved, cells move into S phase where they encounter replicative stress due to nucleotide imbalance and finally DNA damage. We have begun to elucidate the molecular mechanisms by which these checkpoints are interacting, which will provide insights into the treatment of c-Myc driven tumors by inhibition of GMP synthesis. No conflict of interest.

Sunday 10 July 2016

10:45−12:30

Symposium

Cancer Immunotherapy 4 Proffered Paper: Immunological changes during tumor progression from primary to recurrent glioblastoma C. Herold-Mende1 , C. Rapp2 , R. Warta3 , A. Unterberg4 , P. Beckhove5 . 1 University of Heidelberg, Experimental Neurosurgery, Heidelberg, Germany, 2 University Hospital Heidelberg, Division of Experimental NeurosurgeryDepartment of Neurosurgery, Heidelberg, Germany, 3 University Hospital Heidelberg, Division of Experimental Neurosurgery- Department of Neurosurgery, Division of Experimental Neurosurgery- Department of Neurosurgery, Germany, 4 University Hospital Heidelberg, Department of Neurosurgery, Heidelberg, Germany, 5 University Medical Center of Regensburg, Regensburg Center for Interventional Immunology, Regensburg, Germany Frequently novel therapies such as immunotherapy are developed based on knowledge obtained from primary glioblastoma (pGBM), but are then also applied to treat recurrent tumors (rGBM). However, knowledge about expression changes, immunological changes and adaptations of the tumor microenvironment during GBM progression is still limited. Therefore, we performed a comprehensive analysis of pGBM and corresponding

rGBM tissues of 60 patients regarding changes in the transcriptome, their immunogenicity and intratumoral immune cell infiltration. Multi-colorimmunofluorescent stainings and subsequent TissueFAXS-based evaluation showed a significant accumulation of immunosuppressive M2-polarized microglia cells in recurrent tumors. These changes have been accompanied by a concomitant decrease of immune-relevant chemokines and cytokines as determined by Luminex analyses. On the other side, an age-dependent increase of CD8+ effector T cells was observed during tumor progression in association with a better survival. Subsequent microarray analyses revealed marked changes on the transcriptome level occurring from pGBM to rGBM suggesting unbeneficial tumor cell selection processes. To finally identify immunogenic antigens as well as changes in the immunogenic repertoire between pGBM and rGBM, a combination of protein fractionation (PF2D) and T cell activation assays (IFN-g ELISpot Assay) was employed. In most of the patients severe changes regarding the spontaneous recognition of distinct protein fractions were observed. However, out of 1500 proteins identified by mass spectrometry to be present in immunogenic protein fractions of pGBM and rGBM, we recognized four immunogenic proteins to show frequent spontaneous T cell responses in glioblastoma patients but not in healthy donors. Overexpression of these proteins and an inverse relation to survival strengthen their suitability as vaccine antigens for future immunotherapies not only in pGBM but also in rGBM. Altogether our analysis might help to select for patients who are suited for immunotherapy even at the time of recurrence and to select for promising vaccine antigens in this setting. No conflict of interest.

Sunday 10 July 2016

10:45−12:30

Symposium

Mechanisms of Drug Resistance 5 Proffered Paper: Utilising non-mutational drug-tolerance prolongs and improves targeted melanoma therapy M.P. Smith1 , H. Brunton1 , E.J. Rowling1 , M.R. Girotti2 , R. Marais2 , R. Dummer3 , K.T. Flaherty4 , Z.A. Cooper5 , J.A. Wargo5 , C. Wellbrock1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom, 2 CRUK Manchester Institute for Cancer Research, Molecular Oncology Group, Manchester, United Kingdom, 3 University Hospital Zurich, Department Dermatology, Zurich, Switzerland, 4 Massachusetts General Hospital Cancer Center, Department of Medicine, Boston, USA, 5 University of Texas MD Anderson, Surgical Oncology, Houston, USA Background: Harnessing the prevalence of activating mutations in the MAPkinase (MAPK) pathway in melanoma led to the wide use of inhibitors targeting BRAF and MEK. These therapies produce impressive responses in melanoma patients, but are set back by the mutational heterogeneity present in relapsed patients. Once melanoma patients have progressed, treatment plans become more complex and suffer from combinatorial toxicities. To overcome this issue we have studied the initial therapy-phase, when the response to MAPK pathway inhibition is thought to be more universal, to identify tolerance mechanisms. Material and Methods: We analysed patient samples treated with either BRAF or BRAF and MEK inhibitors for changes present early on-treatment. These observed changes were further confirmed in xenograft and allograft mouse models. A FDA approved drug library was screened for drugs targeting these survival mechanisms. Extensive in vitro and in vivo pre-clinical studies were performed including cell death and gene expression analyses. Results: By examining samples from patients on treatment with MAPK inhibitors and samples from corresponding melanoma mouse models, we identified the lineage specific transcription factor MITF as a driver of drug tolerance. Importantly, we show using ex vivo and in vitro experiments that this tolerant state is both reversible and dependent on MITF. This tolerant state is induced directly by the effects of MAPK inhibitors, that cause increased expression of PAX3 an activator of MITF expression. A drug screen identified the HIV1-protease inhibitor nelfinavir as a significant suppressor of MITF and PAX3 expression. Nelfinavir profoundly synergises with MAPK pathway inhibitors and enhances cell death both in vitro and in vivo in BRAF mutant melanoma. Moreover, by targeting MITF and PAX3 nelfinavir can synergise with MAPK inhibitors also in NRAS mutant melanoma. This treatment strategy is even effective in cells isolated from patients that progressed on MAPK inhibitor therapy with RAS mutations and the nelfinavir and MAPKi combinatorial therapy is also potent in BRAF/NRAS/PTEN mutant melanoma in vivo. Conclusions: We have identified a therapy phase that occurs on treatment and which results in enhanced tolerance to MAPK inhibitors. This tolerance phase is maintained by a PAX3/MITF axis and targeting this axis with nelfinavir can prolong and improve responses to MAPK inhibitor treatments. Thus we have identified not only a promising new drug combination but also a new

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EACR24 Oral Presentations, Sunday 10 July 2016 / European Journal of Cancer 61, Suppl. 1 (2016) S1–S4 strategy for delaying the onset of resistance [Smith et al., Cancer Cell, 2016, Article in Press]. No conflict of interest.

Sunday 10 July 2016

14:00−15:45

Symposium

Sunday 10 July 2016

10:45−12:30

Symposium

Epigenetics 6 Proffered Paper: The rRNA epigenetic hypothesis: role of ribosome heterogeneity in tumorigenesis V. Marcel1 , F. Nguyen Van Long1 , N. Pion1 , J. Erales1 , J.C. Bourdon2 , A. Puisieux1 , I. Motorine3 , P. Bouvet1 , F. Catez1 , J.J. Diaz1 . 1 Cancer Research Center of Lyon, Cancer Cell Plasticity, Lyon, France, 2 Jacqui Wood Cancer Center, Division of Cancer Research, Dundee, United Kingdom, 3 University of Lorraine, IMoPA, Nancy, France Unexpected characteristics of the ribosome, the machinery that translates mRNA into proteins, have been recently revealed that open up novel perspectives in the understanding of its biological function. First, it appears that ribosome plays key role in tumorigenesis since specific inhibition of ribosome biogenesis using anti-RNA polymerase I inhibitor targets cancer cells without affecting healthy cells. In addition, it emerges that ribosome is present as different variants, which displays distinct translational activity. The major source of ribosome heterogeneity comes from the ribosomal RNA (rRNA). Like proteins, rRNA exhibits chemical modifications, including 2 -Oribose methylation (or methylation). These modifications have a key role in maintaining ribosome structure and thus affect its translational activity. We demonstrated for the first time that alteration of rRNA methylation occurs during tumor initiation and progression. In p53 inactivated mammary cells, modification of rRNA methylation is associated with change in translational control of a subset of mRNAs encoding oncogenic proteins that promote tumorigenesis [1]. Since then, development of RiboMeth-Seq, a genomewide approach to study rRNA methylation, allowed us to determine that change in rRNA methylation occurs only at some given sites. Recently, we firmly demonstrate that alteration of the rRNA methylation pattern and the associated change in translational control is driven by alteration of expression of fibrillarin (FBL), the unique rRNA methyl-transferase. In addition, RiboMethSeq technology allowed us to identify specific alterations occurring in the rRNA methylation pattern during epithelial-to-mesenchymal transition. The importance of ribosome heterogeneity in cancer development is supported by our clinical data. Indeed, alterations in rRNA methylation patterns were observed using RiboMeth-seq in breast cancer. Moreover, it appeared that FBL is a marker of poor outcome in breast cancer patients. We also identified additional ribosome biogenesis factors as prognostic marker of different cancers. We accumulated several evidences demonstrating for the first time that rRNA methylation pattern is affected in different tumoral models. Our data support the emerging notion of rRNA epigenetics claiming that chemical modifications of rRNA participate in gene-specific expression by modulating translation. They also support the major role of ribosome in tumorigenesis. Finally, it appears that the ribosome can be used as a clinical marker and that ribosome heterogeneity could provide novel and original anti-cancer therapeutic opportunities. Reference(s) [1] Marcel et al. (2013) Cancer Cell 24, 318–330. No conflict of interest.

The Tumour Microenvironment 7 Proffered Paper: Mechanical induction of the tumourogenic beta-catenin pathway by tumour growth pressure 1 2 M.E. Fernandez-S ´ anchez ´ , , S. Barbier1 , J. Whitehead1 , G. Bealle ´ A. Michel2 , H. Latorre-Ossa3 , C. Rey4 , L. Fouassier4 , A. Claperon4 , L. Brulle´ 5 , E. Girard6 , N. Servant6 , T. Rio-Frio7 , H. Marie8 , S. Lesieur8 , 2 C. Housset4 , J.-L. Gennisson3 , M. Tanter3 , C. Menager , S. Fre9 , S. Robine10 , ´ E. Farge1 . 1 CNRS UMR 168 Physicochimie Curie Mechanics and Genetics of Embryonic and Tumour Development INSERM, PSL Research University, Fondation Pierre-Gilles de Gennes, Institut Curie − Centre de Recherche, ´ Laboratoire PHENIX France, 2 UPMC Univ Paris 06, Sorbonne Universites, ` Physico-chimie des Electrolytes et Nanosystemes InterfaciauX, CNRS 3 UMR 8234, F-75005, Paris, France, Langevin Institut − Waves and Images ESPCI ParisTech, PSL Research University, CNRS UMR7587, ´ UPMC Univ Paris 06 & Inserm U979, France, 4 Sorbonne Universites, INSERM, UMR-S 938, CDR Saint-Antoine, F-75012, Paris, France, 5 CNRS UMR3666/INSERM U1143 Endocytic Trafficking and Therapeutic Delivery Institut Curie − Centre de Recherche, France, 6 Bioinformatic platform, U900 − Institut Curie–Mines-ParisTech, France, 7 Next-generation sequencing platform, Institut Curie, France, 8 CNRS UMR 8612, Laboratoire ` ´ Institut Galien Paris-Sud, LabEx Physico-Chimie des Systemes Polyphases LERMIT, Faculte´ de Pharmacie − Universite´ Paris-Sud, France, 9 CNRS ´ etique ´ ´ UMR 3215/Inserm U934 Unite´ de Gen et Biologie du Developpement Notch signaling in stem cells and tumors Institut Curie - Centre de Recherche, 10 France, CNRS UMR144 Compartimentation et dynamique cellulaires, Morphogenesis and Cell Signalling Institut Curie − Centre de Recherche, France

The tumour microenvironment may contribute to tumourogenesis due to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here, we explore the contribution of the mechanical pressure exerted by the tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch+ APC1638N/+ mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, associated with increased Ret and b-catenin signalling. We thus developed a method that allows to deliver defined mechanical pressure in vivo, by subcutaneously inserting a dorsal magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes (UML), stabilized in the mesenchymal cells connective tissue surrounding colonic crypts after intravenous injection. The pressure induced magnetically quantitatively mimicked the endogenous early tumour growth stress in the order of 1200 Pa, without affecting tissue stiffness, as monitored by acoustic strain imaging and shear wave elastography. Exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of b-catenin on Tyrosine 654, which impairs its interaction with the E-cadherin in adherens junctions, and which was followed by b-catenin nuclear translocation after 15 days. As a consequence, elevated expression of b-catenin target genes was observed at one month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci (ACF). Mechanical activation of the tumourogenic b-catenin pathway suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity. Reference(s) Fernandez-Sanchez, M.E. et al. Mechanical induction of the tumorigenic b-catenin pathway by tumour growth pressure. Nature 523, 92−95, doi:10.1038/nature14329 (2015). No conflict of interest.

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EACR24 Oral Presentations, Sunday 10 July 2016 / European Journal of Cancer 61, Suppl. 1 (2016) S1–S4

Sunday 10 July 2016

14:00−15:45

Sunday 10 July 2016

14:00−15:45

Symposium

Symposium

Liquid Biopsies in Cancer

Senescence and Ageing

8 Proffered Paper: Enhancing sensitivity for analysis of circulating tumour DNA C. Massie1 , N. Rosenfeld1 . 1 Cancer Research UK Cambridge Research Institute, Molecular and Computational Diagnostics Laboratory, Cambridge, United Kingdom

9 Proffered Paper: Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence

Background: Plasma can be used to sample and measure a range of biomarkers for cancer, including proteins, circulating tumour cells, and cellfree DNA. In recent years, analysis of cell-free DNA has shown great promise through the application of genomic methods of analysis that enable extraction of information specifically from the circulating tumour DNA (ctDNA). In previous work, we have shown that cell-free DNA in plasma can be used for non-invasive genomic analysis of cancers, to identify cancer mutations (Forshew et al., Sci Transl Med 2012), to study cancer heterogeneity and evolution (Murtaza et al., Nat Comm 2015), and to uncover mechanisms of resistance to therapies (Murtaza et al., Nature 2013). We, and others, have also shown that ctDNA levels can be quantified in plasma to track tumour response and progression, and can provide prognostic information (Dawson et al., N Engl J Med 2013). However the utility of these different approaches has been limited by the sensitivity of the analysis methods. Materials and Methods: We developed improvements to methods for noninvasive analysis of cancer, including efficient protocols for analysis of cell-free DNA on a genome-wide scale, and more sensitive methods for identification of mutations and quantification of low levels of ctDNA. Results: Improved resolution for genome-wide analysis allows us to make in-depth comparison of ctDNA to the tumour genomic profiles at various stages along the cancer progression and evolution. Focused analysis of selected regions shows excellent concordance with tumour profiles for ‘actionable’ mutations. Methods with enhanced sensitivity can enable accurate quantification of ctDNA levels in early cancers or after treatment. Conclusions: In relapsed cancer, mutations found in cell-free DNA reflect stem mutations that are more prevalent across multi-clonal cancer. Targeted analysis of plasma DNA with appropriate methods can be used for molecular charaterisation, which can aid in clinical stratification for molecular therapies. Sensitive quantification of ctDNA in low-burden disease can provide more accurate detection of residual disease or earlier indication of disease relapse. Accurate estimates of ctDNA levels in early cancers can provide data to guide development of future applications for earlier diagnosis. Conflict of interest: Ownership: Inivata Ltd. Board of Directors: Inivata Ltd. Corporate-sponsored Research: AstraZeneca. Other Substantive Relationships: Co-founder and CSO of Inivata Ltd.

B. Morancho1 , A. Martinez-Barriocanal1 , J. Villanueva1 , J. Arribas1 . 1 Vall d’Hebron Institute of Oncology, Preclinical Research, Barcelona, Spain Background: Cellular senescence is a terminal cell proliferation arrest that can be triggered by oncogenes. One of the traits of oncogeneinduced senescence (OIS) is the so-called senescence-associated secretory phenotype or senescence secretome. Depending on the context, the non-cell autonomous effects of OIS may vary from tumor suppression to promotion of metastasis. Despite being such a physiological and pathologically relevant effector, the mechanisms of generation of the senescence secretome are largely unknown. Material and Methods: We analyzed by label-free proteomics the secretome of p95HER2-induced senescent cells and compared the levels of the membrane-anchored proteins with their transcript levels. Then, protein and RNA levels of ADAM17 were evaluated by using Western blot and reverse transcription-polymerase chain reaction, its localization by using biotin labeling and immunofluorescence, and its activity by using alkaline phosphatasetagged substrates. The p95HER2-expressing cell lines, senescent MCF7 and proliferating MCF10A, were analyzed to study ADAM17 regulation. Finally, we knocked down ADAM17 to determine its contribution to the senescenceassociated secretome. The effect of this secretome was evaluated in migration assays in vitro and in nude mice by assessing the metastatic ability of orthotopically co-injected non-senescent cells. Results and Discussion: Using breast cancer cells expressing p95HER2, a constitutively active fragment of the proto-oncogene HER2 that induces OIS, we show that the extracellular domains of a variety of membrane-bound proteins form part of the senescence secretome. We determine that these proteins are regulated transcriptionally and, in addition, that their shedding is limited by the protease ADAM17. The activity of the sheddase is constrained, at least in part, by the accumulation of cellular cholesterol. The blockade of ADAM17 abrogates several prometastatic effects of the p95HER2-induced senescence secretome, both in vitro and in vivo. Conclusion: Considering these findings, we conclude that ectodomain shedding is tightly regulated in oncogene-induced senescent cells by integrating transcription of the shedding substrates with limiting ADAM17 activity. The remaining activity of ADAM17 contributes to the non-cell autonomous protumorigenic effects of p95HER2-induced senescent cells. Because ADAM17 is druggable, these results represent an approximation to the pharmacological regulation of the senescence secretome. No conflict of interest.




Monday 11 July 2016 Monday 11 July 2016

08:30−10:15

Monday 11 July 2016

ESMO Symposium

Symposium

ESMO Symposium: Clinical Developments in the Diagnosis and Treatment of Solid Tumors

Stem Cells

10 Proffered Paper: Radiation-induced lung fibrosis: from molecular mechanisms to novel treatment concepts 1

1

1

1

2

F. Wirsdorfer ¨ , S. De Leve , F. Cappuccini , A.V. Meyer , L.F. Thompson , H. Karmouty-Quintana3 , D. Klein1 , M.R. Blackburn3 , M. Stuschke4 , V. Jendrossek1 . 1 University of Duisburg-Essen Medical School- Institute of Cell Biology Cancer Research, Department of Molecular Cell Biology, Essen, Germany, 2 Oklahoma Medical Research Foundation- OMRF, Immunobiology and Cancer Program, Oklahoma City, USA, 3 University of Texas Health Science Center, Department of Biochemistry and Molecular Biology, Houston, USA, 4 University Hospital Essen, Department of Radiotherapy, Essen, Germany Introduction: Radiotherapy is an integral part of standard treatment for thorax-associated neoplasms. Unfortunately, adverse late effects in the highly radiosensitive lung limit the radiation dose resulting in suboptimal local control, metastases and decreased quality of life. However, the molecular details of radiation-induced pneumopathy are still unclear and no effective treatment is available to date. Material and Methods: To gain more insight into the pathogenesis of radiationinduced lung fibrosis we studied radiation-induced alterations in the activity of the immunomodulatory ecto-5 -nucleotidase (CD73)–adenosine system as well as radiation-induced immune changes, vascular dysfunction and fibrosis in ithe lung tissue upon single high dose whole thorax irradiation of CD73+/+ and CD73−/− mice (C57BL/6 background) using flow cytometry, immunohistochemistry, and high pressure liquid chromatography (HPLC). Pharmacologic modulation of CD73 and adenosine was performed by treating mice with the anti-CD73 antibody (TY/23) or pegylated adenosine deaminase (PEG-ADA) as of week 16 post-irradiation. Results and Discussion: Whole-thorax-irradiation (WTI) with 15 Gy triggered a progressive increase in CD73 activity in the lung and of adenosine levels in bronchioalveolar lavage fluid (BALF) prior to histological evidence for progressive lung fibrosis at 25 weeks post-irradiation. Loss of CD73 in CD73−/− mice or treatment of irradiated CD73+/+ mice with pegylated adenosine deaminase (PEG-ADA) or the CD73 antibody TY/23 resulted in significantly reduced lung fibrosis upon WTI (Wirsdorfer ¨ et al., accepted for publication in Cancer Research 2016). Further data reveal, that the failure of irradiated CD73−/− mice to accumulate adenosine is associated with a reduced accumulation of pro-fibrotic cytokines and of myeloid cells and CD4+ T cells with immunosuppressive phenotypes compared to CD73+/+ mice. Conclusions: Our new findings on the pathogenesis of radiation-induced lung fibrosis suggest that pharmacologic strategies for modulating CD73, adenosine or radiation-induced immune changes may limit radiation-induced adverse late effects presumably without increasing radiation resistance of tumour cells. Acknowledgement: Supported by grants of the DFG GRK1739/1 and JE 275/4-1. No conflict of interest.

08:30−10:15

11 Proffered Paper: Exploring prostate progenitor compartment complexity as a route of targeting prostate cancer heterogeneity J.D. Barros-Silva1 , D. Linn2 , I. Steiner1 , G. Guo3 , G.C. Yuan4 , S. Orkin3 , Z. Li2 , E. Baena1 . 1 Cancer Research UK Manchester Institute, Prostate Oncobiology, Manchester, United Kingdom, 2 Brigham and Women’s Hospital and Department of Medicine, Division of Genetics, Boston, USA, 3 Boston Children’s Hospital and Dana-Farber Cancer Institute, Division of Pedriatic Hematology/Oncology, Boston, USA, 4 Dana-Farber Cancer Institute, Department of Biostatistics and Computational Biology, Boston, USA Background: Prostate cancer (PCa) is a very heterogeneous disease both clinically and biologically. PCa patients exhibit highly variable clinical behaviour; some have indolent disease whilst in others it is lethal. Failure in developing effective therapeutic protocols is a consequence of PCa genetic and cellular heterogeneity and the lack of pathological subtypes that predict patient outcome. Thus, tumour initiation from distinct cell types in the lineage hierarchy gives rise to tumour subtypes with different prognoses and/or treatment responses. Tumor relapse following androgen-deprivation therapy is a strong indicator that within the prostate tumour there are subpopulations of castration resistant (CR) progenitor cells capable of driving aggressive tumour progression. Recently, several elegant lineage-tracing approaches demonstrate that basal and luminal lineages within the adult prostate are largely maintained separately by their unipotent progenitor cells under physiological homeostatic conditions, supporting the existence of both multiple stem/progenitor cells that can serve as effective cell-of-origin of PCa. Material and Methods: Using a Fluidigm multiplex quantitative PCR-based single cell expression analysis platform we have analyzed the expression profiles of individual prostate cells sorted from hormone-na¨ıve and castrated mice, identifying novel prostate subpopulations by their cell surface markers. We are currently characterizing these novel subpopulations and their role in tumour initiation/progression in vitro (Organoid cultures) and in vivo (mouse lineage-tracing). FACS and immunofluorescence were also performed to further assess the identity of these prostate epithelial cells. Results and Discussion: Although most prostate luminal cells are sensitive to castration, some are CR. Single-cell expression profiling from hormonally na¨ıve (HN) and CR prostate cells showed that CR luminal cells and a subset of HN luminal cells exhibited a similar “intermediate” expression pattern, including high expression of prostate stem/progenitor marker genes. We validated LY6D as a novel marker to link CR luminal cells to luminal progenitors and confirmed LGR5 as a stem/progenitor marker on the prostate. Moreover, coupling 3D organoid culture and mouse lineage tracing, these novel luminal progenitors show multipotent capacity in normal and tumorigenic prostate. Conclusions: Although luminal or basal cell-derived prostate cancer ultimately results in a luminal phenotype, recent studies reveal that these tumors are molecularly distinct and display different kinetics in cancer progression. Our study suggests that this cell-of-origin model may also be extended to distinct subsets of cells within the luminal lineage (e.g., castration-resistant compared to castration-sensitive luminal cells). No conflict of interest.


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EACR24 Oral Presentations, Monday 11 July 2016 / European Journal of Cancer 61, Suppl. 1 (2016) S5–S8

Monday 11 July 2016

08:30−10:15

Symposium

Breakthrough Tools in Cancer Genetics 12 Proffered Paper: NGS-based 3D reconstruction reveals copy number changes as the major source of genomic heterogeneity in colorectal cancer S. Mamlouk1 , L. Childs2 , C. Oliveira3 , H. Daniel4 , F. Klauschen4 , D. Aust5 , M. Morkel6 , T. Wolf3 , R. Schafer ¨ 6 , C. Sers6 . 1 German Consortium for ´ Laboratory of Molecular Translational Cancer Research- Partner Site Charite, Tumor Pathology, Berlin, Germany, 2 Institute of Informatics- Humboldt University Berlin, Knowledge Management in Bioinformatics, Berlin, Germany, 3 Institute of Pathology- University of Heidelberg, Pathology, Heidelberg, ´ Pathology, Germany, 4 Institute of Pathology − University Medicine Charite, Berlin, Germany, 5 Institute of Pathology- Technical University Dresden, 6 Pathology, Dresden, Germany, Institute of Pathology − University Medicine ´ Laboratory of Molecular Tumor Pathology, Berlin, Germany Charite, Background: Tumor heterogeneity between and within individual patients is a major obstacle in clinical analysis of genetic alterations for personalized therapy. Material and Methods: To assess heterogeneity in colorectal cancer (CRC), we conducted targeted NGS using a custom gene panel encompassing the 100 most frequently mutated and amplified genes in CRC. We investigated genetic variants (SNVs) and copy number alterations (CNVs) in a cohort of 27 CRC patients with a primary tumor and up to four metastases per patient. Results: Non-synonymous SNVs were detected in 20/100 genes and a strong concordance was found between primary and metastatic samples within one patient in the main driver. Discordance was detected in 4 patients in TCF7L2, CARD11, TP53, MMP9 and GNAS. In contrast to SNVs, discordance was frequently detected in CNV analysis, such as ploidy differences between primary and metastatic samples of individual patients and even between adjacent cells within an individual sample. This observation stimulated a broadened investigation of intra-sample heterogeneity. Directed disassembly of a single primary CRC sample was performed, followed by deep sequencing and validation via FISH and PCR. We observed clearly distinct CNV patterns within the tumor, while SNVs were uniform. 3D reconstruction revealed 2 CNV clusters along the proximal-distal axis of the tumor. Conclusion: Our results indicate that CNV heterogeneity in CRC and its functional consequences need to be taken into account for personalized targeted therapies. No conflict of interest.

Monday 11 July 2016

10:45–12:30

Symposium

Diagnosing Cancer Earlier 13 Proffered Paper: A comparison of four methods of mammographic density measurement in the UK Predicting Risk Of Cancer At Screening (PROCAS) study − on behalf of the PROCAS Study team S. Astley1 , E. Harkness1 , J. Sergeant2 , P. Stavrinos3 , R. Warren4 , M. Wilson3 , A. Brentnall5 , J. Cuzick5 , A. Howell6 , G. Evans7 . 1 University of Manchester, Centre for Imaging Sciences, Manchester, United Kingdom, 2 University of Manchester, Arthritis Research UK Centre for Epidemiology Centre, Manchester, United Kingdom, 3 University Hospital of South Manchester, Genesis Breast Cancer Prevention Centre and Nightingale Breast Screening Centre, Manchester, United Kingdom, 4 University of Cambridge, Cambridge Breast Unit, Cambridge, United Kingdom, 5 Queen Mary University of London, Centre for Cancer Prevention- Wolfson Institute, London, United Kingdom, 6 The Christie NHS Foundation Trust, Breast Oncology, Manchester, United Kingdom, 7 Central Manchester Foundation Trust, Genomic Medicine, Manchester, United Kingdom Objectives: This study compares several methods of mammographic density estimation to determine which best predicts women subsequently diagnosed with breast cancer and which is most strongly associated with cancer in the contralateral breast at the time of screening. Such comparisons are important to enable selection of appropriate strategies for stratifying women in risk adapted breast screening programmes. Materials and Methods: Women participating in a study of cancer risk prediction provided personal information for computation of the Tyrer-Cuzick risk score; mammographic density was measured with Quantra and Volpara and assessed visually by two independent readers using visual analogue scales (VAS). In two case–control studies, cancer cases were each matched to three cancer-free controls. Density was assessed in the contralateral breasts

of 366 cases and matched controls, with a subset of 311 cases and their controls assessed by a trained reader using Cumulus. Density estimates were also obtained for 338 women with cancer detected at a later screening round or during the interval between screening rounds, and matched controls. Analysis was performed using conditional logistic regression. Results: Visually assessed density and % density by Volpara were both significantly associated with screen-detected cancer in the contralateral breast, and showed a dose response relationship with increasing density (c2 trend 35.6, p < 0.001 and 11.2, p < 0.001 respectively). The strongest association with cancer was found for VAS, with an odds ratio (OR) of 4.64 (95% CI 2.84– 7.56) in the highest quintile of density compared with the lowest. Volpara % density gave an OR for the highest quintile of 2.96 (95% CI 1.78–4.93). Density measured by Cumulus was also significantly associated with cancer, but to a lesser extent, and for Quantra no significant association was found. The strongest association with the future development or detection of cancer was found for VAS, with an OR of 4.85 (95% CI 3.00–7.83) in the highest quintile of density compared with the lowest. Volpara % gave an OR for the highest quintile of 4.04 (95% CI 2.33–7.01). For Quantra, those with % density in the highest quintile had an OR of 1.52 (95% CI 1.04–2.23) when adjusted for breast volume and parity. Conclusions: The results show that visual assessment of breast density, recorded on VAS and averaged between two mammographic readers, demonstrates the strongest relationship with cancer. This is the case for mammograms both before the detection of cancer and in the opposite breast at the time of detection. Percentage density measured by Volpara provided the strongest relationship amongst the automated measures, and provides a practical method for risk stratification. No conflict of interest.

Monday 11 July 2016

10:45–12:30

BACR Supported Symposium

Invasion and Metastasis 14 Proffered Paper: Cell density, Her2 and progesterone signaling regulate dissemination of breast cancer cells H. Hosseini1 , K. Harper2 , M. Obradovic1 , M. Soledad Sosa2 , L.K. Nanduri1 , M. Hoffmann3 , C. Werno3 , J.A. Aguirre-Ghiso2 , C.A. Klein1 . 1 University of Regensburg, Experimental Medicine and Therapy Research, Regensburg, Germany, 2 Mount Sinai School of Medicine, Department of Oncological Sciences, New York, USA, 3 Fraunhofer Institute for Toxicology und Experimental Medicine, Project group “Personalized Tumor Therapy”-, Regensburg, Germany Introduction: Current cancer therapies generally assume cancers to progress linearly from primary to metastatic sites. The model postulates a stepwise accumulation of genetic and epigenetic alterations within the primary tumor, and therefore, primary tumors may be used as surrogate markers for disseminated cancer cells. However, this model is challenged by (i) the insufficient therapy success and (ii) the genomic disparity between disseminated cancer cells (DCCs) and their matched primary tumors when isolated at surgery. The genomic disparity suggests that metastatic dissemination may occur early during tumor formation, however the mechanisms of early metastatic spread are unknown so far. Material and Method: To uncover mechanisms of early metastatic dissemination we microdissected mammary tissue from a Her2-driven mouse model (Balb-NeuT) of breast cancer before and after microscopic invasion and performed gene expression analysis. Identified pathways were tested in a series of in vitro and in vivo experiments. Finally, the findings were validated in a panel of human breast cancer cell lines and investigation of more than 2000 patient samples. Results and Discussion: Gene expression profiling identified a distinct signature at the time point of maximal seeding when only pre-invasive mammary lesions could be identified. The morphology of pre-invasive lesions coincided with a transient time window of progesterone receptor (PgR) and Her2 co-expression. Upon functional testing progesterone-induced signaling triggered migration of stem-like cells from early lesions shortly after Her2 activation, but promoted proliferation in advanced primary tumor cells. The switch from migration to proliferation was regulated by elevated Her2 expression and increased cell density involving miRNA-regulated PgR down-regulation. The combined result was a differential response of cancer cells to progesterone and its paracrine-signal mediators Wnt4 and Rankl imposing stemness and migration at low and proliferation at high cellular density. Exploring human breast cancer cell lines we confirmed the observed phenotypes and mechanisms. Molecular-genetic and transcriptomic analysis of DCCs isolated from the bone marrow of breast cancer patients provided strong support for early metastatic dissemination. Finally, we identified a subtype of breast cancer mimicking the Balb-NeuT model closest.

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EACR24 Oral Presentations, Monday 11 July 2016 / European Journal of Cancer 61, Suppl. 1 (2016) S5–S8 Conclusion: We identify microenvironmental signaling (PgR signaling), oncogenic signaling (Her2) and cellular density as central regulators of early dissemination and metastasis. In light of the patient-derived data we suggest that (i) dissemination is mostly early and (ii) clones that evolved over time at the primary site and became pre-dominant are less able to disseminate. We predict the findings not only to be relevant for Her2-driven cancers but for many other cancers as well. No conflict of interest.

Monday 11 July 2016

10:45–12:30

Symposium

Tumour Suppressors 15 Proffered Paper: In young women with atypical hyperplasia, high ERb expression in background breast lobules correlates with decreased risk of future breast cancer T. Hieken1 , J. Carter2 , J. Hawse3 , T. Hoskin4 , M. Bois2 , M. Frost5 , L. Hartmann5 , D. Radisky6 , D. Visscher2 , A. Degnim1 . 1 Mayo Clinic, Surgery, Rochester- Minnesota, USA, 2 Mayo Clinic, Laboratory Medicine and Pathology, Rochester- Minnesota, USA, 3 Mayo Clinic, Biochemistry and Molecular Biology, Rochester- Minnesota, USA, 4 Mayo Clinic, Health Sciences Research, Rochester- Minnesota, USA, 5 Mayo Clinic, Oncology, Rochester- Minnesota, USA, 6 Mayo Clinic, Biochemistry and Molecular Biology, Jacksonville- Florida, USA Background: Estrogen receptor beta (ERb), a putative tumor suppressor, is highly expressed in normal breast epithelia. ERb is distinct from ERa and shows promise as a prognostic biomarker and target for anticancer therapy. Although ERb expression is lost in ~60% of breast cancers (BCs), its expression in the remaining 40% of cases is associated with enhanced therapeutic response and improved survival. However, its role as an inhibitor of carcinogenesis remains unknown. Atypical hyperplasia increases future BC risk 4-fold but further risk stratification is needed to improve patient care. Thus we investigated ERb expression as a potential biomarker in these high risk women. Methods: We examined ERb expression by immunohistochemistry in breast tissues from a cohort of 171 women with atypical hyperplasia diagnosed from 1967 to 1991. Nuclear ERb percent staining and intensity were scored in the atypia and background normal lobules. A calculated sum score (percent + intensity) was used to categorize ERb expression as low, moderate or high. Competing risks regression was used to assess associations of ERb expression with BC risk. Results: After 15 years median follow-up, 36 women developed BC. ERb expression was lower in the atypia lobules than in background lobules, by percent staining and intensity (both p < 0.001). Higher ERb expression in the atypia or background lobules decreased the risk of subsequent BC by 2 (p = 0.04) and 2.5-fold (p = 0.006) independent of other risk biomarkers. To explore the hypothesis that ERb may be more effective in an “estrogenrich” environment, we examined the association within age groups of premenopause (age 55). Interestingly, we observed a dramatic increase in BC risk in women < age 45 with low-moderate ERb expression compared to high ERb expression in background lobules (HR 15.2, 95% CI: 1.8 to >100), vs HR 1.5 (95% CI: 0.6−3.8) in women age 45−55 and HR 2.3 (95% CI: 0.8−7.4) for age >55. The 20-year cumulative BC incidence was 0% (0/15) for women < age 45 at biopsy with high ERb in background lobules vs 40% (6/15) for women < age 45 with low-moderate ERb expression (p = 0.0008). Conclusion: These data suggest ERb may be a useful BC risk biomarker and potential target for pharmacologic risk reduction. High background lobule ERb expression conferred the strongest protective effect in pre-menopausal women indicating that the tumor suppressive effects of ERb may be conferred through paracrine signaling, effects that are more robust in an estrogen-rich environment. Further investigation of the functions of ERb is warranted to understand the mechanisms by which it suppresses BC development in benign breast disease. No conflict of interest.

Monday 11 July 2016

14:00–15:45

Symposium

Novel Targeted Therapies 16 Proffered Paper: Targeting PA2G4, a novel MYCN co-factor, for the treatment of neuroblastoma J. Koach1 , J.E. Murray1 , J. McCarroll1 , G. Milazzo2 , G. Perini2 , M. Haber1 , M.D. Norris1 , J.I. Fletcher1 , B.B. Cheung1 , G.M. Marshall3 . 1 Children’s Cancer Institute of Australia for Medical Research, Lowy Cancer Research Centre, Sydney, Australia, 2 University of Bologna, Department of Pharmacy and Biotechnology, Bologna, Italy, 3 Kids Cancer Centre, Sydney Children’s Hospital, Sydney, Australia Introduction: MYCN oncogene amplification is found in one third of primary neuroblastoma at diagnosis, and correlates with poor prognosis. We have identified Proliferation-associated protein 2G4 (PA2G4) as a direct binding partner for MYCN oncoprotein. PA2G4 belongs to a family of DNA/RNA binding proteins already implicated in cell growth, apoptosis and differentiation. The long isoform of PA2G4 (p48) has a known oncogenic function, whereas the short isoform (p42) acts as a tumour suppressor. However, the role of PA2G4 in neuroblastoma and its link with MYCN is currently unknown. Material and Method: Using a panel of human neuroblastoma cell lines and 477 patient tumour samples, we analysed the expression of PA2G4 by real-time PCR and Western blotting. Co-immunoprecipitation and chromatin immunoprecipitation were performed to assess the interaction between MYCN and PA2G4. Protein half-life was measured by cycloheximide chase assays. PA2G4 and MYCN expression were suppressed using siRNAs specifically targeting these genes. PA2G4 overexpressing cells were xenograft into nude mice to assess tumorigenicity while the TH-MYCN mouse model was used to assess the effectiveness of a PA2G4 inhibitor, WS6. Nanoparticles were used to deliver siRNA targeting PA2G4 intratumorally into xenograft mice. Results and Discussion: For the first time, we have identified ProliferationAssociated protein 2G4 (PA2G4) as a direct protein-binding partner of MYCN which acts in a forward feedback expression loop with MYCN to drive neuroblastoma tumorigenesis. We found high expression of PA2G4 was a strong independent clinical predictor of poor survival and positively correlated with MYCN expression. Suppression of PA2G4 by siRNAs decreases neuroblastoma cell growth, cell migration and colony formation. Most significantly, our in vivo data showed stable overexpression of PA2G4 in a non-tumorigenic neuroblastoma cell line was able to induce tumour growth. Conversely, using nanoparticles to deliver siRNA targeting PA2G4, we were able to delay neuroblastoma tumour growth significantly in a mouse xenograft model. Furthermore, a small molecule known to bind PA2G4, WS6, significantly decreased PA2G4 levels in tumour tissue and tumorigenicity in TH-MYCN mice. Conclusion: Collectively, our data suggest that PA2G4 acts as an oncogenic co-factor, binding and protecting MYCN from proteolysis, thus increasing MYCN levels. Our research provides strong evidence demonstrating PA2G4 is a driver of tumorigenicity and for the first time identifies PA2G4 as a novel therapeutic target for the treatment of neuroblastoma. No conflict of interest.

Monday 11 July 2016

14:00–15:45

Symposium

Inflammation and Cancer 17 Proffered Paper: Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy J. Incio1 , P. Suboj2 , S. Chin1 , H. Liu1 , R. Soares3 , Y. Boucher1 , D. Fukumura1 , R. Jain1 . 1 Massachusetts General Hospital, Radiation Oncology, BostonMassachusetts, USA, 2 MGH, Radiation Oncology, Boston, USA, 3 Porto Medical School, Biochemistry, Porto, Portugal Background: With the current epidemic of obesity, the majority of pancreatic cancer patients are overweight or obese at diagnosis. Importantly, obesity worsens treatment outcomes in pancreatic cancer patients. Therefore, understanding the mechanisms that underlie the poorer prognosis of obese cancer patients is of paramount importance. Obesity causes inflammation and fibrosis in the normal pancreas due to the accumulation of dysfunctional hypertrophic adipocytes. Importantly, desmoplasia − a fibroinflammatory microenvironment − is a hallmark of pancreatic ductal adenocarcinoma (PDAC), and we have shown that activation of pancreatic stellate cells (PSCs) via angiotensin-II type 1 receptor (AT1) pathway is a major contribution to tumor

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desmoplasia. Whether obesity affects desmoplasia in PDACs, and interferes with delivery and response of chemotherapeutics is currently unknown. Material and Methods: Using both human samples and mouse models of PDAC − multiple syngeneic models of PDAC: PAN02, AK4.4, KPC, iKRAS in diet-induced and genetic obese mouse models −, we determined the effects of obesity on desmoplasia and inflammation/immune cell infiltration, tumor growth and delivery and response to chemotherapy. Results: We found that obesity aggravates desmoplasia in PDACs in both patient samples and multiple mouse models. In addition, tumors in obese mice presented with elevated levels of activated PSCs and fibrosis, as well as inflammatory cytokines and TANs. These alterations in the tumor microenvironment in obesity associated with accelerated tumor growth, reduced tumor blood perfusion and increased hypoxia, and impaired delivery and efficacy of chemotherapeutics. Genetic ablation and pharmacological inhibition (losartan) of AT1 signaling reversed obesity-augmented desmoplasia and tumor growth, and improved the response to chemotherapy to the level observed in lean mice. We further discovered the underlying mechanisms: (1) obesity increases intra-tumor adipocytes and IL-1b secretion by these cells; (2) increased IL-1b induces TAN recruitment; (3) recruited TANs activate PSCs; and (4) activated PSCs enhance desmoplasia. Conversely, activated PSCs also secrete IL-1b that recruits further TANs. Of clinical relevance, we found that metformin not only normalizes the abnormal systemic metabolism, but also reprogramms PSCs and immune cells and alleviates the fibroinflammatory microenvironment in pancreatic cancer in obesity/diabetes. Importantly, the strategies described above were not effective in the normal weight setting. Conclusions: Here we successfully demonstrated that targeting desmoplasia, including immunomodulation with anti-IL-1b, or treatment with generic drugs such as losartan and metformin are potential strategies to potentiate treatments in PDAC patients with excess weight. Conflict of interest: Ownership: RJ: Enlight, Ophthotech, SynDevRx, and XTuit. Advisory Board: RJ: Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund and Tekla World Healthcare Fund. Board of Directors: RJ: XTuit. Corporate-sponsored Research: No reagents or funding from these companies was used in these studies.

Monday 11 July 2016

14:00–15:45

Symposium

Breakthrough Cancer Models 18 Proffered Paper: Establishment of resectable transgenic mouse models of pancreatic ductal adenocarcinoma for investigations on adjuvant therapies E. Guerlevik1 , B. Fleischmann-Mundt1 , J. Brooks1 , N. Woller1 , M. Manns1 , S. Kubicka1 , F. Kuehnel1 . 1 Hannover Medical School, GastroenterologyHepatology- and Endocrinology, Hannover, Germany Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor that disseminates at early stages of carcinogenesis. Despite surgical resection and adjuvant chemotherapy prognosis of patients is still dismal. However, animal models of PDAC with a resectable primary tumor and early metastasis for preclinical investigations on adjuvant therapies are still missing. Material and Methods: To allow for surgical resection, a single primary PDAC nodule was induced in mice by intrapancreatic injection of oncogenic plasmids followed by local electroporation. Reflecting genetic alterations in human PDAC, a basic genetic setup with KRasG12 mutations and deletion of p53 was applied, using either sleeping beauty-based transposons or transgenic mice. Furthermore, additional delivery of a transposon for activated Akt2 was investigated since we found Akt2 frequently activated in human PDACs. Tumor growth was monitored and histologically characterized. After tumor formation, the primary tumor was removed by surgery and adjuvant therapies such as gemcitabine were investigated by analysis of survival and recurrence patterns. Results: Local induction of KRasG12 mutations and p53 deletion resulted in development of a single PDAC. Fluorescence tracking and cell-type-specific stainings as well as electron microscopy after electroporation suggested that these PDACs most likely transdifferentiate from transduced acinar cells. Precancerous lesions such as PanINs could be observed. Additional activation of Akt2 resulted in early metastasis as visualized by bioluminescence and confirmed by survival analysis after tumor resection at different time points. The resulting PDACs showed aggressive tissue infiltration, neural invasion and widespread dissemination reflecting the clinical characteristics of PDAC in humans. Recapitulating the findings of the phase III clinical trial (CONKO-001) and thus confirming the preclinical value of our model, we found that adjuvant gemcitabine resulted in improved survival, due to a predominant effect on local recurrence whereas distant metastasis was less affected. Interestingly, we showed that adjuvant gemcitabine inhibited MDSC subsets and enhanced

local NK cell infiltration. Depletion of NK cells, but not of T-cells abrogated therapeutic benefit thus confirming that the innate immune response plays an important role in preventing disease recurrence after surgery and adjuvant gemcitabine. Conclusions: We established a novel murine model of resectable PDAC that reflects histopathological features and clinical characteristic of human PDAC including important observations in clinical trials of adjuvant chemotherapy. Furthermore, we found evidence that innate immune responses play a role after adjuvant gemcitabine suggesting additional NK-cell activation as an attractive therapeutic strategy after tumor resection. No conflict of interest.




Poster Sessions (Sunday 10 July and Monday 11 July 2016) Sunday 10 July 2016 Poster Session

Cancer Genomics, Epigenetics and Genome Instability I 100 Understanding estrogen receptor transcription in breast cancer J. Carroll1 . 1 University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom Background: Estrogen Receptor (ER) is the defining feature of luminal breast cancers, where it functions as a transcription factor. ER requires associated proteins to interact with the DNA, including the pioneer factor FoxA1 and GATA3, both of which mediate where in the genome ER resides. In the absence of FoxA1, ER binding and transcriptional activity is diminished, even in endocrine resistant contexts. The loss of GATA3 results in a complex redistribution of ER binding, but similar to FoxA1, cell proliferation is reduced when GATA3 is inhibited. Both GATA3 and FoxA1 are mutated in breast cancer and we have explored the functional consequences of these mutations on ER activity and assessed whether they contribute to endocrine resistance. Materials and Methods: We performed genomic and proteomic analyses to characterise the role of GATA3 and FoxA1 in ER+ breast cancer. We conducted chemical library screens to identify FoxA1 inhibitors. Results: We are interested in regulating FoxA1 and GATA3, with the goal of indirectly blocking ER activity, even in drug resistant patients. To this end, we have initiated screens to identify and characterize upstream regulatory kinases that modulate FoxA1 and GATA3 function. In addition, we have screened chemical libraries to identify specific inhibitors of FoxA1. In parallel, we have sought to discover novel ER associated proteins that are involved in endocrine resistance and to achieve this, we have established a method for rapid unbiased discovery of protein interacting complexes, which we have applied to discover ER and FoxA1 associated proteins. We find an unexpected interaction between ER and progesterone receptor in ER+ breast cancer. We show that PR is a negative regulator of the ER complex, where it is important for modulating cellular growth. Conclusions: We reveal new insight into how GATA3 and FoxA1 influence ER activity and we identify a novel inhibitor that blocks FoxA1 activity. We also reveal new insight into cross-talk between parallel nuclear receptor pathways. These findings help delineate the complexes that influence ER transcriptional activity and ultimately impinge on tumor progression and drug sensitivity. No conflict of interest. 101 A distinct epigenetic state sensitizes enzalutamide-resistant prostate cancer cells to BET bromodomain inhibition N. Shah1 , V.K. Arora2 , W. Karthaus1 , J. Wongvipat1 , D. Zheng3 , C. Sawyers1,4 . 1 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA, 2 Division of Medical Oncology, Washington University School of Medicine, St. Louis, MO, USA, 3 Departments of Neurology, Genetics and Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA, 4 Howard Hughes Medical Institute, Chevy Chase, MD, USA Introduction: We have previously shown that the glucocorticoid receptor (GR) is an important mediator of enzalutamide (Enz) resistance in a pre-clinical model of prostate cancer. GR is necessary for resistance, and is able to bypass the androgen receptor (AR) blockade by binding to and driving the expression of a subset of AR target genes. However, the mechanism by which GR expression is induced and maintained in these resistant cells is unknown. Here, we propose an epigenetic basis by which GR is able to be induced upon AR-inhibition in prostate cancer cells.

Results and Discussion: As previously described, LREX’ resistant cells (GRhigh) and LNAR’ sensitive cells (GR-low) were derived from an in vivo mouse xenograft model. LREX’ resistant cells have a distinct loss of the repressive H3K27me3 mark around the GR locus, thus allowing for robust GR expression upon AR-inhibition. This epigenetic regulation of GR expression is also seen in prostate organoid cultures, and primary prostate tissues. Furthermore, we describe a prostate-tissue specific enhancer that is important in regulating GR expression in this model. Using BET bromodomain inhibitors, we are able to target GR expression via this putative enhancer, and re-sensitize these resistant tumours to Enz. Conclusion: We propose an epigenetic basis for resistance to Enz which allows for robust GR expression, and a subsequent bypass of the AR blockade. These resistant tumours can be re-sensitized to Enz by treatment with a BET bromodomain inhibitor, which effectively inhibits GR expression by targeting a tissue-specific GR enhancer. No conflict of interest. 102 Comprehensive sequencing-based characterisation of the DNA methylation landscape of 1300 breast tumours R.N. Batra1 , A.T. Vidakovic1 , S.F. Chin1 , H. Clifford1 , M. Callari1 , A. Bruna1 , A.S. Batra1 , S.J. Sammut1 , O.M. Rueda1 , C. Carlos1 . 1 University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom Introduction: Breast cancer is one of the leading causes of cancer death in women, and is unanimously considered a heterogeneous disease displaying distinct therapeutic responses and outcomes. While recent advances have led to the integration of the genomic and transcriptomic architecture of breast cancers to refine the molecular classification of the disease, the epigenetic landscape has received less attention. We are conducting a large Next-generation sequencing-based breast cancer methylome study in order to provide a comprehensive investigation of the DNA methylation landscape of breast cancer. Materials and Methods: Reduced Representation Bisulfite Sequencing (RRBS) was performed on 1300 primary breast tumours (and 300 matched normal tissue samples) from the METABRIC cohort. For this purpose, a robust RRBS pipeline was designed that is not only suitable for high-throughput, but also maximises the information content yield. Statistical methods accounting for spatial correlation of neighbouring CpG sites were used to identify differentially methylated regions (DMRs) between tumours and normals, as well as between different tumour subtypes. A similar pipeline was also implemented for a panel of patient-derived tumour xenografts (PDTXs). Results and Discussion: We identified hyper and hypo DMRs between tumours and normals in different genomics features (such as gene promoters and enhancers) that illuminate the regulatory role of methylation alterations in tumorigenesis. We also determined that DNA methylation contributes to breast cancer heterogeneity by identifying DMRs between Immunohistochemicalbased subtypes as well as the more recently established molecular Integrative Clusters (IntClust subtypes). In addition, gene expression was used to functionally characterise the DMRs in the IntClust subtypes, that led to the identification of subtype-specific candidate targets in breast cancer. Next, the role of DNA methylation was explored in the presence and absence of copy number aberrations (deletions and gains/ amplifications) as well as mutations (activating and inactivating). Our findings revealed complementary epigenetic and genomic aberration patterns associated with transcription across breast cancer patients. Finally, I discuss the investigation of DNA methylation markers using RRBS in a panel of PDTXs, that constitute one of the best pre-clinical models available today and are able to recapitulate inter and intra-tumour heterogeneity observed in patients. Conclusions: We present compelling evidence that methylation alterations can impact tumorigenesis and provide further insights into breast cancer subtype differentiation. No conflict of interest.


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103 Functional genomic screening identifies USP11 as a novel therapeutic target in breast cancer

106 High expressed miR-183 as a potential biomarker of poor survival in tongue cancer patients

L. Dwane1 , A. O’Connor2 , L. Mulrane2 , R. Klinger1 , A. Dirac3 , K. Jirstrom4 , J. Crown5 , R. Bernards3 , W. Gallagher2 , D. O’Connor1 . 1 Royal College of Surgeons in Ireland, Molecular and Cellular Therapeutics, Dublin, Ireland, 2 UCD Conway Institute, School of Biomolecular and Biomedical Sciences, Dublin, Ireland, 3 Netherlands Cancer Institute, Division of Molecular Carcinogenesis, Amsterdam, Netherlands, 4 Malmo¨ University Hospital, ¨ Sweden, 5 St Vincent’s University Department of Laboratory Medicine, Malmo, Hospital, Breast Clinic, Dublin, Ireland

K. Zeljic1 , G. Supic2 , A. Divac Rankov3 , A. Nikolic3 , R. Kozomara4 , N. Jovic4 , D. Radojkovic3 , Z. Magic2 . 1 University of Belgrade- Faculty of Biology, Chair for Genetics and Evolution, Belgrade, Serbia, 2 Military Medical Academy, Institute for Medical Rresearch, Belgrade, Serbia, 3 University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia, 4 Military Medical Academy, Clinic for Maxillofacial Surgery, Belgrade, Serbia

Background: Approximately 70% of breast cancers overexpress the estrogen receptor a (ERa) and depend on this key transcriptional regulator for growth and differentiation. The discovery of novel mechanisms controlling ERa function represent major advances in our understanding of breast cancer progression and potentially offer attractive new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating transcriptional activity of ERa in breast cancer. Materials and Methods: We performed an RNAi loss-of-function screen using an arrayed library of 432 shRNA vectors (4 shRNAs per gene) targeting all 108 known or putative human DUB genes. We found that suppression of the BRCA2-associated DUB, ubiquitin-specific protease 11 (USP11), repressed the activity of an estrogen-response-element (ERE). This was subsequently validated using five individual shRNAs to stably knockdown USP11 in the ZR\erhyphen;75-1 breast cancer cell line. Expression of ERa target genes was quantified by qRT-PCR, while growth assays and Western blot analysis were used to determine changes in cell growth and survival. Immunoprecipitation techniques were used to determine an interaction between USP11 and the receptor. To determine the prognostic relevance of USP11, immunohistochemical staining of a 144-patient breast cancer tissue microarray (TMA) was performed. Results: Reduced expression of endogenous ERa target genes was observed in USP11 knockdown cell lines. Preliminary growth assays revealed a global decrease in cell survival and increased sensitivity to DNA-damaging agents following USP11 suppression. USP11 knockdown had no effect on the stability of ERa, suggesting that the enzyme does not regulate ERa turnover and may regulate the receptor either indirectly or by deubiquitination of a nondegradative ubiquitin chain. Immunoprecipitation also revealed a physical interaction between USP11 and ERa. TMA staining revealed a significant relationship between high USP11 expression and poor prognosis in ER+ patients (p = 0.03). This was further validated in silico by analysis of publically available breast cancer gene expression datasets, consisting of 639 ER+ patients (HR 1.39, CI 1.1–1.76, p = 0.006). Conclusion: This data suggests a role for USP11 in driving cellular growth and identifies USP11 as novel therapeutic target in breast cancer. No conflict of interest. 104 Frameshift mutations of rapamycin pathway genes and their regional heterogeneity in sporadic colorectal cancers C. An1 , J.I. Lee1 , N.J. Yoo2 , S.H. Lee2 . 1 Uijongbu St. Mary’s hospital, surgery, Uijongbu City, Korea, 2 The Catholic University of Korea, Pathology, Seoul, Korea Introduction: Mammalian target of rapamycin (mTOR) pathway is known to be involved in cancer pathogenesis. The aim of our study was to find whether mTOR-related genes were mutated and expressionally altered in colorectal cancers (CRCs). Through public database searching, we found that PIK3CB, IRS1, RPS6, EIF4B, RPS6KA5 and PRKAA2 that were known as mTORrelated genes possessed mononucleotide repeats in DNA coding sequences that could be mutated in cancers with microsatellite instability (MSI). Material and Method: We analyzed 124 CRCs by single-strand conformation polymorphism analysis and DNA sequencing. In addition, we analyzed intratumoral heterogeneity (ITH) of PIK3CB, IRS1, RPS6, EIF4B, RPS6KA5 and PRKAA2 frameshift mutations in 16 CRCs. We also analyzed IRS1 expression in the CRCs by immunohistochemistry. Results and Discussion: There were seven (8.9%), eight (10.1%) and three (3.8%) of 79 CRCs with high MSI (MSI-H) harbored IRS1, EIF4B and RPS6KA5 frameshift mutations, respectively. These mutations were not identified in stable MSI/low MSI (MSS/MSI-L) (0/45). We also found that IRS1, EIF4B and RPS6KA5 mutations had regional ITH in two, two and one CRCs, respectively. Loss of IRS1 expression was identified in 31% of the CRCs. The loss of expression was more common in those with IRS1 mutation than those with wild-type IRS1. Conclusion: Our data indicate that mTOR-related genes harbored not only somatic mutations but also mutational ITH and loss of expression, which together might play a role in tumorigenesis of CRC, especially with MSI-H. Our data also suggest that mutation analysis in multi-regional areas is needed for a precise evaluation of mutation status in CRC with MSI-H. No conflict of interest.

Introduction: Tongue cancer, a subset of oral carcinomas, presents one of the most aggressive types of head and neck cancer. Mortality rate due to oral cancer is remaining high despite major efforts in improved treatment and therapy. Thus, identification of novel and accurate biomarkers with prognostic and predictive value is warranted. Micro RNAs (miRs) are class of small non-coding RNA molecules, negative regulators of gene expression, often deregulated in cancer. Deregulation of miR-183 expression was reported in various types of solid tumors. The aim of the current study was to determine expression level of miR-183 in tongue cancer patients and to explore potential association of miR-183 gene expression with clinicopathological features and survival. Material and Method: The study group has included 60 patients diagnosed with tongue cancer (age median 58, median follow-up time 36 months). The relative expression level of mature miR-183 was quantified using commercially available TaqMan based RT-qPCR assays. RNU6 was used as an endogenous control. All reactions were performed in triplicate, and relative expression levels were determined with the DDCt method. The expression level of miR-183 gene was dichotomized as high or low, with a 2.7-fold change of normalized miR-183 expression used as the cut-off value, according to ROC (Receiver Operating Characteristic) curve analysis. Kaplan–Meier survival curves were compared by the log-rank test. All p values less than 0.05 were considered as significant. Results: Over-expression of miR-183 was observed in 36 (60%) tongue cancer patients. There was no association between miR-183 expression level and clinicopathological characteristics, except noticed statistical trend for association with tumor stage (p = 0.068). Over-expression of miR-183 was significantly associated with worse overall survival in tongue cancer patients (p = 0.019). Conclusion: Our results suggest potential use of miR-183 expression level as a molecular biomarker of survival in tongue cancer patients. No conflict of interest.

107 p53-associated microRNAs and MDM2 expression in the biology and prognosis of neuroblastoma M. Inomistova1 , N. Khranovska1 , O. Skachkova1 , G. Klymnyuk2 , N. Svergun1 , N. Adamchuk3 . 1 National Cancer Institute, Laboratory of Experimental Oncology, Kiev, Ukraine, 2 National Cancer Institute, Department of Pediatric Oncology, Kiev, Ukraine, 3 Taras Shevchenko Natioanal University of Kiev, SRC “Institute of Biology”, Kiev, Ukraine Background: Neuroblastoma (NB) is TP53-wild type malignant childhood tumor. In addition to MDM2, many genetic and epigenetic regulators of p53 activity and its signaling pathway are known. Currently changes in p53 pathway and its deregulation arising in NB are not well known. We have investigated changes of p53, MDM2 genes and miRNA miR-34 a/b/c, miR-137, miR380-5p, miR-885-5p expression in NB and their impact in NB progression and survival. Material and Methods: We have analyzed tumor tissue samples of 61 patients with NB. Nucleic acids extraction was performed with NucleoSpin MiRNA, Machery-Nagel (Germany). MDM2 and p53 expression levels (EL) were detected with real-time PCR using TaqMan primers and probes and microRNAs with TaqMan MicroRNA Assay, Applied Biosystems (USA). Results were normalized to relevant controls. FISH method was used for MYCN amplification (MNA) detection. Results: Higher MDM2 EL correlated with unfavorable clinical NB features (MNA, p < 0.03; disease stage, p < 0.001), indicating a link between MDM2 overexpression and the high-risk phenotype. MDM2 overexpression was associated with a significant decrease in event-free survival of NB patients (F-Cox criterion: 3.22, p < 0.001). We have established that miR-34 a/b/c and miR-380-5p don’t have significant independent effect on the clinical behavior of NB, whereas low expression of miR-885-5p and miR-137 EL is associated with unfavorable disease course. Thus, low miR-885-5p and miR-137 EL in NB tumor cells correlated with MNA (p < 0.05 and p < 0.001), MDM2 overexpression (p < 0.02 and p < 0.001) and was significantly more common in patients with advanced NB stages (p < 0.04 and p < 0.04). In addition, low miR-137 expression was associated with chemotherapy resistance (p < 0.05). We have established significant decrease of event-free survival rates of patients with low miR-885-5p and miR-137 expression (F-Cox criterion: 3.54, p < 0.001 and F-Cox criterion: 2.89, p < 0.02).

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Conclusions: Deregulation of 53-mediated pathway due to gene alterations of its regulators and microRNAs expression is associated with unfavorable outcome in NB. Changes in aforementioned genes and microRNAs expression can be recommended as auxiliary predictive markers in NB. No conflict of interest. 108 Endogenous Ago2 PAR-CLIP reveals novel target genes of deregulated miRNAs in DLBCL M. Fernandez-Mercado1 , E. Larrea1 , I. Goicoechea1 , I. Ceberio2 , M. Landthaler3,4 , M. Arauzo-Bravo5,6 , C.H. Lawrie1,6,7 . 1 Molecular Oncology, Biodonostia Research Institute, San Sebastian, Spain, 2 Department of Haematology, Hospital Universitario Donostia, San Sebastian, Spain, 3 RNA Biology and Posttranscriptional Regulation, Max-Delbruck ¨ Center for Molecular Medicine, Berlin, Germany, 4 Chemistry and Biochemistry Institute, 5 ¨ Berlin, Berlin, Germany, Computational Biology and Freie Universitat Systems Biomedicine, Biodonostia Research Institute, San Sebastian, Spain, 6 IKERBASQUE, Basque Foundation for Science, Bilbao, Spain, 7 Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, United Kingdom Background: Aberrant expression of microRNAs (miRNAs) is a widespread phenomenon in cancer. However, the functional significance of such deregulation is poorly understood as the target genes (targetome) of miRNAs are notoriously difficult to predict computationally and moreover differ according to cellular context. An alternative approach is to directly sample the targetome in situ using immunoprecipitation (IP) techniques such as PARCLIP. The drawback however of such techniques is the need for exogenously produced tagged proteins. We decided to adapt this technique to allow nonengineered cells to be used based on IP of endogenous levels of Ago2. Furthermore, to reduce the number of false positives we compared the targetome of cells over-expressing a specific miRNA with those containing an inhibitor of that miRNA. We used diffuse large B-cell lymphoma (DLCBL) and miR-155 as our model as the most common form of lymphoma which typically aberrantly expresses this miRNA. Material and Methods: Two DLBCL cell lines ABC-type (Riva) and GC-type (SU-DHL10) were transfected with lentiviral vectors that encoded miR-155, or an inhibitor of miR-155, or a scrambled sequence. Cells were then stably selected with puromycin, and grown in the presence of 100 mM 4SU for 18 h. These cells were then irradiated to cross-link the RNA to RNA-binding proteins. PAR-CLIP was then performed on cell lysates using anti-Ago2 mAbs for IP. The original protocol by Hafner et al. was modified to avoid radioactive labelling. The recovered RNA was used for library building using TruSeq Small RNA Sample Kit. Sequencing was performed on a Illumina HiScanSQ system. Sequence reads were aligned to the human genome (GRCh37). After variant calling, T-to-C variants were matched to annotated genomic regions using Ensembl’s VEP. Results: Endogenous Ago2 IP, followed by a radioactive-free modified PARCLIP protocol yielded sufficient RNA for building NGS libraries. Samples gave an average 33×106 aligned reads/library. There were an average of 4480 T-to-C variants in UTRs or CDSs corresponding to 1416 (SU-DHL10) or 1702 (RIVA) genes. A number of these genes corresponded to experimentally validated target genes of miR-155. Ontogeny analysis demonstrated an enrichment of genes involved in hematopoietic and/or lymphomagenesis pathways. Conclusions: To fully understand the role of a particular miRNA in a specific cancer, it is essential to identify its target genes in a relevant cellular context. We have developed an optimised method for interrogating the miRNA:mRNA (targetome) interface within a cellular system without the need of ectopically express tagged Ago2, keeping physiological levels of the core component of the RISC complex unaffected. Giving our success we are presently working towards reducing the number of input cells, in view of interrogating the targetome of patient primary samples. No conflict of interest. 109 Investigating the impact of telomere dysfunction on the cancer genome L. Escudero1 , C. Fegan1 , C. Pepper1 , K. Ashelford1 , K. Norris1 , K. Cleal1 , K. Liddiard1 , D.M. Baird1 . 1 Cardiff University, Division of Cancer and Genetics, Cardiff, United Kingdom Introduction: Telomeres are structures that cap the ends of linear chromosomes and prevent them from being recognised as double strand DNA breaks (DSBs). Short dysfunctional telomeres can result in chromosome ends that are prone to fusion with each other or non-telomeric DSBs. In this project, we aim to understand the role that telomere dysfunction plays in driving the evolution of the cancer genome. Focusing on Chronic Lymphocytic Leukaemia (CLL), we will characterise genomic loci that fuse with dysfunctional telomeres in patient lymphocytes. Translocations involving hTERT, one of the most distal genes on 5p, have previously been detected in CLL and may impact on telomerase activity and disease progression. We will examine whether

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dysfunction at the 5p telomere could lead to chromosomal instability that affects telomerase expression. Materials and Methods: We have adapted STELA (Single TElomere Length Analysis) and a telomere fusion assay, high-resolution single-molecule approaches, to precisely measure telomere length (TL) and to detect unique fusion events at the 5p telomere on patient CLL-B cells. Moreover we will be using paired-end NGS of telomere fusion amplicons, as well as WGS for a biclonal CLL patient detected with STELA. Results and Discussion: Our data are consistent with the 5p telomere displaying similar TL profiles to other unique telomeres, including 17p and XpYp. We also identified rare telomere fusions involving 5p in 47 out of 279 CLL patient samples. To investigate further telomere-driven chromosomal rearrangements in CLL, we have screened 279 samples from patients with short telomeres (TL < 3.81 Kb). We detected telomere fusions in 199 samples (71.3%) and selected the 15 samples with the highest fusion frequency (>4.20×10−5 per diploid genome) for high-resolution paired-end sequencing of single-molecule amplified telomere fusion events. Conclusion: STELA at the 5p telomere is a unique and robust assay that has allowed us to determine that the 5p TL is similar to other unique telomeres. The modified fusion assay has allowed the detection of telomere fusion events involving the 5p chromosome arm, which may provide insights into the mechanism of hTERT translocation in cancer. Data generated from the paired-end NGS of telomere fusion amplicons will allow us to determine a telomere recombination signature that may be informative of the molecular bases of cancer initiation, progression and drug resistance. This work is ongoing and our recent findings will be presented. No conflict of interest.

110 Follicular lymphoma cases harbour recurrent mutations in microRNA binding sites of genes associated with lymphomagenesis ˜ 3, E. Larrea1 , M. Fernandez-Mercado1 , I. Ceberio2 , J.A. Guerra-Assun¸cao J. Okosun4 , J. Fitzgibbon4 , C. Lawrie1,5 . 1 Biodonostia Institute, Oncology, San Sebastian − Guipozcoa, Spain, 2 Hospital Universitario Donostia, Hematology Department, San Sebastian, Spain, 3 Barts Cancer Institute, Bioinformatics Unit, London, United Kingdom, 4 Barts Cancer Institute, Haemato-Oncology, London, United Kingdom, 5 University of Oxford, Radcliffe Department of Medicine, Oxford, United Kingdom Introduction: Follicular lymphoma (FL) is the most common low grade B cell malignancy accounting for ~20% of all non-Hodgkin lymphomas. Approximately 30% of the FL cases suffer a histological transformation to a much more aggressive subtype of lymphoma drastically reducing the overall survival from 10 years to just 14 months. Despite being a critical event during disease progression it is molecularly poorly understood and no biomarkers exist to predict this phenomenon. MicroRNAs (miRNAs) are small endogenously produced non-coding RNAs that play a key regulatory role in many physiological and pathological mechanisms. We hypothesised that mutations in either the miRNAs or the binding sites of miRNAs could be implicated in this process. Material and Method: We interrogated whole genome sequencing (WGS HiSeq) data from sequentially obtained samples of 6 FL patients that underwent transformation using a bespoke bioinformatics pipeline in order to identify mutations in putative miRNA binding sites. Once identified, in order to validate them and test their recurrence in an extended cohort (56 samples from 27 FL patients who underwent transformation) we designed an Ampliseq (Ion Torrent) NGS custom panel to interrogate 503 positions. In order to assess the variant effect on the miRNA:mRNA interaction we used a combination of in silico predictive algorithm and in vitro luciferase assays. Results: 36% of somatic variants from WGS data arose in 3 UTR and 68% of these were putative miRNA-binding sites (525 mutations in 497 genes). Interestingly, the ontogeny analysis showed that these mutations were not randomly distributed but rather there was enrichment in genes associated with haematological malignances. We then validated 85% of these mutations using targeted resequencing. We found 35 of those to be recurrently mutated in an extended cohort of 27 cases, and further identified 68 new variants that were not present in the discovery cohort. Therefore, a total of 103 recurrent variants were identified. QC criteria filtering led us to prioritise 38 variants in 25 genes to be functionally tested. Furthermore, ontogeny analysis showed that these genes were highly enriched for GC-like B-cell lymphoma genes, strongly suggesting that this variants may have a biological significance in FL. Interestingly, preliminary data shows functional relevance of a number of the selected mutated binding sites and the corresponding miRNA. These data deepens on our previous observations on the relationship of aberrant miRNAs expression with FL pathophysiology. Conclusion: Our data show that the identified mutations do not occur randomly, but preferentially in putative microRNA binding sites of genes related to haematological malignances, supporting their being a role in FL. These

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preliminary results might help deepening our knowledge on the molecular mechanisms implicated in FL. No conflict of interest. 111 Tumour promoting HER2 splice variant D16HER2: Regulation and implication in breast cancer A.L. Dittrich1 , H. Gautrey1 , A. Tyson-Capper1 . 1 Newcastle University, Institute of Cellular Medicine, Newcastle, United Kingdom Introduction: Results from high-throughput studies of the last decade have shown important connections between regulated alternative pre-mRNA splicing and tumorigenesis as well as therapy resistance. To date only relatively few such interactions have been studied in depth. The human epidermal growth factor 2 (HER2) is an important biomarker in cancer, especially as 20−30% of breast cancer cases overexpress HER2. This subset of breast cancers is treated in addition to conventional therapy with HER2 targeting drugs. Although, these therapies are very successful, some patients with advanced metastatic breast cancer resistance can be observed. The underlying mechanisms are not well understood. One potential cause is the alternative splicing of HER2, yielding protein variants which can evade binding of the therapeutics. This study focuses on identifying the underlying mechanisms regulating the production of HER2 variants and their effect on cancer progression. A special focus lies on D16HER2, which has been shown to have a high oncogenic potential and resistance to the anti-HER2 agent trastuzumab. Material and Methods: To identify proteins, splicing factors that bind the key pre-mRNA sequence in D16HER2 production RNA-chromatography and mass spectrometry was used. The findings were verified by transient knockdowns followed by qPCR. A D16HER2 minigene construct was also designed to identify RNA recognition sequences that regulate this splicing process. Results and Discussion: From ten splicing factors that bind the key region of D16HER2 splicing two repressors and two promoters of D16HER2 generation were identified. In silico-predicted binding sites for different splicing factors were confirmed by the minigene assay. Conclusion: Key players in the splicing event causing the production of the oncogenic D16HER2 have been identified. Additionally, this study has indicated a potential feedback loop between HER2 and its splicing regulators. Ongoing work is comparing these cell line-based findings to expression patterns observed in a panel of different breast tumours. These findings are important in understanding how cellular processes regulate key biomarkers in breast cancer and eventually how this affects the cancer progression and therapy resistance. No conflict of interest. 112 Whole-genome sequencing of circulating tumor DNA reveals relevance of focal amplifications for the management of metastatic prostate cancer J. Belic1 , P. Ulz1 , R. Graf1 , M. Auer1 , K. Fischereder2 , G. Hoefler3 , T. Bauernhofer4 , J.B. Geigl1 , E. Heitzer1 , M.R. Speicher1 . 1 Medical University of Graz, Institute of Human Genetics, Graz, Austria, 2 Medical University of Graz, Department of Urology, Graz, Austria, 3 Medical University of Graz, Institute of Pathology, Graz, Austria, 4 Medical University of Graz, Division of Oncology, Graz, Austria Introduction: Prostate cancer is the most common malignancy in males and is treated with androgen deprivation therapy (ADT) as a first-line therapy option. Whole-genome sequencing studies have shown the prognostic power of copy number alteration analysis in prostate cancer in comparison to mutational analysis, as it was found that even cases of heavily pretreated metastatic prostate cancer had a low overall mutation rate. Despite all improvements in NGS technology for studying the tumor genome, the plasticity and evolution of the prostate cancer genome is greatly unknown. Methods: We analyzed 493 prostate cancer cases from the TCGA database and performed whole-genome plasma sequencing of 95 plasma samples derived from 43 patients with metastatic prostate cancer. Whole-genome plasma sequencing was performed using our recently published plasma-Seq method in order to establish genome-wide single copy number aberrations (SCNAs). Furthermore, we used a targeted resequencing approach to screen for mutations in a set of 58 cancer-associated genes and for the presence of TMPRSS-ERG fusions. Follow-up samples were available from a total of 30 patients, which enabled us to monitor the constantly changing tumor genomes. Results: We identified established driver aberrations in a cancer-related gene in 97.7% of all cases, including driver gene fusions (TMPRSS2:ERG), driver focal deletions (PTEN, RYBP, SHQ1), and driver amplifications (AR, MYC). Furthermore, when analyzing follow-up samples, we observed the occurrence of novel copy number aberrations and clonal shifts in one-third of the patients The mean time interval between new alterations was 26.4 weeks, suggesting that these copy number changes are rapid adaptations to selection pressure. In few cases, we observed increases of neuron-specific

enolase (NSE) and decreases of prostate-specific antigen (PSA) accompanied by drastic clonal pattern changes in the tumor genome, most consistent with subclonal diversification of the tumor. Conclusions: In summary, our analysis showed that metastasized prostate cancer has numerous focal amplifications whose status can change rapidly to selection pressure and which appear to be a driving force in progression. Decrease in PSA and increase in NSE may indicate phenotype transition which is associated with shifting clonal patterns. The high tumor genome plasticity has significant consequences for clinical trials and our results have tremendous implications for studies testing novel second-generation of ADTs. No conflict of interest. 113 Identification of candidate predisposing factors in familial polycythemia vera with exome sequencing 1 E. Hirvonen1 , E. Pitkanen ¨ , K. Hemminki2 , L.A. Aaltonen1,3 , O. Kilpivaara1 . 1 University of Helsinki- Research Programs Unit- Genome-Scale Biology Research Program, Department of Medical and Clinical Genetics, Helsinki, Finland, 2 German Cancer Research Center DKFZ, Division of Molecular Genetic Epidemiology, Heidelberg, Germany, 3 Karolinska Institutet, Department of Biosciences and Nutrition, Stockholm, Sweden

Polycythemia vera (PV) is a chronic, BCR-ABL—negative myeloproliferative disorder characterized by clonal expansion of red blood cells in peripheral blood. PV may further progress to acute myeloid leukemia (AML) or myelofibrosis. Global annual incidence of PV is 2−3 in 100 000. Although most PV cases appear to be sporadic, familial clustering is also observed in a subset of cases. The cause of PV is not fully understood. Most PV patients (95%) carry a somatic mutation in a non-receptor tyrosine kinase-coding gene JAK2, JAK2V617F. The mutation causes the kinase to be constitutively activated, which leads to excess production of erythrocytes. Familial clustering of PV suggests the presence of susceptibility alleles. High-penetrance predisposition genes have, however, remained unidentified. We have here studied a Finnish family with four confirmed PV cases by whole exome sequencing. Exome sequencing was carried out in the index case and two affected family members (germline), and in peripheral blood DNA of the index case. We analyzed their shared germline variants and filtered against an in-house control set of 542 Finns. The effect of the candidate variants to protein function was predicted with two computational methods (SIFT, PolyPhen). To exclude common variants we further filtered set against public database (ExAC) setting the minor allele frequency (MAF) limit in Finnish population to 0.001. We then validated the variants predicted to be damaging by Sanger sequencing in one additional family member with PV. We also screened the identified variants in six other Finnish familial PV cases (2 PV cases in each). After filtering against controls, we were left with 11 rare shared variants in the family; 10 missense mutations and one splice site mutation. We also observed a rare, although predicted benign, variant in BCORL1. This is of interest since somatic mutations in BCORL1 have previously been reported in myeloid malignancies. The identified candidate variants are being screened in the six other PV families. Identification of predisposing genes and mutations would be important to these families and acknowledging pathological events occurring in hematopoiesis also helps us to understand normal physiological process in the blood cell development. No conflict of interest. 114 Expression of hypoxia related miRNAs in laryngeal squamous cell carcinoma S. Giragosyan1 , G. Stancheva1 , T. Popov2 , D. Konov2 , J. Rangachev2 , O. Stoyanov2 , V. Mitev1 , S. Todorov2 , R. Kaneva1 . 1 Medical University-Sofia, Department of Medical chemistry and Biochemistry / Molecular Medicine Center, Sofia, Bulgaria, 2 UMHAT “Tsarica Yoanna-ISUL”, Department of EarNose and Throat / Clinic of Ear- Nose and Throat, Sofia, Bulgaria Introduction: Laryngeal squamous cell carcinoma (LSCC) is the second most common head and neck cancer with poor prognosis. Since 2000, miRNAs became new players on the scene of cancer research. miRNAs, small non-coding RNAs, are involved in various pathological processes, as well as hypoxia. The aim of the study was to investigate the expression levels, discriminative pattern and correlation between hypoxic miRNAs and hypoxy-inducible factor-1a (HIF1a) and vascular endothelial growth factor A (VEGFA). Material and Methods: The expression of three hypoxia-related miRNAs, miR-31-5p, miR-155-5p and miR-210-3p, was evaluated in 38 freshly-frozen tumour and adjacent normal laryngeal squamous tissues, and in 7 metastasis tissue by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Data from RT-qPCR in our previous study on the same patient samples was used for correlation analysis of HIF1a and VEGFA and miR-31-5p, miR-155-5p and miR-210-3p. Statistical analysis was performed using SPSS 17.0 software program. A p-value less than 0.05 was considered statistically significant.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Results: In the current study, we observed increased expression levels of miR31-5p, miR-155-5p and miR-210-3p, respectively 60.55%, 50.00%, 39.47% in tumor tissue in comparison with paired normal tissue. In order to evaluate the miRNAs as biomarker for differentiation between tumour and metastatic tissue we performed the receiver operating characteristic (ROC) curve analysis. miR31-5p and miR-155-5p showed stronger discriminative accuracy, respectively with AUC = 0.808, p = 0.01 (95% CI: 0.650–0.967) and AUC = 0.744, p = 0.042 (95% CI: 0.575–0.913). miR-210-3p did not show statistically significant values for discriminative tumour tissue versus metastatic tissue potential. Spearman’s rho was performed in order to evaluate the expression levels of the three hypoxic miRNas, and HIF1a and VEGFA. The correlation analysis showed statistically significant correlation coefficient between miR-210-3p with HIF1a (0.452, p = 0.02) and VEGFA (0.349, p = 0.019). Moreover expression of miR-210-3p correlate with miR-31-5p (0.421, p = 0.04). However, miR-31-5p showed significant correlation with HIF1a, rho = 0.394, p = 0.007, but not with VEGFA. None of the studied microRNAs (miRNAs) showed correlation with age and lymph node metastasis. Conclusion: The current findings suggest miR-31-5p, miR-155-5p and miR210-3p have major role in hypoxic mechanisms. These results may contribute to future studies on elucidating the mechanisms of contribution of hypoxic miRNAs to LSCC. No conflict of interest. 115 Antitumor effect of pharmacological and genetic ablation of selected histone modifying enzymes in human glioma cells 1 1 . Nencki Institute of ´ S.K. Krol ´ 1 , M. Maleszewska1 , B. Wojta´s1 , B. Kaminska Experimental Biology, Laboratory of Molecular Neurobiology- Neurobiology Center, Warsaw, Poland

Background: Glioblastoma (GBM, WHO grade IV) is the most frequent, diffusive tumor, highly resistant to radio- and chemotherapy. Despite significant understanding the molecular mechanisms of GBM pathobiology, clinical prognoses remain poor and median survival of patients is 14 months. Recent findings from exome and RNA sequencing revealed many aberrations in sequences and/or expression of genes coding for epigenetic enzymes in GBM suggesting the important role of epigenetic dysfunction in GBM pathogenesis. Histone modifications and global aberrations at the histone level may result in dysregulated patterns of gene expression, and malfunction of proteins that regulate chromatin modification and remodeling. Histone modifications are reversible and the possibility of “resetting” of their abnormal patterns in cancer by genetic or pharmacological compounds is a promising, therapeutic strategy. Material and Methods: We determined the expression of epigenetic enzymes and chromatin modifiers in primary and established glioma cell lines using RT2 Epigenetic Profiler. The expression of selected enzymes and global levels of modified histones were analyzed by Western Blotting. We studied the effect of selected inhibitors (VPA − valproic acid, TSA − trichostatin A, 3DZNep − 3-Deazaneplanocin A) and siRNA-mediated knockdown of epigenetic enzymes: HDAC1, HDAC2, EZH2 on histone modifications by immunofluorescence staining and Western Blotting. Cell viability and proliferation of treated human glioma cells (LN18, U87) were studied using MTT metabolism and BrdU incorporation tests, respectively. Results: We demonstrate global downregulation of epigenetic enzymes in glioma cells lines when compared with non-transformed astrocytes. Only the levels of HDAC1, HDAC2 and EZH2 were significantly increased. We found that siRNA-mediated knockdown of HDAC1, HDAC2 and EZH2 results in inhibition of glioma cells proliferation. Similarly, inhibition of HDAC1, HDAC2 and EZH2 activities with VPA, TSA and 3DNZep has antitumor effects and reduced viability and proliferation of cultured glioma cells in dose- and time-dependent manner. Conclusions: Epigenetic mechanisms are significantly deregulated in glioma cells in a global scale. Inhibition of expression/activity of HDAC1, HDAC2 and EZH2, which are overexpressed in glioma cells, affects growth of tumor cells. These results provide a rationale for applying inhibitors of selected epigenetic enzymes in glioma therapy. No conflict of interest. 116 Identification of chemotherapeutic resistant mutations in castration-resistant prostate cancer F. Ozgun ¨ 1 , H. Sarac1 , N. Lack1 . 1 Koc University, Molecular Biology and Genetics, Istanbul, Turkey Introduction: Prostate cancer is an extremely common disease in Turkish and European men. The standard care for late-stage cancer is designed to inhibit the activation of Androgen Receptor (AR). One class of therapeutics is antiandrogens that directly inhibit AR activation. While this therapeutic approach is initially efficient, the cancer almost always develops resistance. When therapy fails, the median survival of patients who suffer from castrationresistant prostate cancer (CRPC) is less than 24 months. Enzalutamide has been shown to dramatically increase the survival of CRPC patients who have failed hormone therapy. Despite this success, recent clinical studies have

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demonstrated that cancer also develops resistance to enzalutamide. Point mutations on androgen receptor are one of the main causes of resistance. In an effort to identify those patients that will benefit from enzalutamide treatment we are developing a chemical screen to identify nearly all point mutations that cause drug resistance. Material and Method: We modified a well-validated screening technique that has been used to identify mutations that cause resistance to kinase inhibitors to study antiandrogens. We combine random mutagenesis of AR with a cellular selection in AR mutagenesis screen. AR mutants are tested in a cell line which survives only if there is functional AR in the presence of puromycin. In the presence of antiandrogen, AR function would be inhibited and the cells get the puromycin sensitivity back. We test more than 2 million individual mutants in the presence of antiandrogen and puromycin by using reporter cell line. AR gene from the resistant cells will be PCR amplified and sequenced to identify mutants. Results and Discussion: Initially, puromycin resistance of reporter cell line was confirmed by cell viability assay. It has been shown that the cells undergo death with increasing concentrations of enzalutamide. AR mutations were done by using XL-1 Red mutator strain. We performed blue/white screening taking advantage of LacZa gene to identify colonies that include AR mutation and calculate mutation rate. Conclusion: Chemical genetic technique offers a powerful technique to identify drug resistant mutations before it is given to patients. Once further optimized, this approach could be used to investigate the resistance mechanism for many clinical drugs. No conflict of interest. 117 Somatic mutation of the ATRX and gene in the ERBB4-Akt/mTOR pathway are frequent in cervical small cell neuroendocrine tumors M. Choi1 , S. Cho2 , H.J. Ban3 , C.H. Lee4 , H.K. Kim1 , C. Kim5 , J.Y. Lee1 . Korea University, Pathology, Seoul, Korea, 2 Seoul National University, Laboratory of Developmental Biology and Genomics, Seoul, Korea, 3 Hanyang University, 3Division of Molecular and Life Sciences, Ansan, Korea, 4 Thermo Fisher Scientific Corporation, Life Science Solutions Group, Seoul, Korea, 5 Department of Biotechnology, Konkuk University, Seoul, Korea 1

Background: Cervical small cell neuroendocrine tumors (CSCNETs) are extremely rare and aggressive form of neuroendocrine tumors (NETs). Because there is no sufficient standardized diagnostic or prognostic marker and consensus can be used so far, it is still difficult to diagnose and predict disease progress with limited treatment strategy. Material and Methods: This study included 16 formalin fixed paraffin embedded (FFPE) tissue blocks of CSCNET, which were diagnosed and collected between 1997 and 2012 in South Korea. Of 16 samples, five tumornormal paired CSCNETs were used to perform whole exome sequencing (WES) and subsequent immunohistochemistry staining to verify the finding of WES. Results: We first provide mutation profile of five tumor-adjacent normal paired CSCNETs using by whole exome sequencing. The most frequently mutated gene was the ATRX and genes in the Akt/mTOR pathway, which were indicated common mutation signature across NETs. Other frequently mutated gene was the ERBB4 encoding a receptor tyrosine kinase that is a member of the epidermal growth factor receptor (EGFR) and has diverse functions in the development of the nervous and neuroendocrine system by affecting downstream signaling pathway including Akt/mTOR pathway. Increased expression of ERBB4 was examined in the tumor tissue comparing normal counterpart by immunohistochemistry staining. Conclusion: This result suggested CSCNETs shares genetic characteristics of NETs and provided new insight for sufficient clinical management for patient, especially targeting ERBB4 and Akt/mTOR pathway. No conflict of interest. 118 Evolutionary trajectory of Asian EGFR mutation positive lung adenocarcinomas leads to “high intratumor heterogeneity” R. Nahar1 , W. Zhai2 , T. Zhang2 , A. Takano3 , A.J. Khng1 , Y.Y. Lee1 , X. Liu1 , C.H. Lim4 , T.K.H. Lim3 , T.P.T. Koh4 , Z.W. Aung5 , A.S.M. Teo1 , C.X. Chan1 , C.K. Toh6 , W.T. Lim6 , B. Lim7 , W.L. Tam7 , E.H. Tan6 , D.S.W. Tan6 , A.M. Hillmer1 . 1 Genome Institute of Singapore, Cancer Therapeutics and Stratified Oncology, Singapore, Singapore, 2 Genome Institute of Singapore, Human Genetics, Singapore, Singapore, 3 Singapore General Hospital, Department of Pathology, Singapore, Singapore, 4 National Heart Centre SIngapore, Department of Cardiothoracic Surgery, Singapore, Singapore, 5 National Cancer Centre Singapore, Department of Clinical Trials and Epidemiological Sciences, Singapore, Singapore, 6 National Cancer Centre Singapore, Division of Medical Oncology, Singapore, Singapore, 7 Genome Institute of Singapore, Cancer Stem Cell Biology, Singapore, Singapore Background: In contrast to western populations, activating mutations in EGFR are more frequent in Asian lung adenocarcinomas (LUADs) which are enriched for female gender and never-smokers. Although EGFR tyrosine

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kinase inhibitors (TKIs) commonly elicit significant tumor shrinkage in about 70% patients, differential responses are often observed that are ultimately short lived with a median progression free survival (PFS) of about 10 months. Intra-tumor heterogeneity (ITH) has been implicated in drug resistance in multiple cancers and previous studies have attempted to describe the extent of ITH in NSCLC, though predominantly in smokers. The mutational landscape of Asian EGFR-mutant never-smokers still remains unclear. Material and Methods: Multi-sector whole exome sequencing was performed on 16 treatment-na¨ıve EGFR-mutant Asian LUADs (15 never smokers) with 3−11 sectors from each tumor (79 sectors in all, mean depth 114x). Exonic and splice site variants identified were further subjected to amplicon based deep sequencing (mean depth 3860x) and fraction of ubiquitous (trunk) mutations were calculated as an inverse measure of ITH. To remove technical biases in comparisons, we also reanalysed the published Caucasian LUAD multiregion sequencing data through our pipelines. Results and Discussion: EGFR mutations were identified as truncal events in all 16 patients regardless of the specific mutation type (L858R, exon 19 deletions and exon 20 insertions). However despite the high response rates to TKIs and low mutations burdens, we counter-intuitively identify “high ITH” (median of 37.7% trunk mutations) in these non-smoker dominated EGFRmutant patients compared to smoker dominated Caucasian patients (median 75% trunk mutations). While subclonal mutations failed to explain the observed differences in ITH, we show using a “genetic driverness score” that EGFR mutations are strong drivers in LUADs and their early occurrence in context of low mutation burdens is likely responsible for shorter trunks and hence proportionately higher ITH in Asian EGFR-mutant LUADs. Conclusion: In summary our study provides a novel insight into determinants of ITH which possibly is a reflection of driver “dominance”. We show in a coherent framework that mutation rates, timing of alterations and probably ethnicity influence the dominance of a driver mutations which in turn shape the evolutionary trajectories of individual tumors. No conflict of interest. 119 Overexpression of TMPRSS2:ERG variants activates TGF-b signaling and promotes invasion of prostate cancer cells 1 1 ¨ . German L. Ratz1 , M. Laible1 , P. Altevogt1 , S.M. Klauck1 , H. Sultmann Cancer Research Center, Cancer Genome Research, Heidelberg, Germany

Introduction: TMPRSS2:ERG (T/E) gene fusions are detectable in approximately 50% of all prostate cancer cases and can be considered as biomarkers for molecular subtypes. Several T/E variants have been identified and expression of specific fusion mRNAs was associated with adverse clinicopathologic parameters. However, the underlying molecular processes induced by distinct T/E variants still remain unclear. We therefore aimed to characterize the molecular and cellular profile and specific transcriptional modulation induced by different T/E gene fusion variants. Material and Methods: A modified LNCaP cell model was used that stably overexpresses the T/E variants III and VI in a doxycycline-inducible (TetOn) promoter system. T/E expressing cells were functionally characterized by assaying viability, migration and invasion and cell cycle pattern. Illumina microarray gene expression profiling was performed to identify transcriptional changes accounting for cellular phenotypes. qPCR was performed to validate the expression of selected genes. Results and Discussion: Upon doxycycline induction, T/E overexpressing cells showed increased viability and migration. T/E variant VI expressing cells further showed an increased invasive phenotype. Cell cycle analysis revealed accumulation in G1 phase in both variants. This may indicate a cellular program switch to migration and invasion accompanied by a G1 phase arrest induced by T/E overexpression. Global gene expression changes identified a high overlap of transcriptional programs of T/E III and VI that were consistent with the expected transcriptional response to ERG-overexpression in publicly available mRNA expression profiling studies. Functional annotation using Ingenuity pathway analysis indicated a role for T/E in migration and invasion. Decreased ‘Estrogen-mediated S-phase Entry’ was found among the top canonical pathways confirming our previous cellular analysis. T/E variant VI specific modulation of TGF-b signaling was identified as a regulator of epithelial to mesenchymal transition characterized by significant upregulation of SNAI1, SNAI2, and downregulation of CDH1. Conclusion: Activation of TGF-b signaling was identified as a determinant of mesenchymal transformation and invasion of T/E overexpressing cells. We suggest that T/E variant-specific transcriptional modulation accounts for the malignant cellular phenotypes which correspond to a more aggressive phenotype of tumors expressing T/E variant VI. No conflict of interest.

120 TMPRSS4 protein overexpression and its promoter hypomethylation predict poor prognosis in squamous lung cancer patients M. Villalba1 , A. Diaz-Lagares2 , M. Redrado3 , A.L. de Aberasturi1 , M.J. Pajares3 , R. Pio3 , L.M. Montuenga3 , M. Esteller2 , J. Sandoval4 , A. Calvo1 . 1 Private University of Navarra, Histology and Pathology, Pamplona, Spain, 2 Bellvitge Biomedical Research Institute, Cancer Epigenetics and Biology Program, Catalonia, Spain, 3 Center for Applied Medical Research, Program of Solid Tumors and Biomarkers, Pamplona, Spain, 4 Medical Research Institute La Fe, Laboratory of Personalized Medicine- Epigenomics Unit, Valencia, Spain Introduction: TMPRSS4, a serine protease overexpressed in some solid tumors, promotes proliferation, invasion and metastasis. However, mechanisms leading to TMPRSS4 overexpression are unknown. The purpose of our study was to assess whether epigenetic changes may regulate TMPRSS4 expression in NSCLC patients and cell lines and whether both protein expression and promoter methylation status could be used as biomarkers with prognostic value. Material and Methods: We studied TMPRSS4 protein expression in a TMA containing samples from 79 stage I-II NSCLC patients. Infinium 450k methylation array was used for analysis of promoter methylation in NSCLC patients (n = 444) and DNA bisulfite pyrosequencing was performed in a validation cohort (n = 88). Methylation analyses were also confirmed in silico using public data from TCGA. The prognostic role of both TMPRSS4 protein expression and promoter methylation was evaluated by bioinformatic and statistical analyses. A panel of 46 lung cancer cell lines was used to study TMPRSS4 expression and methylation status of its promoter. In vitro studies with a demethylating agent (5-aza-2 -deoxycitidine) were carried out in order to confirm that TMPRSS4 promoter hypomethylation leads to TMPRSS4 expression. Results: Increased TMPRSS4 protein levels were found in tumors when compared to non-malignant samples. High TMPRSS4 expression was significantly associated with reduced relapse free survival (RFS) and overall survival (OS) in patients with squamous cell carcinoma (SCC). Multivariate analysis showed TMPRSS4 as an independent prognostic factor in SCC. Aberrant TMPRSS4 promoter hypomethylation was found in tumoral samples of all the cohorts analysed. SCC patients with high promoter methylation presented a better outcome, as shown by Log-Rank curves. Inverse association between TMPRSS4 expression and promoter methylation status was confirmed by correlation analyses performed in data from TCGA and also in cell lines. Treatment with the demethylating agent showed increased levels of TMPRSS4 mRNA, suggesting that TMPRSS4 expression is regulated by promoter methylation. Conclusion: NSCLC is characterized by epigenetic DNA hypomethylation of the TMPRSS4 promoter, a mechanism that may explain TMPRSS4 overexpression in these tumors. Both TMPRSS4 protein expression and promoter methylation status are useful biomarkers to predict prognosis in early stages of squamous NSCLC. No conflict of interest. 121 Upregulated lncRNA-HNGA1, a target of miR-375, contributes to aerobic glycolysis of head and neck squamous cell carcinoma through increasing levels of the glucose transporter protein SCL2A1 Y. Wang1 . 1 Sun Yat-sen University, Oral Medicine Institute, Guangzhou, China Background: Long noncoding RNAs (lncRNAs) have been regarded as key regulators in cancer. Aerobic glycolysis, also known as the Warburg effect, was recently added as an emerging hallmark of cancer which could contribute to cancer malignancy. However, the role and function of lncRNAs in Head and Neck Squamous Cell Carcinoma (HNSCC) glycolysis remain unclear. Here, we report a novel lncRNA, HNGA1, which could promote HNSCC glycolysis and malignancy by competing for miR-375 binding to regulate SCL2A1. Material and Methods: Microarrays were performed to explore the lncRNA/miRNA profiles in 4 HNSCC samples and adjacent non-tumor tissues. qRT-PCR and functional analysis were used to confirm the expression and role of lncRNA/miRNA. Bioinformatics approach, luciferase assay and western blot were used to verify the miRNA target gene and the interation between lncRNA and miRNA. Nude mouse model was utilized to observe the effect of lncRNA/miRNA in vivo. Tissue array was performed to explore the association between lncRNA and postoperative survival. Results: 1. LncRNA HNSCC glycolysis-associated 1 (HNGA1) was up-regulated in tumor tissues, while miR-375 was down-regulated significantly. These observations were confirmed in 60 pairs of HNSCC/non-tumor tissues samples and 7 cohorts of HNSCC cell lines. 2. Functional analysis showed that silencing of HNGA1 inhibited HNSCC cells proliferation and glycolysis, while overexpression of HNGA1 had the opposite effect.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 3. Ectopic expression of miR-375 repressed HNSCC cells proliferation and glycolysis, whereas miR-375 inhibition resulted in the opposite effect. Bioinformatics tools showed that miR-375 could target to SCL2A1. MiR-375 could repress the SCL2A1 expression by binding to the 3 -UTR region of SCL2A1 directly. 4. After comparing the sequence, HNGA1 was found to be a target of miR-375 and there was an inverse correlation between HNGA1 and miR-375 in HNSCC specimens. Then, the functional and luciferase assay showed that miR-375 suppressed HNGA1’s expression and function by directly binding to HNGA1. In addition, HNGA1 could reverse the inhibitory effect of miR-375 on HNSCC cells, which might act as an endogenous ‘sponge’ by competing for miR-375 binding to regulate SCL2A1. 5. The xenograft mouse model unveiled the suppressive effects of miR-375 on HNSCC tumor growth and glycolysis, while HNGA1 could accelerate this process. 6. The clinicopathological findings suggested that the up-regulation of HNGA1 in HNSCC was associated with poorly differentiated degree and more metastasis. Moreover, the results of tissue array showed that HNGA1 was correlated with postoperative survival. Conclusions: Taken together, our data highlights the pivotal role of HNGA1 in HNSCC glycolysis. More importantly, we elucidate a novel lncRNA-miRNAmRNA regulatory network that is HNGA1-miR-375-SCL2A1 axis in HNSCC malignancy and progression. No conflict of interest. 122 Exploiting loss of heterozygosity for allele-selective cancer chemotherapy 1 1 ¨ . Uppsala University, V. Rendo1 , I. Stoimenov1 , R. Svensson2 , T. Sjoblom Science for Life Laboratory- Department of Immunology- Genetics and Pathology, Uppsala, Sweden, 2 Uppsala University, Drug Optimization and Pharmaceutical Profiling Platform UDOPP- Department of Pharmacy, Uppsala, Sweden

Background: Due to frequent chromosomal aberrations, cancer cells are often found with only one copy of an allele, a feature known as loss of heterozygosity (LOH). These losses in genetic variation can be exploited in the context of cancer chemotherapy, implementing drugs that selectively kill tumor cells with reduced allele content. We have mined data for human genetic variation from the 1000 Genomes project to identify single nucleotide variants (SNVs) causing amino acid substitutions near catalytic or substrate binding sites of enzymes. A top candidate resulting from this screen was a highly polymorphic enzyme involved in the metabolism of xenobiotics, considered as a putative target for a LOHbased therapeutic approach. Material and Methods: We aimed to generate a suitable cell model system representing different activity variants of the enzyme of interest. We therefore transfected RKO human colorectal cancer cells with expression vectors generated to encode the variants with rapid and slow activity that result from the identified SNV. Next, the catalytic activity of the obtained positive clones was quantified in cell lysates by measuring through liquid chromatographymass spectrometry the velocity at which two enzyme-specific substrates become processed. Results: Both enzyme-specific substrates were metabolized by rapid and slow variants, whereas no catalytic activity was identified in parental RKO cells and vector controls lacking endogenous expression of the enzyme of interest. The processing rates of both substrates were 15-fold and 6-fold higher in rapid activity clones when compared to slow variants. These results motivated the use of the generated cell model system as a tool for drug discovery efforts, aiming to identify compounds selective for one enzymatic variant. In a preliminary screening of 176 putative substrates, one compound showed to significantly impair the viability of parental RKO cells (EC50 = 0.34 mM) while leaving the growth of rapid clones unaffected (EC50 = 1.8 mM). Conclusions: In this study we have generated a cell model system expressing rapid and slow variants of our candidate enzyme as a tool to prove that agents targeting loss of allelic variation can be conceived for cancer chemotherapy. Ongoing studies are now focused on determining specificity of the identified hit compound as well as toxicity and efficacy in mouse models of cancer. No conflict of interest. 123 Histone H2B monoubiquitylation − the Ying and Yang of breast cancer O. Tarcic1 , R. Granit2 , I. Ben-Porath2 , M. Oren1 . 1 Weizmann Institute of Science, Molecular Cell Biology, Rehovot, Israel, 2 The Hebrew University-Hadassah Medical School, Department of Developmental Biology & Cancer Research, Jerusalem, Israel Introduction: Breast cancer is a heterogeneous disease that can be stratified into several subtypes, each displaying unique clinical behavior and distinctive gene expression patterns. Estrogen Receptor positive (ER+) tumors of the Luminal subtypes are mostly associated with favorable clinical outcome. Triple

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negative (TN) tumors of the basal-like subtype are typically poorly differentiated and highly aggressive. RNF20 is an E3 ubiquitin ligase, which with its partner RNF40 monoubiquitylates histone H2B (H2Bub1); this chromatin modification plays a pivotal role in transcriptional regulation. H2Bub1 was implicated as a tumor suppressor in many cancers, including breast cancer. Yet, other studies concluded that H2Bub1 plays an oncogenic role in breast cancer. In an attempt to resolve this controversy we explored the role of H2Bub1 in different subtypes of breast cancer. Materials and Methods: Human breast cancer expression data was from TCGA. Additionally, a human breast cancer TMA was stained for H2Bub1 and H2B. Basal-like and luminal cell lines were subjected to RNF20 knockdown and analyzed for growth, migration, and ability to form tumors and metastases in mice. Additionally, the effect of H2Bub1 on the expression of cytokines and ER target genes was assessed by qRT-PCR. Results and Discussion: Data analysis revealed that TN breast cancer patients tend to have low RNF20 and RNF40 mRNA as well as low H2Bub1, while luminal breast cancer patients tend to have higher levels. Moreover, in TN patients, lower H2Bub1 was associated with shorter survival, while luminal breast cancer patients showed the opposite trend. Employing human breast cancer cell lines, we found that H2Bub1 manifests tumor suppressive features in basal-like cancer cells, while in luminal cancer cells H2Bub1 actually supports cancerous features. The tumor suppressive role of H2Bub1 in basallike breast cancer is partly achieved through restricting the expression of inflammatory cytokines. On the other hand, in luminal breast cancer H2Bub1 supports the expression of estrogen receptor target genes. Thus, by modifying the key transcriptional programs in each cancer subtype, H2Bub1 exerts opposite roles in luminal versus basal-like breast cancer. Conclusion: In the present study, we sought to resolve the controversy between studies suggesting a tumor suppressive role of RNF20/H2Bub1 in breast cancer and the proposal that they actually contribute to breast cancer. We now report that whereas in TN and basal-like breast tumors H2Bub1 has a tumor suppressive role, it supports luminal breast cancer. This may be due, at least in part, to the fact that RNF20 facilitates the transcriptional activity of the estrogen receptor, which supports luminal tumors, while restricting the expression of cytokines that underpin aggressive features of TN tumors. No conflict of interest. 124 TP53 variations in human cancers: new lessons from the IARC TP53 Database and genomic studies M. Olivier1 , L. Bouaoun2 , D. Sonkin3 , M. Ardin1 , M. Hollstein4 , G. Byrnes2 , J. Zavadil1 . 1 International Agency for Research on Cancer, Molecular Mechanisms and Biomarkers Group, Lyon, France, 2 International Agency for Research on Cancer, Biostatistics Group, Lyon, France, 3 National Cancer Institute, Division of Cancer Treatment and Diagnosis- Biometric Research Program, Rockville, USA, 4 University of Leeds, Faculty of Medicine and Health, Leeds, United Kingdom Background: Mutations in the TP53 gene are among the most frequent somatic events in cancer. The IARC TP53 Database (http://p53.iarc.fr/) compiles occurrence and phenotype data on TP53 germline and somatic variations linked to human cancer. The database is a popular resource appreciated for the quality and scope of its data and is actively used worldwide. The deluge of data from cancer genomic studies generates new data on TP53 variations and attracts a growing number of database users for the interpretation of TP53 variants. We will present an overview of the current contents of the IARC TP53 Database and the results of a systematic comparative analysis of data extracted from the IARC database and from genomic data repositories. Material and Methods: TP53 mutation data were extracted from the IARC TP53 Database (Sanger sequencing studies) and from TCGA and ICGC data portals (next generation sequencing − NGS − studies). Mutation frequencies per cancer types and mutation distributions by protein location and by cancer types were computed and plotted using R software. Results: Data on somatic mutations available in the IARC TP53 Database are more extensive than what is currently available in genomic repositories, with larger number of TP53 mutations and more cancer types covered. However, the higher sensitivity and more complete screening achieved by NGS highlighted some overlooked facts about TP53 mutations, such as the presence of a significant number of mutations occurring outside the DNAbinding domain in specific cancer types. The database also provides a list of variants that should be considered as neutral frequent variations, a list of celllines with their updated TP53 status, a compilation of inherited variants and their phenotype, and various annotations on the predicted and experimentallyassessed functional impacts of missense mutations. Conclusions: The IARC TP53 Database provides a broad scope of data on TP53 gene variations that can be used to interpret the role of specific TP53 variations in human cancers. Our comparative analysis of new data from genomic repositories with data from the IARC TP53 Database provides an updated knowledge on TP53 variations in human cancers. No conflict of interest.

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125 Genomic analysis of multi-site fresh prostate samples M. Parry1 , A. Cannistraci1 , V. Ramani2 , M. Lau2 , J. Shanks3 , D. Nonaka3 , N. Clarke2 , N. Dhomen1 , E. Baena4 , R. Marais1 . 1 CRUK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 The Christie NHS Foundation Trust, Urology-Surgery, Manchester, United Kingdom, 3 The Christie NHS Foundation Trust, Histopathology, Manchester, United Kingdom, 4 CRUK Manchester Institute, Prostate Oncobiology, Manchester, United Kingdom Background: Prostate cancer is the most common cancer in males in the UK with >40,000 cases diagnosed every year and >10,000 deaths. Despite a high ten-year survival rate of 83.8%, this widespread disease remains a large burden for men and the healthcare system, and diagnostic and prognostic tools are sorely in need of improvement. Recent multi-platform genomic studies have revealed a very complex picture of the disease, with no clear subcategories linking to the histopathological markers currently used in clinical practice. To stratify patients more effectively and identify new therapeutic targets requires a fuller understanding of the genetic basis of prostate cancer development and progression. The multifocal and heterogeneous nature of the disease suggests that single biopsy sites may be missing valuable subclones which contribute to the aetiology of the disease. Materials and Methods: To address this, we obtained fresh core biopsies from multiple sites (7−10) in the prostate from 14 high-risk and 12 intermediate risk patients undergoing prostatectomies at the Christie NHS Foundation Trust. The tissues were immediately processed by the pathology department and each core divided in two: one for genomic analysis and one for parallel disease modelling (PDXs, patient-derived cell lines and organoids). Pathological and clinical data for these patients was also obtained, as well as plasma for cfDNA. The cores were cryo-sectioned and H and E analysis performed at the top, middle and bottom of each core. These were reviewed by a pathologist and tumour tissue was microdissected prior to simultaneous DNA and RNA extraction of normal and tumour tissue. Results: These samples are currently undergoing comprehensive genomic study, consisting of WES, CNA profiling and transcriptomic analysis. Each will be compared in parallel to their own cell lines, organoids and PDXs (where available) for a functional characterisation of the genomic aberrations they carry. Conclusion: The clinical course of prostate cancer is highly heterogeneous and treatment is not always required or effective, leading to unnecessary interventions or conversely, to metastatic spread. The impact of the multifocal and genomically heterogeneous nature of prostate cancer on our understanding of disease progression will be addressed by this comprehensive study. No conflict of interest. 126 New software for detecting somatic mutations at low level in Sanger sequencing traces E. Schreiber1 , H. Leong1 , S. Berosik1 , S. Schneider1 , J. Marks1 , W. George1 , Y.P. Lim1 , E. Chan1 , A. Gerstner1 . 1 Thermo Fisher, Life Sciences Solutions, South San Francisco, USA Introduction: With the rapid adoption of next generation sequencing (NGS) and its use for characterization of mutations in tumor samples, a need has emerged to establish an orthogonal technology for reliable and sensitive detection of somatic mutations which may occur at proportions of 10% or lower compared to the normal allele. Until now, somatic variants with an allelic proportion of 25% or less are often undetected by automated Sanger sequencing software. We have developed Minor Variant Finder software that calls 5% minor variants at 95.3% sensitivity and 99.8% specificity in a test data set. The improved variant detection software calls variants de novo, without a priori knowledge of location or presence, and affords all the advantages of Sanger sequencing, of robustness, low error rate, ease of use, human interpretable visual displays of the output data, and low cost per sample and target. Materials and Methods: New algorithms were developed to compare and subtract the baseline noise signals of a control sample from a test sample enabling the detection of peaks representing candidate minor variants which are confirmed in the complementary strand. To test the algorithms, synthetic mixtures of minor alleles in the range of 2.5%, 5%, 10% and 20% were prepared by combining genomic DNAs containing known mutations. Using an optimized PCR/sequencing protocol to sequence cell line and FFPE derived DNA, 13 different amplicons and seven different genes, including P53, KRAS, BRAF, FLT3, RB1, CDH1, and ERBB2, were sequenced on 3500, 3730, and 3130 Applied Biosystems Genetic Analyzers. Results and Discussion: 632,452 total base positions and 2334 total variant positions, spanning variants at 2%, 5%, 10%, and 20%, were interrogated. The software achieved 95.3% sensitivity and 99.8% specificity for automated detection of 885 variants present at the 5% level in 238,179 high quality base positions. Sensitivity was 99.8% for variants between 7.5−10%, 98.7% for variants between 12.5−25%, and 100% for variants 25%. The software

displays a noise-minimized trace view to facilitate visual inspection for confirmation or rejection. Further, reference sequences and NGS vcf files can be imported and aligned side by side with the Sanger sequencing data for orthogonal verification. Hyperlinks to dbSNP for known rsSNP variants are provided in the software. Conclusions: We have developed new Minor Variant Finder software that detects and reports minor variants by Sanger sequencing. The new software used with standard protocols for fluorescent dye terminator Sanger sequencing enable the identification of de novo somatic mutations to a level of 5% with 95.3% sensitivity and 99.8% specificity. This technology will also be useful for the confirmation of minor variants identified by NGS. For Research Use only − Not for use in diagnostic procedures. Conflict of interest: Corporate-sponsored Research: All authors are employees of Thermo Fisher Scientific. 127 Direct input of biological samples into highly multiplexed target amplification reactions for next-generation sequencing (NGS) C. Van Loy1 , T. Biorac1 , M. Allen1 , D. Topacio1 , M. Carvallo1 , J. Kilzer1 , K. Rhodes1 , M. Andersen1 . 1 Thermo Fisher Scientific, Research and Development, Carlsbad, USA Introduction: Standard library preparation methods for next-generation sequencing research applications require purified DNA as input. However, purification methods are time consuming, costly, and result in variable sample quality, which lengthen the time required to progress from sample to answer. In addition, much of the DNA is lost during the purification process, meaning that extremely small samples cannot be used. Methods: Herein, we describe the use of biological samples as direct input in a highly multiplexed target amplification to generate libraries for subsequent analysis with the Ion Torrent™ NGS platform. We have developed a Direct Reagent for simple processing of retrospective (or archived) samples of formalin-fixed paraffin embedded (FFPE) samples, saliva, buccal swab, and blood prior to library generation. This method is resistant to inhibitors that are present in biological samples therefore allowing their direct use in the amplification reaction. The Direct Reagent is compatible with Ion AmpliSeq™ panels, no purification is required. We evaluated each type of sample using Ion AmpliSeq DNA research panels with 207, 1267 or 3900 assays designed to known oncogenes. Results: Libraries generated directly from FFPE tissue slices, and retrospective samples from whole blood, saliva, and buccal swab samples all resulted in similar sequencing performance as compared to DNA purified from FFPE using commercial nucleic acid purification kits. For the 207 assay DNA panel, uniformity of base coverage was >95%, base reads on target was >95%, and target bases with no strand bias was >97%. Additionally, greater than 98% of the assays were covered at >500× base read depth with a minimum of 2500× average base coverage depth when 6 libraries were included per sequencing run. For the 1267 and 3900 assay DNA panels, libraries made directly from biological samples also resulted in similar metrics as those observed for purified DNA controls. We were able to generate and successfully sequence DNA libraries from single FFPE slices as small as 2 mm × 2 mm × 5 mm and from a single microliter of archived blood or saliva samples. Conclusions: We developed a research method to directly use biological samples and low input samples for generating libraries to reduce the time from sample to analysis on an NGS platform. Extremely small samples were successfully processed with this method, which reduces the total time from sample to sequence to 24 hours. For Research Use Only. Not for use in diagnostic procedures. No conflict of interest. 128 Genetic profiling of mutations in pheochromocytoma and paraganglioma by targeted next generation sequencing − A pilot study S. Pillai1 , V. Gopalan1 , A. King Y. Lam1 . 1 Cancer Molecular Pathology of Menzies Health Institute Queensland- Griffith Univ, School of Medicine, Gold Coast- Southport, Australia Background: A neuroendocrine tumour with properties of chromaffin cells in adrenal medulla is termed phaeochromocytoma whereas that outside adrenal gland is known as paraganglioma. Overall, known genetic mutations account for the genesis of approximately 60% of PCC/PGLs. At least 22 genes (PHD2, VHL, SDHx, IDH, HIF2A, MDH2, FH, RET, NF1, KIF1Bb, MAX, TMEM127, GDNF, H-RAS, K-RAS, GNAS, CDKN2A (p16), p53, BAP1, BRCA1&2, ATRX and KMT2D) have been associated with predisposition to pheochromocytoma or paraganglioma. Materials and Method: A targeted next-generation sequencing approach on an Ion Torrent PGM instrument was used for mutation analysis in formalin fixed paraffin embedded tissues from 10 phaeochromocytomas and paragangliomas. Primers were designed for the 14 target genes − using ion ampliseq designer. The genes included in the study were NF1, RET, VHL,

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, HRAS, KIF1Bb, IDH and PHD2. Analysis was done with the help of Ion Reporter software and Ingenuity® Variant Analysis™ software (www.ingenuity.com/variants) from Ingenuity Systems. Results and Discussion: Overall, 636 variants were identified in ten patients, the variants identified ranged from 64 to 161 per patient. Single nucleotide variants (SNV) were the most common. Further annotation with the help of Ingenuity variant analysis revealed 29 variants which were found to be deleterious. Of the 29 variants detected, 6 were considered to be benign and 4 were pathogenic. The remaining 19 variants were of uncertain significance. The most frequently altered gene in the cohort was KIF1B followed by NF1. Chromosome 1 showed the presence of maximum number of variants. Conclusions: Use of targeted next-generation sequencing is a sensitive method for the genetic analysis of pheochromocytoma and paraganglioma. The new technology could analysis tumours with a high degree of genetic heterogeneity and heritability. No conflict of interest. 129 High sensitivity Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples in precision oncology A. Gerstner1 , E. Schreiber1 , S. Jackson1 , K. Varma1 . 1 Thermo Fisher Scientific, Genetic Analysis, South San Francisco, USA Background: Deleterious sequence variants play an important role in the initiation and progression of many different cancer types. These alterations could also predict prognosis, sensitivity or resistance to specific therapies and has been in the focus of personalized cancer therapy/precision oncology. Next-generation sequencing (NGS) is a valuable solution for high-throughput applications, however, the workflow and the data analysis can be complex, lengthy (often >40 hours) and cumbersome. Moreover, NGS is not a costeffective approach when only a limited number of targets need to be screened. Finally, NGS results often require a reliable and sensitive confirmatory method. There is clearly a need for a robust, fast, simple and affordable screening and/or confirmatory method for detecting low level somatic variants. The detection of germline variants by the gold standard Sanger sequencing has been well established; however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging. Material and Methods: PCR and sequencing reactions were performed using BigDye™ Direct Sanger Sequencing Kit in Veriti™ Thermal cycler. Sequencing reactions were cleaned-up using BigDye XTerminator Purification Kit. Reactions were separated on the Applied Biosystems™ 3500xL Genetic Analyzer. Results: To facilitate analysis of somatic mutations in tumor samples, we have developed Sanger sequencing panels that cover the entire coding regions of specific genes (e.g. TP53) as well as an extended panel encompassing 66 frequently detected oncogenic alleles from 18 genes. We have also developed companion software, Minor Variant Finder (MVF), that facilitates detection of low levels of somatic mutations by Sanger sequencing. To demonstrate the workflow of these panels, we analyzed FFPE DNAs from 12 different cancer tissue types. We initially determined variants in these samples using Ion Torrent™ Personal Genome Machine (PGM™) next generation sequencing. We confirmed the identity and variant allele frequency of these variants by Sanger sequencing using 1 ng, 0.5 ng or 0.1 ng DNA. Finally, we made serial dilutions of one of these samples to establish limit of detection (LOD) at as little as 3% of a minor variant in an FFPE sample. Conclusions: Sanger sequencing is the gold standard for confirmation of minor variants detected by NGS. In this study, we show that Sanger sequencing of limited number of targets, in conjunction with the MVF software, can also be an ideal first line screening choice for tumor FFPE samples where limited amount of DNA is available. For Research Use only − Not for use in diagnostic procedures. No conflict of interest. 130 Functional assessment of low-frequency mutations in breast cancer A. Leonidou1 , S.L. Maguire1 , P.T. Wai1 , P. Huang2 , B. Peck1 , R.C. Natrajan1 . 1 Institute of Cancer Research, Division of Breast Cancer Research, London, United Kingdom, 2 Institute of Cancer Research, Division of Cancer Biology, London, United Kingdom Background: Next-generation sequencing efforts have unveiled a vast complexity in the mutational repertoire of breast cancer. Such efforts have revealed that there are few mutations that are highly recurrent in unselected breast cancers aside from TP53 and PIK3CA and that the majority occur at low frequency. Whilst some of these low frequency mutations occur in known oncogenes and have been shown to be oncogenic and subsequently targetable, the majority remain uncharacterised. Our aims were to functionally interrogate low-frequency missense mutations identified in breast cancer sequencing data in order to identify novel drivers and therapeutic targets.

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Methods: We compiled a database of mutations identified in publicly available and in-house sequencing data from breast cancer. High confidence variants were run through bioinformatic prediction algorithms FATHMM and CHASM to predict which mutations are most likely to have an oncogenic impact, and CANSAR was used to assess which genes are druggable using existing inhibitors. Following several exclusion criteria and manual curation, we identified 8 tyrosine kinases and a total of 22 mutations which were predicted to be oncogenic. Single missense mutations predicted to be drivers from above were engineered using site-directed mutagenesis of full length cDNA constructs. Mutant and wild-type constructs were transduced into a panel of cell lines (HEK293, MCF10A, MDA-MB-231 and MCF7) to generate stable clones in order to characterise the impact of mutations on signalling capacity. The phenotypic impact of mutations was assessed using in vitro 2D and 3D spheroid and Matrigel assays. Results and Discussion: We found FGFR2 hotspot mutations to be constitutively active and showed a high global phosphorylation state, consistent with reports in endometrial and ovarian cancer. Furthermore cells carrying this mutation were highly sensitive to siRNA-mediated gene silencing of FGFR2 as well as chemical inhibition with AZD4547 and PD173074. Mutations in other genes such as INSRR were found context-dependent phenotypes. Kinase domain mutations were shown to confer a proliferative advantage when cells were grown in 2D tissue culture, and this effect was significantly more pronounced in 3D spheroids, suggesting that features such as 3D cell-cell contact, oxygen and nutrient gradients, as well as pH gradients across the spheroids better recapitulate physiological conditions found in tumours and may present a more appropriate system in which moderateimpact driver mutations can be unveiled. Conclusion: In conclusion, we found some lower frequency kinase mutations to be oncogenic. However, the effects of moderate-impact driver mutations can only be fully understood in conditions closely resembling in vivo conditions, such as 3D culture. No conflict of interest. 131 Assessing the interplay of RNA-fusion events with cancer driver mutations in melanoma patients A. Mandal1 , M.R. Girotti1 , N. Dhomen1 , A. Viros1 , G. Gremel1 , E. Galvani1 , F. Baenke1 , R. Lee1 , K.H.J. Lim1 , P. Lorigan2 , R. Marais1 . 1 CRUK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 University of Manchester, The Christie NHS Foundation Trust, Manchester, United Kingdom Introduction: Treatment options for cutaneous melanoma in recent years have undergone major improvement, primarily due to development of targeted and immunotherapies. Still patient heterogeneity remains a challenge and studies looking for predictors of response by profiling DNA-level somatic alterations in large-scale cohorts have failed to identify consensus biomarkers. In the present study we examine RNA-fusion events to complement other genomic alterations. We hypothesize that this will assist better clinical decision-making (targeted or immunotherapy) in individual patients. Materials and Methods: We performed whole exome sequencing (WES) and complimentary RNA-seq of 28 tumor tissue samples from 28 patients. Of these, ten were wild type for BRAF or NRAS hotspot mutations. We profiled RNA-seq datasets for fusion events. Briefly, after quality-based trimming, fastq reads were aligned using STAR aligner to detect chimeric transcripts. Initial candidates were annotated and filtered for spurious calls using STAR-Fusion and FusionAnnotator. Results and Discussion: We detect two in-frame BRAF fusions, one in BRAF V600E (RABEP1-BRAF) and other in a BRAF kinase-dead mutation (AGKBRAF) sample. As in previous reports, BRAF is the 3prime partner in both the cases with its first 8 and 7 exons deleted respectively. The exon loss may increase BRAF kinase domain dimerization. AGK is a (lipid) kinase that is previously reported as a BRAF fusion partner in melanoma. We also detect an in-frame G12D KRAS fusion (CCDC91-KRAS) in a patient lacking BRAF/ NRAS hotspot mutations. This fusion retains exon 2 and beyond of KRAS, and effectively constitutes the entire protein coding region of G12D KRAS. Conclusion: BRAF fusions are reported not to respond to BRAF inhibitors, so the V600E BRAF fusion is contra-indicated for these therapies. Thus, it is crucial to combine somatic alteration and fusion events as part of the standard NGS-based profiling for melanoma patients. We are further analysing additional fusion candidates detected, and examining the individual contributions when hotspot somatic mutation and gene fusion events are combined. No conflict of interest.

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132 Circulating tumour DNA analysis for monitoring and stratification of patients with small cell lung cancer S. Mohan1 , M. Ayub1 , H.S. Leong2 , S. Sahoo2 , N. Smith1 , C. Miller3 , F. Blackhall4 , D. Rothwell1 , C. Dive1 , G. Brady1 . 1 CRUK Manchester Institute, Clinical and Experimental Pharmacology, Manchester, United Kingdom, 2 CRUK Manchester Institute, Computational Biology Support, Manchester, United Kingdom, 3 CRUK Manchester Institute, Computational Biology Support and RNA Biology Group, Manchester, United Kingdom, 4 Christie NHS Foundation Trust, University of Manchester, Manchester, United Kingdom Background: Tumour tissue genotyping for actionable information is the current practice in medical oncology. Increasingly, and in view of tumour evolution and the almost universal emergence of resistance to targeted treatments, clinical management of cancer patients is becoming dependant on serial monitoring to chart complex tumour dynamics. However, several limitations such as invasiveness of the biopsy procedure, localisation, size and inherent heterogeneities of the tumours make serial tissue biopsies a difficult choice. ‘Liquid biopsies’, either circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA) measured in a patient’s peripheral blood sample allow the detection of tumour associated alterations such as copy number aberrations (CNA), single nucleotide variations and loss of heterozygosity. We present an evaluation of ctDNA analysis applied to peripheral blood from limited stage (LS, confined to 1 hemithorax) and extensive stage (ES, with distant metastases) small cell lung cancer (SCLC) patients obtained through the course of their treatment to establish its utility for monitoring disease progression. Methods: Following plasma preparation and ctDNA isolation, next generation sequencing (NGS) libraries were prepared and whole genome sequencing carried out at a low depth (0.1−0.2×) using Illumina MiSeq platform to establish genome wide CNA from 25 LS SCLC patients and 25 ES SCLC patients. From the same NGS libraries, targeted enrichment of 110 SCLC associated genes was performed using Agilent SureSelect and subsequently sequenced at a higher depth (500x) on the Illumina NextSeq platform. Results and Conclusions: Characteristic SCLC associated mutations were detected in RB1, TP53, TP73, Notch and PIK3CA genes along with loss of copy number on 3p and 17p as well as increased copy number of chromosome 20 in both LS and ES disease. Comparison of mutation and CNA burden in the LS and ES datasets will provide key information on the course of the disease. For three patients with ES disease, the presence of detectable CNA and the frequency of mutations changed in sequential blood samples indicating the suitability of ctDNA analysis for routine monitoring of SCLC patients. Our study highlights that ctDNA analysis provides an easy tool for the identification of novel determinants of disease progression, providing much needed genetic follow-up data for clinical trials. No conflict of interest.

when considering all CD44 promoter methylation, gene expression and ADT (p = 0.185). Conclusions: Both CD44 promoter methylation and gene expression seems to be affected by ADT in PC. In particular, we observed lower methylation levels and higher mRNA levels in ADT treated cases. Considering that CD44 is a well-known stem-cell marker, we hypothesize that ADT could select a population of cells with stem-cell-like behaviour. CD44 may thus represent a powerful prognostic marker and may help in evaluating treatment response in PC. No conflict of interest. 134 Overexpression of PBK/TOPK relates to tumor malignant potential and poor outcome of gastric carcinoma T. Ohashi1 , S. Komatsu1 , D. Ichikawa1 , M. Miyamae1 , W. Okajima1 , T. Imamura1 , J. Kiuchi1 , K. Okamoto1 , H. Tsuda2 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Digestive Surgery, Kyoto, Japan, 2 National Defense Medical College, Basic Pathology, Tokorozawa, Japan Background: PBK/TOPK (PDZ binding kinase//T-LAK cell originated protein kinase) is serine-threonine kinase and overexpressed in various types of cancer by inhibiting the transactivation activities of p53 and PTEN. In this study, we tested whether PBK/TOPK acts as a cancer-promoting gene through its activation/overexpression in gastric cancer (GC). Material and Methods: We analyzed five GC cell lines and 144 primary tumors, which were curatively resected in our hospital between 2001 and 2003. An immunohistochemical, cell proliferation, fluorescence activated cell sorting, and transwell migration and invasion analyses were performed. Results: Overexpression of the PBK/TOPK protein was frequently detected in GC cell lines (four out of five lines, 80.0%) and was detected in primary tumor samples of GC (24 out of 144 cases, 16.6%), and significantly correlated with venous invasion, deeper tumor depth and higher recurrence rate. PBK/TOPKoverexpressing tumors had a worse survival rate than those with nonexpressing tumors (P = 0.0009, log-rank test) in both intensity and proportiondependent manner. PBK/TOPK positivity was independently associated with worse outcome in multivariate analysis [P < 0.0001 hazard ratio 6.40 (2.71– 14.49)]. In PBK/TOPK overexpressing GC cells, knockdown of PBK/TOPK using several specific siRNAs inhibited the cell proliferation through the activation of p53 in a TP53 mutation-dependent manner, and inhibited the migration and invasion through the up-regulation of PTEN in a TP53 mutationindependent manner. Conclusions: These findings suggest that PBK/TOPK plays a crucial role in tumor malignant potential through its overexpression, and highlight its usefulness as a prognostic factor and potential therapeutic target in GC. No conflict of interest. 135 Methylome profiling of BRCA-positive and BRCA-negative MBCs

133 CD44 mRNA expression in hormonally treated and non-treated prostate cancer cases A.S. Navazio1 , P. Rizzolo1 , V. Zelli1 , V. Silvestri1 , V. Valentini1 , R. Santi2 , G. Nesi2 , L. Ottini1 . 1 University of Rome La Sapienza, Molecular Medicine, Rome, Italy, 2 University of Florence, Division of Pathological Anatomy, Florence, Italy Background: Prostate cancer (PC) is the most common non-cutaneous cancer in men. Neoadjuvant androgen deprivation therapy (ADT) is the main therapy for advanced PCs, although tumors often develop resistance to ADT. Alterations in the cell-surface hyaluronan receptor CD44 have been demonstrated in PC and related to PC aggressive behaviour, although the mechanisms involved in these changes are still not well defined. Methylation of CD44 promoter has been proposed to regulate CD44 expression in PC. In this study, we aimed to investigate CD44 promoter methylation and mRNA expression in a selected series of PCs including ADT treated and non-treated cases. Material and Methods: 84 PC cases, 42 ADT treated and 42 non-treated samples, were included. CD44 promoter methylation levels were evaluated by pyrosequencing. CD44 standard and variants mRNA expression was investigated by quantitative Real-Time PCR with TaqMan probes. Pearson product-moment correlation between methylation and gene expression levels was established. Possible associations between methylation levels, ADT and gene expression status were evaluated by ANOVA, Mann–Whitney and Welch Two Sample t-test. Results: Overall, 44% of the cases showed low and 56% high CD44 promoter methylation levels. A statistically significant difference in methylation levels between ADT treated and non-treated cases emerged (p = 0.0005), with lower levels in treated cases. With regard to CD44 expression, 67.8% of the cases showed low and 23.8% high CD44 mRNA levels. A statistically significant difference in CD44 expression between ADT treated and non-treated cases emerged (p = 0.038), with higher mRNA levels in treated cases. A correlation between CD44 promoter methylation and mRNA levels was observed in 58.3% of cases. However, no statistically significant association emerged

P. Rizzolo1 , V. Silvestri1 , V. Licursi2 , A.S. Navazio1 , V. Valentini1 , V. Zelli1 , S. Bianchi3 , D. Palli4 , S. Fox5 , L. Ottini1 . 1 University of Rome La Sapienza, Molecular Medicine, Rome, Italy, 2 University of Rome La Sapienza, Biology and Biotechnologies “Charles Darwin”, Rome, Italy, 3 University of Florence, Medical and Surgical Critical Care, Florence, Italy, 4 Cancer Research and Prevention Institute ISPO, Molecular and Nutritional Epidemiology Unit, Florence, Italy, 5 Peter MacCallum Cancer Institute, Pathology, Melbourne, Australia Background: There is growing evidence that the characterization of tumorspecific methylome profiles may lead to the identification of specific breast cancer (BC) subgroups. Methylome analysis of female BC allowed for the identification of gene methylation profiles associated with molecular subtypes via ER/PR, HER2 and BRCA-mutation status. To date, whole methylation profiling in MBC characterized for BRCA1/2-mutation status has not yet been performed. In this study, we performed methylome profiling of BRCA1/2positive and BRCA1/2-negative MBCs in order to identify specific MBC subgroups by molecular subtype. Materials and Methods: Genome-wide methylation profiling was conducted on 52 FFPE samples, including 16 BRCA1/2-positive and 34 BRCA1/2negative MBCs and 2 normal male breast tissues. Analysis was performed using the Illumina Infinium HumanMethylation450 BeadChip (450K). Data analysis was performed using Minfi Bioconductor package and the biological relevance of the differentially methylated genes, functional annotation and gene enrichment analyses were performed using DAVID (http://david.abcc.ncifcrf.gov/). Unsupervised hierarchical clustering analysis was performed on the most significantly differentially methylated CpG loci between samples, using Ward’s method. Results: Compared to normal breast tissues a distinctive methylation profile of MBCs, was observed. By unsupervised hierarchical clustering two distinct tumor subgroups emerged: one including the great majority of BRCA-positive MBCs and the great majority of high grade (G3) tumors, and the other including the great majority of BRCA-negative and low grade (G1/2) MBCs. Pathwaybased analyses showed different methylation profiles between BRCA1/2-

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 negative and BRCA1/2-positive MBCs, for genes involved in cell differentiation and cell-cell signaling pathways. Conclusion: Our results indicate that MBCs exhibit a tumor-specific methylation profile and that BRCA1/2-mutation status may define genomewide DNA methylation profiles for MBC. Our data may allow the identification of epigenetic signatures that could define molecular subgroups of MBCs, thus elucidating pathways underlying BC development. Acknowledgement: Study supported by AIRC (IG 16933) to L.O. No conflict of interest. 136 Confirming variants discovered by Next Generation Sequencing (NGS) with Sanger sequencing using innovative bioinformatics tools E. Schreiber1 , S. Berosik1 , M. Wenz1 , S. Chang1 , S. Jackson1 , J. Zhai1 , S. Schneider1 , P. Brzoska1 . 1 Thermo Fisher Scientific, Genetic Sciences Division, South San Francisco, USA Introduction: Next-generation sequencing (NGS) has revolutionized our understanding of biological processes. In basic or applied research studies, insights are made by comparing the primary DNA sequence of genes in different groups of subjects. Researchers identify sequence variations between the groups that may cause disease susceptibility, progression, or phenotypic variation. The accurate identification of sequence variants is instrumental in insuring experimental success. However, NGS library preparation and chemistries are prone to low, but detectable, levels of errors that can lead to confusing or incorrect sequencing interpretations. Materials and Methods: Many labs confirm potential variants identified by NGS by using an orthogonal method such as Sanger sequencing. This is a robust and inexpensive method with chemistries and analysis tools that are easy to use and well understood and due to its high accuracy and ease of data interpretation is considered the gold standard for DNA sequence analysis. It is therefore an ideal system for validating variant calls made on NGS platforms. Results and Discussion: To streamline the validation workflow we have developed a web-based Primer Designer tool for PCR and Sanger sequencing that provides pre-designed PCR primer pair sequences for the human exome in an easy to search format. Using these primers researchers can readily PCR amplify and Sanger sequence variants of interest discovered by NGS. The resulting Sanger sequencing data can then be analyzed and compared against the NGS vcf variant file using novel software termed NGC (next generation confirmation). The software aligns the NGS variant finding directly with the variant found in the Sanger sequencing trace for visual inspection and produces a consolidated report for matches or mis-matches as well as a joint vcf file with genome coordinates for downstream bioinformatic processing. Conclusion: The Primer Designer and NGC analysis tools enable a seamless workflow for NGS variant confirmation by traditional Sanger sequencing. Conflict of interest: Corporate-sponsored Research: All authors are employees of Thermo Fisher Scientific. 137 Effects of histone acetyltransferases GCN5/PCAF knockdown on urothelial carcinoma cells E. Koutsogiannouli1 , C. Hader1 , M. Pinkerneil1 , M.J. Hoffmann1 , W.A. Schulz1 . 1 University Hospital HHU, Urology, Duesseldorf, Germany Introduction: Disturbances in histone acetyltransferases (HATs) and deacetylases (HDACs) are common in cancers. In urothelial carcinoma (UC), e.g., the HATs p300 and CBP are often mutated, whereas the GNAT family HATs GCN5 and PCAF are upregulated in many cases, according to the TCGA data accessed via the cBioPortal database. HDACs are well-established as cancer drug targets, but HATs are less well studied. Here, we investigated the expression of the GNAT HATs and explored the effects of their specific knockdown in UC cell lines. Material and Method: Expression of GCN5 and PCAF was measured by qRT-PCR and Western blotting in UC lines compared to normal proliferating urothelial cells. Following siRNA-mediated knockdown of GCN5, PCAF or both HATs in 4 UC lines, expression of HATs and marker proteins was determined by western blotting and qRT-PCR. Cellular effects were analyzed by ATP assay, caspase assay, colony forming assay and flow cytometry. Results: GCN5 and PCAF mRNA and especially protein were moderately upregulated in many UC lines. Efficient siRNA-mediated knockdown in four cell lines diminished proliferation and inhibited clonogenic growth depending on cell line and HAT, with double knockdown usually most efficient for shortterm effects. Following siRNA-mediated knockdown of GCN5, compensatory upregulation of PCAF occurred, but not vice versa. Flow cytometry revealed an increased G1 fraction indicating cell cycle arrest after GCN5, PCAF and double knockdown; whereas caspase activity increased after PCAF or double knockdown. Conclusions: GCN5/PCAF upregulation is also common in many UC cell lines. The effects of specific down-regulation differ between the two HATs in a cell-dependent fashion. Typically, even double knockdown resulted in only moderate effects on cell proliferation and apoptosis. Our results predict that

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developing drugs targeting individual HATs for the treatment of UC, as already available for HDACs, may be challenging. Acknowledgement: Supported by the Bodossaki foundation. No conflict of interest. 138 Combined circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer G. Brady1 , M. Ayub1 , D. Rothwell1 , S. Mohan1 , J. Chudziak1 , C. Miller2 , S. Gulati1 , A. Lamarca3 , J.W. Valle3 , C. Dive1 . 1 Cancer Research UK Manchester Institute, Clinical & Experimental Pharmacology, Manchester, United Kingdom, 2 Cancer Research UK Manchester Institute, RNA Biology Group, Manchester, United Kingdom, 3 Christie NHS Foundation Trust, Medical Oncology, Manchester, United Kingdom Introduction: The challenge to improve outcomes for patients diagnosed with advanced pancreatic cancer remains very real with only small improvements in median survival gained by the use of systemic chemotherapy and little improvement in 5-year survival over the past decades. The advent of next generation sequencing (NGS) of tumor nucleic acids has opened up the possibility of improving outcomes through personalized therapies selected on the basis of tumor genetics. Whilst NGS biomarkers can be measured in tumor biopsies sampled shortly before and after treatment this is often not practical for ethical, logistical and simple lack of availability. To circumvent problems associated with tumor sampling we have developed and evaluated blood-borne nucleic acids biomarkers for patients diagnosed with advanced pancreatic cancer. Material and Method: We developed a NGS circulating free DNA (cfDNA) analysis pipeline based on the generation of whole genome NGS libraries followed by sequencing of over 600 cancer-associated genes using Agilent SureSelect. Using whole blood collected with Streck cell free DNA blood collection tubes (cfDNA BCT) we optimised combined circulating tumor cell (CTC) enrichment and cfDNA isolation. Quantitative measurements of KRAS mutations using both NGS and droplet digital PCR (ddPCR) were used to compare the tumor component present in both CTCs and cfDNA. We evaluated the overall approach using External Quality Assessment (EQA) controls and over 50 advanced pancreatic cancer patient samples. For CTC analysis we also compared epitope dependent (CellSearch) and independent (Parsortix) enrichment. Results and Discussion: After applying our NGS pipeline to 5 EQA genomic controls we identified all 14 known mutations correctly indicating high sensitivity and specificity. Both ddPCR and NGS identified KRAS mutations in patient cfDNA with a higher success rate seen for ddPCR consistent with the higher sensitivity of this methodology. From the NGS output additional mutations were detected in samples which either harboured or lacked detectable KRAS mutations. Consistent with previous observations CellSearch CTCs were detected at low levels in around 20% of patients with a similar frequency seen in initial analysis of CTCs obtained by epitope independent enrichment (Parsortix). In general KRAS mutations were detected at a higher level in patient cfDNA compared to enriched CTCs although some patients showed detectable CTC KRAS mutations with no KRAS mutations detected in their cfDNA by either ddPCR or NGS. Ongoing analysis is aimed at establishing if the molecular observations correlate with clinical outcome. Conclusion: Combined CTC enrichment and cfDNA isolation is readily achievable using a single Streck cfDNA BCT. Results indicate that combined CTC and cfDNA analysis is more sensitive than either approach alone. No conflict of interest. 139 Genomic characterization of the role of chromatin remodelling genes in human cancer T. Moreno1 , C. Revilla1 , S. Montes2 , L. Cereceda2 , E. Salido3 , A. Astudillo4 , P. Isidro4 , I. Esposito5 , M. Ramirez6 , I. Varela1 . 1 Instituto de Biomedicina y Biotecnolog´ıa de Cantabria CSIC-UC-Sodercan, Departamento de Biolog´ıa Molecular- Universidad de Cantabria, Santander, Spain, 2 Hospital ´ de Valdecilla/IDIVAL, Departamento de Anatom´ıa Universitario Marques Patologica y Nodo de tejidos- Biobanco Valdecilla., Santander, Spain, 3 Centre for Biomedical Research on Rare Diseases CIBERER- Universidad La Laguna, Departamento de Patolog´ıa, Tenerife, Spain, 4 Hospital Universitario Central de Asturias, Biobanco del Principado de Asturias, Oviedo, Spain, 5 Heinrich-Heine-University, Institute of Pathology, Duesseldorf, Germany, 6 ˜ Jesus, ´ Sanitaria de La Princesa, Hospital Nino ´ Instituto Investigacion Madrid, Spain Introduction: The recent burst of sequencing data from cancer samples has allowed the identification of recurrent somatic mutations in different components of the SWI/SNF chromatin remodelling complex like ARID1A, ARID1B, PBRM1, SMARCA4, ARID2 and SMARCA2 in several tumour types. This, together with the concurrence of recurrent mutations in genes for histone modifiers in cancer, support the active role of chromatin structure in cancer development.

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Material and Methods: We have performed target sequencing on a collection of 600 samples coming from several human tumour types, using custom probes designed against the coding sequence of 250 genes involved in chromatin structure remodelling: including ATP-dependent remodelling complexes, histone modifier genes as well as bona fide high recurrent cancer genes. Both tumour and normal DNA samples were extracted from the specimens and targeted sequencing was performed using a 100 paired-end protocol. Validation of potential mutations found in the screen was assessed using high-coverage amplicon sequencing. Results and Discussion: We have detected more than 4900 somatic mutations among all the samples studied. More than 800 of these mutations are located in the genes of the components of the four main ATP-dependent chromatin remodelling complexes (SWI-SNF, ISWI, POLYCOMB and INO80). 29% of the samples studied had at least one mutation on a component of one of these major complexes, specially the SWI-SNF complex. We have also found lack of concurrent mutations on different members of the same complex in a given sample, which indicate functional redundancy for some of these genes. Finally, we have identified recurrent genes with tissue-specific mutations, which indicates a specific role for some of these genes in some tumour types. Conclusion: Our results are in agreement with the notion that chromatin structure plays an important role in tumour progression and that some genes coding for components of ATP-dependent chromatin remodelling complexes are bona fide cancer genes essential for tumour development in specific tumour types. No conflict of interest. 140 Detection and molecular analysis of circulating tumour cells isolated from SCLC patients following short and long term storage B. Mesquita1 , D. Burt1 , M. Carter1 , C. Smowton1 , L. Priest1 , F. Blackhall2 , D.G. Rothwell1 , C. Dive1 , G. Brady1 . 1 CRUK Manchester Institute, Clinical and Experimental Pharmacology, Manchester, United Kingdom, 2 Christie NHS Foundation Trust- Manchester- UK, Institute of Cancer SciencesUniversity of Manchester- Manchester- UK, Manchester, United Kingdom Introduction: Small cell lung cancer (SCLC) is an aggressive, highly metastatic cancer with poor prognosis. Tumour biopsies are difficult to obtain in contrast to (relatively) prevalent circulating tumour cells (CTCs) which can be accessed from a simple peripheral blood draw. As a ‘liquid biopsy’ CTCs hold considerable potential as a source of biomarkers to assist clinical management of SCLC patients. We and others previously reported the prognostic significance of CTC number in SCLC. To exploit clinical utility of SCLC CTC based biomarkers fully, we sought to establish a robust workflow for CTC enrichment, isolation and biobanking to facilitate retrospective, as well as prospective CTC analysis. Methods: We performed CTC enrichment using CellSearch® (Johnson and Johnson), an epitope dependent system that captures EpCAM expressing tumour cells. For blood samples with CTC counts of 5–120, CTCs were isolated by DEPArray (Silicon Biosystems) after storage of CellSearch cartridges containing enriched CTC samples at 4ºC (average storage time, 15 days). Isolated CTCs were stored at −80ºC. For blood samples with CTC counts of 120–200, 50% of the resuspended cell suspension from CellSearch cartridges was stored at −20ºC in glycerol and 50% was processed immediately for CTC isolation. If the CTC count was 200, 33% of the resuspended cell solution was stored at 4ºC, 33% at −20ºC in glycerol and 33% processed for CTC isolation immediately. Following DEPArray image analysis, individual CTCs, pools of CTCs and control white blood cells (WBCs) were isolated from CTC enriched samples after storage in the conditions above and isolated CTCs subsequently subjected to whole genome amplification (WGA). WGA efficacy was evaluated by multiplex PCR (Genome Integrity Index) and next generation sequencing (NGS). Low pass NGS was used to establish genome wide copy number alterations (CNA). Results and Discussion: For both “spike in” controls and clinical samples neither cell image quality nor WGA efficacy (judged by the Genome Integrity Index and CNA analysis) were measurably affected by glycerol storage. We also demonstrated that for a panel of 16 patients long term storage of isolated cells at −80ºC had no detrimental effect on subsequent WGA and NGS data. We are currently evaluating stability of CellSearch® enriched CTC samples at 4ºC to establish at what switching to longer term storage at −20ºC in glycerol is required. Conclusions: In summary, we have established simple yet effective ‘stop off’ points along the workflow for CTC molecular analysis. This provides much needed flexibility and extends utility, for example to multi-site clinical studies, where either real time analysis, CTC sample batch molecular analysis, or selected sample analysis can be performed after patient outcomes are known. No conflict of interest.

141 A novel ESR2 frameshift mutation predisposes to medullary thyroid carcinoma and causes inappropriate RET expression M. Read1 , J. Smith1 , V. Smith1 , N. Morgan2 , N. Wake1 , J. Watkinson3 , Y. Wallis4 , E. Maher5 , C. McCabe1 , E. Woodward6 . 1 University of Birmingham, Institute of Metabolism and Systems Research, Birmingham, United Kingdom, 2 University of Birmingham, Institute of Cardiovascular Sciences, Birmingham, United Kingdom, 3 University Hospitals Birmingham National Health Service Foundation Trust, Queen Elizabeth Hospital, Birmingham, United Kingdom, 4 West Midlands Regional Genetics Laboratory, Birmingham Women’s Hospital, Birmingham, United Kingdom, 5 University of Cambridge, Department of Medical Genetics, Cambridge, United Kingdom, 6 Manchester Centre for Genomic Medicine, St Mary’s Hospital, Manchester, United Kingdom Introduction: Familial medullary thyroid cancer (MTC) and its precursor C Cell hyperplasia (CCH) is associated with germline RET mutations causing multiple endocrine neoplasia type 2 (MEN2). The majority of germline RET mutations predisposing to MEN2 result in single amino acid substitutions causing inappropriate constitutive RET activation. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation (non-RET), suggesting that further predisposing gene alterations remain to be identified. Here, we investigated one kindred and 19 individuals with non-RET MTC/CCH and detected novel mutations in ESR2 encoding the beta subunit of the oestrogen receptor (ERb). Functional studies were performed to determine the role of ESR2 germline mutations in MTC tumorigenesis. Material and Methods: We undertook exome sequencing studies in one family with apparent predisposition to MTC/CCH and identified a novel ESR2 frameshift mutation, c.948delT; p.G318Afs*22, which segregated with histological diagnosis following thyroid surgery. Sequencing of the ESR2 gene in 19 individuals with apparently isolated MTC identified a novel germline missense alteration, c.382G>C; p.V128L in a female who developed MTC age 36 years. In human cell lines (MCF-7, HCT116) the activity of ESR2 mutants was evaluated in the presence of ER agonists (E2, DPN, PPT) by luciferase (LUC) and cell proliferation assays. Results: LUC reporter assays showed that ESR2-V128L retained transcriptional activity with a significant increase in LUC activity in response to ESR2agonist DPN in HCT116 (3.7-fold; P < 0.01) and MCF-7 (1.8-fold; P < 0.05) cells. In contrast, ESR2-G318Afs*22 was incapable of inducing LUC activity in either cell line (P=NS). Furthermore, ESR2-G318Afs*22 failed to inhibit ESR1 driven LUC activity in response to either 17b-estradiol (E2) or ESR1-agonist PPT, or restrain ESR1-driven proliferation of MCF-7 cells (P=NS vs ESR1 alone). In contrast, wild-type (WT) ESR2 and ESR2-V128L inhibited ESR1driven LUC activity (>60%; P < 0.01) and cell proliferation (>30%; P < 0.05). As RET expression is known to be stimulated by oestrogen, we then determined the influence of ESR2 mutants on RET in E2- and PPT-treated HCT116 cells. In contrast to WT ESR2, ESR2-G318Afs*22 was unable to oppose ESR1stimulation of the RET proto-oncogene at both the mRNA and protein level (P=NS vs ESR1 alone). Treatment with anti-oestrogen 4-hydroxytamoxifen was however capable of inhibiting E2-induced RET mRNA expression in cells with ESR2-G318Afs*22. Conclusion: Together these data indicate an emerging role for ESR2 as a novel susceptibility gene in non-RET MTC development, especially as ESR2 mutant G318Afs*22 was associated with elevated RET. These results also suggest that anti-oestrogens might represent a promising therapeutic strategy for MTC individuals with defective ESR2. No conflict of interest. 142 Pan-cancer analysis of mutation hotspots in protein domains M. Miller1 , N. Gauthier2 , E. Reznik2 , C. Sander2 . 1 University of Cambridge, CRUK Cambridge Institute, Cambridge, United Kingdom, 2 Memorial Sloan-Kettering Cancer Center, Computational Biology, New York, USA Introduction: In cancer genomics, frequent recurrence of mutations in independent tumor samples is a strong indication of functional impact. However, rare functional mutations can escape detection by recurrence analysis for lack of statistical power. We address this problem by extending the notion of recurrence of mutations from single genes to gene families that share homologous protein domains. In addition to lowering the threshold of detection, this sharpens the functional interpretation of the impact of mutations, as protein domains more succinctly embody function than entire genes. Methods: We map somatic missense mutations in 22 human cancer types to equivalent positions in multiple sequence alignments of protein domains and perform binomial statistics to detect mutation hotspots in aligned protein domains. Results: We (1) confirm well-known functional mutation hotspots; (2) identify uncharacterized rare variants in one gene that are equivalent to wellcharacterized mutations in another gene, such as uncharacterized ERBB4 (S303F) mutations that are analogous to canonical ERRB2 (S310F) mutations in the furin-like domain; (3) detect previously unknown mutation hotspots, such as hotspot mutations in prolyl isomerase domains and in DNA-binding forkhead

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 domains; and (4) provide hypotheses about molecular mechanisms and downstream effects of domain mutations. We make all results available in an interaction web resource (www.mutationaligner.org), which enables discovery and exploration of mutation hotspots identified in protein domains in more than 5000 cancer patient samples across 22 different tumor types. Conclusion: Using cancer genomics datasets from thousands of tumor samples in 22 tumor types, we analyze somatic missense mutations in protein domains and discover new domain mutation hotspots. By associating mutations in infrequently altered genes with mutations in frequently altered paralogous genes that are known to contribute to cancer, this study provides many new clues to the functional role of rare mutations in cancer. No conflict of interest. 143 Characterization of the genomic profile of pseudomyxoma peritonei using amplicon sequencing combined with exome sequencing 2 ¨ , P. Nummela1 , L. Saarinen1 , A. Thiel1 , R. Lehtonen1 , P. Jarvinen 3 H. Jarvinen ¨ ¨ 1. , L.A. Aaltonen1 , A. Lepisto¨ 3 , S. Hautaniemi1 , A. Ristimaki 1 University of Helsinki, Genome-Scale Biology Research Program, Helsinki, Finland, 2 Helsinki University Central Hospital, Dept. of Urology, Helsinki, Finland, 3 Helsinki University Central Hospital, Dept. of Surgery, Helsinki, Finland

Introduction: Pseudomyxoma peritonei (PMP) is a rare but fatal disease in which mucinous tumor cells grow in the peritoneal cavity and secrete voluminous mucinous ascites. The genomic profile of PMP, typically having quite low tumor cell content among the mucus, has started to open by the advent of the powerful technique of next-generation sequencing. Material and Methods: We analyzed 19 formalin-fixed, paraffin-embedded PMP tumor samples with an amplicon sequencing panel covering the mutational hotspot areas of 48 cancer-related genes (average coverage 3580x). Further, to expand the analysis to genes and pathways outside this panel, we sequenced the whole coding exome (average coverage 120x) of nine of these tumors and validated these results with ultra-deep amplicon sequencing. Results and Discussion: KRAS was mutated in all 19 PMP tumors and GNAS in over half of the cases (63%). Other detected mutations, including AKT1, ATM, PIK3CA, SMAD4, and TP53, were mostly detected in 5% and maximally 16% (SMAD4) of the cases. The genetic background of mainly appendix-originating PMP tumors thus seems to resemble more pancreatic intraductal papillary mucinous neoplasms (IPMNs) than colorectal cancers. In addition to GNAS, our exome sequencing approach revealed also seven other genes of the cAMP-protein kinase A (PKA) pathway to be mutated in our PMP tumors (8/9 tumors having this pathway affected), which may account for the highly increased secretion of mucus. In addition, we validated mutations in six genes belonging to the TGF-beta pathway, which may further enhance cell proliferation from that induced by mutated KRAS. Conclusion: Since cAMP-PKA pathway was mutated in 89% of the tumors, inhibition of this pathway might reduce mucus production in most of the patients, thereby suppressing the progression of the disease. No conflict of interest. 144 DNA damage responses and chemosensitivity of breast cancer tissue slices ex vivo D. Van Gent1 , K. Naipal1 , N. Verkaik1 , T. Meijer1 , J. Hoeijmakers1 , C. Van Deurzen2 , R. Kanaar1 , A. Jager3 . 1 Erasmus MC, Molecular Genetics, Rotterdam, Netherlands, 2 Erasmus MC, Pathology, Rotterdam, Netherlands, 3 Erasmus MC, Medical Oncology, Rotterdam, Netherlands Background: Tumor cell characteristics and sensitivities have been studied extensively in two dimensional cell cultures. Much less is known about therapy response in three dimensional growth conditions in the natural tumor microenvironment. Material and Methods: We optimized growth conditions for patient-derived breast tumor slices with a thickness of approximately 30 cell layers. These conditions allowed maintenance of the tumor slices without loss of viability or proliferation over a period of at least 7 days, allowing extensive analysis of tumor cell parameters, such as DNA damage response characteristics and sensitivity to chemotherapy. Results: We used this experimental set up to determine DNA double strand break repair deficiencies in a series of approximately 150 unselected primary breast tumors. We found Homologous Recombination deficiency in 13% of the breast tumors, in many cases associated with triple negative breast cancer. We also showed proof of principle that HR deficient tumor slices were PARP inhibitor and cisplatin sensitive. We are currently adapting the technology for use on biopsy material, for which we will present the first results comparing ex vivo responses and treatment outcome in the patient. In addition to these mechanistic studies, we also investigated whether chemotherapy response could be predicted from ex vivo reaction to chemotherapy. We found a range of sensitivities to treatment with 5-Fluoro-Uracil/Adriamycin/cyclophosphamide

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combinations. As proof of principle, we found one clinically insensitive tumor to be insensitive ex vivo, as well. Conclusion: Breast tumor tissue slices provide a novel opportunity to monitor tumor response to therapy. Under optimal conditions, these slices can be maintained for more than a week without loss of viability, which enables extended experimental protocols. No conflict of interest.

Monday 11 July 2016 Poster Session

Cancer Genomics, Epigenetics and Genome Instability II 145 Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing J. Demeulemeester1,2 , E.K. Møller3,4 , P. Kumar5,6 , N. Silje3,4 , D.C. Wedge7 , K.P. White8 , B. Naume4,9 , V.N. Kristensen3,4,10 , P. Van Loo1,2 , T. Voet5,6 . 1 The Francis Crick Institute, Cancer Genomics, London, United Kingdom, 2 KU Leuven − University of Leuven, Department of Human Genetics − Human Genome Laboratory, Leuven, Belgium, 3 Institute for Cancer Research − Oslo University Hospital, Department of Genetics, Oslo, Norway, 4 Institute for Clinical Medicine − University of Oslo, K.G. Jebsen Center for Breast Cancer Research, Oslo, Norway, 5 KU Leuven − University of Leuven, Department of Human Genetics − Laboratory of Reproductive Genomics, Leuven, Belgium, 6 Wellcome Trust Sanger Institute, Single-cell Genomics Centre, Hinxton, United Kingdom, 7 Wellcome Trust Sanger Institute, Cancer Genome Project, Hinxton, United Kingdom, 8 Institute for Genomics & Systems Biology − University of Chicago, Department of Human Genetics, Chicago, USA, 9 Oslo University Hospital, Department of Oncology, Oslo, Norway, 10 Oslo University Hospital, Department of Pathology, Oslo, Norway Introduction: Single-cell micro-metastases of solid tumors often occur in bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. Unfortunately, the molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer. Material and Method: We sequenced the genomes of 40 single cells isolated by micromanipulation from the bone marrow of six patients using established immunocytochemical markers and morphologic characteristics for epithelial tumor cells. We compared the cells’ DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, and genotyped somatic mutations called on bulk tumor exomes in the singlecell sequences. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enabled ordering of CNA events in molecular pseudo-time. Results and Discussion: CNA landscape analysis revealed that a quarter of the cells are DTCs disseminating from the observed tumor. The remaining cells represented non-aberrant ‘normal’ cells and ‘aberrant cells of unknown origin’ that have CNA profiles discordant from the tumor. Probing somatic mutations confirmed that these cells did not derive from the same lineages as the observed breast cancers. Evolutionary reconstruction pinpointed the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis. Conclusions: Single-cell sequencing of bone-marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells of unknown origin is prominent in bone marrow. Author contributions: JD, EKM and PK contributed equally to this work. KPW, BN, VNK, PVL and TV are joint senior authors. No conflict of interest. 146 The expression of MNT, a MYC antagonist, is autoregulated at the mRNA and protein level M.C. Lafita1 , J. Liano ˜ 1 , F. Ourique1 , J. Aresti1 , P.J. Hurlin2 , J. Leon1 . 1 Instituto de Biomedicina y Biotecnolog´ıa de Cantabria- UC-CSIC, Department of Molecular and Cellular Signalling, Santander, Spain, 2 Shriners Hospitals for Children- Oregon Health & Science University, Department of Cell and Developmental Biology, Portland, USA Background: MNT is a basic-helix-loop-helix-leucine zipper protein from the MXD family. MNT and other MXD proteins are antagonists of MYC, one of the most frequently altered oncoproteins in human cancer. Both MYC and MXD proteins heterodimerize with MAX and bind to E-boxes within regulatory regions of target genes, resulting in the activation (MYC-MAX) or repression (MXD-MAX) of their transcription. MNT expression is ubiquitous and does not fluctuate during cell cycle, so it overlaps with the expression of MYC at critical

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points. However, little is known about its regulation at the mRNA and protein level. Material and Methods: The cell lines used in this work were MAX-deficient H1417 (human SCLC) and UR61 (rat pheochromocytoma), together with K562 and HEK293T. In addition, we worked with URMAX34 and Kmax12, which are UR61 and K562 cells with a Zn+2 MAX-inducible system. Chromatin immunoprecipitation assays (ChIP) were used to study MNT and MAX binding to MNT promoter. Luciferase reporters of human MNT promoter were constructed and used to analyse its activity. mRNA and protein levels were assessed by RT-qPCR and western blot. Results: MNT expression changed in different conditions and cell lines, suggesting a tight regulation. First, rat (UR61) and human (H1417) MAXdeficient cells showed higher MNT protein levels in comparison to most MAXexpressing cell lines tested. Transient and induced MAX expression in UR61 and K562 showed a decrease in MNT, both at the mRNA and protein level. The opposite occurred when silencing MAX in K562 by siRNAs, pointing out an inverse correlation between MNT and MAX expression. MNT promoter contains two E-boxes where MNT and MAX bind to, as shown by ChIP experiments and confirmed by the ENCODE data in the case of MAX. UR61 cells were then transfected with luciferase reporters that contained either the two E-boxes together or each E-box separately, plus MNT and MAX expression vectors. We found that the expression of MNT and MAX led to a decrease in the luciferase activity, mostly in the non-canonical E-box 2, result that was also reproduced in HEK293T. As for protein regulation, MNT overexpression in UR61 cells could not be achieved unless we treated cells with bortezomib, a proteasome inhibitor. However, mutants lacking the SID domain or different PEST sequences at N- or C-terminal domain did not show this autoregulation, suggesting that these regions are involved in MNT degradation and that the control of MNT levels is driven by proteasome-dependent mechanisms in the absence of MAX. Conclusions: In summary, our data suggested that MNT together with MAX bound MNT promoter and negatively regulated it. At protein level, MNT levels seemed to be tightly regulated through proteasomal-dependent mechanisms. MNT SID domain and PEST sequences could be involved in its autoregulation in MAX-deficient UR61 cells. No conflict of interest. 147 Molecular analysis of circulating tumour cells identifies distinct profiles in treatment na¨ıve chemosensitive and chemorefractory small cell lung cancer patients D.G. Rothwell1 , B. Mesquita1 , L. Carter1 , C. Smowton1 , H.S. Leong2 , D. Burt1 , C. Miller3 , F. Blackhall4 , C. Dive1 , G. Brady1 . 1 CRUK Manchester Institute, CEP, Manchester, United Kingdom, 2 CRUK Manchester Institute, Computational Biology Support, Manchester, United Kingdom, 3 CRUK Manchester Institute, Computational Biology Support and RNA Biology Group, Manchester, United Kingdom, 4 Christie NHS Foundation Trust, ICS, Manchester, United Kingdom Introduction: Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic disease with dismal prognosis. Though response rates to first line chemotherapy are high in 80% patients, progression free survival is short due to acquisition of chemotherapy resistance via unknown mechanisms. 20% SCLC are initially refractory to chemotherapy. Due to the difficulty in collecting tissue biopsies in SCLC, blood, which can be sampled simply and routinely, provides a means of inferring the current genetic status of a patient’s tumour via analysis of circulating tumour cells (CTCs). This offers a minimally invasive approach to study drug resistance mechanisms, evaluate CTC heterogeneity and potentially reveal new drug targets. Here, we molecularly profile CTCs sampled at baseline from a cohort of patients who were shown to be either chemosensitive or chemorefractory (progressing before or after 90 days of treatment respectively). Material and Methods: CTCs (with WBC controls) were enriched and isolated from an initial cohort of 10 SCLC patients at baseline using CellSearch® and DEPArray™ technologies respectively. Following isolation, each cell underwent whole genome amplification (WGA) and copy number profiles were generated using low-depth whole genome sequencing (WGS). Bioinformatic analysis of the patterns of copy number aberrations (CNA) from chemosensitive and chemorefractory patients was then performed. Results and Discussion: To determine the utility of using CTCs to molecularly profile tumours we applied single cell WGA and next generation sequencing (NGS) to 80 CTCs and 24 corresponding white blood cells (WBC). Analysis of CNA patterns of CTCs found high levels of chromosomal aberration and marked differences from corresponding WBCs. Heterogeneity was also observed across patient samples. Analysis of baseline CTCs from sensitive and refractory patients suggested distinct CNA profiles which correlated with patients being chemorefractory or chemosensitive. Bioinformatic analysis of the CTC CNA profiles lead to the generation of a predictive classifier, which correctly called all the samples in the initial patient cohort. This signature is now being further tested in a cohort of chemosensitive and chemorefractory

CTC Derived Xenograft (CDX) models and validation set of SCLC patient samples. Conclusion: Our study highlights the accuracy and utility of the approach described for genetic profiling of single CTCs in SCLC and has led to development of a potential predictive classifier of chemosensitive vs chemorefractory disease to assist patient management. Further studies are underway which aim to validate this predictive classifier and extend the analysis to include whole exome sequencing to identify genetic changes associated with the acquisition of chemotherapy resistance in SCLC. No conflict of interest. 148 The binding profile of proteins is maintained in snap-frozen tissue A. Uwineza1,2 , C. Kenny1,2 , M. O’Sullivan1,2 . 1 National Children’s Research Centre, Cancer Resaerch, Dublin 12, Ireland, 2 Trinity College Dublin, Clinical Medicine Research, Dublin, Ireland Background: Traditionally, fresh tissue or cell lines are used for chromatin immunoprecipitation (ChIP), a technique used to study the interaction between proteins and DNA. We investigated whether snap-freezing of tissue has an influence on the binding profile of proteins by comparing direct (H3K27Ac, H3K27Me3 and H3K4Me1) and indirect (Brg1 and PB1) DNA-associated proteins in fresh and snap-frozen thymus. Material and Methods: Fresh and snap-frozen thymus were homogenized in parallel, followed by fixation in 1% formaldehyde. The cross-linking was stopped by addition of glycine at a final concentration of 0.125M. Fixed tissue was dissolved in SDS buffer with 1 mg/mL Leupeptin, 1 mg/mL Aprotenin, 10mM PMSF and snap-frozen. Before sonication, the fixed tissue was resuspended in IP buffer. Chromatin was sheared to yield DNA fragments with a bulk size of 200–1500bp. For each immunoprecipitation, 1 mL of diluted lysate was pre-cleared by the addition of blocked beads. Blocking was performed in TE buffer containing 0.5 mg/mL fatty acid-free BSA and 0.2 mg/mL salmon sperm DNA. Samples were incubated with the antibodies overnight at 4ºC. Immune complexes were recovered by adding blocked beads, and incubated at 4ºC. Beads were washed sequentially with 1 mL of the following buffers at 4ºC: Mixed Micelle Buffer (3×), Buffer 500 (2×), LiCl Detergent Wash Buffer (2×) and TE buffer (1×). To elute, ChIP elution buffer was added to the beads and incubated at 65ºC. The beads were pelleted and the supernatant was incubated overnight at 65ºC. DNA in the eluate was phenol/chloroform-extracted and DNA resuspended in water. Quantitative realtime PCR was performed using SYBR Green detection chemistry system (Applied Biosystems). Enrichments were normalized to the input. Results: Comparison of the binding profiles of H3K27Ac, H3K4Me1 and H3K27Me3 in fresh and snap-frozen tissue shows that the binding profiles of these histone post-translational modifications (PTMs) are comparable across these tissue substrates. Thus, the binding profiles of these PTMs are maintained in snap-frozen tissue. We found that the binding profiles of the indirect DNA-associated proteins, Brg1 and PB1, are similarly maintained in snap-frozen tissue. We in fact observed higher gene enrichments in ChIPs performed with snap-frozen tissue in comparison to fresh tissue for all proteins except PB1. These results suggest that protein-DNA interactions are slightly better preserved in snap-frozen tissue during ChIP. Conclusion: We have shown that the binding profiles of direct and indirect DNA-associated proteins are retained in snap-frozen tissue. Although fresh tissue and cell lines are the classical substrates for ChIP, these results demonstrate that snap-frozen tissue is also a good ChIP substrate, thus widening applicability of this valuable technique. No conflict of interest. 149 The expressions of genes causing histone modifications in colorectal carcinomas with signet ring cells S. Yalcin1 , Y. Karslioglu2 , E. Celikmakas3 , B. Kurt2 , O. Onguru2 . 1 Ahi Evran University- Faculty of Engineering and Architecture, Department of Food Engineering, Ankara, Turkey, 2 Gulhane Military Medical Academy, Department of Pathology, Ankara, Turkey, 3 Gulhane Military Medical Academy, School of Medicine, Ankara, Turkey Introduction: Histone proteins are essential for packaging DNA segments and can affect gene expression considerably. Histones can be the subject of various posttranslational modifications like acetylation, methylation, phosphorylation, and ubiquitylation. Among these modifications, acetylation and methylation constitute the majority. These changes are mediated by some special enzymes. Chromosomal functions can be altered extensively as a result of histone modifications. It has been proven in several studies that these alterations play critical roles in tumorigenesis of many types of cancer. In this study, our aim was to analyze the expression of specialized enzymes which are important for histone modifications in colorectal cancers with signet ring cells. Material and Method: Twenty-eight (28) resected specimens of colorectal cancers with signet ring cells (CRC-SRC) were retrieved from Gulhane Military Medical Academy between the years of 1990 and 2015. Eleven (11) cases of

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 normal colonic mucosa which obtained from various non-neoplastic conditions were used as control. We studied the following histone modifying enzymes: Enhancer of zeste homolog 2 (EZH2; histone-lysine N-methyltransferase), E1A binding protein p300 (EP300; histone acetyltransferase), DNA topoisomerase 1 (TOP1), Lysine-specific histone demethylase 1A (KDM1A) and 4B (KDM4B). We used quantitative RT-PCR arrays to assess the fold-changes of expression in terms of these enzymes’ genes. Results: Based on the RT-PCR data, we observed increased expressions of TOP1 (~0.9X), EP300 (~0.6X), and EZH2 (~0.5X) genes while KDM1A (~−1.1X) and KDM4B (~−2.3X) showed much lower expression levels, as distinct from normal tissue counterparts. Conclusion: Our data have revealed that the genes responsible for histone modifications are being shown differential expressions in adenocarcinomas with signet ring cells compared to the nonneoplastic cells. We think that up or down-regulation of different sets of histone modification enzymes can have vast effects on the whole genome by making various lengths of chromatin sealed i.e. unable to be expressed. Variations in the patterns of modifications on histone proteins may play pivotal roles in tumor initiation and evolution. As a consequence, this event can be one of the several phenomena underlying different morphological characteristics and biological behavior of malignant tumors. Their roles must be clarified by further studies on different types of tumors. No conflict of interest. 150 Deregulation of IGF2, FZD10, MAPK3, SMAD4 and SRF expression in colorectal cancer M. Ibarrola-Villava1 , N. Tarazona1 , V. Gambardella1 , C. Mongort2 , S. Navarro2 , S. Garcia-Botello3 , S. Rosello1 , A. Cervantes1 , G. Ribas1 . 1 Biomedical Research Institute INCLIVA − University of Valencia, Medical Oncology Unit, Valencia, Spain, 2 Biomedical Research Institute INCLIVA − University of Valencia, Department of Pathology, Valencia, Spain, 3 Clinic University Hospital of Valencia, Department of Surgery, Valencia, Spain Introduction: Colorectal cancer (CRC) is a frequent lethal disease with heterogeneous outcomes and drug responses. Understanding the roles that genetic and epigenetic alterations play in CRC pathogenesis has increased over the last decade and emerging genes have been described related with the disease. The aim of our study was to determine the expression levels of five CRC related genes: IGF2, FZD10, MAPK3, SMAD4 and SRF. Material and Methods: We compared IGF2, FZD10, SMAD4, SRF and MAPK3 mRNA expression levels among colorectal tumour and normal adjacent mucosa samples using quantitative real-time reverse transcription PCR. A total of 56 paired-samples were included in the study. Furthermore, the analysis of 238 mutations across 19 common oncogenes was performed using the Sequenom Oncocarta Panel v1.0 (Sequenom Hamburg, Germany). All study subjects gave written informed consent, and the study was approved by the Biomedical Research Institute INCLIVA Ethics Committee. Results: When compared tumour tissues with adjacent non-tumour tissues a lesser MAPK3 mRNA expression was found in 35 (72.91%) tumours (P = 1.69×10−3 ). Similarly, SMAD4 and SRF expression was slightly lesser in 29 tumour samples although no statistical difference was found (P = 0.19 and P = 0.40, respectively). Although an increased IGF2 expression was detected in tumour tissues, it did not remain statistically significant (P = 0.10). FZD10 expression was null among all the samples analysed. Regarding mutational status, 68.3% of the samples harboured at least one mutation, mainly in the KRAS gene. One expression cluster was identified and most of the samples included are mutated in KRAS G12 position (55%). Conclusions: Our data suggest that MAPK3 may play an important role in CRC and may serve as potential target for therapy. No conflict of interest. 151 Methylation profile of candidate genes in gastric cancer with microsatellite instability using high-throughput MALDI-TOF mass array technology: The role of RUNX3 in cancer progression 1 1 , , T. Fleitas1 , M. Ibarrola-Villava1 , M.C. Pena-Chilet ˜ M.J. Llorca Cardenosa ˜ C. Mongort2 , L. Navarro2 , S. Navarro2 , G. Ribas1 , A. Cervantes1 . 1 Biomedical Research Institute INCLIVA- University of Valencia, Department of Medical Oncology, Valencia, Spain, 2 Biomedical Research Institute INCLIVAUniversity of Valencia, Department of Pathology, Valencia, Spain

Introduction: Epigenetic alterations through DNA methylation are known to play a key role in inhibiting the expression of tumour-related genes. Gastric cancers (GC) with microsatellite instability (MSI) are a distinct subgroup, presenting differential molecular, clinicopathological and prognostic patterns, and have been associated with female gender, antral tumour location, hypermethylation (CpG Island Methylation phenotype, CIMP), better survival rate, underexpression of MLH1, mutations in ARID1A, KRAS, HER2 and affection of PIK3/PTEN/mTOR pathway. The aim of our study has been to perform a methylation panel of candidate genes to study the correlation of

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MSI with the hypermethylation of CIMP-related genes and the association with other clinicopathological features. Material and Method: The methylation status of 47 promoter-CpG islands of 5 genes was studied through MALDI-TOF mass spectrometry analysis of basespecifically cleaved amplification in 34 microsatellite stable (MSS) GC samples, 24 MSI, 18 cancer-free GC, 6 MSS cell lines and 4 MSI cell lines. HER2, HER3, ARID1A and RUNX3 expression was determined by immunohistochemistry (IHC). MSI status was analyzed by IHC and by a multiplex PCR amplification of a 5 microsatellite-marker panel and subsequently sequenciation of denaturalized PCR products. Validation of RUNX3 expression by IHC in tumour, normal adjacent mucosa and inflammatory peritumoural infiltrate was performed in 40 GC samples. Results and Discussion: Unsupervised hierarchical clustering of methylation levels showed no aggrupation of MSI samples. Methylation of APC, CDH1, MLH1 and CDKN2A was higher in GC compared to normal samples, showing 15 statistical significant different CpGs. Conversely, RUNX3 was highly methylated in normal tissue comparing to tumour, presenting statistical significant differences in 10 CpGs. Correlated to these findings, IHC analysis of RUNX3 expression showed protein silencing in cancer and normal mucosa, but a high expression in inflammatory peritumoural infiltrate in almost all cases. Methylation analysis of peritumoural infiltrate compared to tumour and normal mucosa will be discussed, in order to correlate epigenetic silencing and protein expression, and to find the roles of RUNX3 in cancer progression by its expression in the inflammatory cells. Conclusion: Hypermethylation of APC, CDH1, MLH1 and CDKN2A genes is associated with GC. RUNX3 high expression in inflammatory infiltrate confirms the importance of RUNX3 expression in GC through inflammatory processes. No conflict of interest.

152 The Shiga Toxin receptor Gb3/CD77, a promising therapeutical target in gastrointestinal cancer, is epigenetically regulated M. Perl1 , M. Gehrmann2,3 , U. Nitsche1 , G. Multhoff2,3 , L. Johannes4,5 , ¨ Munchen, K.P. Janssen1 . 1 Klinikum rechts der Isar − Technische Universitat ¨ Surgery, Munich, Germany, 2 Klinikum rechts der Isar − Technische 3 ¨ Munchen, Universitat ¨ Radiation Oncology, Munich, Germany, Helmholtz Zentrum Munchen ¨ − Deutsches Forschungszentrum fur ¨ Gesundheit und Umwelt, Clinical Cooperation Group CCG “Innate Immunity in Tumor Biology”, Munich, Germany, 4 Institut Curie, CNRS UMR3666, Paris, France, 5 Institut Curie, INSERM U1143, Paris, France Introduction: Gastrointestinal cancer comprises some of the tumor entities with worst prognosis. Therefore, new tools for screening and targeted drug delivery are urgently needed. We have previously shown that the non-toxic B subunit of the bacterially derived Shiga Toxin (StxB) can be exploited for tumor targeting, since it binds with high affinity to the glycosphingolipid Gb3/CD77 at the cell surface. Following receptor binding, StxB gets taken up rapidly along a retrograde Golgi-ER route, thereby avoiding lysosomal degradation. Gb3 is overexpressed in colon cancer and its metastases, stomach and pancreatic cancer, and StxB coupled to radioactive or fluorescent probes can be used for tumor detection in murine models. Moreover, we could demonstrate targeted delivery of cytotoxic compounds to tumor cells by StxB. Thus, the Gb3-ligand STxB is a promising tool for cancer therapy. However, the molecular mechanisms leading to aberrant biosynthesis of Gb3, as well as the consequences of elevated Gb3 levels for malignancy are yet unknown. Material and Method: Regulation mechanisms of Gb3 biosynthesis were analyzed in patient samples of gastric (n = 50) and colon cancer (n = 66), and established cell lines. We quantified Gb3 expression by flow cytometry, fluorescence microscopy and thin layer chromatography, and compared the results to clinical and molecular parameters. Additionally, cells were treated in vitro with pharmacological agents, or with StxB coupled to the topoisomerase I inhibitor SN38. Results and Discussion: SN38 coupled covalently to StxB showed greatly enhanced cytotoxicity, when compared to the clinically approved SN38 prodrug irinotecan in cancer cells overexpressing Gb3 on their surface while sparing Gb3 negative cells. However, we found that only a fraction of clinical samples, and cancer cell lines from gastric, pancreatic, esophageal or colon cancer express Gb3. We could show that Gb3 surface exposure is critically dependent on expression of the enzyme Gb3-synthase (A4GALT), and Gb3 levels are tightly correlated with A4GALT expression in cancer cell lines. A4GALT presents several CpG-islands in its presumed regulatory region. In Gb3 deficient gastric (AGS, MKN45) and colon cancer (DLD1) cell lines, A4GALT expression as well as Gb3 surface levels could be restored by inhibition of DNA methylation or histone deacetylation. In contrast, inhibition of glucosylceramide synthase or histone methylation had no effect on A4GALT expression. Preliminary evidence indicates aberrant CpG methylation of the A4GALT promoter in the Gb3-deficient cell line DLD1. Conclusion: These findings not only allow to stratify patients for future STxB-based targeted therapies, they offer a possible explanation for

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deregulated biosynthesis of glycosphingolipids in cancer cells by epigenetic reprogramming. No conflict of interest. 153 Exploiting DNA damage repair defects for effective targeting of acute myeloid leukaemia by PARP inhibitors M.T. Esposito1 , L. Zhao2 , T.K. Fung2 , J. Rane2 , A. Wilson2 , N. Martin3 , J. Gil4 , A.Y. Leung5 , A. Ashworth6 , C.W.E. So7 . 1 University of East London, Health Sport and Bioscience, London, United Kingdom, 2 King’s College London, Haematological Medicine Leukemia and Stem cell biology group, London, United Kingdom, 3 Centre de Recerche en cancerologie de Lyon, Senescence Escape Mechanisms Lab, Lyon, France, 4 Imperial College London, Medical Research Council Clinical Sciences Centre- Cell Proliferation group, London, United Kingdom, 5 The University of Hong Kong, Department of Medicine, Hong Kong, Hong Kong, 6 University of California- San Francisco UCSF, Hellen Diller Family Comprehensive Cancer Center, San Francisco, USA, 7 King’s College London, Haematological Medicine- Leukemia and Stem cell biology group, London, United Kingdom Introduction: Inhibitors of Poly-ADP-Ribose Polymerase (PARPi) have been successfully developed and approved by FDA for treatment of ovarian cancer carrying mutations in DNA damage response (DDR) genes BRCA1/BRCA2. In Acute Myeloid Leukemia (AML) patients, these mutations are extremely rare. However, chromosomal rearrangements generate chimeric oncofusion proteins that, by acting as transcriptional regulators, impair DDR gene expression. This prompted us to test the efficacy of using PARPi in AML. Materials and Methods: PARPi were tested in vitro and in vivo. In vitro experiments were carried out in mouse and human leukemic cell lines. Mouse leukemic cells expressing the oncofusion gene of interest were generated by Retroviral Transduction Transformation Assay (RTTA). Results: Leukemic cells driven by the oncofusion genes, AML1-ETO and PML-RARa, are sensitive to PARP inhibition whereas cells harbouring MLLAF9 translocation are resistant. Treatment of AML1-ETO and PML-RARa leukemic cells with PARPi induces apoptosis, senescence, cell cycle arrest and differentiation in vitro and significantly prolongs survival in vivo. By using gH2AX and RAD51 as markers of DNA damage and HR (Homologous Recombination) we showed that AML1-ETO and PML-RARa cells accumulate DNA damage and are defective in recruiting RAD51 to DNA damage foci upon PARPi treatment. Further analysis revealed that the expression of a number of genes that are involved in the HR pathway are reduced in AML1-ETO and PML-RARa cells including Rad51, Brca2 and Rpa1. This suggests that AML-ETO and PML-RARa are sensitive to PARPi as result of defective DDR. We showed that HOXA9, a key downstream target of MLL-fusions plays a critical role in promoting expression of HR genes and thus providing evidence by which MLL-AF9 are resistant to PARPi. Depletion of Hoxa9 reduces the expression of Rad51 and Brca2 in MLL-AF9 cells and confers PARPi sensitivity in MLL-AF9 leukemic cells, compromising its ability to form colonies, repair DNA damage and prolongs survival in mouse models. Conversely, HOXA9 overexpression rendered AML1-ETO and PML-RARa cells resistant to PARPi. Likewise, pharmacological suppression of Hoxa9, using GSK3 inhibitor LiCl, can also sensitize MLL-AF9 cells to PARPi and prolongs survival in our mouse model. Discussion: Our data indicate that PARPi might offer a new therapeutic strategy for patients with AML1-ETO or PML-RARa translocations. More importantly, we showed for the first time that HoxA9 can activate a potential DNA repair back-up pathway. PARPi in combination with pharmacological inhibitors of HOXA9 may represent a novel avenue for tailored therapeutic targeting of the aggressive MLL leukaemia. Conflict of interest: Ownership: Alan Ashworth: Patents related to the development of PARP inhibitors with AstraZeneca. Advisory Board: Alan Ashworth: Consulting or Advisory Role: Genentech, Sun Pharma, GlaxoSmithKline, Novartis. 154 Targeted next generation sequencing of Bulgarian prostate cancer patients finds new somatic mutations and reflects disease heterogeneity D. Kachakova1 , A. Vlahova2 , K. Mihova1 , A. Mitkova1 , I. Popov1 , E. Popov3 , S. Christova2 , C. Slavov3 , V. Mitev1 , R. Kaneva1 . 1 Molecular Medicine Centre, Department of Medical Chemistry and Biochemistry- Medical University of Sofia, Sofia, Bulgaria, 2 General and Clinical Pathology Clinic- Alexandrovska University Hospital, Department of General and Clinical Pathology- Medical University of Sofia, Sofia, Bulgaria, 3 Clinic of Urology- Alexandrovska University Hospital, Department of Urology- Medical University of Sofia, Sofia, Bulgaria Background: Prostate cancer (PC) continues to be most frequently occurring cancer in men after lung cancer and it is second leading cause of cancer related deaths among men worldwide. Despite its clinical significance little is known about its etiology. Novel therapeutic approaches are needed. PC shows biological heterogeneity which likely reflects underlying genomic

diversity. The oncogenic landscape of PC has become clearer with nextgeneration sequencing (NGS) studies. NGS could help in investigations of the mechanisms of prostate cancerogenesis and finding new therapeutic targets. Material and Methods: Twelve paraffin embedded radical prostatectomy (RP) tissue samples from patients with PC were used in this study. DNA was extracted from part of the RP samples containing more than 75% tumour tissues using QIAamp DNA FFPE Tissue kit. NGS was performed on MiSeq sequencer. Sequencing amplicon libraries were prepared using TruSeq Amplicon Cancer Panel (TSACP), according to the manufacturer’s instructions. For variant calling human genome build 19 was used as reference. Variants were filtered according the following criteria: sequencing quality (GQX above 30), consequence of variants and alternative variant frequency above 5%. Then pathogenic or likely pathogenic variants were considered only. Results and Discussion: Variants passing sequence quality were between 34 and 259 per sample. Among them between 2 and 28 variants per sample have some consequence and are with alternative allele frequency above 5%. In three of the samples there were no pathogenic or likely pathogenic variants meeting the criteria. In the rest of the samples these variants vary between 1 and 10. Six pathogenic or likely pathogenic new mutations (2 missense, 3 stop gain and 1 frameshift) were found in RB1 gene in 4 of the patients. Such variants were also found in PTEN and ABL1 in 3 of the patients. Pathogenic and likely pathogenic variants were detected in APC, ATM, TP53, CDKN2A, FBXW7 and others. Most of these variants were new. Conclusion: The results from this pilot study show that most of the variants with protein consequence are with frequency below or around 5%. This observation could be explained with the heterogenic nature of the prostate tumours. New pathogenic or likely pathogenic variants were discovered which could direct to possible new therapeutic targets. Larger sample size is needed to fully understand the mutation landscape and evaluate the role of the newly identified somatic mutations as possible therapeutic targets and/or prediction biomarkers. No conflict of interest. 155 CRISPR/Cas-9-mediated targeting of TP53 and MYC to investigate antimitotic mode of action S. Littler1 , H. Whalley1 , S. Sousa1 , S. Taylor1 . 1 University of Manchester, MCP, Manchester, United Kingdom Background: Antimitotic drugs, e.g. the taxanes, are used widely to treat breast, ovarian and lung cancer. By inhibiting microtubule dynamics, antimitotic drugs disrupt cell division, causing cells to then die, either directly in mitosis or following “slippage” back into the next interphase. However, the molecular mechanisms governing cell fate following mitotic disruption are poorly understood. It is clear that p53 plays a major role in terms of postmitotic behavior and recently, we showed that MYC is also a major determinant of mitotic cell fate (Topham et al (2015, Cancer Cell)). To determine how p53 and MYC modulate antimitotic chemotherapy responses, we are using CRISPR/Cas-9 approaches in order to correlate p53 and MYC function with mitotic behavior and subsequent cell fate. Material and Method: Utilizing CRISPR/Cas-9 technology, we screened a panel of sgRNAs to identify guides that efficiently target TP53 and MYC in established cell lines. We used immunoblotting to validate gene targeting and characterized the resulting null lines using a variety of assays. We then introduced cDNAs to generate transgenic lines where p53 and MYC are under tetracycline control. Finally, we used donor constructs to introduce exogenous sequences into both TP53 and MYC via homologous-recombination-mediated repair. Results: We identified sgRNAs that efficiently inactivate TP53 and MYC by introducing mutations near the ATG. We have introduced tet-responsive p53 and MYC cDNAs allowing us to fine tune p53 and MYC levels. And finally, we have successfully introduced various fluorescent protein tags into the N-terminus of both p53 and MYC. We have started to analyze these lines using a variety of microscopy-based approaches and we will present our latest results. Conclusions: CRISPR/Cas-9 technology can efficiently target both TP53 and MYC, providing us with an opportunity to measure gene expression dynamics at a single-cell level, both in unperturbed mitosis and in response to antimitotic agents. Future experiments will hopefully enhance our understanding of the mechanisms regulating mitotic cell fate. No conflict of interest. 156 Search for predisposing alleles in Hungarian non-BRCA breast and ovarian cancer families 1 ´ 1 . 1 National Institute of , A. Bozsik1 , J. Papp1 , T. Vaszko´ 1 , E. Olah T. Pocza ´ Oncology, Molecular Genetics, Budapest, Hungary Introduction: Germline mutations in the BRCA1 and BRCA2 genes account for about 7% of all breast cancer cases in Hungary. Near 60% of patients with strong family history of hereditary breast and ovarian cancer (HBOC) syndrome were diagnosed negative for germline mutations in our routine

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 BRCA1/BRCA2 tests. Since next generation sequencing (NGS) opens new avenues to test many genes in several patients, we aimed at identifying novel HBOC susceptibility alleles. Material and Method: Twenty-six patients were tested for the presence of germline mutations in 94 hereditary tumor-associated genes using the Illumina TruSight Cancer Panel kit. For the enrichment of the DNA libraries, the TruSight Rapid Capture kit was used, whereas sequencing was carried out on an Illumina MiSeq platform. An in-house-built workflow was created using online available tools for secondary and tertiary data analysis. The results were compared to the outcome of the manufacturer’s MiSeq Reporter software analysis. In silico prediction tools were used to test for pathogenicity of selected variants. Results and Discussion: We have detected 3 clearly pathogenic variants in 3 genes in 4 patients. A nonsense mutation was found in the FANCM gene (c.1972C>T; p.Arg658*) in 2 patients, an insertion in FANCL (c.1096_1099dup; p.Thr367Asnfs*13) and a deletion in CHEK2 (c.277del; p.Trp93Glyfs*17) in one patient each. Using the XHMM software, we also detected a deletion in FANCM, whose validation is in progress. Additionally, 4 splice site variants, 10 missense variants and 2 in-frame micro-indels were predicted pathogenic by several in silico tools. Most of these candidate genes are related to DNA repair pathways and suspected to be involved in HBOC predisposition. Conclusion: Our study underlie the applicability of NGS in clinical diagnostic use, as we could identify rare susceptibilty alleles by this method. Three protein truncating variants and a copy number variant were found in three genes. These pathogenic alterations accounting for approximately 20% of the cases studied. We also detected several candidate variants, whose function as well as the magnitude of their risk modifying effect deserve further investigation. Acknowledgement: Our study was supported by the Hungarian Research Grant OTKA K-112228 to Edith Olah. ´ No conflict of interest. 157 Genetic and epigenetic evaluation of potentially important subtypes of clear cell sarcoma of kidney (CCSK) 4 ¨ , S.L.M. Gooskens5 , C. Kenny1,2 , S. Bausenwein3 , A. Lazaro2 , R. Furtwangler M. Van den Heuvel Eibrink5 , C. Vokuhl6 , I. Leuschner6 , N. Graf4 , M. Gessler3 , M.J. O’Sullivan1,2,7 . 1 University of Dublin- Trinity College, Oncology Division, Dublin 2, Ireland, 2 The National Children’s Research Centre- Gate 5- Our Lady’s Children’s Hospital- Crumlin-, Oncology Division, Dublin 12, Ireland, 3 Wuerzburg University, Theodor-Boveri-Institute/Biocenter and Developmental Biochemistry and Comprehensive Cancer Center Mainfranken-, Wuerzburg, Germany, 4 Saarland University Hospital, Department of Paediatric Oncology and Hematology, Homburg, Germany, 5 Princess Maxima Center for Pediatric Oncology/Hematology, Oncology/Hematology, Utrecht, Netherlands, 6 Christian Albrechts University, Paediatric Cancer Registry, Kiel, Germany, 7 Our Lady’s Children’s Hospital- Crumlin-, Histology Laboratory-, Dublin 12-, Ireland

Introduction: Clear Cell Sarcoma of the Kidney (CCSK), the second most common renal malignancy of childhood, is an aggressive tumour of unknown cellular origin. It typically arises in children below the age of five years, and has a propensity for bone, lung and brain metastases. The oncogenetic mechanisms underpinning CCSK are poorly understood and until recently, the only recurrent genetic aberration was t(10;17)(q22;p13), identified in up to 12% of cases and which was previously characterized in our group to show a 14-3-3e-NUTM2 fusion product encoded by YWHAE-NUTM2. Recently, an Internal Tandem Duplication (ITD) within the last exon of BCOR, encoding the PUFD domain, important in the interaction with PCGF1 and in the formation of the variant Polycomb Repressor Complex 1, was described in 20/20 CCSKs. In addition, BCOR was reportedly overexpressed in all cases. Interestingly, none of these 20 cases expressed the YWHAE-NUTM2 transcript. This observation raised consideration for distinct mutational subsets of CCSK. Material and Methods: We studied 159 CCSKs for BCOR ITD and YWHAE-NUTM2 fusion by RT-PCR followed by Sanger sequencing. Clinical data were available for 130/159 cases. We screened for alternative or additional oncogenic drivers in 10 CCSK tumours with matched germline DNA by Whole Genome Sequencing (WGS). Furthermore, targeted epigenetic profiling of CCSK was determined using antibodies to detect posttranslation modifications (PTMs) by Chromatin Immunoprecipitation followed by sequencing (ChIP-Seq). Results and Discussion: With our analysis of these 159 cases, we show for the first time that there are indeed distinct subsets of CCSK with these BCOR and YWHAE mutations, but furthermore, we demonstrate that a substantial proportion (11.8%) of CCSKs bear neither mutation when tested by these assays, raising the possibility of distinct aetiologies for subsets of CCSK. Clinical and gene expression differences observed between these subsets support this notion. BCOR mutations were all in-frame and consisted of 5 main forms of duplication with additional minor variations of these, resulting in a total of 15 mutation types, all sharing a common 13 amino acid motif. Moreover, cross-correlation between WGS and ChIP-Seq data provides new

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insights into the genomic and epigenomic landscape of this potentially lethal tumour. Conclusion: Our studies have revealed distinct subsets of CCSK with initial indications of divergent molecular biology and clinical behavior. These observations prompt further exploration of CCSK biology with a view to development of individualized treatment strategies for distinct subsets of CCSK in the future, in an effort to replace more toxic regimens currently in use. No conflict of interest. 158 Deep resequencing of the regions assigned by GWAS reveals new candidate breast cancer susceptibility variants 1 A. Bozsik1 , J. Papp1 , T. Vaszko´ 1 , T. Pocza ´ , T. Gyuris2 , B.L. Balint ´ 2 , E. Olah ´ 1. National Institute of Oncology, Molecular Genetics, Budapest, Hungary, 2 University of Debrecen, Biochemistry and Molecular Biology, Debrecen, Hungary 1

Introduction: Breast cancer is a common complex tumour type with high incidence and strong heritable components in 5−10% of all breast cancer cases. Known causative genes together with the associated regions identified by several GWA surveys performed in the last decade altogether explain only 20−25% of the germline susceptibility background of the hereditary breast cancer. One theory claims that “missing heritability” might be partially explained by the sum of rare, even individual variants with modest effects. Along these lines, we subjected the chromosome loci designated by the most relevant GWAS to massively parallel resequencing in order to detect candidate variants with possible pathogenicity. Material and Method: We performed next-generation Illumina deep resequencing of 53 targeted genes positioned in a 1 Mb neighbourhood of each of the most significant SNPs at 12 GWAS loci in 120 early-onset female and 60 male primary invasive breast cancer patients. Libraries were prepared using the Agilent SureSelect XT2 enrichment method and sequenced on an Illumina HiScan SQ platform. The secondary bioinformatics evaluation was carried out by optimized in-house scripts. The yielded variant calls were appropriately quality-controlled and subjected to extensive in silico annotations and predictions (including missense, splice, expression regulation computations) to assess their possible pathogenicity. Results and Discussion: The exhaustive sequencing led to the identification of 2845 high-confidence variants, 724 of them were unique, not listed in variant databases at the time of evaluation. Annotation of the variants resulted in the detection of 13 clear-cut mutations (7 nonsense, 4 canonical splice, 2 frameshifts) as well as 30 predicted pathogenic variants with minor allele frequency 0.05 in a total of 20 genes. Eight of them (SYNE1, ATE1, RTKN2, ZFYVE26, DAP, ROPN1L, CHD9, ZNF365) are of especially high interest, with several putatively causative variants genotyped within. Conclusion: These candidate genes may contribute to the complex architecture of genomic susceptibility to breast cancer. Follow-up studies as well as functional evaluations are required to confirm the inferred pathogenic nature of their variants. Acknowledgement: This study was supported by the Hungarian Research Grant KTIA-OTKA K112228 to Edith Olah. No conflict of interest. 159 In vivo overexpression of EMI1 promotes chromosome instability and tumorigenesis P. Duijf1 . 1 University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia Background: The anaphase-promoting complex/cyclosome (APC/C) controls cell cycle progression by sequentially targeting various substrates for degradation. Many cell cycle genes, such as Early Mitotic Inhibitor 1 (EMI1 or FBXO5), which inhibits the APC/C from S phase to early mitosis, are often aberrantly expressed in cancer. However, if and how their misexpression may drive tumorigenesis in vivo mostly remains unclear. Materials and Methods: In a combined total of over 8,800 tumor and matched normal control tissue samples, we compared EMI1 mRNA and protein levels by, among others, Western blot analysis and immunohistochemistry on tissue microarrays. In addition, we performed patient survival analysis. We also generated a transgenic mouse model in which we overexpressed Emi1 in vivo. Its cancer phenotype was analyzed and the levels of aneuploidy, chromosome missegregation and mitotic abnormalities were assessed in the tumors using fluorescence in situ hybridization and H&E staining. Live-cell imaging was performed to study the effects of EMI1 overexpression on mitotic progression and chromosome segregation in real time. Finally, we determined the extent to which EMI1 expression correlates with the degree of aneuploidy in human cancers. Results: We find that EMI1 overexpression is widespread in human solid tumors but not in blood cancers. In solid cancers, EMI1 overexpression is a strong prognostic marker for poor patient outcome. We demonstrate causality in vivo, as Emi1-overexpressing animals develop a wide variety of solid tumors, in particular adenomas and carcinomas with inflammation and lymphocyte

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infiltration, but not blood cancers. These tumors are significantly larger and more penetrant, abundant, proliferative and metastatic than control tumors. In addition, they are highly aneuploid with tumor cells frequently being in early mitosis and showing mitotic abnormalities, including lagging and incorrectly segregating chromosomes. We further show in vitro that EMI1 overexpression promotes chromosome instability following delayed chromosome alignment and anaphase onset. In human solid tumors, EMI1 is co-expressed with many markers for chromosome instability and EMI1 overexpression is a stronger marker for chromosome instability than most well established ones. Conclusions: Emi1 overexpression promotes chromosome instability and the formation of solid cancers in vivo. This indicates that Emi1 overexpression actively drives solid tumorigenesis. These novel mechanistic insights have important clinical implications. No conflict of interest. 160 Understanding loss of SDF1 expression in colon tumors R. Benbrika Nehmar1 , B. Romain1 , L. Marisa2 , C. Bour1 , I. Duluc3 , E. Pencreach1 , D. Guenot1 . 1 EA3430, Bas Rhin, Strasbourg, France, 2 Ligue contre le cancer, ˆıle-de-France, Paris, France, 3 U1113, Bas Rhin, Strasbourg, France Background: SDF1 is a chemokine implicated in carcinogenesis for proliferation and migration during metastatic dissemination. In colon cancer, SDF1 is highly expressed in organs known to be metastatic sites. According to the literature, SDF1 expression in the primary tumor is either overexpressed or lost. Therefore, the SDF1 expression was evaluated in a gene expression data set (n = 519) colon carcinomas and demonstrated a decrease in 75% of the tumors. Our aim was to confirm the loss of SDF1 on a new cohort, to analyze whether default in SDF1 promoter methylation and/or acetylation could induce this loss and propose that the tumor cells not expressing SDF1 will follow a SDF1 gradient to disseminate in metastatic organs. Material and Methods: The SDF1 expression was measured in 40 human colon tumors by qRT-PCR and immunohistochemistry. SDF1 promoter methylation was analyzed with whole-genome DNA methylation ChIP (n = 85). SDF1 promoter acetylation was evaluated by ChIP on colon cell lines. APCD14 mice were treated with valproate, a histone deacetylase (HDAC) inhibitor. Finally, dedicated PCR arrays were used to detect epigenetic deregulated genes. Results: As compared to the normal mucosa, we confirmed the loss of SDF1 expression in 97% of the tumors of the new cohort both at the transcript and protein level. Interestingly, a similar loss was found in tumors that spontaneously developed in the intestine of APCD14 mice. Methylome analysis demonstrated that the SDF1 promoter was methylated in only 30% of the MSS carcinomas. Therefore, the promoter hypermethylation does not fully explain SDF1 gene extinction. We then next evaluated whether SDF1 promoter acetylation default would be involved. Treatment of colon cell lines with HDAC inhibitors, valproate and butyrate, increased SDF1 promoter acetylation and reestablishes SDF1 expression in the three cell lines and inhibited cell migratory proprieties. Importantly, valproate treatment of APCD14 mice resulted in a decreased tumor number and re-expression of SDF1 in epithelial cells. These results suggest that SDF1 loss results from two epigenetic processes, partly methylation and mainly to a SDF1 promoter acetylation defect in 70% of the tumors. We also evaluated 20 colon carcinomas for the expression of 92 genes implicated in regulation of epigenetic processes. This screening allowed identifying overexpressed genes (like AURKA, DNMT3B) and down-regulated genes (like HDAC9, CIITA, KAT2B, RPS6KA5). These genes are described as regulating methylation and acetylation but also cell cycle and mitosis. Conclusion: In conclusion, these findings suggest that SDF1 silencing is mainly associated to histone deacetylation and to a lesser extend to a promoter meythylation, and that its re-expression has a major anti-tumor effect that could lead to new preventive or therapeutic strategies in colon cancer. No conflict of interest. 161 Functional evaluation of breast cancer case-associated non-coding variants in BRCA1/2 J. Sevcik1 , M. Janatova1 , Z. Kleibl1 , P. Kleiblova1 , M. Borecka1 , P. Zemankova1 , K. Zdarilova1 , L. Stolarova1 , M. Brown2 . 1 First Faculty of Medicine- Charles University, Institute of Biochemistry and Experimental Oncology, Prague, Czech Republic, 2 University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, Australia Background: Breast cancer (BC) is the most frequent malignancy in the female population worldwide. The high penetrant Mendelian pattern of familial BC indicates the presence of inherited genetic events that predispose to the disease. This possibility has been supported by the identification of two BC predisposition genes, BRCA1 and BRCA2. Following their discovery, the prevalence and penetrance of mutations in these genes has been studied extensively. Whilst the absolute majority of described disease-causing mutations have been found in the coding regions of these genes, it is becoming apparent that these only account for a relatively small proportion of

mutations underlying BC risk. Despite the relatively subtle impact of mutation in regulatory region to the gene and its product, such regulatory variants in many genes have been shown to contribute to cancer risk. The aim of this work was to functionally evaluate the consequences of BRCA1/2 non-coding variants, in order to help predict their contribution to BC risk. Material and Methods: The promoter and 5 UTR of both BRCA1 and BRCA2 were sequenced in a cohort of 600 BC cases, with no known coding region mutations, and 600 healthy controls. A subset of detected variants was prioritized for experimental analysis, based on the results of bioinformatics analysis, including the presence of transcription binding sites, histone modifications and the position of the active promoter. To determine the influence of particular sequence variant on the BRCA1/2 promoter activity, we conducted a luciferase-based reporter assay, using a set of human BC-derived cell lines. The effect of particular mutations on transcription factor/protein binding capacity was also determined, using electrophoretic mobility shift assay (EMSA) with whole nuclear extract. Result and Discussion: Our results show that analyzed variants cause significant decrease of BRCA1/2 promoter activity compared to the BRCA1/2 wild type promoter. Moreover, this decrease in activity is associated with qualitatively and/or quantitatively altered binding capacity of proteins to the affected promoter region. These results suggest that analyzed variants may alter the occupation of BRCA1/2 promoters by proteins/transcription factors, which in turn could result in altered expression of gene products. We conclude that analyzed BRCA1/2 non-coding variants could negatively affect the levels of BRCA1/2 tumor suppressors and thus contribute the BC risk. Acknowledgement: Supported by: AZV NV16-33444A. No conflict of interest. 162 Identification of pancreatic cancer susceptibility genes in the Czech Republic ´ a´ 1 , L. Stolarova1 , K. Zdarilova1 , M. Borecka1 , M. Janatova1 , P. Zemankov J. Soukupova1 , J. Sevcik1 , P. Kleiblova1 , Z. Kleibl1 . 1 Charles University in Prague- First Faculty of Medicine, Institute of Biochemistry and Experimental Oncology, Prague, Czech Republic Introduction: The Czech Republic belongs to countries with the highest incidence of pancreatic ductal adenocarcinoma (PDAC) worldwide. PDAC has the worst prognosis among common solid cancer diagnosis and only 7% of the patients survive 5 years after diagnosis. About 10% of PDAC cases have a familial component. Identification of inherited risk factors could help to reveal high-risk individuals and contribute to detection of the disease in the early stages when surgical treatment combined with chemotherapy is the only potentially curative treatment. In our study we aimed to identify new possible PDAC predisposition genes. Material and Method: We analyzed peripheral blood DNA samples of 19 highrisk PDAC patients using custom-designed panel next-generation sequencing (NGS; panel CZECANCA), targeting 219 genes. We also studied exon 6 of the NBN gene by HRM analysis in 241 unselected PDAC patients and in a group of 915 non-cancer control samples. Results and Discussion: We found two carriers of pathogenic germline mutations in genes previously described as PDAC predisposition genes: BRCA2 c.8755−1G>A, CHEK2 c.1100delC. In addition, we identified two carriers of mutations in genes not associated with PDAC susceptibility thus far. One patient harbored RET c.2136+2T>G. She was diagnosed at 71 years and had personal and family history of breast cancer (BC). The RET oncogene is usually associated with multiple endocrine neoplasia and medullary thyroid carcinoma. Another patient harbored NBN c.657del5 mutation. She was diagnosed at 64 years and had a family history of two cases of ovarian cancer, gastric cancer, and Hodgkin lymphoma. The mutation was found to be present in the proband’s sister deceased from gastric cancer. The NBN gene was recently found to predispose to various cancers including BC and prostate cancer. The NBN c.657del5 variant is the most frequent pathogenic NBN mutation and recurrent Slavic founder mutation. The analysis revealed overall frequency of c.657del5 mutation (5/241; 2.1%) significantly different from that in non-cancer controls (2/915; 0.2%; P = 0.006). Conclusion: Our results indicate that the NBN c.657del5 variant represents a novel PDAC-susceptibility allele increasing PDAC risk (OR = 9.7; 95% CI: 1.9 to 50.2). Therefore, it should be included in multi-gene panel testing of PDAC patients. Moreover, implementing panel NGS to genetic testing of PDAC patients can reveal carriers of pathogenic mutations in genes contributing to a wider range of cancer syndromes. These mutation carriers with PDAC can undergo a specific therapy and their relatives can be offered appropriate screening methods for associated tumors. Acknowledgement: Supported by grants: AZV NV15-28830A, AZV NV1629959A, PRVOUK-P27/LF1/1, and SVV-UK 260256/2016. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 163 CHEK2 gene analysis in 1020 high-risk breast and ovarian cancer patients in the Czech Republic L. Stolarova1 , P. Kleiblova1 , P. Zemankova1 , M. Janatova1 , M. Borecka1 , K. Zdarilova1 , J. Sevcik1 , J. Soukupova1 , Z. Kleibl1 . 1 First Faculty of Medicine- Charles University, Institute of Biochemistry and Experimental Oncology, Prague, Czech Republic Introduction: Breast cancer (BC) is the most frequent oncological diagnosis in Czech female population. Hereditary breast cancers (HBC) account for 5−10% of all cases. BRCA1 mutations represent the most frequent genetic alterations in Czech HBC patients while mutations in BRCA2 and other known BC-susceptibility genes occur with lower frequency. Our previous analysis showed that the recurrent CHEK2 variant c.1100delC affects 0.1. A total of 213 SNPs in 206 pre-miRNAs were analyzed in a cohort of patients (n = 74) and their corresponding controls (n = 160) using Goldengate Veracode technology. c2 or Fisher and FDR were used. Results: The most remarkably finding was the association detected between 4 SNPs located at 14q32 miRNA cluster. Interestingly, miRNAs in this cluster have been associated with MYC deregulation. Therefore, genetic variations in these miRNAs could lead to their downregulation, which in turn would lead to the overexpression of MYC. This result supports the idea that this region is a hotspot for the development of the disease. Conclusion: In conclusion, different variants in 14q32 miRNA cluster could be implicated in osteosarcoma susceptibility. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI 11/35) and Basque Government (IT66113). No conflict of interest. 173 Genetic variant in miR-618 in the risk of chronic lymphocytic leukemia I. Mart´ın-Guerrero1 , A. D´ıaz-Navarro1 , A. Gutierrez-Camino1 , A. Garc´ıa-Orad1,2 . 1 University of the Basque Country, Genetics- Physical Anthropology and Animal Physiology, Bilbao, Spain, 2 BioCruces Health Research Institute, Pediatrics, Barakaldo, Spain Background: Recent Genome Wide Association Studies (GWAS) have found new genetic variants associated with chronic lymphocytic leukemia (CLL) susceptibility, several of which were located in non-coding regions. In fact, nowadays it is known that more than 40% of significant variants associated with cancer risk are in non-coding regions, where non-coding RNAs are located. MicroRNAs (miRNAs) are one of the most studied non-coding RNAs that have been involved in the risk of CLL. In a previous study of our group including 46 SNPs in 42 pre-miRNAs, we observed rs11614913 and rs2114358 associated with the risk of CLL. Considering these results, we aimed to determine the involvement in the risk of CLL of all the SNPs described in pre-miRNAs with a MAF >1% in a larger population. Material and Methods: A total of 213 SNPs in 206 miRNAs were genotyped in 164 CLL patients and 237 cancer-free controls using Veracode GoldenGate Technology (Illumina). Results: One polymorphism at miR-618 showed the most significant association under the dominant model (OR = 0.49; 95% CI: 0.29–0.81; p = 0.005). Remarkably, this polymorphism was previously found to be associated with follicular lymphoma, altering the expression of miR-618. In addition, this miRNA was observed, in silico, to regulate genes associated with CLL, such as BCL2, LEF1 or QPCT. Conclusion: These findings suggest that polymorphisms in pre-miRNAs contribute to the risk of CLL. Large-scale studies are needed to validate the current findings. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI11/35) and Basque Government (IT661-13, S-PE13UN079). No conflict of interest.

Background: Recently, several Genome wide associations studies (GWAS) have found genetic variants associated with pediatric acute lymphoblastic leukemia (ALL) risk. The interest of these studies was mainly focused in coding regions. However, nowadays it is known that more than 40% of significant variants associated with cancer risk are situated in non-coding regions, where non-coding RNAs are located. One of the non-coding RNAs more related with cancer are microRNAs (miRNAs), which have been shown to be dysregulated in ALL, suggesting that they may have a role in ALL risk. Therefore, variants in miRNAs that may alter its function could contribute to childhood B-ALL predisposition. The aim of this study was to determine if SNPs in miRNAs are involved in B-ALL susceptibility. Material and Methods: Blood samples of 296 B-cell ALL patients in complete remission and 426 healthy controls of Spanish and a Slovenian cohort were analyzed. We selected all the SNPs described in pre-miRNAs with a MAF >1% (213 SNPs in 206 miRNAs). VeraCode GoldenGate platform was used. Results: Among the most interesting results, a variant in the seed region of miR-3117 was associated with B-ALL in both populations (p = 0.006). Of note is that in silico analyses show that miR-3117 could regulate genes of the MAPK pathway, which is known to promote leukemogenesis. Conclusion: Our results suggest that a SNP in miR-3117 may be involved in B-ALL susceptibility and give new keys to understand its biology. Acknowledgement: This project was supported by RETICS (RD12/0036/0060, RD12/0036/0036), UPV/EHU (UFI 11/35) and Basque Government (IT661-13, S-PE12UN060). No conflict of interest.

175 The KDM5B demethylase in the normal and malignant mammary gland F. Kogera1 , C. Steven1 , B. Spencer-Dene2 , G. Picco1 , V. Tajadura-Ortega1 , Y.H. Chen3 , E. Bennett3 , J. Quist4 , J. Taylor-Papadimitriou1 , J. Burchell1 . 1 KCL, Research Oncology, London, United Kingdom, 2 Cancer Research UKCrick Institute, Experimental Histopathology Lab, London, United Kingdom, 3 University of Copenhagen, Copenhagen Centre for Glycomics, Copenhagen, Denmark, 4 KCL, Breast Cancer Now Unit, London, United Kingdom Background: It is becoming clear that changes occurring in cancer, including breast cancer reflect a dysregulated epigenome. Histone marks play a major role in determining the chromatin state of a cell, and the H3K4me3 mark is associated with active transcription. The KDM5 family of histone demethylases specifically demethylate the H3K4me3 mark and therefore act as transcriptional repressors. KDM5B, a KDM5 family member, was cloned in our lab as being down regulated when a cell over expressing HER2 was treated with Herceptin, and has been found to be widely expressed in breast cancer. Here, we investigate the role of KDM5B in the normal gland and in breast cancer, using breast cancer cell lines and a transgenic mouse model. Materials and Methods: Expression of KDM5B in breast cancers has been documented in samples from Cancer databases. Cell lines derived from different breast cancer subtypes have been analysed for KDM5B expression and the effect of a KO and KD of KDM5B examined. While constitutive KO of KDM5B in the mouse is embryonic lethal, a transgenic strain carrying a demethylase −ve mutant (D ARID domain deleted) is viable. This strain has been used to document an important function for KDM5B in the developing and pregnant mammary gland. Results: High expression of KDM5B was observed in the ER+ and HER2+ breast cancer subtypes. In the DARID mouse mammary gland, a transient delay in mammary tree expansion at mid pregnancy is observed. At this stage, expansion of the luminal progenitor cells is evident and involves the JAK/STAT5 signalling pathway. However, in the DARID mouse, STAT5 activation is dramatically reduced, implicating a role of KDM5B in this activation. We find that, decreased STAT5 activation in the DARID gland is due to increased levels of Caveolin1 (CAV1), an inhibitor of STAT5 activation. Thus in the normal gland KDM5B represses CAV1 expression during development of the luminal cell lineage. The effect of CAV1 on STAT5 activation must be mediated in a paracrine fashion, as CAV1 is not expressed in the luminal cells where STAT5 is functional but is limited to stromal and myoepithelial cells. KDM5B is expressed in these cells as well as in the luminal cells and thus could repress CAV1 expression.

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Conclusions: The importance of CAV1 expression in stromal cells in breast cancer is well documented and high expression is associated with good prognosis. Conversely, expression of CAV1 is indicated to be a marker of triple negative cancers. A CRISPR KO of KDM5B in a HER2+ cell line shows increased CAV1 expression, signifying regulation of CAV1 by KDM5B. We are currently setting up methods to examine the effect of KDM5B on CAV1 expression in breast cancer associated fibroblasts to understand the role of cell phenotype in the interactions between KDM5B and CAV1 in modifying JAK/STAT signalling pathways in breast cancer. No conflict of interest. 177 Comprehensive characterization of matched pre-treatment biopsies and residual disease of chemotherapy treated breast cancer M. Hoogstraat1 , E. Lips2 , L. Mulder2 , P. Nederlof3 , G. Sonke4 , S. Rodenhuis4 , J. Wesseling2 , L.F.A. Wessels1 . 1 Netherlands Cancer Institute, Molecular Carcinogenesis, Amsterdam, Netherlands, 2 Netherlands Cancer Institute, Molecular Pathology, Amsterdam, Netherlands, 3 Netherlands Cancer Institute, Pathology, Amsterdam, Netherlands, 4 Netherlands Cancer Institute, Medical Oncology, Amsterdam, Netherlands Introduction: Neoadjuvant chemotherapy is standard of care for locally advanced breast cancer, unfortunately not all patients benefit from this treatment. Even after decades of research, we still cannot predict which tumors will and which ones will not respond. This may in part be due to tumor heterogeneity, as the sample taken before treatment not necessarily represents the tumor cell population that causes therapy resistance. Methods: To test this hypothesis and to study potential mechanisms of therapy resistance, we collected matched blood, pre-treatment and residual disease samples from 21 breast cancer patients treated with doxorubicin and cyclophosphamide in a neoadjuvant setting. Specifically, tumors were selected with a tumor percentage >50% after chemotherapy to enrich for resistant samples and ensure high quality data. RNA and whole exome sequencing data were generated to characterize somatic mutations, copy number alterations and gene expression profiles, and histopathological characteristics were determined to obtain a comprehensive profile of all tumor samples. Results: Both the comparison of somatic variants and copy number alterations revealed a very diverse image: in several cases, high-level amplifications, large genomic gains or losses, and mutations in known oncogenes or tumor suppressors such as MAP3K1 and RUNX1 were either lost or gained during treatment, while in other cases no such changes were detected. We observed a remarkable number of genetic alterations involved in cell cycle progression and DNA damage checkpoints, including amplification of MDM2, CCND1 and CDK4, and copy number loss or mutations in CDKN1B and ATM. Strikingly, both cases of CDKN1B loss were identified in pre-treatment samples and no longer detectable in the surgery specimen. In contrast, CCND1, CDK4 and MDM2 amplifications were retained, although CCND1 expression decreased significantly in CCND1 amplified tumors. In addition, eighty percent of tumors showed a decreased proliferation rate after chemotherapy, where the high-proliferative ER+ (Luminal B) tumors were most severely affected. This trend was also visible in a validation cohort of 94 ER+ samples, but the prognosis of Luminal B tumors that showed a decrease in proliferation was still significantly worse than that of Luminal A tumors that did not show an altered proliferation rate. Conclusion: Our results confirm that biologically relevant genomic alterations can differ between pre- and post-treatment samples, which greatly impedes biomarker discovery. In addition, our findings emphasize the aggressiveness and chemotherapy insensitivity of CCND1 amplified ER+ breast cancers, and stress the need for better treatment regimens for these patients. In contrast, genomic loss of CDKN1B may be a marker for (partial) sensitivity to chemotherapy. No conflict of interest. 178 Epigenetic control of CD24 expression in colorectal cancer M. Ayub1 , W. Bodmer1 . 1 University of Oxford, Oncology, Oxford, United Kingdom Understanding the mechanism and maintenance of heterogeneity in Colorectal Cancer (CRC) is of paramount interest. By cloning CRC cell lines we have demonstrated that CD24 based heterogeneity is quite common and methylation is directly involved in the evolution and maintenance of heterogeneity in Colorectal Cancer. LS174T, a CRC cell line was cloned using FACS and two clones were found to have differential expression of CD24. They differ in morphology, clonogenicity and growth rate. Later SW480, DLD1, RKO and HCT116 were also found to be heterogeneous with clones of different CD24 level. To understand the regulation of CD24, Bisulfite modified DNA from clones were sequenced. Our study has revealed a region near CD24 promoter region, which is directly methylated. When treated with 5-azacytidine, the CD24 negative clones re-expressed CD24 again. We have also found that CD24 is dynamically regulated and the expressions in clones change towards the expression profile

of their parents over time. This is a clear evidence of convergent evolution in cancer cell lines. CD24 is an important marker of cancer stem cell. So far there has been no clear understanding of the mechanism of its regulation. Here we have reported that CD24 is regulated by direct methylation in Colorectal Cancer (CRC) cell lines. We have also shown that clones with differential expression of CD24 can be isolated and stably maintained from CRC cell lines. No conflict of interest. 179 Digital sorting enables whole-exome and low-pass whole-genome sequencing from low tumor-content formalin-fixed paraffin embedded (FFPE) biopsies C. Forcato1 , J. Laliberte2 , C. Bolognesi1 , C. Schumacher2 , G. Buson1 , C. Mangano1 , P. Tononi1 , G. Medoro1 , T. Harkins2 , N. Manaresi1 . 1 Silicon Biosystems spa, R&D, Bologna, Italy, 2 Swift Biosciences Inc, R&D, Ann Arbor, USA Introduction: Recent data from ongoing basket trials show that for 20% of patients, biopsies are discarded for precision oncology because of too small sample size and/or low-tumor content. Here we describe a workflow for whole-exome (WES) and low-pass whole-genome (WGS) sequencing of pure populations of tumor cells obtained from very low tumor-cellularity FFPE samples using DEPArray™ (Silicon Biosystems) sorting technology. Materials and Method: A FFPE 50 mm thick section from breast infiltrating ductal carcinoma, with 10% tumor cellularity, was dissociated into a cell suspension. Using DEPArray™ digital sorter, 419 cells from 100%-pure tumor and 497 cells from normal stromal subpopulations were recovered based on Keratin/Vimentin immunofluorescence and DNA content. After lysis, Illumina® compatible libraries were prepared from cell-sorted or DNA-extracted samples using Accel-NGS® 2S DNA Library Kit from Swift Biosciences, amplified, enriched using SeqCap EZ MedExome enrichment kit (Roche) and sequenced on a HiSeq 2500 at 29x mean coverage producing 100x2 paired-end reads. An aliquot of pre-capture libraries were used for a low-pass (0.06x mean coverage) WGS on MiSeq to analyze copy number alterations (CNA). Sequences were aligned to hg19 genome and preprocessed using open source software (BWA, Picard, GATK). Variant calls were obtained using samtools mpileup. In lowpass WGS analyses, Control-FREEC algorithm was used to obtain copy number calls, using the mode without control sample. Results and Discussion: Matched stromal/tumor analysis of B-allele frequency of heterozygous SNPs precisely identified Loss-of-Heterozygosity (LOH) regions, as well as Copy-gain regions, both undetectable on unsorted genomic DNA (gDNA). Genetic analyses readily revealed a clinically relevant homozygous (23/23 = 100% reads) TP53:p.L111R somatic mutation in a LOH region in sorted-tumor fraction, missed in unsorted gDNA, where the mutation was not called as present in only 1 read out of 20 (5%). Due to the low tumor cellularity, no copy number alterations were detected in low-pass WGS of unsorted gDNA, showing a flat profile undistinguishable from DEPArray™ purified stromal cells, whereas in sorted tumor cells we detected numerous copy-number alterations in form of gains and losses. Conclusions: DEPArray™ sorting combined with Accel-NGS® 2S library kit allow to obtain whole-exome and low-pass whole-genome data on pure tumor and stroma of FFPE samples, offering a clear picture of tumor-specific variants including LOH and copy-numbers independently of original tumor content. Conflict of interest: Other Substantive Relationships: Employees at Silicon Biosystems spa: C. Forcato, C. Bolognesi, G. Buson, C. Mangano, P. Tononi, G. Medoro, N. Manaresi. Employees at Swift Biosciences Inc: J. Laliberte, C. Schumacher, T. Harkins. 180 An oncogenomics-based in vivo screen identifies novel melanoma tumor-suppressors M. Olvedy1 , J.C. Tisserand2 , F. Luciani1 , B. Boeckx3 , F. Rambow1 , J. Wouters4 , J. Van den Oord4 , D. Lambrechts3 , P. De Sepulveda2 , J.C. Marine1 . 1 VIB, Center for the Biology of Disease, Leuven, Belgium, 2 ´ Institut National de la Sante´ et de la Recherche Medicale, Centre de ´ Recherche en Cancerologie de Marseille, Marseille, France, 3 VIB, Vesalius Research Center, Leuven, Belgium, 4 KU Leuven, Department of Pathology, Leuven, Belgium Introduction: Human cutaneous melanomas are notoriously known as the most aggressive and treatment-resistant cancers. Their complex cytogenetic profiles, due to extremely high mutation burden and frequent aneuploidies, however, hamper the discovery of genes that drive this cancer and thus obstruct the development of novel therapeutics to combat melanoma. There is therefore a pressing need for designing biologically meaningful approaches that would facilitate the identification and validation of the driver lesions. We have developed an innovative screen combining the oncogenomic analysis with cross-species comparison and in vivo validation using a refined mouse model system to discover novel genes driving melanoma. Methods: We collected 59 melanoma lesions from various genetically induced mouse melanoma models driven either by activating mutations in BRaf or

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 NRas and performed whole-exome sequencing to search for novel mutations and low coverage whole-genome sequencing (LC-WGS) to analyze copynumber variations (CNVs). Results: Although the average time of tumor onset in our melanomas was close to one year we found on average only 1 non-synonymous/indel mutation per lesion. Analysis of LC-WGS identified focal deletions in loci targeting wellknown tumor-suppressor genes (TSGs) such as Cdkn2a, Trp53 and Nf1, and focal amplifications of oncogenes such as Braf and Smo. In the p53-null melanomas we observed deletions targeting several mouse chromosomes. Using cross-species comparison we identified 17 TSGs which expression positively correlated with the overall survival of melanoma patients in The Cancer Genome Atlas (TCGA). In addition to the known TSGs such as Pten, Fbxw7 and Tet2 we identified TSGs that were not previously linked with melanoma. One gene was profoundly expressed in normal melanocytes and benign lesions, but was lost in a subset of human melanomas carrying poor prognosis. Notably, genetic inactivation of the gene in mice resulted in acceleration of melanomagenesis on the Tyr:CreERT2;BRafCA/+;Ptenl/l background, supporting its tumor-suppressor role in melanoma. Conclusions: Our results indicate that rather than mutations, focal and/or whole-arm aberrations are likely to drive melanomagenesis in spontaneous melanoma mouse models. Importantly, we identified several potential novel melanoma TSGs. No conflict of interest.

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transcripts were detected using the ChimeraScan algorithm. Samples were molecularly characterised by immunohistochemical (IHC) receptor, PAM50, IntClust and Triple-Negative (TNBC) subtyping, genomic instability measures, and gene signatures. We identified a total of 106 disrupted transcripts with proximal copy number alterations, and recurrent in at least five breast cancers. RNA-seq of BCCL and TCGA data, depicted 33/106 to be potential gene fusion partners. IHC receptor subtype-specificity was observed for 7 aborted transcripts (nTNBC = 1, nNon-TNBC = 6). Aborted transcripts were significantly less prevalent in the Luminal Androgen Receptor (LAR) subtype among TNBC subtypes (Fisher’s exact test p-value: 0.034). While 13 aborted transcripts had one fusion partner, 20 were involved in multiple fusion events (range 2−31, median = 3). Many full length genes of the disrupted transcripts have putative oncogenic roles (n = 31) and were previously found as fusion partners (n = 26). Three fusions (CLDN12-CDK14, FOXP1-EIF4E3 and BC090059-RIN2), recurrent across all datasets and with multiple junction-spanning and uniquely-aligned reads, are experimentally interrogated by the Archer FusionPlex assay in tumour samples. By integrating exon expression and copy number data, genes with disruption in their transcriptional units can be identified, some of which represent fusion genes. Characterisation of these transcripts, their partners, and their prevalence with certain molecular features will provide further knowledge to their biological significance in breast cancers. No conflict of interest.

181 Novel regulators of ESR1 activity T. Campbell1 , B. Ponder1 , K. Meyer1 . 1 CRUK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom Background: Studying transcription factor (TF) interactions and gene regulatory networks in breast cancer, we have recently identified two distinct and opposing groups of TFs associated with enhanced breast cancer risk, defining estrogen receptor-positive and estrogen receptor-negative breast cancer. We refer to these as group 1 and 2 TFs, respectively. The relative activity of these two groups of TFs has a dramatic effect on patient outcomes and is likely to influence the phenotypic plasticity observed in breast cancer. Here, we examine the molecular mechanisms underlying the opposing functions of the two networks. Material and Methods: We examined proteins that interact with the estrogen receptor (ESR1) using Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) and identified TFs known to bind ESR1 (FOXA1 and GATA3) as well as novel interactors that belong to the group 2 TFs. The interactions were confirmed using co-immunoprecipitation and microscopy experiments. The effect of the identified TFs on ESR1 transcriptional activity was analysed using qRT-PCR, proteome/transcriptome analysis and luciferase reporter assay experiments. Results and Discussion: We demonstrate for the first time that two TFs associated with estrogen receptor-negative disease progression interact with the ESR1-FOXA1 TF complex and are able to influence the transactivational potential of ESR1. Moreover, signalling through FGFR2, a known risk factor in breast cancer development, appears to augment this interaction and further repress ESR1 target gene expression. This finding is consistent with our novel observation that risk SNPs in the FGFR2 gene lead to a decrease in FGFR2 expression and activity, which is associated with greater estrogen responsiveness. Conclusions: We show that members of two opposing groups of risk TFs, associated with estrogen receptor-positive and -negative breast cancer, respectively, physically interact. Two TFs associated with estrogen receptornegative disease bind to the ESR1-FOXA1 TF complex, repress ESR1 activity and drive cells towards a less estrogen-dependent cancer phenotype. No conflict of interest. 182 The characterisation of potential fusion genes in breast cancer A. Mohd Noor1 , S. Maguire2 , J. Watkins1 , J. Quist1 , H. Mirza1 , K. Ougham1 , A. Tutt1 , C. Gillett3 , R. Natrajan2 , A. Grigoriadis1 . 1 King’s College London, Research Oncology, London, United Kingdom, 2 Institute of Cancer Research, Breast Cancer Now Research Centre, London, United Kingdom, 3 King’s College London, King’s Health Partners Cancer Biobank, London, United Kingdom Breast cancer is characterised by widespread structural, numerical aberrations and chromosomal rearrangements. These genomic alterations can influence transcription, leading to aborted transcripts, gene inactivation, or fusion genes. This study aims to identify such transcripts with underlying genomic alterations and to characterise the molecular features of breast cancers harbouring these events. Affymetrix SNP6.0 and Exon1.0ST arrays of 123 in-house primary breast tumours were analysed to identify disrupted transcripts with proximal genomic breakpoints. A walking student’s t-test was developed to determine aborted transcription. SNP- and exon-array, and RNAseq data of 51 breast cancer cell lines (BCCL) and 152 TCGA BRCA samples were used for validation. Fusion

183 Genetic analyses of early-onset gastric cancer P. Kapusta1 , J. Machlowska1 , A. Bogdali1 , P. Radkowski1 , F. Morsink2 , W. Polkowski3 , J. Offerhaus2 , P. Wołkow1 , R. Maciejewski4 , R. Sitarz3 . 1 Center for Medical Genomics, Jagiellonian University Medical College, Krakow, Poland, 2 Department of Pathology, University Medical Center, Utrecht, Netherlands, 3 Department of Surgical Oncology, Medical University of Lublin, Lublin, Poland, 4 Department of Human Anatomy, Medical University of Lublin, Lublin, Poland Introduction: Gastric cancer (GC) is the fourth most common cause of cancerrelated death in Europe and the third worldwide. GC can be divided into early-onset gastric carcinoma (EOGC) (10% of cases, occurring in patients at the age of 45 years or younger) and conventional GC (80% of cases, older than 45 years). Recently, it has been claimed that research on EOGC may contribute to the unravelling of the mystery of GC because younger patients are less exposed to environmental carcinogens, and their neoplasms rely more on genetic factors. In spite of the falling stomach cancer incidence rates in the older age groups, little change has been observed among the youngest patients. In addition, adolescent and young adult patients with stomach cancer, show less progress in survival rates, compared to older age groups. Taking all things together, genetic predisposition appears to play an important role in the development and treatment of GC. Our working hypothesis in this pilot study is that young patients with gastric cancer have a different spectrum of cancer predisposing mutations compared to the older ones. Material and Method: DNA was isolated from 8 formalin-fixed, paraffin embedded tumor fragments from stomach cancer patients, 4 diagnosed below 45 years of age and 4 above. Libraries for targeted next generation sequencing (NGS) were prepared for sequencing of a panel of over two hundred genes, in search for mutations specific for early onset gastric cancer. Bioinformatic pipeline was used to find the mutations and to identify pathways enriched in mutations, comparing EOGCs and conventional GCs. Results: NGS results were generated for 7 patients (3 EOGCs and 4 conventional GCs). One patient was excluded due to low coverage and poor data quality in a sample. In total, we identified 514 different mutations in 208 genes, of which 2 were identified as variants with high impact, and 133 as variants with medium impact according to GATK software. Among high impact variants we identified a splice acceptor variant of RECQL4 gene, that was present in all patients, and a frameshift variant of FANCD2 gene, which was present only in 6 patients, although all patients has several moderate mutations in this gene. We also identified several mutations in ALK, ERCC5, HNF1A, ATM, BRCA2, PMS2 and MEN1 genes, of moderate impact, present in all patients. Conclusion: In this pilot study, in a small group of patients, we did not identify mutations characteristic of early onset gastric cancers, however we have identified several genes mutated in all gastric cancer patients, regardless of the age of onset. No conflict of interest.

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184 Deregulated microRNAs in breast ductal carcinoma in situ (DCIS) with invasive propensity S. Volinia1 , V. Bertagnolo1 , F. Brugnoli1 , S. Grassilli1 , M. Galasso1 , C. Scatena2 , F. Lessi2 , C.M. Croce3 , S. Capitani1 . 1 University of Ferrara, Dipartimento di Morfologia- Chirurgia e Medicina Sperimentale, Ferrara, Italy, 2 University of Pisa, Division of Surgical- Molecular and Ultrastructural Pathology- Department of Translational Research and New Technologies in Medicine and Surgery, Pisa, Italy, 3 Ohio State University, Wexner Medical Cencer, Columbus, USA Background: The lack of prognosticators for the malignant potential of noninvasive lesions is an unresolved problem in the management of patients with breast cancer. This paper addresses the need to improve the approach to ductal carcinoma in situ (DCIS), which accounts for 20 to 25% of breast tumors. The objective of our work was to identify and characterize the miRNAs that play key roles in the progression from DCIS to invasive ductal carcinoma (IDC). Material and Methods: This study includes 3 different cohorts of formalinfixed samples for miRNA measurement: Cohort 1 (pure DCIS, n = 30), Cohort 2 (adjacent DCIS and IDC, n = 30), Cohort 3 (pure DCIS from patients with and without IDC recurrence in contralateral breast, n = 22). A DCIS cell line was used for molecular and cellular studies. The association between DCIS miRNAs and outcome in IDC was studied in the Cancer Genome Atlas (TCGA, n = 918), METABRIC (n = 796) and UK (n = 210) cohorts. Complete miRNome profiles were determined by short RNAseq in pure DCIS samples and compared with those of IDCs. The miRNAs up-modulated only in pure DCIS vs IDC were investigated in vitro for their impact on the invasive properties of DCIS-derived cells. The levels of selected miRNAs were correlated with invasive recurrence in DCIS patients and relapse-free or overall survival in IDC patients. Results: We detected a few miRNAs that were up-regulated in pure DCIS with respect to unrelated IDC, while had comparable levels in adjacent DCIS and IDC lesions. In vitro, inhibitors of this DCIS miRNAs impacted on epithelial-tomesenchymal transition (EMT) markers. miRNAs were evaluated for promoting invasiveness in vitro and were associated with IDC recurrence in patients with primary pure DCIS. Some of the miRNAs differentially regulated in the DCIS also correlated with outcomes across multiple IDC cohorts. Conclusions: In a DCIS in vitro model, we identified miRNAs that repressed EMT, a key process in invasion and metastasis. Furthermore, some of the miRNAs deregulated in DCIS played a robust protective role in the outcomes of IDC patients. High levels of a handful of miRNAs might characterize breast lesions with low-invasive potential and might represent novel biomarkers for risk assessment.cted miRNAs were correlated with invasive recurrence in DCIS patients and relapse-free or overall survival in IDC patients. No conflict of interest. 185 Targeted enrichment method using molecular barcodes to improve low frequency allele detection K. Zobeck1 , L. Forsmark1 , C. LeCocq1 , H. Tao1 , B. Arezi1 , H. Johansson1 . 1 Agilent Technologies, R&D, Santa Clara, CA, USA Agilent’s HaloPlex HS workflow is a next generation target enrichment method that enables enrichment of thousands of targets in a single tube and uses molecular barcodes within the primer cassettes to detect tens of thousands of unique molecules. The protocol utilizes specificity gained from restriction enzyme digestion, hybridization and DNA ligation to capture the target region. The HaloPlex HS workflow has been optimized so that it takes less than 5 hours to complete and requires only 50 ng input. Improvements in the probe design algorithm in Agilent’s SureDesign, results in increased coverage of custom designs. In our test designs we demonstrate >85% specificity and above 90% of target regions covered at >10% average depth. Using the improved algorithm, we have designed a panel covering the regions commonly mutated in Acute Myeloid Leukemia, and experimentally demonstrate that the Illumina design has a specificity of 99.8% and above 99% of target regions covered at >10% average depth, while the Ion Torrent design has a specificity of 99.4% and above 98% of target regions covered at >10% average depth. For both sequencing platforms, the targeted region includes: ASXL1, CBL, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, MPL, NRAS, NPM1, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1. Molecular barcodes in HaloPlex HS provide a unique method for identifying PCR duplicates and unique molecules present in the sequencing reaction. These molecular barcodes can aide in calculating efficiency of the protocol and assist in correcting sequencing errors, improving the detection of low frequency alleles and confidence of variant calling. Here we show the HaloPlex HS protocol with molecular barcodes can detect variants down to 0.5% allelic fraction. Finally, HaloPlex HS is compatible with Agilent’s FFPE QC assay, which quantifies the amount of amplifiable DNA and generates a DDcq score that can be used in order to determine the amount of sequencing capacity to apply to the prepared library. Using a range of FFPE samples we have developed

a set of recommendations for adjusting the HaloPlex HS protocol to provide optimal sequencing results for differing quality of samples. In conclusion, Agilent’s HaloPlex HS has been improved to include molecular barcodes, which allows low allele frequency detection using the molecular barcodes. It also provides recommendations for using FFPE samples, increasing the clinical applications for HaloPlex HS. Furthermore we have developed a targeted panel for Acute Myeloid Leukemia for both Illumina and Ion Torrent sequencing panels. Conflict of interest: Corporate-sponsored Research: Agilent Technologies. 186 “Melanomics”: analysis and integration of whole genomes, transcriptomes and miRNomes of primary melanoma patients S. Reinsbach1 , A. Wienecke-Baldacchino2 , A. Ginolhac1 , L. Vallar3 , A. Muller4 , A. Krishna2 , P. Nazarov3 , P. May2 , S. Kreis5 . 1 University of Luxembourg, LSRU, Belvaux, Luxembourg, 2 University of Luxembourg, LCSB, Belvaux, Luxembourg, 3 LIH, Genomics Research Unit, Luxembourg, Luxembourg, 4 LIH, Genomcis Research Unit, Luxembourg, Luxembourg, 5 University of Luxembourg, Life Sciences Research Unit LSRU, Belvaux, Luxembourg Introduction: Melanoma is the most fatal skin cancer with increasing case numbers worldwide. So far, most published studies describing genetic landscapes of melanoma rely on exome sequencing of metastatic lesions as they are easier to obtain than primary tumours. The so far largest sequencing effort was undertaken by the TCGA consortium, which has confirmed mutations in most of the previously described driver genes and have classified melanoma into 4 genetically distinct subtypes (BRAF, NF1, NRAS and triple wild type) that are useful for tailoring subsequent treatments. Material and Methods: Here, we generated whole genomes (Illumina paired end DNA Seq data), transcriptomes (Illumina paired end RNA-Seq data) and miRNomes (Qiagen whole miRNome qPCR array data) of 5 primary melanoma tumours alongside matching normal skin. After extensive QC and DNA/RNA read alignments, small nucleotide variants (SNVs) were called and annotated. Next, mutational signatures (motifs), DNA repair pathways and SNVs affecting potential phosphorylation sites were investigated in more detail, followed by comparisons to public data sets. Finally, we developed new approaches to integrate whole genomes, transcriptome and miRNome data sets. Results and Discussion: On top of UV-induced mutations, a hypermutated patient with 569 SNVs/Mb (average melanoma: ~100 SNVs/Mb) was found to have a gene signature indicative of smoking habits and accumulation of changes in DNA repair pathways. Furthermore, our data confirm the enormous heterogeneity in melanoma and we found amongst known driver genes, new potentially interesting players that are currently being validated: AHNAK2, a large nucleoprotein and assumed tumour suppressor was identified as a gene with a “high regulatory load”. Conclusion: In order to gain a deeper understanding of the events driving early neoplastic transformation, we analysed genomes, transcriptomes and miRNomes of primary melanoma tumours and found several interesting explanations for a hypermutated genotype detected in one of the patients. Although good data integration tools are still scarce especially when analysing more than 2 layers of data, we showed that new pathways and potentially interesting genes and miRNAs can be identified by a combined analysis of the different data sets. In a next step, we will focus on a more detailed investigation of the non-coding parts of melanoma genomes. No conflict of interest. 187 Investigation on Korean gastric tumorigenesis by performing whole transcriptome and miRNA sequence analysis Y.H. Koh1 , H.J. Seul2 , J.B. Seo3 , H.M. Kim4 , K. Ahn4 , H.S. Kang5 , B. Han6 , H.S. Kim7 , G. Jang8 , J. Seo9 , K.C. Kim10 , H.S. Na11 , S.E. Choi11 , J.W. Cho10 , D.Y. Zang6 . 1 Hallym University, Ilsong Institute of Life Science, Anyang, Korea, 2 Hallym University Scared Hospital, Translational Research Team, Anyang, Korea, 3 Korea Basic Research Institute, Omics team, Seoul, Korea, 4 Theragen Etex, NGS Research Team, Suwon, Korea, 5 Hallym University Scared Hospital, Department of Gstroenterology, Anyang, Korea, 6 Hallym University Scared Hospital, Hematology-oncology, Anyang, Korea, 7 Hallym University Kangnam Sacred Heart Hospital, Hematology-oncology, Seoul, Korea, 8 Hallym University Kangdong Sacred Heart Hospital, Hematology-oncology, Seoul, Korea, 9 Hallym University Scared Hospital, Pathology, Anyang, Korea, 10 Hallym University Scared Hospital, Surgery, Anyang, Korea, 11 Ministry of Food and Drug Safety, Clinical trials, Chungju, Korea Background: To initiate pharmacogenetics researches in Korea gastric tumorigenesis, we started to search Korean specific gastric cancer genes. Materials and Methods: We have analyzed whole transcriptomes and micro (mi) RNAs from 34 pairs of normal and cancer tissues from gastric cancer patients to identify somatic mutations and differentially expressed coding and long non-coding (lnc) RNAs and miRNAs.


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Results: Among 49,023 single nucleotide polymorphisms (SNPs) identified from 34 cancer tissues, highly defective 181 SNPs were revealed. In addition, we found 446 highly defective insertion/deletion mutations (In-dels) from 29,682 In-dels. Among identified 1,578 fusion genes, 166 fusion genes were observed from at least four cancer tissues. Furthermore, we identified differentially expressed miRNAs and lnc RNAs and analyzed differential expression of their target coding RNAs. Conclusion: In the future study, we will investigate how mutations and altered miRNA and lncRNAs identified from this study contribute on tumorigenesis. This work was supported by a grant (15182HanImPeyong512) from Ministry of Food and Drug Safety. No conflict of interest.

Consequently, alternative strategies are critically needed in order to identify and reduce the noise generated in SCS experiments. Here, we expand the census-based variant discovery principle (Zhang et al., 2015) to study the sensitivity in variant discovery and genotyping accuracy from whole-genome (WGS) and wholeexome (WXS) SCS data in an unbiased manner. Making use of publicly available datasets, our aim is to quantify the specificity of different sequencing strategies to somatic variant detection, and to determine the degree to which sequencing depth affects false positive (FP) contamination. Furthermore, the ability to resolve clonal heterogeneity, as well as to detect ADO events, will be analyzed by estimating the genotype accuracy in SCS data sequenced to pre-defined depths. No conflict of interest.

188 A pre-operative, diagnostic gene panel for guiding primary treatment choices in endometrial cancer: Advancing beyond the decades-old technology of dilation and curettage (D&C)

Sunday 10 July 2016

J. Martignetti1 , B. Reva1 , P. Elena1 , O. Camacho-Vanegas1 , D. Rykunov1 , S. Kendall1 , H. Shah1 , N. Nair1 , M. Strahl1 , W. Hamou1 , T. Kalir1 , E. Schadt1 , R. Sebra1 , P. Dottino1 . 1 Icahn School of Medicine at Mount Sinai, Genetics and Genomic Sciences, New York City, USA

Carcinogenesis I

Endometrial cancer is the fourth most common cancer in women worldwide and its incidence is rising. Approximately 300,000 women will be diagnosed this year. These cancers have historically been classified by histopathologic criteria into two groups and this has provided some insight towards patient treatments and outcomes. Type I cancers are generally more benign, have a favorable outcome following treatment by surgery and radiotherapy. By contrast, Type II cancers are more aggressive and have a worse outcome despite combination treatment with surgery, radiotherapy and chemotherapy. Currently, the diagnosis of type I and II endometrial cancer is based on H&E staining of tissue which is collected through endometrial biopsy and dilation and curettage (D&C). Resulting ambiguity and errors in the pathology interpretation can stem from the subjective nature of the presently used test, limited access to tissue, and/or heterogeneous histologies present within a single sample. These shortcomings can result in incorrect treatments for a significant fraction of patients. Having an exact molecular subtype diagnosis is critical for selecting the appropriate treatment and avoiding over and undertreatment. Recently, the TCGA provided a comprehensive, multiplatform analysis of 373 endometrial carcinomas spanning type I and II disease. We performed a retrospective analysis using this information to define a genome-guided molecular signature that can more precisely classify the two endometrial cancer treatment subtypes. We have designed and synthesized a custom endometrial typing panel and demonstrated the panel’s utility against two independent cohorts of samples wherein the pathologic diagnosis and clinical outcomes of patients was used to recapitulate the robustness of this assay. These results were then tested on an orthogonal sequencing platform. The gene selection criteria, panel design, workflow, sensitivity and specificity of this new molecular diagnostic will be discussed along with the implications and examples of this work for pre-symptomatic diagnosis of endometrial cancer. Conflict of interest: Other Substantive Relationships: For a part of the molecular analysis discussed, Swift Biosciences provided sequencing and sequence analysis at no cost to the investigators. None of the investigators received any financial compensation, considerations nor remuneration of any kind.

F. Ebrahimi1 , V. Gopalan2 , A. Lam2 . 1 Cancer molecular pathology- Menzies Health Institute Queensland- Griffith University- Australia, Molecular Pathology school of medicine, Gold Coast, Australia, 2 Griffith University, Molecular Pathology school of medicine, Gold Coast, Australia

190 Identifying effective single-cell sequencing strategies for intra-tumor heterogeneity estimation J. Alves1 . 1 Universidad de Vigo, Vigo, Spain Recent advances in next-generation sequencing technologies have revealed that the large majority of cancer genomes are genetically heterogeneous despite its monoclonal origin, with the continuous expansion of tumor mass contributing to the accumulation of somatic mutations within malignant cells through time. While quantifying this heterogeneity remains a difficult task, as standard methods in cancer genomics generally rely on population-level analysis from pooling experiments, single cell sequencing (hereon SCS) approaches are now widely viewed as a promising alternative to explore clonal evolution. However, several technical challenges surrounding current SCS methodologies greatly limits one’s ability of obtaining reliable genomic information from single cells. For instance, the multiple rounds of whole genome amplification (WGA), required prior to genome sequencing, are known to introduce a high number of sequence artifacts that can be confounded with genuine biological variation. Other technical errors, such as insufficient physical coverage, uneven genome amplification, and allelic dropout (ADO), may also generate substantial artificial variability in cancer genomes, compromising the accuracy to detect real somatic heterogeneity from SCS data.

Poster Session

191 miR-15a expression and deregulation in colorectal cancer biology and its impact on clinicopathological features

Aim: The specific aim of the research was to determine whether miR-15a expression is found altered in neoplastic tissues with and without metastasis and to identify how variation correlates to specific pathological characteristics of colorectal cancers. Material and Method: Colorectal tissues from 60 primary cancers with 52 matched lymph nodes, and 25 non-neoplastic tissues were recruited from 85 patients. The expression levels of miR-15a in colon cancer tissues and cell lines (SW480, SW48, and HCT116) and one normal colonic cell line (FHC) were investigated by using qRT-PCR. Additionally, various downstream functional assays such as cell proliferation, colony formation assay, invasion assay as well as the target predictions were performed. Results and Discussion: Expression of miR-15a was down-regulated in colorectal cancer (CRC) tissues compared to normal colonic tissues. In microsatellite instability (MSI) positive adenocarcinomas, notable downregulation of miR-15a (P = 0.018) was observed. Also, miR-15a downregulation was noted to be significantly predominant in CRCs with larger size tumours (>40 mm) (P = 0.014). Following miR-15a overexpression, colon cancer cells showed reduced cell proliferation, low colony formation and low invasion potential compared to control cells. The oncogenic protein BCL2 and tumour suppressor gene P53 protein expressions were significantly downregulated in colon cancer cells with miR-15a overexpression. Conclusion: This study suggests that miR-15a is a potential tumour suppressor in colorectal cancer and that its dysregulation may assist in regulating various biological and pathogenic processes in colon carcinogenesis via modulating its target proteins such as BCL2 and P53. No conflict of interest. 192 Conditional transgenic expression of PIM1/PIM2 kinases in hormone-dependent tissues induces mammary gland and female reproductive system tumours M.P. Jimenez-Garc´ ´ ıa1 , A. Lucena-Cacace1 , B. Felipe-Abrio1 , E.M. Verdugo-Sivianes1 , D. Otero-Albiol1 , M. Perez1 , S. Munoz-Galvan ´ 1, J. Peinado-Serrano1 , J.M. Garc´ıa-Heredia1 , M. Narlik-Grassow2 , C. BlancoAparicio2 , A. Carnero1 . 1 Instituto de Biomedicina de Sevilla IBiS. Hospital Universitario Virgen del Roc´ıo HUVR. Consejo Superior de Investigaciones Cient´ıficas CSIC. Universidad de Sevilla US, Avenida Manuel Siurot s/n. 41013, Seville, Spain, 2 Experimental Therapeutics Programme. Spanish ´ National Cancer Centre CNIO., C/Melchor Fernandez Almagro 3. 28029, Madrid, Spain Background: PIM proteins (Proviral Insertion site in Moloney murine leukemia virus), form a highly evolutionarily conserved family of serine/threonine kinases composed of three different isoforms (PIM1, PIM2 and PIM3). These proteins are regulated by transcription and stability through pathways that are controlled by JAK/STAT transcription factors. PIM family proteins have been implicated in regulation of apoptosis, metabolism, cell cycle, homing and migration processes, and they have been found to be overexpressed in hematological malignancies and solid tumours. This led to the postulation of these proteins as interesting targets for anticancer drug therapy. Although PIM kinases have been identified as oncogenes, they have weak transforming abilities on their own. However, they greatly enhance the capacity of other genes or chemical carcinogens to induce tumours. The effects vary depending on affected tissues and pathways activated. Proto-oncogene PIM1 is a novel estrogen receptor target associated with high grade breast tumours. Furthermore,

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PIM1 expression levels were elevated in most mammary carcinoma cell lines, and progesterone increased PIM1 protein expression in mammary epithelia, matching its expression pattern with PIM activity in mouse mammary glands. Material and Method: To characterize the proto-oncogenic role of PIM1/PIM2 in mammary gland tissues, we generated two conditional transgenic murine models: MMTV-Cre/PIM1 and MMTV-Cre/PIM2. We confined expression to hormone-dependent tissues by regulating Cre through MMTV promoter. The phenotype was fully characterized, analysing all tumoral disturbances. Results: Conditional transgenic MMTV-Cre/PIM1 and MMTV-Cre/PIM2 models showed a reduced survival, a higher percentage of tumours at clinical endpoint and a higher incidence of total tumours percentage, noticing more statistically significant data of tumor incidence at 700 days, possibly because PIM1/PIM2 expression induces alterations in hormone-dependent tissues that increase proliferation rate of cells, leading to physiological mechanisms that shortens life. PIM1/PIM2 conditional models generated hyperplasia in mammary glands and also in sexual organ tumours (oviduct, uterus and ovary). TgPIM1 generated mammary gland tumours, which indicates that PIM1/PIM2 genes are involved at tumour initiation level. Conclusions: As a conclusion, over-expression of transgenic human isoforms PIM1/PIM2 induces a neoplastic phenotype in mammary gland and female reproductive system, indicating a role at tumor initiation in these tissues. No conflict of interest. 193 Determination of the proto-oncogenic role of PIM1/PIM2 kinases in male reproductive system pre-neoplastic lesions by using conditional transgenic murine models M.P. Jimenez-Garc´ ´ ıa1 , M.J. Robles-Frias1 , B. Felipe-Abrio1 , A. LucenaCacace1 , D. Otero-Albiol1 , E.M. Verdugo-Sivianes1 , J. Peinado-Serrano1 , M. Perez1 , S. Munoz-Galvan1 , J.M. Garc´ıa-Heredia1 , M. Narlik-Grassow2 , C. Blanco-Aparicio2 , A. Carnero1 . 1 Instituto de Biomedicina de Sevilla IBiS. Hospital Universitario Virgen del Roc´ıo HUVR. Consejo Superior de Investigaciones Cient´ıficas CSIC. Universidad de Sevilla US, Avenida Manuel Siurot s/n. 41013, Seville, Spain, 2 Experimental Therapeutics Programme. ´ Spanish National Cancer Centre CNIO, C/Melchor Fernandez Almagro 3. 28029, Madrid, Spain Background: The Proviral Insertion site in Moloney murine leukemia virus proteins (PIM) are an evolutionarily conserved family of serine/threonine kinases composed of three different isoforms (PIM1, PIM2 and PIM3), which are regulated primarily by transcription and stability through pathways that are controlled by JAK/STAT transcription factors. The PIM proteins have been implicated in regulation of apoptosis, metabolism, cell cycle, homing and migration, and they have been found to be over-expressed in haematological malignancies and solid tumours. For those reasons they have been proposed as targets for anticancer drug therapy. It has been suggested that PIM family members are weak oncogenes that can contribute to tumorigenesis by selectively enhancing tumorigenic properties. Although PIM kinases have been identified as oncogenes, they have weak transforming abilities on their own. However, they greatly enhance the capacity of other genes or chemical carcinogens to induce tumours. Germinal tumours are among the solid tumours in which both PIM1/PIM2 proteins have been found to be over-expressed. Previous data determined that PIM1 increased the severity of mouse prostate intraepithelial neoplasias (mPIN) and might contribute to progression rather than initiation in prostate neoplasia. Material and Method: In order to characterize the proto-oncogenic role of PIM1/PIM2 in the male reproductive system, we generated two MMTV-Cre conditional transgenic mice with confined expression of human PIM1 or PIM2 in steroid-dependent tissues. We fully characterized tumoral response to these genetic alterations in both models. Results: Conditional transgenic MMTV-Cre/PIM1 and MMTV-Cre/PIM2 models showed reduced survival, and higher percentage of tumours at clinical endpoint, possibly because PIM1/PIM2 expression induces alterations in steroid-dependent tissues that increase proliferation rate and tissue atrophy of certain cyto-architectonic structures embedded in those tissues. PIM1/PIM2 conditional models generated hyperplastic changes in testicle and prostate, and areas of epithelial adenomas in seminal vesicle, but no carcinomas of male reproductive system, which indicates that PIM1/PIM2 genes are involved at tumor initiation level. Additionally, enhanced inflammation surrounding target tissues were found in PIM1/PIM2 models, which it can be a tumour initiation mechanism led by PIM deregulation of cellular JAK/STAT signalling, since STAT proteins have been tied to controlling development of hematopoietic cells that regulate inflammation, and mediating the responses of target cells to inflammatory cytokines. Conclusion: Together our data indicate that PIM1/PIM2 over-expression induces a pre-neoplastic phenotype in testicle, seminal vesicle and prostate, pointing a role at oncogenic initiation in these tissues possibly related to PIMdependent immune activation. No conflict of interest.

194 Expression and localization of GAEC1 oncogene in peripheral blood from patients with colorectal adenocarcinoma V. Gopalan1 , M. Orr1 , S. Pillai1 , A. Lam1 . 1 Menzies Health Institute Queensland- Griffith University, Cancer Molecular Pathology, Gold Coast, Australia Introduction: GAEC1 is previously reported to have oncogenic potential invivo in gastrointestinal carcinomas. The current study aims to examine the expression profiling and localization of GAEC1 within peripheral blood samples of patients with colorectal adenocarcinoma. Materials and Method: Peripheral blood samples of 43 patients with colorectal adenocarcinoma and 3 healthy individuals (control) were collected. GAEC1 mRNA expression from these blood samples was performed using a real time PCR. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood samples using ficoll separation. GAEC1’s protein expression was localized to a leukocyte sub-population using intracellular staining in suspension and flow cytometric analysis. Various sub-populations of PBMC’s were identified using CD3-FITC, CD16-PE-Vio770, CD19-APC, CD56-PerCPVio700 and CD45-VioBlue antibodies. GAEC1 monoclonal antibody was directly conjugated to Anti-IgG-PE. Results and Discussion: GAEC1mRNA overexpression was noted in 40% (17/43) of the colorectal cancer patients. Blood samples from high grade (moderate or poorly differentiated) carcinomas showed lower GAEC1 mRNA expression when compared to those from low grade (well-differentiated) adenocarcinoma (p = 0.030). CD45+ subpopulations showed a marked increase in GAEC1 expressed cells in colorectal cancer patients when compared to those from healthy blood samples (61% vs 2%) confirming the localization of GAEC1 in leukocytes. Also, when compared to controls, blood samples from patients with colorectal adenocarcinoma showed significant increase in GAEC1 cell populations in CD3+ sub-populations of PBMCs indicating the expression localization of GAEC1 in T lymphocytes (44% vs 0.2%). Conclusion: This study was the first to present significant data on the role of the novel oncogene GAEC1 in the peripheral blood of patients with colorectal adenocarcinoma, and provides substantial direction for future studies. Furthermore, the successful localization of GAEC1 protein in T lymphocytes provides vital information that can be utilized for future molecular targeting based experiments to investigate GAEC1’s therapeutic potential. No conflict of interest. 195 YAP signalling promotes aggressive tumour development over heterotopia in the brain M. Mayrhofer1 , V. Gourain1 , M. Reischl2 , P. Affaticati3 , A. Jenett3 , J.S. Joly3 , D. Sieger4 , M.C. Mione1 . 1 Karlsruhe Institute of Technology, Institute for Toxicology and Genetics, Eggenstein-Leopoldshafen, Germany, 2 Karlsruhe Institute of Technology, Institute for Applied Informatics, EggensteinLeopoldshafen, Germany, 3 Paris-Saclay Institute of Neuroscience- CNRSUniversite´ Paris-Saclay, Tefor Core Facility, Gif-sur-Ivette, France, 4 The University of Edinburgh, Centre for Neuroregeneration, Edinburgh, United Kingdom Introduction: Somatic mutations activating MAPK/PI3K signalling have recently been reported in 88% of glioblastoma (TCGA, 2008) and in brain developmental disorders/heterotopia. Even though a progression from heterotopia to tumours is possible, the mechanism and preventive treatments are unknown. Materials and Methods: We developed a progressive zebrafish model of glioma based on somatic expression of oncogenes that activate MAPK/PI3K signalling in neural progenitor cells. Histological and immunological analysis as well as RNA sequencing were used to characterise the developing lesions. Manipulation of this model through an active form of YAP (YAPS5A) was used to clarify the molecular events leading to malignant tumours instead of benign lesions. Results and Discussion: Oncogenic HRAS (HRASG12V) was the most effective in inducing ERK phosphorylation and caused the development of different types of growth disorders in juvenile fish: from benign dysplasia/heterotopia to invasive tumours of the telencephalon, midbrain and cerebellum. Specific signatures distinguish benign heterotopia from tumours and establish that tumours require persistent activation of phospho (p)ERK. Moreover, analysis of global RNA expression showed that brain tumours expressed a gene signature similar to the human mesenchymal glioblastoma subtype, with a strong YAP component. Application of a signature of 8 genes derived from our NGS data to human benign lesions and tumours established that YAP activation is a hallmark of tumours versus heterotopia, in zebrafish and in human samples. Further, we tested whether YAP could play an obligatory role in the development of aggressive tumours induced by oncogenes activating pERK. An active form of YAP (YAPS5A) was able to induce tumour development on its own and co-operates with HRASG12V in inducing very aggressive tumours, while no heterotopia was observed in these brains. Conclusion: We propose that YAP1 activation in pERK+ brain tumours is necessary to promote definitive malignant growth. No conflict of interest.


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196 Reprogramming bladder cancer cells for studying cancer initiation and progression

199 The role of Fanconi Anaemia pathway in sporadic non-FA associated head and neck squamous cell carcinoma

B. Iskender Izgi1 , K. Izgi2 , H. Canatan1 . 1 Erciyes University Faculty of Medicine, Department of Medical Biology/ Betul-Ziya Eren Genome and Stem Cell Centre, Kayseri, Turkey, 2 Erciyes University Faculty of Medicine, Department of Medical Biochemistry/ Betul-Ziya Eren Genome and Stem Cell Centre, Kayseri, Turkey

F. Alsobahi1 , S. Collis2 , K. Hunter1 . 1 University of Sheffield, School of Clinical Dentistry, Sheffield, United Kingdom, 2 University of Sheffield, Department of Oncology and Metabolism, Sheffield, United Kingdom

Background: The induced pluripotent stem cell (iPSC) technology is the forced expression of specific transcription factors in somatic cells resulting in transformation into self-renewing, pluripotent cells which possess the ability to differentiate into any type of cells in the human body. The iPSC technology not only offers an invaluable source for cell replacement therapies but also provides a system to study the disease mechanism and drug development/toxicity testing. Malignant cells could also be reprogrammed into iPSC-like cells with lower efficiency but due to the genetic and epigenetic barriers in cancer cells only a limited number of cancer cell types could be successfully reprogrammed until today. In the present study we aimed at reprogramming bladder cancer cells and this is the first study focusing on the reprogramming susceptibility of two different bladder cancer cell lines to reprogramming. Materials and Methods: By using non-integrating Sendai virus (SeV) system we transfected two bladder cancer cell lines (T24 and HTB-9) with reprogramming factors − OCT4, SOX2, c-MYC and KLF4. Pluripotencyassociated features were demonstrated using immunofluorescence staining, qRT-PCR and western blotting. In vitro differentiation ability of iPS-like cells was assessed by embryoid body assay. Results: We generated iPSC-like cells from T24 bladder cancer cell line which represented pluripotent features both at gene and protein level. Reprogrammed T24 cells were demonstrated to differentiate into cells representing three embryonic germ layers in vitro. Using the same technique, we have also transfected HTB-9 cells with reprogramming factors which were unable to give rise to iPS-like cells but only upregulated some pluripotencyassociated marker expression. Conclusion: To the best of our knowledge, this is the first study describing the generation of pluripotent stem cells using iPSC technology from bladder cancer cells. Our study provides a valuable tool for cancer studies with the potential to represent the early steps of bladder carcinogenesis and for understanding the origin of cancer stem cells. No conflict of interest. 198 COX-2 gene variations and risk of developing breast cancer G. Ozhan1 , Z. Kara2 , H. Kara3 . 1 Istanbul University, Faculty of Pharmacy, Istanbul, Turkey, 2 Istanbul University, Pharmacology, Istanbul, Turkey, 3 Acibadem University, Faculty of Medicine, Istanbul, Turkey Introduction: Breast cancer is the most frequent cancer and responsible for the deaths from 33% of all cancers and from 20% of all cancer related deaths in women. Beside the general risk factors such as gender, age, age of menarche and menopause, obesity, hormone replacement therapy, oral contraceptive use, high and low penetrance genes, and epigenetic and modifier genes have a important role in its carcinogenesis. Cyclooxygenases (COXs), central enzymes in the prostaglandin pathway, play important role in several biologic processes relevant to cancer development and progression such as inflammation, tumour cell invasion and metastasis. COX-2 over-expression in carcinomas has been associated with a more aggressive behavior and a worse prognosis. While expression of COX-2 is nearly undetectable in normal breast tissue, the gene is over-expressed in 40% of invasive breast tumors. There are some studies on the association between COX-2 genetic variants and breast cancer risks, however, the findings have been inconsistent. Therefore, our aim was to investigate the relationship between COX-2 genotypes and haplotypes in a Turkish population-based case–control study on breast cancer susceptibility. Material and Method: It was evaluated COX-2 (G765C, T8473C, G306C) genotypes in 104 patients and 100 healthy controls by using RT-PCR. Result and Discussion: It was observed that only COX-2 T8473C might be associated with breast cancer risk (OR = 1.86±0.79–4.38), however the association did not reach statistical significance (p = 0.151). Clinicopathological characteristics and haplotype analysis were not associated with breast cancer risk. Conclusion: We believe that the findings are the first results of COX-2 allele distributions in the Turkish population and may provide an understanding of aetiology in breast cancer. No conflict of interest.

Introduction: Fanconi Anaemia (FA) is a rare inherited disease caused by mutations in genes of the Fanconi Anaemia pathway. The FA pathway is involved in the DNA damage response and the mutilations result in chromosomal instability. Patients with FA who survive bone marrow transplantation are found to be at high risk of developing Head and Neck squamous cell carcinoma (HNSCC) with 500–700 times the risk of normal individuals. Given the relative specificity of this effect, we hypothesize that alterations in the expression and posttranslational activation of FANC genes may contribute to the pathogenesis of sporadic non-FA HNSCC in addition to FA-HNSCC. Aims: To determine whether FANC genes are altered in non-FA HNSCC, and to define the effect of such alterations on the DNA damage response in HNSCC. Materials and Methods: The expression of FANCA, FANCC, FANCD2, and KI67 proteins was assessed normal oral mucosa and HNSCC tissue using immunohistochemistry. Expression of FANCA, FANCC and FANCD2 was assessed by qPCR and Western blot in a panel of Cisplatin treated and untreated sporadic HNSCC and FA-HNSCC cells. Immunofluorescence was used to assess DNA damage (53BP1, gH2AX) and repair and FANC pathway activation (FANCD2 foci) while Comet assay was used to assess DNA strand breaks and repair. Results: Higher FANCD2, FANCA and FANCC expression was found in HNSCC tissues compared to normal tissue and this correlates with the proliferation marker Ki67. The expression of FANCA, FANCC and FANCD2 are higher in HNSCC and FA-HNSCC cells compared to normal cells and this increased further after treatment with Cisplatin. FANCD2, 53BP1 and gH2AX nuclear foci and DNA fragmentation were increased after cisplatin treatment in all cells and the DNA damage was largely repaired in non-FA HNSCC cells over 48 hours, but not in FA-HNSCC cells. Conclusions: These data demonstrate that functional activation of the FA pathway in response to DNA damage occurs in non-FA HNSCC, but not FAHNSCC after treatment with Cisplatin. No conflict of interest. 200 HER2 cooperates with YWHAZ to promote to invasive esophagogastric cancer by inducing epithelial–mesenchymal transition S. Komatsu1 , D. Ichikawa1 , M. Miyamae1 , T. Kosuga1 , H. Konishi1 , A. Shiozaki1 , H. Fujiwara1 , K. Okamoto1 , H. Tsuda2 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan, 2 National Defense Medical College, Department of Basic Pathology, Saitama, Japan Background: Co-overexpression of HER2 and YWHAZ was significantly correlated with poor outcomes in breast cancer patients (Lu et al., Cancer Cell 2009). Material and Methods: We used MKN74 cells, which present cooverexpression of HER2 and YWHA Z and 104 primary tumors of esophagogastric adenocaricinoma, to clarify the clinical significance and biological functions of co-overexpressions. Results: In MKN74 cells, knockdown of both HER2 and YWHAZ using each specific small interfering RNA more strongly inhibited migration and invasion with enhancing E-cadherin expression and reducing Vimentin and ZEB1 expressions than knockdown of either. Importantly, patients whose tumor was overexpressed both HER2 (H) and YWHAZ (Y) significantly had higher rates of metastatic recurrence and death than those whose tumors overexpressed only one (5-year survival rate; H+Y− vs. H−Y− vs. H−Y+ vs. H+Y+: 78% vs. 80% vs. 56% vs. 22%). Conclusion: Co-overexpression of YWHAZ in HER2-overexpressing esophago-gastric cancer contributes to tumor progression and poor outcomes. YWHAZ may be a key molecule for selecting prospective patients with malignant outcomes in patients undergoing chemotherapy for HER2 overexpression. No conflict of interest. 201 Plasma microRNA profiles; down-regulation of tumor suppressive miR-X level in plasma relates to poor outcomes and is a novel treatment target in gastric cancer S. Komatsu1 , D. Ichikawa1 , T. Imamura1 , W. Okajima1 , T. Ohashi1 , J. Kiuchi1 , T. Arita1 , H. Konishi1 , A. Shiozaki1 , E. Otsuji1 . 1 Kyoto Prefectural University of Medicine, Division of Digestive Surgery- Department of Surgery, Kyoto, Japan Background: This study aimed to explore decreased tumor suppressive microRNAs (miRNAs) in plasma of gastric cancer (GC) patients and detect their possible roles as a treatment target as well as a biomarker for GC.

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Methods: Through the Toray® 3D-Gene miRNAs microarray-based approach to compare plasma miRNA levels between GC patients and healthy volunteers, we identified a novel plasma biomarker and treatment target. Results: Five down-regulated miRNAs (miR-V, miR-W, miR-X, miR-Y and miR-Z) in plasma were selected using high-resolution miRNAs array approach. Test- and large scale analyses using 130 GC patients revealed that plasma miR-X level was most down-regulated among candidates compared to healthy volunteers (p < 0.0001). A low level of plasma miR-X was significantly associated with advanced T-, N-stage and peritoneal recurrence, and significantly associated with a worse cause-specific survival after curative gastrectomy. Multivariate analysis identified a low level of miR-X as an independent poor prognostic factor in GC patients (p = 0.017, Hazard ratio 7.99 (95% CI: 1.39–152.0)). Enforced expression of miR-X in GC cell lines inhibited cell proliferation, migration, and invasion. In vivo, we demonstrated that the restoration of plasma miR-X could suppress tumor growth in mice with peritoneal metastasis. Conclusion: Down-regulation of plasma miRNA-X, which serves as tumor suppressive functions in GC, contributes to disease progression and poor outcomes in GC. Maintaining high level of miR-X in body fluid may be a novel treatment strategy using nucleic acid medicine for GC. No conflict of interest.


Carcinogenesis II 202 Comparative analysis of human and mouse expression data identifies distinct proto-oncogene PTTG- and PBF-associated genes in thyroid cancer M. Read1 , J. Fong1 , W. Imruetaicharoenchoke1 , B. Modasia1 , H. Nieto1 , J. Watkinson2 , K. Boelaert1 , V. Smith1 , A. Turnell3 , C. McCabe1 . 1 University of Birmingham, Institute of Metabolism and Systems Research, Birmingham, United Kingdom, 2 University Hospitals Birmingham National Health Service Foundation Trust, Queen Elizabeth Hospital, Birmingham, United Kingdom, 3 University of Birmingham, Institute of Cancer and Genomic Sciences, Birmingham, United Kingdom Introduction: Whilst the proto-oncogene PTTG and its binding partner PBF (PTTG1IP) have previously been shown to be up-regulated in differentiated thyroid cancer (DTC), there is a paucity of information regarding their coexpression and specific roles in tumour progression. PTTG and PBF have both been reported to modulate the tumour suppressor p53, whose activity is impaired in most human cancers. Hence, the role of PTTG and PBF in thyroid tumorigenesis may involve the disruption of p53 pathways that are central to DNA-damage repair (DDR), cell growth and apoptosis. In the present study we investigated whether PTTG and PBF expression are associated with p53related genes in the TCGA thyroid cancer dataset, as well as in a bi-transgenic murine model overexpressing PTTG and PBF specifically in the thyroid gland. Material and Methods: A bi-transgenic model (Bi-Tg) of thyroid-specific PTTG and PBF overexpression was generated by crossing two FVB/N murine models of human PBF (PBF-Tg) and PTTG (PTTG-Tg) transgenes under the control of the bovine thyroglobulin promoter. Gene expression in primary murine thyrocytes was analysed using a DNA damage signalling pathway-focussed PCR array. Expression of PTTG, PBF and p53-related genes in human thyroid carcinomas was analysed in the TCGA publically available dataset. Results: Characterisation of primary murine Bi-Tg thyrocytes revealed that co-expression of PTTG and PBF caused extensive repression of DDR genes (39/82 genes; P < 0.05). Of these, 31 genes were down-regulated >1.5-fold, including genes with key roles in maintaining genomic integrity such as Pms2 and Brca1. Irradiation exposure to increase intracellular p53 further showed that the most significant difference in overall DDR gene expression (n = 82 genes) was between irradiated Bi-Tg and wild-type (WT) thyrocytes (P = 0.0002) compared with either PBF-Tg (P = 0.0015) or PTTG-Tg thyrocytes (P=NS). By comparison in the TCGA dataset, there were striking correlations observed with PTTG and PBF in well-characterised DDR- and p53-related gene panels (>60% of genes; P < 0.05; 82−96 genes per panel; n = 322 unmatched TCGA tumour samples). Importantly, more than half of the significant DDR gene alterations observed in Bi-Tg thyrocytes (20/39 genes) were also present in TCGA (matched tumour/normal specimens) when samples with either low or high PTTG/PBF mRNA levels were compared. Furthermore, the overall survival (P = 0.0002) and disease-free survival (P = 0.02) was poorer for TCGA patients with high tumoral PTTG/PBF expression (n = 20) than for all other patients (n = 255). Conclusion: We have identified a distinct panel of p53-related genes associated with PTTG and PBF co-expression in different thyroid cancer models. Together our findings provide important insights into the specific role of PTTG and PBF in thyroid tumorigenesis. No conflict of interest.

204 Rab20 and Rab30 regulate hepatocellular carcinoma (HCC) for tumour suppression H.M. Liu1 , S.K. Tey1 , J.W.P. Yam1 . 1 The University of Hong Kong, Department of Pathology, Pokfulam, Hong Kong Ras related in brain 20 (Rab20) and Ras related in brain 30 (Rab30) are members of the Rab family of small GTP-binding proteins. Rab proteins contribute to the control of vesicular traffic in eukaryotic cells, by which they mainly regulate the precision of vesicle transportation along the biosynthesis/secretary pathway. Rab20 and Rab30 are low-weight GTPase that localise in the Golgi apparatus. Rab20 participates in apical endocytosis and phagosome functions, while Rab30 is important for controlling the structural integrity of the Golgi apparatus. Functions of Rab20/30 in cancer development have not been fully understood. Rab20 was identified to be upregulated in pancreatic cancer, and should be associated wih early stage of pancreatic carcinogenesis. However, roles of Rab30 has not been published in any cancer research. This drives us to investigate the potential roles of Rab20/30 in the development of hepatocellular carcinoma (HCC). In order to evaluvate the expression of Rab20 and Rab30 in HCC, 30 clinical cases were randomly selected for Real-time quantitative PCR using Rab20/30 TaqMan Gene Expression Assays (Applied Biosystem® ). HPRT was selected as the internal control for normalization. Clinicopathological correlation and statistical analysis were performed to determine their expression ratios of tumorous to non-tumorous samples and potential roles in HCC development. Expessions of Rab20/30 were also determined in nine HCC cell lines through qPCR. Selected cell lines included MIHA, BEL7402, Hep3B, HLE, Huh7, PLC/PRF/5, SMMC7721, MHCC97L and MHCC-LM3. Our qPCR results reflected both Rab20 and Rab30 were signficantly downregulated in tumorous samples when compared to non-tumourous samples, with the p-value of 0.0115 and 0.0004, respectively. Regarding to tumor stage, underexpresssion of Rab20 was mainly observed in patients with stage III-IV, which is regarded as late stage. In contrast, apart from patients with stage III-IV, underexpression of Rab30 was also detected in patients with stage I-II. This suggested Rab20 maybe responsible for HCC progression by inhibiting invasive and metastatic properties of HCC, while Rab30 may play roles in the initiation of hepatocarcinogenesis. None of the clinicopathological parameters tested was significantly associated to the expression of Rab20/30. For HCC cell line panel, Rab20 was highly expressed in MIHA and BEL7402, but slightly expressed in other cell lines. There was no obvious expression pattern of Rab30 in HCC cell lines. Through qPCR, Rab20 and Rab30 were underexpressed in HCC, implying their potenital role in tumor suppression. Rab20 was mainly downregulated in late stages, while underexpression of Rab30 may be an early event in hepatocarcinogenesis. Further experiments were needed to determine the functional significance of Rab20/30 in HCC initiation and progression. No conflict of interest. 205 UNG deficient BALB/c mice do not develop hyperplasia or B cell lymphomas L. Alsøe1,2 , A.B. Wennerstrom ¨ 1 , T. SenGupta1,2 , H. Nilsen1,2 . 1 Akershus University Hospital, EpiGen, Nordbyhagen, Norway, 2 University of Oslo, Institute of Clinical Medicine- Department of Clinical Molecular Biology, Nordbyhagen, Norway UNG deficient mice suffer from Hyper IgM type V syndrome. It was previously reported that aging mice on a mixed 129SV/C56Bl/6 background developed hyperplasia of lymphoid organs or B cell lymphomas reflecting the role of UNG in the humoral immune response. We backcrossed UNG deficient mice onto the BALB/c background to gain knowledge about the immunological background in which B cell lymphomas may develop. At 3 to 18 months, we examined the composition of the immune cells in the spleen by flow cytometry. Contradictory to previous findings, UNG deficient mice on the BALB/c background did not develop lymphoid hyperplasia or B cell lymphomas. Furthermore, we could not reproduce the previously reported genotype specific changes in the immune cell composition of CD4+ T cells and NK/NKT cells. We speculate that housing of the UNG deficient mice in an SPF (specific pathogen free) facility did not provide sufficient immunological challenge to promote lymphoid hyperplasia or B cell lymphoma development. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 206 Mutation screening of Bulgarian hereditary breast and ovarian cancer patients with multi-gene cancer panel A. Mitkova1 , R. Dodova1 , D. Pencheva1 , A. Vlahova2 , M. TaushanovaHadjieva3 , S. Valev3 , K. Timcheva3 , S. Christova2 , V. Mitev1 , R. Kaneva1 . 1 Medical University of Sofia, Molecular Medicine Center- Department of Medical Chemistry and Biochemistry- Medical Faculty, Sofia, Bulgaria, 2 Medical University of Sofia, General and Clinical Pathology Clinic- University Hospital “Alexandrovska”/Department of General and Clinical PathologyMedical Faculty, Sofia, Bulgaria, 3 Nadezhda Women’s Health Hospital, Clinic of Medical Oncology Chemotherapy, Sofia, Bulgaria Background: It has been estimated that about 5% to 10% of breast cancers and 15 to 20% of ovarian cancers are hereditary. Germline mutations in the highly penetrant cancer susceptible genes BRCA1/2 account for the majority of the cases with Hereditary Breast and Ovarian Cancer (HBOC). Other less common genes have been also associated with high (TP53, PALB2, PTEN), moderate (CHEK2, ATM, NF1, NBN), elevated, but imprecise (CDH1, STK11) breast and ovarian cancer risk (MMR genes, RAD51D, BRIP1). Advances in next generation sequencing technologies make massive parallel sequencing more feasible and afford an efficient hereditary gene panel testing of HBOC patients. Methods: In the present study we included a group of 75 Bulgarian high-risk HBOC patients, selected following the recognised BCLC and NCCN criteria for genetic testing. The mutation screening was performed by NGS in panel of 94 cancer related genes (TruSight Cancer Sequencing Panel, Illumina) on a MiSeq platform (Illumina). All detected mutations and variants of unknown clinical significance (VUSs) were confirmed by Sanger sequencing. Results: Twenty six pathogenic mutations (15 frameshift, 8 missense, one nonsense, one splice, one 5 UTR) and one likely pathogenic missense variant were found in 31 out of 75 HBOC patients. Damaging BRCA1/2 mutations (12 frameshift, two missense, one splice variant) were observed in 58% (18/31) of the mutation carriers, followed by two missense mutations in AIP (12.9%, 4/31) and RET (9.7%, 3/31), respectively. Pathogenic variants in other cancer related genes were detected in 29% (9/31) of the carriers: 3 frameshifts in ATM, BLM and WT, one nonsense mutation in MLH1, one 5 UTR in BULB1B and 5 missense mutations in SDHD, NBN, TSC1, CHEK2 and MUTYH. Six of the patients harboured two pathogenic variants in two different genes. In addition 215 VUSs were found. Conclusions: The mutation screening of Bulgarian HBOC patients with TruSight Cancer Sequencing Panel lead to identification of clinically relevant pathogenic variants in 41.33% of the cases. It is evident that hereditary cancer multi-gene panel testing offers new opportunities in the diagnosis and clinical management of cancer susceptibility, with improved power to identify more HBOC patients who are carrying undiagnosed pathogenic mutations. While panel testing is more likely to provide a more complete capture of an individual’s genetic landscape, there are several unanswered questions and areas of future research. As the number of tested genes increases, the likelihood of detecting VUSs increases as well. Another challenge for the genetic counselling is the interpretation of pathogenic variants in cancer related genes that have never been associated with HBOC syndrome. Acknowledgements: This work was supported by Grant No. 95, Contract No. 7-C/2015 Medical University of Sofia, Bulgaria. No conflict of interest. 207 Protective effects of Sanicula europaea extracts on nuclear DNA damage ˘ 1 , L. Dalyan1 , N. Arda1 . 1 Istanbul E. Onay U¸car1 , M. Pekmez1 , E. Mertoglu University, Molecular Biology and Genetics, Istanbul, Turkey Background: Sanicula europaea is widely used for the treatment of many diseases in traditional and complementary therapies. It is known that Sanicula europaea extracts have antiviral, antifungal and antimicrobial activities. The aim of the present study is to examine the comparative effects of different Sanicula europaea extracts on H2 O2 -induced DNA damage in HeLa cells. Material and Method: In this study, the effects of ethanolic and water extracts of Sanicula europaea are compared. Oxidative damage in two specific nuclear regions [transcriptionally active APEX (apurinic/aprimidinic endonucleases) gene and transcriptionally inactive insulin gene] are assessed by QPCR assay. The HeLa cells are pretreated with the extracts for 48 hours, followed by the treatment with 750 mM H2 O2 for 1 hour. Intracellular ROS level of the cells are evaluated by using a fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA). Results: DNA damage was significantly induced by H2 O2 while it was inhibited by Sanicula europaea extracts. All extracts completely protected against nuclear DNA damage, especially the ethanolic extracts were significantly inhibited DNA damage. While the intracellular ROS level increased in HeLa cells treated with H2 O2 , on the other hand ROS level decreased in the cells pretreated with Sanicula europaea extracts. Our results suggest that the extracts of Sanicula europaea can prevent oxidative DNA damage. Conclusion: Taken together, our results suggest that Sanicula europaea extracts can protect against DNA damage via direct inhibition of ROS formation

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and/or indirectly by other mechanisms. Therefore, dietary intake of Sanicula europaea extract may lower the risk of oxidative stress-mediated diseases such as some cancers via reduction of oxidative DNA damage and intracellular levels of ROS. No conflict of interest. 208 Prognostic significance of TERT promoter mutations and association with other common genomic aberrations in Bulgarian glioblastoma patients G. Stancheva1 , T. Goranova1 , M. Laleva2 , A. Mitkova1 , G. Poptodorov2 , N. Velinov2 , M. Kamenova3 , V. Mitev1 , N. Gabrovsky2 , R. Kaneva1 . 1 Medical University of Sofia, Molecular Medicine Center- Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria, 2 University Multiprofile Hospital for Active Treatment and Emergency Medicine “N.I. Pirogov”, Department of Neurosurgery, Sofia, Bulgaria, 3 University Multiprofile Hospital for Active Treatment and Emergency Medicine “N.I. Pirogov”, Department of Pathology, Sofia, Bulgaria Background: In the recent years many comprehensive studies were focused on genomic characteristics of gliomas. This led to the discovery of a variety of genes that were not associated with glial carcinogenesis before. Mutations in genes encoding isocitrate dehydrogenase isoforms 1 (IDH1) and 2 (IDH2) have been found in a large proportion of gliomas. IDH1/2 genetic alterations occur early in tumour progression of brain neoplasias, but rarely in other solid tumours and are associated with better outcome. Two mutations (c.1–124G>A and c.1–146G>A) affecting the promoter region of the telomerase reverse transcriptase (TERT) gene, have been recently associated with poor prognosis as well as with EGFR amplification, CDKN2A homozygous deletion and loss of 10q chromosome harbouring PTEN. Materials and Methods: To determine the prevalence of pTERT and IDH1/2 mutations in Bulgarian patients and their impact on prognosis, 30 primary glioblastomas were examined by sequencing for mutations in exon 4 of IDH1/2 as well as in the promoter region of TERT. Further alterations in EGFR, TP53, PTEN and CDKN2A genes were investigated by MLPA kit P105. Results: The two known hot spot mutations c.1–124G>A and c.1–146G>A in TERT promoter were found in 13 (43%) and 7 (23%) patients, respectively. Both TERT mutations have tendency of association with higher age of onset (median age 59.9 vs 44 in non-mutated cases; p = 0.142) while c.1– 146G>A was significantly associated with worse patient survival (median survival 4.27 months vs 16.43 in non-mutated cases; p = 0.05). IDH1 genetic alteration R132H was observed in 6 (20%) tumour samples. Mutations in IDH2 were not detected. The IDH1 mutation was found in younger patients (median age 43 vs 60.5 in non-mutated cases; p = 0.04) and was associated with an increased overall survival (median survival 28.4 months vs 8.07 in non-mutated cases; p = 0.002). In 56.6% (17) of the patients EGFR amplification was observed whereas PTEN, CDKN2A and TP53 deletions were identified in 63.3% (19), 43% (13) and 13% (4) of the patients, respectively. Patients with PTEN deletions revealed significant worse survival than patients without such aberrations (median survival 18.07 months vs 8.33 in non-mutated cases; p = 0.023). Multivariate regression analysis determined the IDH1 mutation as an independent prognostic factor of better survival (p = 0.023, HR = 0.104) whereas the mutation c.1–146G>A in the TERT promoter (p = 0.026, HR = 2.347) as such decisive for worse survival. Conclusions: The genetic alteration affecting IDH1 and the c.1–146G>A mutation in the promoter region of TERT could be used as an independent specific marker for prognosis of Bulgarian patients with primary glioblastoma. Acknowledgements: This work was supported by Grants: 66/2015, Medical University of Sofia and DUNK01/2/2009, National Science Fund, Ministry of Education and Science, Bulgaria. No conflict of interest. 209 Genome-wide sequencing identifies genetic relationship between first and late-onset second cancers in aristolochic acid nephropathy patients X. Castells1 , M. Ardin1 , S. Rorive2,3 , N. Broeders4 , A. Heguy5 , P.P. Bringuier6 , T. Quackels7 , T. Roumeguere7 , J. Nortier4 , J. Zavadil1 . 1 International Agency for Research on Cancer, Molecular Mechanisms and Biomarkers Group, Lyon, France, 2 Universite´ Libre de Bruxelles, Department of Pathology − ´ Erasme University Hospital, Brussels, Belgium, 3 Academie Universitaire Wallonie-Bruxelles, DIAPath − Center for Microscopy and Molecular Imaging 4 CMMI, Gosselies, Belgium, Universite´ Libre de Bruxelles, Department of Nephrology − Erasme University Hospital, Brussels, Belgium, 5 New York University Langone Medical Center, Genome Technology Center, New York, ˆ Edouard Herriot, Laboratoire de Biologie des Tumeurs- Service USA, 6 Hopital Central d’Anatomie et Cytologie Pathologiques, Lyon, France, 7 Universite´ Libre de Bruxelles, Department of Urology − Erasme University Hospital, Brussels, Belgium Introduction: Exposure to aristolochic acid (AA, IARC Group 1 carcinogen) present in some Chinese traditional medicines leads to aristolochic acid

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nephropathy (AAN), often complicated by development of multiple urothelial carcinomas of sequential onset. We used deep sequencing of multiple urinary tract tumours of AAN cases from a unique, well-characterized women population in Belgium, to characterize the relationships of the patients’ lateonset second cancers to the exposure to AA as well as to the first cancers. Materials and Methods: Aristolactam-DNA adduct-positive AAN patients (n = 4) who developed cancer within 8 years following the initial exposure to AA were chosen for analysis of their first cancers (upper tract urothelial carcinomas, UTUC) and second cancers with delayed onset (1−9 years after first-cancer diagnosis, involving the bladder or ureteral meatus). One case developed a third malignancy in the bladder, 3 years after the second cancer. All patients had received a kidney transplant before developing the second cancers and had a functional renal graft prior to prophylactic nephroureterectomy. Genomic DNAs were isolated from FFPE sections of the renal cortex and from the upper and lower tract tumours of each patient using laser capture microdissection or macrodissection of the tumour areas. Lowcoverage (~15x) exome 100-bp paired-end sequencing was performed using Illumina HiSeq2500. Customized variant calling identified somatic variants absent in non-tumour tissues. Non-negative matrix factorisation was applied to extract exome-wide mutational signatures in the tumour tissues. Results and Discussion: In all cases, we established the mutational signature of AA (the COSMIC signature 22) in the first UTUC as well as second cancers involving the bladder or lower ureter (meatus). Additionally, the first and second cancers of all cases harboured considerable overlaps in exposure-specific (A>T) somatic mutations. This finding provides evidence that the delayed onset of bladder urothelial carcinomas in AAN patients is likely due to distal seeding of cancer cells originating from the primary UTUC tumours. Conclusions: Our first-of-its-kind study addresses the risk as well as mechanistic factors leading to the second, late-onset bladder urothelial carcinomas following kidney transplantation and primary UTUC development. Our results underline the importance of long-term bladder follow-up in high-risk populations with established or suspected AA exposure. Acknowledgments: Funding from IARC; CMMI is supported by the European Regional Development Fund and the Walloon Region. We thank Ms. C. Carreira and Dr Y. Song for expert assistance with histopathology and Dr S. Villar and O. Aminova for sample processing and sequencing. The NYU Genome Technology Center is partially supported by the NIH/NCI (P30CA016087). No conflict of interest. 210 Reactivation of thyroid hormone receptors in fully developed rat hepatocellular carcinomas causes their regression and prevents lung metastasis A. Perra1 , M.A. Kowalik1 , L. Cabras1 , E. Puliga1 , G.M. Ledda-Columbano1 , A. Columbano1 . 1 University of Cagliari, Department of Biomedical Sciences, Cagliari, Italy Introduction: Hepatocellular carcinoma (HCC) is the second cause of cancer related deaths Worldwide. To date, the only curative treatment available is surgery. Our previous data demonstrated that a short treatment with T3 causes a rapid regression of rat preneoplastic nodules, suggesting that T3 induces a differentiation program towards a normal phenotype. Moreover, repeated administrations of T3 to rats bearing preneoplastic nodules, resulted in reduction of HCC incidence by 50%. Based on these results we evaluated whether fully developed HCCs are still responsive to the anti-tumorigenic and pro-differentiating effects of T3. Materials and Methods: F-344 rats were subjected to Resistant-Hepatocyte rat model of hepatocarcinogenesis, considting of a single dose of the carcinogen diethylnitrosamine (DENA) and a 2 week exposure to 2-acetylaminofluorene coupled with partial hepatectomy (PH). Ten month after DENA all rats developed early HCC (eHCC) and were divided in 2 groups. Group 1 was fed a T3-supplemented diet a week every three weeks and rats were sacrificed after 1 or 5 cycles; group 2 was maintained on a basal diet and rats were sacrificed at the same time points as T3 treated animals. After sacrifice liver was either snap frozen or formalin fixed. Histopathological analysis and immunohistochemical staining for the prognostic marker KRT-19 were done. Frozen samples were cryosectioned and HCC were lasermicrodissected (LMD) after histo-pathological classification. Total RNA was extracted from LMD samples and used for gene expression analysis by qPCR. Results and Discussion: Results showed that eHCCs are still able to respond to T3, as shown by increased expression of the TRs target genes Dio1 and Spot14 after 1 week. Reactivation of the T3/TRs axis was associated with a significant reduction of the more aggressive KRT19+ HCCs. Fourteen months after initiation, while 100% of the rats not treated with T3 showed well- or poorly-differentiated HCCs, the liver of rats treated with 5 cycles of T3 did not show macroscopic tumors and, microscopic analysis confirmed the absence of HCCs, with only a few adenomas being visible. Moreover, T3 treatment totally abolished the occurrence lung metastases.

Conclusion: These data demonstrate a strog anti-tumorigenic effect of T3 and suggest that thyroid hormone may induce a reprogramming of neoplastic cells towards a more benign phenotype. The evidence of a strong anti-neoplastic effect associated to the T3/TRs axis activation in already developed HCC could lead to new therapeutic strategies aimed to reactivate TRs signaling. No conflict of interest. 211 Breast cancer and Epstein–Barr virus infection Y. Shlyakhtunou1 , A. Savchenko1 . 1 Vitebsk State Order of Peoples’ Friendship Medical University, oncology, Vitebsk, Belarus At the present time we do not stop looking for new predictors of tumor flow in breast cancer. Association of viruses and neoplastic processes in one body continues to interest and excite many of the scientific community worldwide. Objective: To investigate persistence of Epstein–Barr virus in the tumor tissue of breast carcinoma as a possible new prognostic factor in tumor progression. Materials and Methods: The study included all 178 tumor tissue samples of breast carcinomas. Of these, 40 tumor samples for retrospective analysis, and 138 samples for prospective study. Conducted research presence core antigen of Epstein–Barr virus (EBNA-1) in breast carcinoma tissue immunohistochemistry (IHC) method (n = 40 retrospectively, n = 70 prospectively) and (n = 68) by the polymerase chain reaction (PCR) you searched the DNA of the virus. Results of the study: It is found that in 53.5% of tumor tissue samples occurs presence of viral nuclear antigen confirmed by IHC and in 42.6% of samples − confirmed by PCR. Most often the presence of EBNA-1 and DNA of Epstein–Barr virus was determined in patients materials having metastasized to regional lymph (N +) components, as well as tumor-high and medium grade (G2-G3), having lymphovenous invasion (LVSI «+») as well as Her2-neooverexpressing cancers and high proliferative activity index (Ki-67 >50%). The risk of progression of tumor after completion of special treatment in terms of up to 21 months increased in 69.2–96.6% of women who have the persistence of Epstein–Barr virus in the tumor tissue. In 87.5% of patients with the presence of the viral genome (EBNA-1), confirmed by IHC method has a risk of dying from breast cancer within 5 years after diagnosis. Conclusion: Association of Epstein–Barr virus can be considered as a negative prognostic factor contributing to the progression of the tumor process, increasing the risk of death from breast cancer. So far, not established pathogenetic mechanism of influence of Epstein–Barr virus in the tumor tissue of breast carcinoma and the ability of the virus to influence the development of drug resistance to different groups of chemotherapeutic agents used to treat cancer of this localization. Further investigation of the mechanisms of tumor cells using the culture will optimize approaches to breast cancer treatment. No conflict of interest. 212 New approach to analyzing gene expression in neuroendocrine tumors M.V. Comanescu1 , M. Dobre2 , F. Vasilescu3 . 1 INCD Victor Babes, Pathology, Bucuresti, Romania, 2 INCD Victor Babes, Pathology, Bucharest, Romania, 3 Military Central Hospital, Pathology, Bucharest, Romania Neuroendocrine tumors (NETs) are epithelial neoplasms arising mainly from the gastrointestinal tract, pancreas and the bronchial tree that have different behavior, from indolent tumors to clinically aggressive, poorly differentiated carcinomas. The objective of the study was to investigate NETs in order to identify an alternative method for the immunohistochemical testing by performing a qRT-PCR reaction and to create a PCR array that will be used as a virtual device with direct application in molecular diagnosis, prognosis and therapy of NETs. Materials and Methods: We studied tumoral and peritumoral fragments fixed in formalin and embedded in paraffin from 26 patients diagnosed with NET (pulmonary, gastrointestinal and pancreatic) by histopathological and immunohistochemical techniques. In order to assess gene expression a PCRarray method was used (Qiagen) which identified relative expression of 18 genes of interest by comparing the tumour with the peritumoral tissue, the normalization being carried out by means of two reference genes. The quality control test was performed using primers for the control of genomic DNA contamination, reverse transcription control and control of the efficiency of amplification. Results: In cases of pulmonary NETs, VGF, MGMT and SSTR1 gene were strongly over-expressed, GAST, SYP, and CCND1 genes were moderately overexpressed while mTOR, INS, CHGA, MKI67, SSTR5 and SLIT2 genes had a low overexpression. In gastrointestinal NETs, CHGA and SSTR1 genes were moderately overexpressed, whileGRPR, mTOR, SSTR3 and SSTR5 genes had a low overexpression. sStr2 and M KI67 genes were downregulated. In pancreatic tumors, GAST, VGF and TYMS genes were moderately overexpressed, SSTR3 and SSTR1 genes had a low overexpression and MGMT and SLIT2 genes were downregulated. We identified different gene profiles for the three anatomical locations of NET studied. Somatostatin receptor (SSTR) overexpression represents a predictive marker for the therapy

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 with somatostatin analogues. According to the literature, the expression of somatostatin receptors, especially SSTR1, is positively correlated with the patient survival rate which justifies the use of these receptor genes as prognostic markers in NETs. Conclusions: Identification of gene profile may represent a sensitive tool for diagnosis and therapy in NETs. The virtual device will allow the pathologist, instead of using a broad panel of antibodies, to detect and quickly quantify the degree of differentiation, specific hormone production expression of somatostatin receptor, the potential of proliferation and angiogenesis. Acknowledgement: The study was supported by Project RENET − PN-II-PTPCCA-2011-3.2.0623 91/20012. No conflict of interest.

Sunday 10 July 2016 Poster Session

Cell and Tumour Biology I 215 Frequent and functional derepression of the Iroquois homeobox gene IRX3 in human acute leukemia T. Somervaille1 , T. Somerville1 . 1 Cancer Research UK Manchester Institute, Leukaemia Biology, Manchester, United Kingdom Background: Transcription factors are master regulators of cell fate and their tissue-inappropriate mis-expression is an important and emerging mechanism of oncogenic transformation. Methods and Results: Using bioinformatics methods and quantitative PCR, we identified the Iroquois homeobox transcription factor gene IRX3 as highly expressed in ~30% of cases of primary human acute myeloid leukemia (AML), but minimally expressed in prospectively purified normal human hematopoietic populations (stem, progenitors and mature). High IRX3 expression in AML is strongly associated with mutated NPM1, FLT3-ITD and high concomitant HOXA gene expression. High IRX3 expression is also observed in both T-acute lymphoblastic leukemia (T-ALL) (~50% of cases) and B-ALL (~20% of cases), also in association with high HOX gene expression. Iroquois members have essential developmental roles in mesodermal tissues such as heart, fat and bone, but to date there is no reported functional role for IRX3 in leukemic hematopoiesis. Compared with control cells, forced expression of IRX3 in murine CD117+ bone marrow hematopoietic stem and progenitor cells (BM HSPCs) resulted in enhanced serial replating in semisolid culture and significantly impaired morphologic differentiation. In transplantation experiments, murine recipients of IRX3-expressing CD117+ BM HSPCs exhibited a significant expansion of LY6C+ T-cells and later developed donor-derived T-cell leukemias with incomplete penetrance. In coexpression experiments, Hoxa9/IRX3 double transduced CD117+ BM HSPCs exhibited significantly greater clonogenic cell frequencies and morphologic differentiation block by comparison with control Hoxa9/empty vector (MTV) cells. In vivo, in the post-transplant period Hoxa9/IRX3 BM HSPCs exhibited multilineage differentiation but with skewing away from the myeloid lineage and towards the B-lineage by comparison with control Hoxa9/MTV cells. Nevertheless, donor-derived AML subsequently developed in all mice in both cohorts. Unexpectedly, recipients of control Hoxa9/MTV cells developed AML significantly more rapidly than recipients of Hoxa9/IRX3 cells (median 125 days versus 270 days). However, mice receiving Hoxa9/IRX3 cells developed leukemias with a much more pronounced differentiation block by comparison with Hoxa9/MTV recipients, as evidenced by immunophenotype, morphology and transcriptome. IRX3 knockdown in both human and murine AML cells induced differentiation and loss of clonogenic potential, whereas similar experiments in primary human CD34+ cells did not affect clonogenic potential. Conclusions: De-repression of the mesodermal transcription factor gene IRX3 in human acute leukemia is both frequent and functional, contributing to leukemic transformation through skewing differentiation, enhancing selfrenewal and modulating the cellular consequences of HOX gene expression. No conflict of interest. 216 Tumour suppressor genes of common fragile sites: Active players in DNA damage response?! R. Aqeilan1 . 1 Hebrew University-Hadasah Medical School, Immunology & Cancer Research, Jerusalem, Israel The role of common fragile sites (CFSs) in cancer is controversial. Two main views dominate the discussion: one view suggests that CFSs are inert structures associated with genomic instability leading to inactivation of genes associated within them. The opposing view suggests that CFSs are functional units harboring genes and other elements that when altered lead to selective pressure contributing to cancer development and/or other diseases. This view is supported by emerging evidence showing that inactivation of these residing tumour suppressor genes leads to dysregulation of the DNA damage response

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(DDR) and increased genomic instability. Emerging evidence from our lab and others directly links CFS gene products with the DDR, genomic instability, carcinogenesis and other disease states. These findings underscore the critical roles of these genes and how their inactivation represents selective advantage in pathogenesis. No conflict of interest. 218 Study of the biomarker potential of circulating non-coding PRNCR1 & CCAT2 RNAs in plasma of breast cancer patients E. Babaei1 , V. Montazeri2 . 1 Genetics, School of Natural Sciences, Tabriz, Iran, 2 Noor Najat Hospital, Torax, Tabirz, Iran Introduction: Despite recent breakthroughs in the treatment of breast cancer, early non-invasive detection and prognosis remain poor. Emerging evidence shows that long non-coding RNAs (lncRNAs) play an important role in cancer initiation and progression. Here, we evaluated the expression of PRNCR1 & CCAT8 as novel lncRNAs in plasma of breast cancer patients. Materials and Methods: The expression levels of PRNCR1 & CCAT8 in 50 pairs of breast cancer patients and normal women were detected by quantitative real-time PCR (qRT-PCR). Results and Discussion: Our data showed that both lncRNAs are upregulated in plasma of breast cancer patients in comparison with normal ones. Furthermore, statistical data revealed that lncPRNCR1 & lncCCAT2 are significantly expressed in advanced stages rather than lower stages. Also, ROC analysis demonstrated the biomarker potential of circulating CCAT2. Conclusion: Our data provides the first evidence detecting lncRNAs; PRNCR1 & CCAT2 in blood of cancer patients. Meanwhile, the positive correlation between their expression levels and carcinomas as well as their TNM stage highlights PRNCR1 & CCAT2 as a biomarker for detection and prognosis of breast malignancies. No conflict of interest. 219 Determination of pyruvate metabolic fate regulates head and neck tumourigenesis W.C. Li1,2,3 , T.Y. Chen1 , J.M. Huang1 , C.W. Chang1 , C.Y. Chen1 , W.J. Chang1 , C.J. Liu1,2,4,5 , H.M. Chen6 , J.F. Lo1,2,3,7 . 1 Institute of Oral Biology, School of Dentistry- National Yang-Ming University, Taipei, Taiwan, 2 Department of Dentistry, School of Dentistry- National Yang-Ming University, Taipei, Taiwan, 3 Genome Research Center, National Yang-Ming University, Taipei, Taiwan, 4 Department of Medical Research, MacKay Memorial Hospital, Taipei, Taiwan, 5 Department of Oral and Maxillofacial Surgery, MacKay Memorial Hospital, Taipei, Taiwan, 6 Department of Dentistry, National Taiwan University Hospital- College of Medicine, Taipei, Taiwan, 7 Department of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan Introduction: Most cancer cells preferentially obtain cellular energy and biomacromolecules via aerobic glycolysis over mirochondrial oxidative phosphorylation. Indeed, previous studies have shown that suppression of glycolytic enzymes could modulate cell malignancy in various cancers. Pyruvate is the main glycolytic metabolite that could be sequentially converted into either lactate or acetyl-CoA by Lactate Dehydrogenase A (LDHA) and Pyruvate Dehydrogenase Complex (PDC), repectively, under various physiological conditions. In tumour cells, lactate is largely procuded and exported out of cells resulting in acidic environment whereas the conversion of pyruvate to acetly-CoA, a key reactant for TriCarboxylic Acid (TCA) cycle in mitochondrion, is often less active. Owing to this variance, the determination of pyruvate metabolism might be of great potential to regulate tumorous phenotype in cancer cells. Material and Method: The cellular and molecular regulations of LDHA and Pyruvate dehydrogenase E1 component a subunit (PDHA1), a catalytic subunit of mitochondrial PDC, for cell malignancy in Head and Neck Squamous Cell Carcinoma (HNSCC) were examined. The multifaceted cancer phenotypes were identified in vitro in shRNA-mediated LDHA and PDHA1 deficient HNSCC cells while in vivo growth of LDHA and PDHA1 deficient HNSCC derived xenografic tumours were analysed. Results and Discussion: In vitro analysis showed that decreased cell growth and cell migration/invasion capacity as well as up-regulated cell differentiation and drug sensitivity to chemotherapeutic agents in LDHA deficient HNSCC cells were observed. Loss of PDHA1, on the contrary, enhanced cell growth, cell motility, drug resistance but inhibited cell differentiation. The xenografic tumours deficient for LDHA grew slower than control tumours whereas PDHA1 knockdown resulted in larger tumour mass in vivo. In molecular level, the protein array analysis demonstrated that LDHA/PDHA1 mediated regulation for HNSCC tumourigenesis could possibly be through the modulation of ERK/JNK/p38-c-jun pathway. Conclusion: Taken together, the choices of pyruvate metabolic fates regulated cell malignancy in HNSCC cells revealing that the balance between lactate production and oxidative phosphorylation activity plays a significant role for HNSCC development. No conflict of interest.

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220 Multiple myeloma cells reprogram bone marrow mesenchymal stem cells’ translation initiation thereby promoting their migration M. Dabbah1,2 , O. Attar-Schneider2 , S. Tartakover Matalon2 , V. Zismanov2 , L. Drucker2 , M. Lishner2 . 1 Tel Aviv University, Sackler Faculty of Medicine, Tel Aviv, Israel, 2 Meir Medical Center, Oncogentic Laboratory, Kfar Saba, Israel Background and Purposes: Myeloma cells’ (MM) interaction with the bone marrow (BM) microenvironment is integral to disease progression and drug resistance. Mesenchymal stem cells (MSCs) are an important component of BM niche. Protein syntheses mostly regulated at the initiation phase is critically important to the cells’ phenotype. Previously, we showed co-modulation of translation initiation in BM-MSCs from normal donors (ND) co-cultured with multiple myeloma. Here, we characterized the time line of MSCs conditioning by multiple myeloma cells, the persistence of this effect, and the consequences on cell phenotype. Methods: MSCs were isolated and propagated from BM of normal donors (ND). ND-MSCs were co-cultured with MM cell lines (U266, ARP1) (MM conditioned MSCs) (1.5 h,12 h, 24 h, 48 h and 3 days) assayed for translation initiation status (eIF4E, eIF4GI; regulators: mTOR, MNK, 4EBP; targets: SMAD5, NFkB, cyclinD1, HIF1, cMyc) (immunoblotting) and migration (scratch assay, inhibitors). Involvement of MAPKs in MSCs conditioning and altered migration was also tested (immunoblotting, inhibitors). MM conditioned MSCs were re-cultured alone (1−7 days) and assayed for translation initiation (immunoblotting). qPCR of extracted RNA was tested for microRNAs levels. Results: MAPKs are activated within 1.5 h of co-culture and responsible for MM conditioned MSCs translation initiation status (↑~>200%, p < 0.05) and elevated migration (16 h, ↑~>400%, p < 0.05). The Bone Marrow MSCs conditioning by MM cells is reversible after only 1 day of MM conditioned MSCs culture alone. Decreased expression of MIR199b and MIR125a (↓~>40%, p < 0.05) in MM conditioned MSCs supports elevated migration. Discussion: Time and proximity dependent conditioning of ND-MSCs underscores the dynamic co-evolution of MM and bone marrow niche. MAPK/translation initiation factors induced MM conditioned MSCs migration may contribute to disease progression and additional studies are warranted to establish its potential as an effective therapeutic target. No conflict of interest. 222 ILK as a signaling nexus for induction of breast cancer stem cells in response to tissue stiffness and hypoxia M.F. Pang1 , M. Stallings-Mann2 , M.J. Siedlik1 , S. Han1 , D.C. Radisky2 , C.M. Nelson1 . 1 Princeton University, Chemical and Biological Engineering, Princeton, USA, 2 Mayo Clinic Cancer Center, Cancer Biology, Jacksonville, USA The microenvironment has been defined as a key determinant of cell phenotype during tumor progression, and breast tumors are both more stiff and hypoxic than nonmalignant breast tissue. Using an innovative engineered culture model, we now demonstrate that stiff and hypoxic microenvironmental conditions promote the formation of breast cancer stem cells (CSCs) through modulation of integrin-linked kinase (ILK). Specifically, stiff and hypoxic microenvironments activate integrin signaling and the expression of CSC markers and induction of CSC phenotypic characteristics in invasive breast cancer cells through the PI3K/Akt pathway. ILK depletion blocks acquisition of the CSC phenotype and marker exrpression, while ectopic expression of ILK stimulates CSC properties under softer or normoxic conditions. Stiff microenvironments are also found to promote tumorigenesis and metastasis in ovo, both of which are abrogated by knocking down ILK. Analysis of breast cancer patient samples reveals an association between matrix stiffness, ILK and CSC markers, as ILK and CD44 double-positive breast cancer cells are found in the regions of tumors predicted to be stiff in breast cancer biopsies. Our data suggest a role for ILK as a critical mechanotransducer that modulates breast CSC phenotype in response to tissue mechanics and oxygen tension. No conflict of interest. 225 Modulation of microRNA-1288 in oesophageal squamous cell carcinoma via targeting tumour suppressor FOXO1 F. Islam1 , S. Pillai1 , V. Gopalan1 , A. King-Yin Lam1 . 1 Cancer Molecular Pathology of Menzies Health Institute Queensland- Griffith University- Gold Coast- Australia, School of Medicine, Gold Coast- Southport, Australia Background: MicroRNA-1288 (miR-1288) is located at 17p11.2 and has been reported to play important role in the pathogenesis of cancers. The expressions and functional roles of miR-1288 have not been studied in oesophageal cancer. Thus, this study aimed to examine the expression profiles and functional significance of miR-1288 in oesophageal squamous cell carcinoma (ESCC). Material and Methods: In total, 120 oesophageal tissues (90 primary ESCCs, 30 non-neoplastic tissues) with surgical resection were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic was used to detect its in-vitro effects on various cellular responses such as cell proliferation, colony formation, cell invasion and migration. Modulatory

changes of miR-1288 targeted proteins, FOXO1 and TAB3, were examined using Western-blot and immunofluorescence. Results and Discussions: Overexpression of miR-1288 was predominantly noted in ESCC tissues and cell lines when compared to normal oesophageal tissues and non-neoplastic cell lines respectively. The miR-1288 overexpression was frequently associated in ESCC patients with advanced pathological stages. However, patients of ESCC with high levels of miR-1288 expression showed a better survival when compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. Ectopic overexpression of miR-1288 in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced cancer promoting features of ESCC cell by targeting FOXO1 implied that miR-1288 acts as an oncogenic miRNA in ESCC. Conclusion: Overexpression of miR-1288 play a key role in the pathogenesis of ESCC and its modulation may have potential therapeutic value in patients with ESCC. No conflict of interest. 226 ZEB1 induces the Wnt antagonist DKK1 to jointly determine poorer survival in colorectal carcinomas 3 2 O. De Barrios1 , B. Gyorffy ˝ , M.J. Fernandez-Ace ´ nero ˜ , E. Sanchez-Till ´ o´ 1 , J.I. Casal4 , D.S. Darling5 , A. Castells6 , A. Postigo7 . 1 IDIBAPS, Group of Transcriptional Regulation- Dept. of Oncology and Hematology, Barcelona, Spain, 2 MTA TTK, Lendulet ¨ Cancer Biomarker Research Group, Budapest, ´ Jimenez ´ Hungary, 3 Hospital Universitario Fundacion D´ıaz, Dept. of Pathology, 4 ´ CIB-CSIC, Dept. of Madrid, Spain, Centro de Investigaciones Biologicas Cellular and Molecular Medicine, Madrid, Spain, 5 Center for Genetics and Molecular Medicine- University of Louisville, Dept. of Oral Immunology and Infectious Diseases, Louisville, USA, 6 Hospital Cl´ınic, Institute of Metabolic and Digestive Diseases, Barcelona, Spain, 7 ICREA, Institut Catala` de Recerca i Estudis Avan¸cats, Barcelona, Spain

Introduction: Aberrant activation of Wnt signaling is involved in the pathogenesis of several carcinomas, most notably colorectal (CRCs). Canonical Wnt signaling can be triggered by the engagement of cell receptors by soluble Wnt ligands, which induce the transcription of Wnt target genes, including the tumor-promoting transcription factor ZEB1. In turn, the Wnt pathway can be inactivated by antagonists such as Dickkopf factors (DKK1DKK4). DKK family members usually act as tumor suppressors but in certain tumors and stages of cancer progression, they can promote oncogenesis. Of note, DKK1 is induced by canonical Wnt signalling and is overexpressed in breast and prostate carcinomas. Materials and Methods: The study has made use of a wide range of technical approaches: transgenic CRC cell lines, high-throughput techniques (gene expression microarrays) and transgenic mouse models. Results: We found that, in contrast to other Wnt antagonists, DKK1 paradoxically associates with poorer prognosis in CRCs. Study of the relapsefree survival associated to different levels of ZEB1 and DKK1 in gene microarray databases from 928 CRC patients revealed that CRC patients displaying high expression of both ZEB1 and DKK1 have lower survival rate than those with high levels of ZEB1 but low DKK1. In other words, the worse survival effect determined by ZEB1 depends on joint expression of DKK1. Both genes also significantly correlated in a gene array of 1557 CRCs and in a panel of CRC cell lines. We also examined ZEB1 and DKK1 in colon samples from wild type and ZEB1-deficient mice, and found that DKK1 expression was downregulated in the latter. Both transient and stable ZEB1 knockdown in several CRC cells resulted in the downregulation of DKK1 protein and mRNA levels. Transcriptional reporter and ChIP assays showed that ZEB1 directly binds to the promoter region of DKK1 gene and that ZEB1 activates its transcription through the recruitment of the co-activator p300 in Wnt-active CRC cells. Discussion and Conclusions: Our data shows that ZEB1 binds to the DKK1 promoter to directly drive its transcription through a mechanism involving p300 and that the maximum effect of ZEB1 as a predictor of reduced survival requires high levels of DKK1. These results indicate that DKK1 mediates some of ZEB1 tumor promoting effects, offering a new approach to target ZEB1 expression and function in cancer therapy. No conflict of interest. 227 The progression of K-ras induced early cancerous lesions is driven by a COPD-like inflammation C. Jungnickel1 , P. Schnabel2 , R. Bohle2 , R. Bals1 , C. Beisswenger1 . 1 Saarland University, Internal Medicine V, Homburg/Saar, Germany, 2 Saarland University, General and Special Pathology, Homburg/Saar, Germany Background: Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer (LC). COPD is characterized by chronic inflammation of the

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 airways and lung infections. Adenocarcinomas are frequently associated with an activating mutation in the K-ras gene. The aim of this study was to determine the effect of COPD-like inflammation on the progression of K-ras induced cancerous lesions. Material and Methods: Mice were exposed to cigarette smoke (CS) 5 days/week. CS- or air exposed mice were exposed to a clinical isolate of heat inactivated non-typeable Haemophilus influenzae (NTHi) 3 days/week. Histologic analyses were performed on formalin fixed and paraffin embedded sections. Pre-cancerous lesions were divided into alveolar hyperplasia (AH), alveolar adenomatous hyperplasia (AAH), and adenocarcinoma. AH were defined by an increase of cells along the interalveolar septa in the alveolar space. AH developed from single-layered lesions into AAH, characterized by a further increase in cellularity. The structure of the alveoli was recognizable, without invasive growth. Cells within adenocarcinoma were charcterized by a round shape, nuclear atypia and less differentiation compared to AH. Results: Mice with an oncogenic K-ras allele in lung epithelium were exposed to CS, NTHi, and the combination of CS and NTHi for 12 weeks. Exposure of mice to NTHi resulted in an increased total burden and in increased numbers of AAH lesions, whereas exposure to CS alone did not affect tumor growth. Combined exposure to CS and NTHi led to larger pre-cancerous lesions and an increased adenocarcinoma formation. Conclusions: CS and NTHi promote the progression of K-ras induced early cancerous lesions resulting in more severe phenotype. No conflict of interest. 228 GLUT1/GLUT4 balance is a marker of androgen-insensitivity in prostate cancer P. Gonzalez-Menendez1 , D. Hevia1 , R. Alonso2 , I. Gonzalez-Pola1 , J.C. Mayo1 , R.M. Sainz1 . 1 Instituto de Oncolog´ıa del Principado de Asturias/University of Oviedo, Morphology and Cell Biology, Oviedo, Spain, 2 Hospital Universitario Central de Asturias, Immunology department, Oviedo, Spain Background: Cancer cells show an increase in glucose uptake, mainly associated with an overexpression and membrane translocation of GLUT transporters. GLUT1 is the main responsible in most of the tumours but others can be involved, as the insulin-dependent GLUT4. On the other hand, glycolytic metabolism differs between androgen-sensitive and insensitive stages in prostate cancer. A downregulation of androgen receptor (AR) levels was found in hyperglycaemia, showing less cases of prostate cancer in diabetic patients. Thus, the aim of this work was to study the balance between GLUT1 and GLUT4 in prostate cancer and the role androgen signalling in glucose metabolism. Material and Methods: Androgen-sensitive LNCaP and androgen-insensitive PC-3 cells were used. Also, androgen-sensitive PC-3AR cells, castrationresistant LNCaP-R cells and cells overexpressing GLUT1 or GLUT4 were stablished. In addition, TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice and prostatic samples from patients were employed in order to study the role of androgenic signalling on GLUT1/4 balance in vivo. Results and Discussion: It was found a direct association between GLUT1 and GLUT4 protein levels and the androgen-sensitivity of prostate cancer. Androgen-sensitive cells upregulated GLUT1 expression under glucose deprivation, being AR predominantly found in nucleus. In addition, cellcycle distribution of GLUT1 was similar to nuclear AR distribution. DHT treatment increased GLUT1 levels, showing that GLUT1 was directly related to AR localization. On the other hand, GLUT4 was downregulated after DHT treatment and played a more important role in glucose uptake by androgen-insensitive cells. PSA (prostate specific antigen) production and Bcl2 levels increase in LNCaPGLUT4 cells despite AR nuclear levels were lower. In TRAMP mice, both GLUT1 and GLUT4 levels increased with tumour progression while castrated mice show lower GLUT4 levels that were restored under testosterone treatment. Finally, GLUT4 levels correlates with PSA and Bcl-2 expression in prostatic samples with Gleason 7 while GLUT1 expression was higher in lower Gleason scores. Conclusions: The balance between GLUT1 and GLUT4 is a potential marker of androgen-sensitivity in prostate cancer, being their expression dependent of nuclear AR expression. No conflict of interest. 229 The effects of aspirin on growth and COX-2 expression in prostate cancer cells R. Wang1 , U.K. Shah1 , G. Jenkins1 , S. Doak1 . 1 Institute of Life Science, Swansea University Medical School, Swansea, United Kingdom Background: Aspirin is widely used in clinical practice and has recently been found to decrease the risk of prostate cancer. However, the exact mechanisms behind its anti-cancer properties are not fully understood. The aim of this project was to identify the effects of aspirin on prostate cancer cells and the differences in response between cells grown in monolayer cultures versus 3D spheroid culture.

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Materials and Method: In this work, the metabolite of aspirin, salicylate, was used. The regulation of salicylate on growth of PC-3 and DU-145 prostate cancer cell lines, and PNT-2, a normal prostate cell line, was firstly explored by cytotoxicity analysis based upon relative population doubling (RPD). In addition, cyclooxygenase-2 (COX-2) expression in response to salicylate was assessed by RT-qPCR. A 3D culture system based on agarose-coated wellplates was developed. The growth between two-dimension (2D) and threedimension (3D) PC-3 and PNT-2 cells models was then evaluated by the MTT assay. Results and Discussion: Low concentrations of salicylate (2 and 4mM) significantly inhibited the proliferation of PC-3 (P < 0.001) and DU-145 (P < 0.001) prostate cancer cells. In contrast, salicylate’s anti-proliferative effect was not as prominent on the normal prostate PNT-2 cells. With the 2D culture method, after 24 hour exposure to salicylate, COX-2 expression was significantly reduced by 30% at 10mM in DU-145 and PC-3 cells (P < 0.001), but PNT-2 did not show any significant reduction with respect to the expression. As cells grown in 3D constructs demonstrate closer to in vivo behaviour, we optimised growth of the PC-3 and PNT-2 cells in spheroid format. The 3D models had a slow growth rate as compared to standard 2D cultured cells. The number of live cells in the 3D spheroids reached a peak by the 4th day after seeding with an initial concentration of 5000 cells/well. Conclusions: Based on the cytotoxicity analysis and COX-2 analysis, salicylate promotes cell death and decreases COX-2 gene expression in prostate cancer cells. These effects were more notable in the cancer cells and were less striking in the normal prostate cells indicating the cancer cells are more sensitive to salicylate induced toxicity. A 3D prostate cancer models has also been developed, which will be valuable for future studies into prostate carcinogenesis. No conflict of interest. 230 MiR-302b improves chemotherapy efficacy in human breast cancer and affects tumor microenvironment A. Cataldo1 , I. Plantamura1 , E. D’Ippolito1 , S. Baroni1 , D. Palmieri2 , M.V. Iorio1 . 1 IRCCS Fondazione Istituto Nazionale dei Tumori, Start Up UnitDepartment of Experimental Oncology, Milan, Italy, 2 Comprehensive Cancer Center- The Ohio State University, 2Department of Molecular VirologyImmunology and Medical Genetics- College of Medicine and Solid Tumor Biology Program, Columbus, OH, USA Introduction: MicroRNAs (miRNAs) are a class of non-coding regulatory RNAs that play key roles in different biological processes including cancer. We have shown that miR-302b over-expression enhances sensitivity to cisplatin in breast cancer cell lines by targeting directly E2F1 and indirectly ATM. Recently the role of microRNAs was also investigated not only in tumor cells but also in tumor microenvironment. Macrophages are present in tumor tissues and constitute a major component of the inflammatory microenvironment of cancer and have been classified in M1 macrophages, that are potent killers of pathogens and tumor cells, or M2 macrophages, that enhance tissue remodeling and repair and promote tumor growth and invasion. M1 and M2 can switch between forms in response to stimuli. M2 macrophages have been also associated with resistance to chemotherapeutic treatments. MiRNAs can regulate macrophages polarization and their gene expression profile. We here investigated whether miR-302b plays a role in macrophage M1 polarization, and if this affects response to chemotherapy. Material and Method: MDA-MB-231 were transfected with miR-302b precursor or control, than cells were treated with doxorubicin (100nM), 5-FU (100uM) and docetaxel (50nM) for 24 h, after cell viability was evaluated. MiR302b expression has been evaluated by qRT-PCR in 35 TNBC treated with chemotherapy in adjuvant setting, and associated with prognosis. Immortalized murine macrophages RAW264.7 were treated with CpG-ODN, a synthetic agonist of TLR9 able to switch macrophages phenotype in M1, for 12, 24 and 72 hours. MiR-302b expression was evaluated by qRT-PCR and macrophages polarization in M1 was evaluated by IL-12 ELISA. Results and Discussion: We found that miR-302b enhances sensibility, affecting cell viability, to doxorubicin, 5-FU and docetaxel. MiR-302b expression was also associated with time to relapse and overall survival in triple negative breast cancer patients treated with adjuvant chemotherapy. Moreover, immortalized murine macrophages RAW264.7, polarized in M1 with CpGODN, increase the expression of miR-302b compared with non treated macrophages. Conclusion: Our data underline that miR-302b affects all chemotherapeutic mechanisms and not only genotoxic drugs such as cisplatin; for this reason miR-302b could be used as a therapeutic tool in breast cancer model in combination with chemotherapy and could be classified as a predictive factor for response to chemotherapy. Furthermore, miR-302b modulation in M1 suggests that this microRNA exerts its effect not only affecting specific processes in tumor cells, but also modulating the activation of tumor associated macrophages. Finally, we hypothesize that miR-302b-mediated M1 induction affects chemotherapy response in breast cancer. No conflict of interest.

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231 Extracellular matrix proteins derived from the tumor microenvironment as circulating breast cancer diagnostic markers M. Giussani1 , E. Landoni1 , G. Merlino1 , F. Turdo1 , S. Veneroni2 , V. Cappelletti1 , M.G. Daidone1 , R. Miceli1 , R. Orlandi1 , T. Triulzi1 , E. Tagliabue1 . 1 Fondazione IRCCS Istituto Nazionale Tumori, Experimental Oncology and Molecular Medicine, Milan, Italy, 2 Fondazione IRCCS Istituto Nazionale Tumori, Tissue Biobank, Milan, Italy Background: An early and well-documented event during tumorigenesis is the generation of activated fibroblasts, which contributes to alterations in the extracellular matrix (ECM). Here, we tested whether the abnormal production of ECM molecules fostered by the cross-talk between an incipient breast carcinoma (BC) clone and the adjacent fibroblasts might lead to blood-released ECM molecules that precede tumor diagnosis. Materials and Methods: Class comparison analysis was performed on two public available datasets (GSE5764 and GSE59595) using the BrB-ArrayTool. Normal human dermal fibroblasts (NHDFs) treated with conditioned media from BC cell lines MDAMB468, SKBr3 and T47D were evaluated for ECM molecule production and release in culture supernatants by gene expression profiling on Illumina platform and by Western blot. Two independent series of human plasma samples from healthy donors, patients with benign breast disease and patients with BC were tested by ELISA for the presence of circulating COL11A1, COMP and COL10A1 molecules. Data quality control, class comparison and class prediction analyses were performed on ELISA data. Results and Discussion: In silico analysis of gene expression profiles of normal and BC tissue samples revealed 11 ECM genes significantly upmodulated in tumor versus normal tissue (p < 0.01, FDR 1.5, adjusted p-value G, c.5263_5264insC in BRCA1, and c.5851_5854delAGTT, c.5946delT, c.5718_5719delCT c.9098_9099insA, c.9908delA in BRCA2. The mutation analysis was carried out by direct sequencing using automated Genetic Analyser ABI 3130xl. Results: Three of the recurrent mutations were found in the HBOC patients: c.181T>G, c.5263_5264insC and c.5946delT. The predominant mutation observed with frequency of 23.73% was BRCA1 c.5263_5264insC, which has been recognised as the most prevalent mutation in high-risk Bulgarian breast

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cancer patients. Among the HBOC patients carrying the mutation 57.14% developed ovarian cancer, 35.71% breast cancer and 7.14% cancer of the fallopian tubes. The mutations c.181T>G in BRCA1 and 5946delT in BRCA2 were observed only once with frequency of 1.79% each in patients with both bilateral breast and ovarian cancer. Altogether inherited BC predisposition was identified in 27.13% of the patients. Conclusions: The most prevalent mutation in Eastern Europe c.5263_ 5264insC was prevalent among the studied Bulgarian HBOC patients with 23.73% overall frequency and appeared to have a founder effect in the Bulgarian population. We suggest initial screening of all HBOC families to specifically detect this mutation and identify the highest proportion of the carriers. Acknowledgements: This work was supported by Grant No. 45, Medical Faculty, Medical University, Sofia, Bulgaria and by Grant DUNK01/2/2009, National Science Fund, Ministry of Education and Science, Bulgaria. No conflict of interest. 283 Stromelysin-3 as a marker of metastasis and predictor of poor survival in oral squamous cell carcinoma S.H. Lin1,2 , S.F. Yang1 , K.T. Yeh2 , C.W. Lin3 . 1 Chung Shan Medical University, Institute of Medicine, Taichung city, Taiwan, 2 Department of Surgical Pathology-, Changhua Christian Hospital, Changhua, Taiwan, 3 Chung Shan Medical University, Institute of Oral Sciences, Taichung, Taiwan Introduction: Stromelysin-3 is reported to be overexpressed in several cancers and may contribute to tumorigenesis. The current study investigated the association between the clinicopathological characteristics and stromelysin-3 levels in oral squamous cell carcinoma (OSCC) patients. Material and Method: In this study, we detected the expression of stromelysin-3 in 279 patients with OSCC using tissue microarrays (TMAs), and evaluated its correlation with clinicopathologic factors and disease prognosis. In addition, the metastatic effects of the Stromelysin-3 overexpression on the oral cancer cells were investigated by cell migration and invasion assay. Results: Stromelysin-3 expression was present in 118/279 (42.3%) cases and was associated with lymph node metastasis (p = 0.034) and tumor differentiation (p = 0.009). Importantly, stromelysin-3 expression was also correlated with a poorer patient prognosis in a univariate survival analysis (p = 0.010, log-rank test). Moreover, the in vitro studies also demonstrated that stromelysin-3 overexpression promoted cell migration and invasion of oral cancer cells. Conclusion: Our study showed that stromelysin-3 levels may be useful for assessment of the disease progression, especially lymph node metastasis, in patients with OSCC. No conflict of interest. 285 Eribulin differentiates cetuximab resistant oral squamous cell carcinoma cells to sensitive by inducing mesenchymal–epithelial transition (MET) H. Kitahara1 , M. Hirai1 , H. Nakamura1 , S. Kawashiri1 . 1 Kanazawa Univ. of Graduate School of Medical Science, Oral and Maxillofacial Surgery, Kanazawa, Japan Background: In recent years, inhibition of EGFR signaling has emerged as new treatment strategy for oral squamous cell carcinoma (OSCC). In our previous study, we found that loss of EGFR expression in OSCC was associated with EMT, and might have functional implications in resistance to cetuximab treatments. The eribulin may also make residual tumours less aggressive and less likely to metastasise by triggering a shift from mesenchymal to epithelial phenotypes via reversal of the EMT state to the MET state. In the present study, we evaluated the effect of eribulin in human cetuximab resistant OSCC cell lines and analyzed with regard to their EMT characteristics and their sensitivity to cetuximab. Material and Methods: The subjects were three human oral squamous cell carcinoma cell lines established from tumor biopsies with different grade of invasive abilities were used: OSC-20 (low grade invasive cells), OSC-19 (low grade invasive cells) and HOC313 (high grade invasive cells). Results: The EMT-associated genes N-cadherin, vimentin and Snail were upregulated in cetuximab resistant high grade invasive HOC313 cells compare to other low grade invasive cells. The eribulin sensitivity of HOC313 cells was high in comparison with other low-grade invasive cell lines, and eribulin induced this HOC313 cells to undergo an EMT-associated gene switch that results in high EGFR expression. In eribulin treated HOC313 cells, the proliferation was significantly inhibited by cetuximab compared to control eribulin untreated cells. Conclusions: These data suggested that eribulin differentiates cetuximab resistant oral squamous cell carcinoma cells to sensitive by reversing phenotype from epithelial–mesenchymal transition (EMT) to mesenchymal– epithelial transition (MET) states. No conflict of interest.

286 The role of TEX19 in growth and maintenance of cancer stem-like cells V. Planells1 , L. Parry2 , J. Wakeman1 , R. McFarlane1 . 1 Bangor University, North West Cancer Research Institute, Bangor, United Kingdom, 2 European Cancer Stem Cell Research Institute, Cardiff University, Cardiff, United Kingdom Cancer Stem-like Cells (CSC) are believed to be responsible for local relapse and metastasis of tumours and may also be the cells that are responsible for resistance to therapies. As such, it is of fundamental importance to study the molecular mechanisms involved in generating cells with properties of CSCs. Cancer Testis Antigen (CTA) genes are a group of genes, with generally unknown function, whose expression is restricted to the testis (and fetal ovary and placenta) in normal individuals, but are also expressed in a wide variety of malignant tumour cells types. CTAs are potentially of great immunotherapeutic importance since the testis is an immune privileged tissue meaning that the immune system does not recognise antigens normally present in this site. TEX19 (Testis Expressed 19) is a mammalian specific gene whose expression was first detected in the testis and ovary in mouse. Since then, it has been shown to expressed in adult human testis (spermatogonia) and also in pluripotent mouse embryonic stem (ES) cells. The function of this gene remains unknown although several studies suggest that it maintains the selfrenewal potential and pluripotency of germ and ES cells. More recently, TEX19 has been reported as a regulator of the expression of transposable elements (TE). TEs are DNA sequences which can change their position within the genome; therefore they can cause deleterious mutations, gene disruption and chromosome rearrangements that may lead to cancer. This research employed different molecular biology techniques as well as in vitro and in vivo experiments to elucidate the role of the TEX19 gene. Results redefine TEX19 as a CTA gene, suggesting that it can regulate the proliferation of cancer cells in vivo/in vitro and it can act as a TEs regulator in human cells. No conflict of interest. 287 Control of epithelial splicing regulatory proteins in epithelial–mesenchymal transitions L. Li1 , S. Oltean1 . 1 University of Bristol, School of Physiology and Pharmacology, Bristol, United Kingdom Introduction: The epithelial–mesenchymal transition (EMT), one of the hallmarks of cancer, is a biological process that allows polarized epithelial cells to undergo multiple biochemical changes to become mobile mesenchymal cells. Epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) are cell-type-specific regulators of transcripts that switch splicing during the EMT. ESRPs drive splicing events of about 200 genes those give epithelial phenotype. One of the well-known genes regulated by tissue-specific alternative splicing is the fibroblast growth factor receptor 2 (FGFR2) which has a switch between two mutually exclusive isoforms − FGFR2-III b and FGFR2-III c during the EMT. ESRP 1 and 2 were identified as regulators of FGFR2 alternative splicing, leading to the FGFR2 isoforms being switched to the epithelial cell-type-specific splicing program. Previous studies established ESRPs as master regulators of EMT and underlie epithelial–mesenchymal transitions during tumour progression and fibrosis. Material and Method: As FGFR2 splicing is a sensor of ESRPs activity and EMT, it may be used for understanding the regulation of ESRP in cancer and fibrosis. Using a splicing-sensitive fluorescent reporter based on inclusion/exclusion of FGFR2 exon IIIc we have performed a screen with the LOPAC library (Library of Pharmacologically Active Compounds) on HEK293 cell line to identify compounds that induce skipping of exon IIIc in FGFR2 and potentially block EMT. Results: We have identified several compounds that are able to switch FGFR2 splicing, and currently being validated as modulators of epithelial– mesenchymal transitions, and show various activities in functional assays (proliferation and migration) in vitro. Conclusions: We used FGFR2-based splicing-sensitive fluorescent reporter to perform a screen with the LOPAC library and identified several compounds that are able to switch FGFR2 splicing and potentially modulate ESRP activity and EMT. No conflict of interest. 288 New insights of mutant calreticulin in myeloproliferative neoplasms M. Morlan Mairal1 , A. Aziz1 . 1 Salford University, Biomedical Research Centre- School of Environment and Life Sciences, Salford, United Kingdom Calreticulin (CALR) is an endoplasmic reticulum (ER) chaperone and calcium binding protein mutation of which has recently being associated with myeloproliferative neoplasms. Insertion and deletion mutations in exon 9 of CALR gene have been shown to be the only genetic anomaly in about 40% essential thrombocythemia (ET) and primary myelofibrosis (PMF) without Janus kinase 2 mutations. However, the cellular behaviour of this mutated

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 protein still being unclear. Here we address this issue by employing a combination of computational prediction tools and molecular techniques. We analysed the predicted secondary structure and the disorder probability of CALR wild-type and CALR mutants using S2D, PONDR-FIT, IUPRED and RONN-FIT serves as molecular structure prediction bioinformatic tools. Further insight of mutant CALR cellular localization was addressed by fluorescent microscopy and the possible link between CALR mutations and altered JAK/STAT pathway was analysed by Real-time polymerase chain reaction. Analysis of CALR mutant secondary structure showed that CALR intrinsical disordered C-terminal domain decreases the disorder probability in CALR mutants and these mutations lead to small changes in the secondary structure of CALR C-terminal domain. Changes in the secondary structure of CARL could lead to changes in the cellular behaviour of this mutant protein. CALR mutant localization showed that CALR mutant is localized out of ER in abundant vesicles within the cytoplasm. Additionally, preliminary experiments show changes in expression of JAK/STAT pathway target genes in CALR mutant expressing cells suggesting a possible dysregulation of this signalling pathway in myeloproliferative neoplasms positive for CALR mutations. C-terminal domain mutations in the endoplasmic reticulum chaperon CALR lead to small changes in the disorder probability and secondary structure of CALR mutant. Moreover, CALR mutations alter the cellular localization of this protein and they possibly lead to a dysregulation of JAK/STAT pathway, a known signalling pathway commonly affected in myeloproliferative neoplasms. No conflict of interest. 289 Gene-specific interactions between ultraviolet radiation and melanoma A. Viros ´ 1 , M. Pedersen2 , S.J. Furney1 , M.R. Girotti1 , K. Hogan1 , G. Saturno1 , E. Galvani1 , B. Sanchez-Laorden1 , C. Ng3 , J.S. Reis-Filho3 , P. Lorigan4 , M. Cook1 , R. Marais1 . 1 CRUK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 Institute of Cancer Research, Targeted therapy team, London, United Kingdom, 3 Memorial Sloan Kettering Cancer Center, Department of Pathology, New York, USA, 4 The Christie NHS Foundation Trust, Medical Oncology, Manchester, United Kingdom Background: There are differences in the epidemiological, clinicopathological and genetic features of melanoma driven by oncogenic BRAF or oncogenic NRAS. NRAS melanoma affects older people more frequently and occurs at sites accruing UVR damage such as the head and neck. The contribution that ultraviolet radiation (UVR) makes to genetically distinct melanoma subtypes is unclear. Material and Methods: We modelled the interaction of UVR with V600EBRAF or G12DNRAS to mimic human disease, using mouse melanoma models and translated our findings to human BRAF and NRAS melanoma. Results: Previously we reported that UVR cooperates with V600EBRAF by targeting the tumour suppressor TP53. In this study we show that, similar to humans, UVR cooperates with G12DNRAS to drive melanoma in older animals that accumulate more UVR damage. Moreover, murine UVR-G12DNRAS melanomas are histologically and genetically heterogeneous, replicating the heterogeneity observed in human NRAS melanoma. As in humans, UVRG12DNRAS melanomas have a higher burden of UVR-induced mutations, a longer latency to tumour development and present secondary driver mutations distinct from UVR-V600EBRAF melanoma despite their similar UVR-induced mutation rate. Our data suggests that the longer tumour latency in NRAS tumours is due to an increased requirement for genetic cooperators to drive melanomagenesis. We also show UVR cooperates differently with other RAS isoforms by exposing animals expressing G12DKRAS in melanocytes to UVR. These animals presented specific cooperating secondary targets distinct from UVR-G12DNRAS. This suggests that different RAS isoforms activate the MAP kinase pathway differently. We then show the relationship between excessive sun exposure and NRAS-driven melanoma by linking the epidemiological, clinical and genetic characteristics of a G12DNRAS-driven human patient exposed to extreme doses of UVR and present preclinical evidence using patient derived xenografts, that targeting the UVR-induced cancer mutations found in the “long tail” of UVR-related mutations in NRAS melanoma can help stratify disease and provide therapeutic options for patients. Conclusions: The insight gained begins to provide a molecular explanation for distinct associations between UVR and individual oncogenes in melanomagenesis. No conflict of interest. 290 PDGFRb inhibition by Gint4.T aptamer prevents recruitment of bone marrow-derived mesenchymal stem cells into breast cancer microenvironment R. Fontanella1 , S. Camorani2 , L. Cerchia2 , A. Zannetti1 . 1 Istituto di Biostrutture e Bioimmagini, CNR, Naples, Italy, 2 Istituto di Endocrinologia e Oncologia Sperimentale “G. Salvatore”, CNR, Naples, Italy Background: Bone marrow-derived mesenchymal stem cells (BM-MSCs) are shown to participate in tumor progression by establishing a favorable

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tumor microenvironment that through a cytokine networks promote metastasis. However, the mechanism of homing and recruitment of BM-MSCs into tumors and their potential role in malignant tissue progression is poorly understood. The aim of this study was to analyze the involvement of PDGFRb in controlling BM-MSC recruitment by Triple Negative Breast Cancer (TNBC) cells, and investigated the role of BM-MSCs within breast cancer microenvironment. In order to interfere with cross-talk between BM-MSCs and TNBC cells, we tested a validated anti-PDGFRb RNA aptamer. Materials and Methods: To determine PDGFR expression in BM-MSC, western blotting and RT-PCR analysis were performed. Activation of liganddependent PDGFR and downstream signaling in BM-MSCs, untreated or treated with Gint4.T, was analyzed by western blot. Cell viability and migration were assessed by MTT assay and transwell Boyden chamber assay, respectively. To evaluate BM-MSC migration toward TNBC cell lines (MDA-MB-231 and BT-549), BM-MSCs were seeded into the upper chamber, in the presence or absence of Gint4.T, and TNBC cells were used as chemoactractans. Furthermore, the differentiation of BM-MSCs into cancer associated fibroblasts (CAF), was analyzed by western blot with a-SMA and fibroblast activation protein (FAP) antibodies. Results: We found that PDGFRb is overexpressed and plays a crucial role in controlling BM-MSC activity. Indeed, targeting PDGFRb in BMMSCs with Gint4.T aptamer, causes a strong inhibition of P-PDGFRb and downstream P-Erk and P-Akt. Gint4.T affects BM-MSC proliferation and migration. Furthermore, the aptamer prevents BM-MSC migration toward TNBC cells and their differentiation in CAF. Conclusions: These findings suggest that the recruitment of BM-MSCs and their activity in breast cancer microenvironment is prevented by aptamermediated PDGFRb inhibition. No conflict of interest. 291 MALDI-MS and [11 C] acetate PET imaging of lipid heterogeneity in non-small-cell lung cancer F. Henderson1 , D. Lewis2 , D. Soloviev2 , K. Brindle2 , A. McMahon3 , K.J. Williams1 . 1 The University of Manchester, Manchester Pharmacy School, Manchester, United Kingdom, 2 The University of Cambridge- La Ka Shing Centre, Cancer Research United Kingdom—Cambridge Institute, Cambridge, United Kingdom, 3 The University of Manchester, The Wolfson Molecular Imaging Centre, Manchester, United Kingdom Background: Tumour heterogeneity poses a major problem for diagnosis and therapeutics. Lipid membrane formation and signalling are altered in cancer, and so it is advantageous to understand lipid heterogeneity in tumours. Lipids consist of fatty acids, which are synthesised de novo by fatty acid synthase (FAS) in humans. FAS is upregulated in numerous cancers, including nonsmall-cell lung cancer (NSCLC)1. [11 C] Acetate positron emission tomography (PET) and matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS) imaging have been used as complementary imaging techniques to reveal inter-tumour heterogeneity of lipids. Material and Methods: [11 C] Acetate PET scans were carried out on a NSCLC mouse model and lung tissue was flash-frozen. Tumours were sectioned and mounted onto indium tin oxide coated slides. Sections were washed and sprayed with matrix (2,5-dihydroxybenzoic acid). MALDI-TOF MS imaging was performed on a MALDI-7090 (Shimadzu, Manchester, UK). Images were processed using BioMap (Novartis). Lipid MS/MS spectra were compared to the LIPID MAPS database for interpretation. Results: [11 C] Acetate PET scans of a NSCLC mouse model have revealed heterogeneity of FAS activity, suggesting metabolic differences between tumours. MALDI-MS images complement these findings by showing clear inter-tumour heterogeneity of lipid profiles, despite hematoxylin and eosin images showing no histological difference. Lipid signals showing inter-tumour heterogeneity include lysophosphatidylcholines and phosphotidylcholines. Conclusions: Lipid heterogeneity in NSCLC tumours has been observed with MALDI-MS imaging and PET. Future work will include comparing autoradiographs with MALDI images to see which lipids are involved with high and low regions of FAS activity, as well as imaging fatty acids in the TCA cycle to elucidate mechanisms occurring within these tumours. No conflict of interest. 292 Metabolic rewiring in melanoma cell lines that acquired resistance to BRAF inhibitors F. Baenke1 , B. Chaneton2 , M. Smith1 , N. Van Den Broek2 , K. Hogan1 , H. Tang1 , A. Viros1 , N. Dhomen1 , E. Gottlieb2 , R. Marais1 . 1 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 Cancer Research UK Beatson Institute, Cancer Metabolism Research Unit, Glasgow, United Kingdom Background: Metabolic reprogramming is one of the hallmarks of cancer as it supports the increased demand in bioenergetics, macromolecule biosynthesis and balance of the redox potential that is needed to maintain continuous proliferation and growth in environments that are deprived of oxygen and

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nutrients. Aerobic glycolysis, a common characteristic of solid tumours, converts glucose to lactate regardless of oxygen availability. This can be directly linked to oncogenes and tumour suppressors, altering metabolic processes in tumour cells. The most common mutation in cutaneous melanoma is a glutamate for valine substitution at position 600 (V600E) in BRAF, which results in an active kinase that drives constitutive MEK/ERK signalling and hyperproliferation. Targeted therapies that inhibit V600EBRAF, such as vemurafenib and dabrafenib, can be effective therapies in BRAF mutant melanoma and they extend progression-free and overall survival. However, most patients eventually develop resistance to these drugs. Material and Methods: To investigate whether acquired resistance to BRAF inhibitors is accompanied by metabolic changes, we generated BRAF resistant melanoma cell lines by chronically exposing a panel of melanoma cells to PLX4720. The parental and drug-resistant cell lines were metabolically profiled and metabolic flux analysis was performed. Results: A subset of the BRAF inhibitor resistant cell lines displayed increased mitochondrial mass and increased oxidative metabolism. Accordingly, these cells were more dependent on mitochondrial function and therefore more sensitive to mitochondrial poisons. Moreover, these resistant cells switched from using glucose to glutamine as their primary carbon source and this rendered them more sensitive to drugs that inhibit glutamine metabolism. We validate these observations in vivo by showing that treatment of tumourbearing mice with glutamine inhibitors delayed the onset of resistance to BRAF inhibitors. Conclusion: Here we show that a subset of BRAF inhibitor resistant melanoma cells are more dependent on mitochondrial function and glutamine metabolism, rendering these cells more dependent on pathways and providing a new therapeutic target. This approach therefore has the potential to provide exciting new treatment opportunities that could delay the emergence of resistance to BRAF inhibitors. No conflict of interest. 293 The role of vasculogenic mimicry in small cell lung cancer S. Williamson1 , F. Trapani1 , B. Abbott1 , M. Galvin1 , R. Metcalf1 , M. Hendrix2 , F. Blackhall3 , K. Frese1 , K. Simpson1 , C. Dive1 . 1 CRUK Manchester Institute, Clinical and Experimental Pharmacology, Manchester, United Kingdom, 2 Northwestern University, Robert H. Lurie Comprehensive Cancer Center, Chicago, USA, 3 The Christie NHS Foundation Trust, Department of Medical Oncology, Manchester, United Kingdom Background: Vasculogenic mimicry (VM) describes the ability of tumour cells with ‘stem-like’ plasticity to adopt an endothelial phenotype and form de novo perfusable vascular structures. We recently discovered that VM occurs in small cell lung cancer (SCLC) and is associated with poorer prognosis in limited stage patients. We asked whether Vascular Endothelial-Cadherin (VE-Cad) was causal for SCLC VM. We also asked whether VM affected tumour growth dynamics in vivo, the delivery and efficacy of cisplatin (the mainstay of SCLC treatment) and the ability of SCLC cells to metastasise. Material and Methods: VM was assessed in vitro with network forming assays on matrigel. qPCR was used to examine a previously reported VM associated gene expression signature. Knockdown (KD) of VE-Cad with shRNA was performed in VM competent SCLC H446 cells. Parental and VE-Cad KD H446 cells were implanted s.c. in NSG immune-compromised mice and the efficacy of cisplatin/etoposide treatment was assessed. Periodic Acid Schiff and CD31 immunohistochemistry (IHC) was used enumerate VM vessels within xenografts and cisplatin delivery was assessed via IHC of platinumbound DNA. Results: VE-cad expression was increased in SCLC cell lines competent for VM network formation in vitro. VE-Cad KD led to loss of VM network formation in vitro and the VM associated gene expression signature and decreased prevalence of VM vessels in vivo. Xenografts of H446 parental cells grew rapidly and mice developed extensive metastases, whereas H446 VE-Cad KD xenografts showed increased latency (mean time to 200 mm2 tumour 20.95 days for H446 parental and 43.27 H446 VE Cad KD) before exponential growth and lost metastatic capacity. VE-Cad KD xenografts contained reduced levels of cisplatin bound DNA compared to parental xenografts. However, despite this impaired intra-tumoural cisplatin delivery H446 VE-Cad KD xenografts were more sensitive to treatment with cisplatin/etoposide (study ongoing). Conclusion: These data suggest that VM affects tumour growth kinetics, metastatic capability, cisplatin delivery and chemotherapy efficacy in SCLC in vivo. Future research will explore the molecular mechanisms that underpin SCLC VM and investigate whether novel therapeutic opportunities in SCLC emerge via a better understanding of this process. No conflict of interest.

294 New models for investigating integrin antagonists A. Elsharif1 , H. Sheldrake1 , L. Patterson1 , S. Shnyder1 . 1 University of Bradford, Institute of Cancer Therapeutics, Bradford, United Kingdom Introduction: Invasion and metastasis of cancer is the main cause of cancer mortality. Therefore, new treatments which will inhibit this progression of the disease are required. Therefore, new treatments which will inhibit this progression of the disease are required. Integrin adhesion receptors, particularly the RGD-binding integrin subfamily (avb3, avb5 and allbb3) are related to progress and spread of cancer with poor prognosis. The overarching principle in this study is that for the development of new treatments, models are needed to test them. Methods: Consequently, the goal of this study was to characterise the expression of av, aIIb, a5, and b3 and b5 integrin subunits using immunofluorescence and flow cytometry in a panel of cell lines, in order to determine whether the cells expressed functional avb3 and aIIbb3. Results: The expression of av subunit was the highest of all integrins investigated, whereas the expression of allb was variable level between the cells lines. In addition, an inducible expression of allb was observed in PMAtreated K562 using immunofluorescence. PMA induction of K562 cells causes a variety of changes in morphology and expression of av, aIIb, a5, and b3 and b5 integrin subunits. There was no significant difference between PMAtreated DU145 and untreated cells. After characterising the expression of avb3 and aIIbb3 integrins in K562 cells, a functional test for cellular adhesion was determined; PMA-treated cells showed increased adhesion on fibrinogen compared to untreated cells. Then cRGDfV (anti avb3/b5) and GR144053 (anti aIIbb3) antagonists were investigated for cytotoxicity and for effects on K562 fibrinogen adhesion with and without PMA. The tested cells adhered to fibrinogen, a b3 substrate, and adhesion was inhibited by anti-avb3 and antiaIIbb3 controls (particularly in PMA-treated cells), suggesting the cell lines would be a useful dual b3-expressing model. A panel of potential novel b3 integrin antagonists was investigated for cytotoxicity and effect in the validated adhesion assay. Conclusion: The identification of compounds with dual anti-b3 activity will be reported. No conflict of interest.

295 Identification of new combination therapies for NSCLC tumours harbouring KRAS mutations M. Molina-Arcas1,2 , D.C. Hancock2 , C. Moore2 , S. Horswell3 , N. Matthews4 , J. Downward1,2 . 1 Institute of Cancer Research, Lung Cancer Group, London, United Kingdom, 2 The Francis Crick Institute, Oncogene Biology Laboratory, London, United Kingdom, 3 The Francis Crick Institute, Bioinformatics core, London, United Kingdom, 4 The Francis Crick Institute, Advanced Sequencing Facility, London, United Kingdom Introduction: Oncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. Thus, new therapeutic strategies are needed to treat these tumours. We have previously shown that NSCLC cells harbouring KRAS mutations are more sensitive to inhibition of KRAS downstream effectors MEK and RAF and to treatment with IGF1R inhibitors. Mechanistically, results showed that PI3K pathway activity is dependent on basal IGF1R activity in KRAS mutant, but not wild-type, lung cancer cells. Materials and Methods: In order identify complementary targets to improve the effect of MEK and IGF1R inhibitor therapies, we performed a whole genome shRNA screen in KRAS mutant NSCLC cells to identify genes that, when inhibited, cooperate with MEK and/or IGF1R inhibition. Validation of the top hits was performed using shRNAs and small molecule inhibitors in a panel of NSCLC cell lines. In vivo efficacy of drug combinations was measured in an autochthonous mouse model of Kras-induced NSCLC using microcomputerized tomography (CT) scanning. Results: The screen results reveal genes that can promote sensitization to MEK and/or IGF1R inhibitors as well as genes that can produce drug resistance. Interestingly, the list of sensitizers to IGF1R inhibitors includes several genes from the mTOR pathway. Viability assays in a panel of cell lines confirms that combinations of IGF1R inhibitors with mTOR inhibitors, both rapalogs and kinase inhibitors, resulted in a synergistic anti-proliferative effect in KRAS mutant NSCLC cells. Mechanistic investigation demonstrates some differences between rapalogs and mTOR kinase inhibitors, however, in both cases combination with IGF1R inhibitors blocks PI3K feed-back loop activation and results in a more effective inhibition of AKT and mTORC1 signalling. This inhibition is stronger in KRAS mutant cells than in wild-type cells. Addition of a MEK inhibitor to the combination of mTOR and IGF1R inhibitors increases apoptosis in KRAS mutant cells and produces marked tumour regression in a mutant Kras-induced, p53 deleted NSCLC mouse model. Conclusions: These findings suggest potential novel therapeutic strategies for NSCLC tumours harbouring KRAS mutations. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 296 Modes of cell death induced by novel tetrahydroisoquinolinonebased analogues in breast- and lung cancer cell lines

299 Apoptosis and proliferation in micropapillary structures of colorectal polyps and carcinomas

M. Verwey1 , A.M. Joubert1 , W. Dohle2 , B.V.L. Potter3 , A.E. Theron1 . 1 University of Pretoria, Department of Physiology, Pretoria, South Africa, 2 University of Bath, Department of Pharmacy and Pharmacology, Bath, United Kingdom, 3 University of Oxford, Department of Pharmacology, Oxford, United Kingdom

M. Patankar1 , S. Vayrynen1 , A. Tuomisto1 , M. Makinen1 , T. Karttunen1 . 1 University of Oulu, Cancer and Translational Medicine Research Unit/Department of Pathology, Oulu, Finland

Background: The A,B-ring of the steroidal microtubule disruptor, 2-methoxyestradiol (2-ME) can be mimicked with a tetrahydroisoquinolinone (THIQ) core. These THIQs are cytotoxic agents with antitumor-and antimicrobial activities, interact with microtubule networks and lead to cell cycle arrest in the G2phase. The aim of this in vitro study was to investigate the modes of cell death induced by four non-steroidal THIQ-based analogues on metastatic breast adenocarcinoma MDA-MB-231 cells and A549 lung carcinoma epithelial cells. Methods and Materials: Cytotoxicity studies were done to determine the half-maximal growth inhibitory concentration (GI50) of 2-ME, and four nonsteroidal THIQ based analogues namely STX 2895, STX 3329, STX 3451 and STX 3450 using spectrophotometric quantification of crystal violet staining and lactate dehydrogenase (LDH) release for necrosis quantification. Light- and confocal microscopy were used to investigate cellular morphological changes and microtubule integrity in response to these analogues. Fluorescent microscopy using monodansylcadaverine-staining was conducted to visualize acidic vesicle accumulation. Flow cytometric quantification of annexin V-FITC and propidium iodide was used to quantify apoptosis as well as cell cycle progression. To determine the signal transduction pathways involved in the induction of apoptosis, flow cytometric analysis of mitochondrial membrane integrity, aggresome detection and cytochrome c release were performed, as well as spectrophotometric quantification of caspase-3, -6 and -8. Results: Cytotoxicity studies revealed that the THIQ-based analogues inhibited 50% of cell growth at nanomolar concentrations. No increase in LDH levels was observed in treated cells, indicating the absence of necrotic cell death. Results obtained from morphology studies indicated that all the compounds induced cell death via apoptosis, with the potential involvement of autophagy and led to microtubule depolymerisation. These THIQ-based analogues caused an increase in cytochrome c release, caspase 3 activation and reduction in the mitochondrial membrane potential indicating the induction of the intrinsic apoptotic pathway. Conclusion: This in vitro study revealed that the novel chimeric THIQ-based analogues exert antiproliferative- and antimitotic effects culminating in the induction of apoptosis in both MDA-MB-231 and A549 cell lines. In vitro and in vivo investigations into the efficacy of these potential anticancer drugs are envisaged. No conflict of interest. 297 Lysyl oxidase regulates EGFR signalling through the extracellular matrix H. Tang1 , L. Leung2 , G. Saturno1 , A. Viros1 , G. Di Leva1 , E. Morrison1 , D. Smith1 , L. Johnson2 , N. Dhomen1 , C. Springer2 , R. Marais1 . 1 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 The Institute of Cancer Research, Gene and Oncogene Targeting Team, London, United Kingdom Cancer cells remodel their microenvironment to promote tumour growth and metastatic spread, and increased expression of enzymes such as lysyl oxidase (LOX), which takes part in these processes, is correlated with poorer outcome in many cancer types. LOX is a secreted enzyme that cross-links collagen and elastin to remodel the extracellular matrix (ECM) but how this contributes to tumour growth, metastasis and tumour progression is unclear. To investigate this we used a PathScan RTK signalling antibody array to reveal LOX dependent signalling pathways and found that EGF receptor (EGFR) phosphorylation was significantly reduced when LOX was depleted in MDA-MB-231 cells. We showed that this was because LOX depletion led to poorly retained EGFR at that cell surface following EGF stimulation, leading to reduced EGFR signalling and consequently reduced proliferation. Using a GFP-trap/mass spectrometry approach, we identified Matrillin2 (MATN2) as a protein that binds to LOX in the extracellular space. We showed that in addition to binding to MATN2, LOX regulates MATN2 expression by suppressing TGFb1 signalling. Interestingly, MATN2 has 10 EGF-like repeats and we showed that purified human MATN2 bound to the cell surface, but that this binding was blocked by an EGFR neutralising antibody and by EGF itself. We conclude that MATN2 binds to EGFR, to promote its retention at the cell surface and enhance EGF-dependent EGFR activation and cell proliferation. We found that LOX depletion, or treatment of cells with a LOX inhibitor suppressed MATN2 expression in tumours, diminished EGFR plasma membrane localisation and suppressed tumour growth. We conclude that through MATN2, LOX regulates EGFR retention at the cell surface to control tumour growth and metastasis. No conflict of interest.

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Background: Micropapillary structures (MIP) can be defined as focal piles of epithelial cells in columnar epithelium. MIP’s are found in many cancers, but they are characteristic for serrated colorectal carcinomas. Suprabasal cells of MIP show no contact with extracellular matrix (unpublished). Our main objective is to evaluate whether MIP cells in colorectal polyps and cancers would show evidence for anoikis inhibition we assessed proliferation and apoptosis indexes (PI and AI, respectively) in MIP and non-MIP epithelium. Materials and Methods: We stained human colorectal lesions: non-serrated adenomas (n = 15), serrated polyps (n = 29), conventional adenocarcinomas (n = 32), and serrated adenocarcinomas (n = 30) with antibodies to Ki67 and M30. To obtain PI and AI two independent observers counted proportions of positive cells separately in the cells of MIP and in non-MIP epithelium. Results: In carcinomas, AI was lower in the cells of MIP (M30; median 0, range 0−40) than in the non-MIP cells (median 2, range 0−40; p < 0.001, Wilcoxon). Similarly, PI was lower in the MIP cells (Ki67; MIP: median 17, range 0−93; non-MIP: median 30, range 1−93; p < 0.001). Similar patterns were evident in subsets of serrated lesions, in both carcinomas (p < 0.001) and polyps (p < 0.001), and in non-serrated carcinomas (p < 0.004). Apoptosis/proliferation (AI/PI) ratio was lower in the MIP cells than in the non-MIP epithelium in both cancers and their precursor lesions (P < 0.01). Conclusion: Apoptosis rate was lower in MIP’s than in other tumor epithelium indicating ability of these cells to survive without matrix contact, i.e. Anoikis is inhibited in them. Lower PI could suggest that cells in the MIP are in some kind of quiescent stage. However, significantly lower AI/PI ratio in the MIP infers that these structures may form a fast growing subpopulation of tumor cells. Further studies are in progress to dissect mechanism of formation of MIP and matrix independent survival of tumor cells in them. No conflict of interest.

300 AICAR, an AMPK activator, sensitizes TRAIL-mediated apoptosis through p38 MAPK-dependent and AMPK-independent signaling pathways in human bladder cancer T24 cells Y.H. Choi1,2 , M.H. Han3 , S.H. Hong1 , J.W. Jeong1,2 , E.O. Choi2,4 , K.Y. Jeon5 , C. Park5 . 1 Dongeui University, Department of Biochemistry- Dongeui University College of Korean Medicine, Busan, Korea, 2 Dongeui University, Anti-Aging Research Center, Busan, Korea, 3 National Marine Biodiversity institute of Korea, Natural Products Research Team- Converging Research Division, Seocheon, Korea, 4 Pusan National University, Department of Food and Nutrition- College of Human Ecology, Busan, Korea, 5 Dongeui University, Department of Molecular Biology- College of Natural Sciences and Human Ecology, Busan, Korea Background: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-superfamily that selectively induces apoptosis in cancer cells, but several cancer cells have acquired potent resistance to TRAIL-induced cell death. 5-aminoimidazole-4-carboxamide riboside (AICAR) is widely used as an activator of adenosine monophosphate-activated protein kinase (AMPK) that regulates the responses of the cell to energy change. Although AICAR-induced AMPK activation can induce apoptosis in various cancer cells, the molecular mechanisms whether AICAR triggers TRAILmediated apoptosis in TRAIL-resistant cancer cells. Here, we investigated the ability of AICAR to sensitize TRAIL-mediated apoptosis in human bladder cancer TRAIL-resistant T24 cells. Material and Methods: The inhibition of cell growth was evaluated by an MTT assay, and the apoptotic cells were determined by the DAPI staining, agarose gel electrophoresis and DNA flow cytometry analysis. JC-1 fluorescence probe was used to examine the mitochondrial membrane potential (MMP, Dym). The protein levels and caspases activity were measured using Western blot analyses and colorimetric assay, respectively. Results: Our data indicated that AICAR sensitized TRAIL-resistant T24 cells to TRAIL-mediated apoptosis, compared with either agent alone. The induction of apoptotic cell death by combined treatment with AICAR and TRAIL was connected with an up-regulation of DR4, down-regulation of Bcl-2 and XIAP, and increased the percentage of cells with a loss of MMP. Combined treatment with AICAR and TRAIL also induced the proteolytic activation of caspases (-3, -8 and -9), and degradation of caspase-3 substrate proteins. Moreover, AICAR treatment triggered phospholylation of p38 mitogen-activated protein kinase (MAPK) and AMPK; however, petreatment with SB203580, an inhibitor of p38 MAPK, resulted in a significant inhibition of apoptosis induced by combined treatment with AICAR and TRAIL, but not compound C, a specific inhibitor of AMPK. Conclusions: Overall, our findings suggest that AICAR enhances TRAILmediated apoptosis in TRAIL-resistant T24 cells via p38 MAPK-dependent

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and AMPK-independent signaling pathways, suggesting that AICAR may offer an effective therapeutic strategy in TRAIL-resistant bladder cancer cells. Acknowledgement: This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2014R1A1A1008460 & 2015R1A2A2A01004633). No conflict of interest. 301 Regulation of AMPK-dependent MDR1 expression by resveratrol in cisplatin-resistant human bladder cancer T24 cells via attenuating NF-úB and CRE transcriptional activity S.H. Hong1 , M.H. Han2 , C. Park3 , S.Y. Ji3 , J.W. Jeong1,4 , E.O. Choi4,5 , Y.H. Choi1,4 . 1 Dongeui University, Department of Biochemistry- Dongeui University College of Korean Medicine, Busan, Korea, 2 National Marine Biodiversity institute of Korea, Natural Products Research Team- Converging Research Division, Seocheon, Korea, 3 Dongeui University, Department of Molecular Biology- College of Natural Sciences and Human Ecology, Busan, Korea, 4 Dongeui University, Anti-Aging Research Center, Busan, Korea, 5 Pusan National University, Department of Food and Nutrition- College of Human Ecology, Busan, Korea Background: The expression of P-glycoprotein (P-gp) encoded by the multidrug resistance 1 (MDR1) gene is associated with the emergence of the MDR phenotype in cancer cells. Resveratrol, a natural polyphenolic compound, has been attracted increasing attention in recent years because of its potent chemopreventive and anti-tumor activities; however, its effects on drugresistant cancer cells are unknown. In this study, we investigated whether resveratrol down-regulates MDR1 expression in the cisplatin-resistance human bladder cancer T24 (T24/Cis) cells. Material and Methods: Cell viability was determined using an MTT assay. The intracellular accumulation of the fluorescently-tagged P-gp substrate using rhodamine-123 was analyzed by a flow cytometer. The mRNA and protein levels were determined by RT-PCR and Western immunoblotting, respectively. Results: Our data indicated that resveratrol suppressed preferentially the growth of T24/Cis cells as compared with T24 cells, and inhibited MDR1 expression, which was associated with the increased intracellular accumulation of the fluorescently-tagged P-gp substrate. Resveratrol also induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK); however, the knockdown of AMPK expression by small interference RNA reversed the inhibition of MDR1 expression in resveratroltreated T24/Cis cells, indicating that the suppression of MDR1 expression by resveratrol was mediated with activation of AMPK signaling pathway. Moreover, resveratrol-induced inhibition of MDR1 expression in T24/Cis cells was mediated with a blockade of nuclear factor-úB (NF-úB) and cAMPresponsive element (CRE) transcriptional activation. Conclusions: The results indicated that resveratrol-induced down-regulation of MDR1 expression was associated with the activation of AMPK, and attenuation of NF-kB and CRE signaling pathways in T24/Cis cells, suggesting a novel function of resveratrol as a multi-drug resistance reversal agent. Acknowledgement: This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2013R1A1A2065537 & 2015R1A2A2A01004633). No conflict of interest. 302 Deregulation of peripheral circadian clock in murine colorectal tumor deregulates the genes of cell cycle and proliferation M. Sotak1 , K. Balounova1 , P. Ergang1 , A. Sumova2 , J. Pacha1 . 1 Institute of Physiology- Czech Academy of Sciences, Epithelial Physiology, Prague, Czech Republic, 2 Institute of Physiology- Czech Academy of Sciences, Neurohumoral Regulations, Prague, Czech Republic Background: Disruption of circadian machinery appears to be associated with the acceleration of tumor development. The circadian oscillators mediate rhythmic physiology through transcriptional regulation by core clock components via E-boxes in the promoters of clock-controlled genes or by circadian regulation of clock-output genes encoding transcription factors such as DBP, TEF and HLF, which in turn regulate the transcription of downstream genes. We have shown recently that circadian clock in murine colon is severely disrupted in colorectal tumors (Sotak ´ et al. Int. J. Cancer 132:1032, 2013). To determine whether the changes of colonic clock during tumorigesis drive downstream changes in the expression of clock-output genes and clockcontrolled genes associated with cell cycle and proliferation, we measured the expression of the clock-output genes Dbp, Tef and Hlf, the established markers of cell cycle transit c-Myc, Ccna1 (cyclin A1), Ccnd1 (cyclin D1), Ccne1 (cyclin E1) and Wee1 (WEE1 kinase) and the genes encoding proteins that are associated with cellular proliferation (Mki67, Klf9) and regulation of cyclin D accumulation (Usp2).

Materials and Methods: Gene expression was measured using real-time RTPCR in colon of healthy mice and in colorectal tumors of mice treated with azoxymethane and dextran sodium sulfate. Results: We found strong and weak circadian oscillations of the studied genes within the same tissue and identified a significant effect of neoplastic transformation on the gene expression. In healthy colon, we found circadian rhythmicity of all studied genes with the exception of c-Myc, Ccnd1 and Wee1 and with the exception of Ccna1 and Ccne1 this rhythmicity was preserved also in the neoplastic tissue. However, the amplitudes of the rhythms of Tef, Dbp, Usp2 and Mki67 were significantly reduced and that of Hlf was largely increased. The expression of the non-rhythmic genes Ccnd1, Wee1 and c-Myc were significantly increased in tumors. Conclusion: The findings indicate that circadian rhythmicity of the clockoutput genes and the genes associated with cell cycle and proliferation are significantly modulated during neoplastic transformation. The expression of genes, which oscillates in healthy colon of mice was mostly decreased (Tef, Dbp, Usp2) or totally disappeared (Mki67, Klf9, Ccna1, Ccne1) in tumor. Only in the case of Hlf expression, the circadian rhythmicity was amplified in tumor; similar effect was observed also in the non-rhythmic genes. Together, our data demonstrate that perturbations of the peripheral colonic clock observed in colorectal cancer are sufficient to deregulate the genes associated with colonic cell cycle and proliferation and via transcription factors of the PARbZip family may modulate other regulatory processes in tumor. Acknowledgement: The study was supported by the Czech Science Foundation (grant 13-08304S). No conflict of interest. 304 Crosstalk of CCL7 and CCR3 promotes metastasis of colon cancer cells via MAPK signaling pathways Y.S. Lee1 , Y.R. Lee1 , S.J. Song1 , B.Y. Oh2 , W.Y. Lee2 , Y.B. Cho2 . 1 Samsung Biomedical Research Institute- Sungkyunkwan University School of Medicine, Department of Surgery, Seoul, Korea, 2 Samsung Medical CenterSungkyunkwan University School of Medicine, Department of Surgery, Seoul, Korea Introduction: The epithelial–mesenchymal transition (EMT), a process in which epithelial cells adopt mesenchymal characteristics through alterations in their morphology, adhesion, and migratory capacity, is a hallmark of metastasis. Chemokine (C-C motif) ligands (CCLs) and their receptors (CCRs) play an important role in tumor growth and angiogenesis; however, the role of CCLs in metastasis has only recently been explored. This study aims to investigate how the complex network of CCL7 and its receptors influences the progression and metastasis of colon cancer. Material and Methods: For CCL7 overexpression on HCT116 and HT29 cells, lentiviral CCL7 and GFP-pCMV plasmids were used at multiplicity of infection (MOI) of 100. Expressions of CCL7 and/or CCRs were analyzed in HCT116 and HT29 cells using FACS, western blot and real-time PCR. We examined the effect of CCL7 on metastasis process using both in vitro approaches such as cell proliferation assay, FACS, migration/invasion assay, proteome profile array as well as western blot in colon cancer cells and in vivo approaches including immunohistochemical analysis and MRI monitoring in ectopic/orthotopic mouse model. Results and Discussion: Using in vitro and in vivo approaches, our data demonstrated that overexpression of CCL7 enhances proliferation and metastasis of colon cancer cells, which mediated through CC chemokine receptor 3 (CCR3). Among the well-known chemokine receptor of CCL7, CCR3 expression was more strongly increased than CCR1, CCR2, or CCR5 following CCL7 overexpression in colon cancer cells. Intriguingly, changes in the expression of EMT markers, such as E-cadherin (epithelial marker) and a-SMA, Snail, and Twist (mesenchymal markers) that were provoked by CCL7 were altered in the presence of the CCR3 inhibitor SB328437. We further showed that CCL7 regulates mitogen-activated protein kinase (MAPK) pathways through CCR3, which may influence the tumorigenesis and metastasis. Of note, metastasis was found only in the CCL7 overexpressing HCT116 cells-injected group. Our results imply that CCR3-mediated MAPK activation may be a molecular link in the induction of metastasis by CCL7 in colon cancer cells. Conclusion: The results provided in this study strongly suggest that targeting the oncogenic CCL7 and CCR3 interaction may be a potential therapeutic strategy for preventing human colon cancer metastasis. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 305 Establishment of three-dimensional culture of cholangiocarcinoma cells S. Sukphokkit1 , P. Kiatwuthinon2 , S. Kumkate3 , T. Janvilisri1 . 1 Faculty of Science- Mahidol University, Biochemistry, Bangkok, Thailand, 2 Faculty of Science- Kasetsart University, Biochemistry, Bangkok, Thailand, 3 Faculty of Science- Mahidol University, Biology, Bangkok, Thailand Cholangiocarcinoma (CCA) is a bile duct cancer associated with liver fluke infection, predominantly found in Thailand. The mortality rate of CCA is high due to the difficulty in the diagnosis as CCA conceals the symptoms until the late stage of progression. At present, the in vitro three-dimensional culture overwhelms the traditional culture because it is similar to in vivo than twodimensional culture. Hence, we compared the characteristics of CCA cell lines between three-dimensional and two-dimensional cultures including cell viability, organization, and migration activity. CCA cells including KKU-100 and KKU-M213 were cultured in Matrigel® to produce multicellular tumor spheroids. We found that the three-dimensional culture of CCA cell lines exhibited the compact clusters of cells and the growth rate was drastically slower than two-dimensional culture. The migration activities of KKU-100 in both culture systems were lower than that of KKU-M213. We successfully established the three-dimensional culture of CCA cells, which will certainly be useful for drug screening for CCA. No conflict of interest. 306 Study of Moesin regulation and effects in breast cancer cells W.M. Abdel-Rahman1 , F. Alam1 , F. Mezhal1 , M.S. Ayad2 , A. El-Serafi2 , H. El Hassasna3 , J. Thiery4 , Z. Noman4 , S. Chouaib4 . 1 University of Sharjah, Medical Lab Sciences, Sharjah, U.A.E., 2 University of Sharjah, Medicine, Sharjah, U.A.E., 3 University of Sharjah, SIMR, Sharjah, U.A.E., 4 Gustave Roussy Cancer Campus, INSERM U1186 formerly U753, Villejuif, France Introduction: p53 is the most significant tumor suppressor protein in the cell. It regulates a number of micro RNAs (miRNAs) which in turn regulate carcinogenesis. This study aimed to analyze the expression of miRNAs in relation to p53 status in breast cancer cells and to delineate the role of the target gene Moesin. Material and Methods: We used isogenic breast carcinoma cell lines MCF7 (with wildtype p53), 1001 (MCF7 with mutated p53), and MCF-E6 (MCF7 in which p53 function was disrupted by transfection with the human papilloma virus type-16 E6 gene). Western blot analysis was performed to confirm the status of p53 protein in each cell line. High quality RNA was extracted by the trizol method and submitted to miRNA microarray in duplicates, using the Agilent 8x60K v16 chips. Results and Discussion: Taking a 2-fold change (2 FC) as a cut-off level, the 1001 clone with mutant p53 showed 21 upregulated and 25 downregulated miRNAs (2 FC). The predicted targets of these 46 miRNAs were 384 human genes belonging to interesting functional groups such as stem cell development and maintenance. The two most significantly down regulated miRNAs were miR141 and miR200c. Here, we focused on miR200c which targets many transcripts that are involved in Epithelial to Mesenchymal Transition (EMT) from which we chose to explore the target protein “Moesin”. Western blotting showed that Moesin was differentially expressed in our model where it was expressed in p53-mutant cell line but not in wild type cell line consistent with the observed mesenchymal features in the p53 mutant cell (vimentin positivity, E-cadherin negativity, ZEB1 positivity, and mesenchymal morphology). This prompted exploring the potential role of Moesin in the EMT phenotype observed in our model. To analyze the functions of Moesin, we silenced its expression in p53-mutant cells by siRNA transfection. Western blotting confirmed about 90% efficiency of the siRNA transfection. Then we looked at the effects of Moesin silencing on the cell morphology by confocal microscopy which showed that after Moesin silencing, the p53-mutant cells changed from mesenchymal phenotype to epithelial phenotype. Moesin silencing did not significantly alter migration and invasion, and did not affect the expression of E-cadherin and vimentin, with loss of expression of ZEB1 and insignificant reduction of SNAIL expression. Interestingly, Moesin silencing restored the 1001 sensitivity to standard chemotherapeutic agent Doxorubicin. Conclusion: The results indicate that loss of miR200c as a consequence of p53 mutation relieves significant target oncogenes from degradation and hence promotes carcinogenesis. Moesin may play a role in metastasis and drug resistance of breast cancer cells. These findings might have diagnostic and therapeutic applications. No conflict of interest. 307 Assessment of WT1 expression as a marker of treatment outcome in karyotype normal acute myeloid leukemia patients in Pakistan Z. Ahmed1 . 1 Aga Khan University Hospital-Karachi-Pakistan, Pathology and Laboratory Medicine, Karachi, Pakistan Background: Currently, there is an effort to predict relapse by follow-up monitoring of MRD and subsequently to begin the treatment of the patients during their clinical and hematological remission prior to overt hematological

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relapse. Expression of WT1 in AML is known to be independently associated with significant inferior response to therapy and short survival outcome. Followup monitoring of WT1 gene expression during or after therapy would be a valuable predictive marker for early recurrence or relapse of AML disease. Objective: To demonstrate prognostic relevance of WT1 expression levels monitored at diagnosis and during follow up of chemotherapy for predicting relapse in AML patients. Methods: For this study, five AML patients registered at the Oncology Clinics of the Aga Khan University Hospital, Karachi were recruited. Blood was collected from these patients and it used to isolate RNA from purified PBMCs. The RNA was reverse transcribed to make cDNA which served as template for quantitative RT-PCR analysis. Results: The WT1 levels were quantified by RT-PCR in five male patients with AML. Relatively higher level of WT1 mRNA (4000–9500 copies/ml) was detected in treatment na¨ıve patients, whereas only (100–250 copies/ml) were seen in two patients (one was receiving induction and the other patient was on consolidation therapy). In addition, one patient who received induction therapy showed raised WT1 mRNA levels (7700 copies/ml). Conclusion: High WT1 burden (>5000 copies/ml) in two patients is indicative of early recurrence of the disease along with shorter disease free and overall survival. The low WT1 expression (5-fold), and catalase protein, suggesting S100A8 reduced the oxidative environment in LLC-bearing mice. This was maintained at 10 days; peroxiredoxin and superoxide dismutase activities were elevated some 2-fold, and nitrite levels in BALF reduced to baseline (P < 0.01) in LLC+S100A8-treated mice. At this time, LLC markedly upregulated IL-4 and IFN-g mRNAs (>15-fold) and proteins in BALF, whereas S100A8 suppressed these to baseline (p < 0.05), suggesting reduced MDSC activation. There was concomitant reduction of splenic neutrophilic MDSC (CD11b+/Gr-1+/CD14−/F4/80-) numbers, indicating reduced MDSC recruitment. Conclusions: S100A8 prolonged LLC survival. It changed the redox microenvironment and suppressed some immunomodulatory cytokines that may impede MDSC activation and recruitment. Its induction of IL-10 within the microenvironment may reduce tumor progression at advanced stages. No conflict of interest. 324 Tumour matrix stiffness regulates metastasis by promoting cancer cell interactions with the endothelium S. Reid1 , A.T. Henze2 , J. Serneels2 , L. Neilson1 , A. Ramon-Fernandez3 , R. Adams4 , K. Blyth5 , D. Bryant3 , M. Mazzone6 , S. Zanivan1 . 1 Cancer Research UK Beatson Institute, Tumour Microenvironment and Proteomics Lab, Glasgow, United Kingdom, 2 VIB Vesalius Research Center, Department of Oncology, Leuven, Belgium, 3 Institute of Cancer Sciences- University of Glasgow, Molecular Control of Epithelial Polarity, Glasgow, United Kingdom, 4 Max Planck Institute for Molecular Biomedicine, Tissue Morphogenesis, Munster, ¨ Germany, 5 Cancer Research UK Beatson Institute, Transgenic Models Lab, Glasgow, United Kingdom, 6 VIB Vesalius Research Center, Oncology, Leuven, Belgium Introduction: The formation of a stiff lump in soft tissue is one of the most common symptoms that people associate with cancer. This is a sign of abnormal tissue stiffness, which derives from changes in the extracellular matrix (ECM) dictated by cancer and stromal cells. This physical property is known to be both cause and consequence of tumour development. While it is well established that high stiffness promotes invasive behaviour of cancer cells, the role of abnormal stiffness on endothelial cells (ECs) is still largely unknown. Material and Method: Here we use unbiased mass spectrometry (MS)-based proteomics to ascertain how increased tissue stiffness affects ECs, and the impact of these alterations in cancer. To do this we used an in vitro system of polyacrylamide gels representing ECM stiffness in normal and tumour tissues. Results and Discussion: Using high resolution MS combined with stable isotope labelling of amino acids in cell culture (SILAC), we quantified the global proteome of ECs cultured under different ECM stiffness and unveiled that many cell adhesion proteins were upregulated by increased tumour stiffness, including N-Cadherin and the matricellular protein CCN1. By silencing and overexpressing CCN1 in ECs, we demonstrated that stiffness-induced CCN1 induces a b-catenin mediated increase in N-Cadherin expression. N-Cadherin is known to play a key role in trans-endothelial migration of cancer cells through the blood vessels, and we show that CCN1 induces cancer cell adhesion to ECs via N-Cadherin in vitro. This indicates that CCN1 can increase cancer cell metastasis by aiding the exit of cancer cells from the primary tumour and entry at distant sites. We confirmed this hypothesis in vivo, by using C57Bl/6 Ccn1loxP/loxP mice and a syngeneic B16 melanoma model. Ccn1 was acutely knocked out in the vasculature by injecting the Tat-Cre fusion protein intravenously, and this showed that knocking out Ccn1 decreased the number of circulating tumour cells and subsequent metastases in the lungs. Thus, CCN1 loss in the vasculature can decrease cancer cell intravasation. Conclusion: Our work determines an unprecedented mechanism through which CCN1 in the stromal cells promotes invasion of cancer cells. This complements the findings that CCN1 can induce invasion of cancer cells, and provides evidence that targeting CCN1 in endothelial cells may decrease the spread of cancer in the clinical use. No conflict of interest. 325 The epigenetic repressor EZH2 controls tumour escape mechanisms during immunotherapy D. Zingg1 , N. Arenas-Ramirez2 , R.A. Rosalia2 , A.T. Antunes1 , J. Haeusel1 , O. Boyman2 , L. Sommer1 . 1 Institute of Anatomy, University of Zurich, Zurich, Switzerland, 2 Department of Immunology, University Hospital Zurich, Zurich, Switzerland Although immunotherapy is a promising approach for advanced cancer, malignancies often escape immune control by loss of tumor antigen expression and immunosuppressive strategies. The molecular mechanisms of tumor escape from immune surveillance remain poorly understood. We now show that several tumor cell-intrinsic and extrinsic immune escape mechanisms are controlled by the Polycomb group protein enhancer of zeste homologue 2 (EZH2), a histone methyltransferase that epigenetically

silences gene expression. In murine melanoma models, interleukin-2 (IL-2) / anti-IL-2 antibody complex (IL-2cx)-based immunotherapy promoted an Ezh2 upregulation in tumor cells, particularly in areas rich in tumorinfiltrating lymphocytes. Enhanced Ezh2 activity fostered a transcriptional dedifferentiation program reminiscent of disease progression and silenced tumor antigen presentation. Importantly, Ezh2 inactivation using RNAi or the EZH2 inhibitor GSK503 reversed these effects in melanoma cells and synergized with IL-2cx immunotherapy to suppress tumor growth. These anti-tumor effects depended on non-exhausted CD8+ T cells, which selectively accumulated intratumorally, produced interferon-g, and maintained low programmed death (PD)-1 levels. This was accompanied by PD-L1 downregulation on melanoma cells. Contrarily, immunosuppressive regulatory T cells and myeloid-derived suppressor cells failed to accumulate within tumors during concomitant immunotherapy and Ezh2 blockade. Hence, Ezh2 serves as a molecular switch to control tumor immune escape, indicating that combination of EZH2 inhibition and immunotherapeutics might be considered for cancer therapy. No conflict of interest. 326 miR-29c modulates chemoresistance in esophageal cancer cells by suppressing FBXO31 A.L. Cheung1 , B. Li1 , W.W. Xu1 , J. Liu1 , K.W. Chan2 . 1 University of Hong Kong, School of Biomedical Sciences, Hong Kong SAR, China, 2 University of Hong Kong, Pathology, Hong Kong SAR, China Background: Esophageal cancer is the 6th most common cause of cancerrelated death worldwide. Multimodal treatment combining surgical resection and the use of chemotherapeutic drugs is commonly used in the management of this disease. However, resistance to chemotherapy drugs may contribute to poor treatment outcome. A better understanding of the molecular mechanisms of chemoresistance will facilitate the development of novel therapies. Material and Methods: Esophageal squamous cell carcinoma (ESCC) cell line models of acquired chemoresistance to 5-fluorouracil (5-FU) were established by exposing ESCC cell lines to increasing concentrations of 5-FU for over 18 months. These 5-FU-resistance (FR) sublines were used to identify microRNAs (miRNAs) and genes associated with 5-FU resistance using an integrative approach that combined Taqman miRNA array, cDNA microarray and in silico analysis. Results and Discussion: Microarray analysis of miRNA expression in FR and parental cells showed that miR-29c was one of the most downregulated miRNAs in the FR sublines. cDNA microarray data showed that FBXO31, a predicted target of miR-29c, was amongst the upregulated mRNAs in the FR cells. We found that overexpression of miR-29c could restore 5-FU chemosensitivity in FR cells and suppress FBXO31 protein expression in ESCC cell lines. Re-expression of FBXO31 in miR-29c-overexpressing ESCC cells abrogated the effects of miR-29c on 5-FU chemosensitivity. Moreover, FBXO31 overexpression enhanced, whereas FBXO31-knockdown reduced, the sensitivity of ESCC cells to cisplatin both in vitro and in vivo. Conclusion: Our data suggest that miR-29c can modulate the chemosensitivity of ESCC cells by regulating FBXO31. Since FBXO31 has prognostic significance in patients with ESCC, this miR-29c/FBXO31 regulatory mechanism may be exploited to develop novel biomarkers and therapeutic strategies to improve patient survival. Acknowledgement: This study was supported by the National Natural Science Foundation of China Research Grants General Program Project no. 81472790 and Research Grants Council of the Hong Kong SAR, China, GRF Project No. HKU17103814. No conflict of interest. 327 Acidosis meets the “hallmarks of cancer”: transcriptome analysis to uncover its role in melanoma S. De Summa1 , A. Ferretta2 , P. Rosamaria1 , P. Orazio3 , M. Carella3 , G. Guida2 , A. Azzariti4 , S. Tommasi1 . 1 IRCCS-Istituto Tumori “Giovanni Paolo II”, Molecular Genetics Laboratory, Bari, Italy, 2 University of Bari, Department of Medical Biochemistry, Bari, Italy, 3 IRCCS Casa Sollievo della Sofferenza, Medical Genetics Unit, San Giovanni Rotondo, Italy, 4 IRCCS Istituto Tumori “Giovanni Paolo II”, Clinical and Preclinical Pharmacology Laboratory, Bari, Italy Background: The tumor microenvironment differs profoundly from the environment found in normal tissue. External pH (pHe) of tumors is frequently acidic not only due to poor perfusion but also to altered metabolic activity of tumor cells. It is known that tumor cells use glycolysis even when there is enough O2 to support mitochondrial function, the so called “Warburg effect”. Melanoma represents one of the most aggressive human cancer and show extracellular acidosis. pHe of human melanoma has been reported to be within range of 6.4 to 7.3. The role of acidosis in metastasization has been demonstrated by many research groups. However, to deeply exploit the role of acidosis in the biology of melanoma, we performed a transcriptome analysis

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 in 4 primary melanoma cell lines stratified by the presence of BRAF V600 alteration. Material and Methods: Melanoma cells, HBL, LND1, hmel-1 and M3, obtained from human sporadic melanoma biopsy, were grown in T-25 culture flasks. Cell culture media used was high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine, 1% (v/v) penicillin/streptomycin, at 37ºC in a humidified atmosphere of 5% CO2. Experiments were carried out with cells in exponential growth, the cells were plated and cultured in normal (pH 7.4) medium, the medium was removed and replaced by normal or acidic medium (pH 6.8) for 24 hours. The pH of the medium was adjusted with HCL 1N. Transcriptome data were obtained through Affymetrix Human Transcriptome Array 2.0 and analyzed by Expression Console and Transcriptome Analysis Console. Results: Firstly, we compared expression levels according to pH conditions in mutated and wild-type cell lines. Transcriptome analysis showed the deregulation of 2590 and 4012 genes in mutated and wild-type cells, respectively. The first observation was that acidosis affects mutated and wildtype cell lines in different ways. In order to have a snapshot of the role of acidosis, we performed pathway enrichment analysis and we merged results from mutated and wild-type cells. Interestingly, we found terms related to the so-called “hallmarks of cancers”. Focusing on “avoiding immune destruction” hallmark whose enabling characteristics are “genomic instability” and “tumorpromoting inflammation”, we found deregulation of genes involved in DNA damage response (e.g., BRCA1, RAD51, H2AX) and inflammation (e.g., IL-16, IL-4, TGF-alpha). Other than enabling features, cell lines in acidic conditions showed enrichment in terms related to immune response. Conclusions: In conclusion, these preliminary results showed that acidosis, a common feature of solid tumors, could be a causal factor of the “hallmarks of cancer”. In particular, it could be an important factor to be addressed in order to improve immunotherapy outcomes. No conflict of interest. 328 The bone metastatic niche promotes breast cancer stem cell activity via IL-1b−Wnt signalling R. Eyre1 , K. Spence1 , D. Alferez1 , A. Santiago-Gomez1 , C. Hart1 , B. Simoes1 , M. Brown1 , A. Gurney2 , G. Farnie1 , R. Clarke1 . 1 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom, 2 Oncomed Pharmaceuticals, Molecular and Cellular Biology, Redwood City- California, USA Introduction: The vast majority of breast cancer-related deaths are due to the spread of tumour cells to distant organs, the most common of which is the bone. It has been demonstrated that breast tumours contain cancer stem cells (CSCs), a rare subpopulation of cells which are capable of giving rise to all other tumour cells. There is evidence that CSCs within a tumour are responsible for metastatic colonisation, where they are dependent on the stromal niche to survive and proliferate. However, little is known about how the bone microenvironment promotes colony formation by metastatic breast CSCs, a fact which is often attributed to a lack of models to study metastases. Materials and Methods: We have developed two novel models to investigate CSC colony formation and signalling in response to metastatic niche factors: 1. An in vitro system utilising patient-derived bone marrow and patient-derived breast cancer samples. 2. An in vivo model utilising dual tomato/luciferase labelled MCF-7 cells injected intra-femorally into NOD SCID IL2gammaReceptor (NSG) mice, then flushed from the femur following tumour colonisation. In both models CSC activity is assessed by mammosphere colony formation. Results and Discussion: Bone marrow-secreted factors stimulate breast CSC colony formation both in 15/17 early breast cancers in vitro and in MCF-7 cells in vivo (p = 0.0001). This is regulated by Wnt signalling, and can be reversed upon treatment with the Wnt inhibitors OMP-18R5 (Oncomed) and Sulforadex (SFX, Evgen), both in early breast cancers in vitro (SFX; p = 0.0014, OMP-18R5; p = 0.0060) and in MCF-7 following systemic treatment of mice in vivo (SFX; p < 0.0001, OMP-18R5; p < 0.0001). Treatment of bone marrow and breast cancer cells with porcupine inhibitor demonstrated that Wnt ligands are produced by breast cancer cells in response to a bone marrow-secreted factor. Cytokine arrays identified that IL-1b is secreted by the bone marrow, and IL-1b induces both Wnt signalling (p < 0.0001) and CSC activity (p < 0.0001) in breast cancer cells. Inhibiting IL-1b reverses the Wnt-dependent induction of CSC activity by the bone marrow in vitro (p < 0.0001). Conclusions: These data demonstrate that bone marrow derived IL-1b activates Wnt signalling in breast cancer cells, promoting metastatic colony formation by breast CSCs. Preventing CSC activity in the metastatic niche via an inhibition of IL-1b−Wnt signalling may provide a novel therapeutic opportunity in breast cancer, both in the adjuvant and advanced settings. No conflict of interest.

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329 Clinical relevance and functional role of galectin-1 in hepatocellular carcinoma Z. Leung1 , F.C.F. Ko1 , J.W.P. Yam1 . 1 The University of Hong Kong, Pathology, Pokfulam, Hong Kong Introduction: Hepatocellular carcinoma (HCC) has one the world’s leading cancer incidences and despite extensive research, the numbers of HCCcausing deaths remain high, accounting for the 3rd leading cause of cancer death. Galectins are b-galactose specific binding proteins, which in turn facilitate the crosslinking of molecules and thus enabling vital cell-cell and cellmatrix interactions and signal transduction. Galectin-1 (Gal1), the first identified member of the galectin family, has been found to regulate cell processes; both intracellularly and extracellularly upon its secretion. The oncogenic role of gal1 in HCC has been increasingly studied, however, ambiguities concerning its non-classical secretion mechanism and any associated signalling pathways remain undisclosed. This study aims to further understand the clinical relevance and functional role of Gal1 in HCC tumourigenesis. Materials and Methods: Clinical analysis of Gal1 was conducted with human HCC cell lines and patient RNA samples by quantitative real time PCR. ELISA analysis further highlighted the clinical relevance of Gal1 in detecting the secretory gal1 levels in patient blood serum samples. The functional role of Gal1 in HCC was subsequently explored in migration, invasion and soft agar assays after the stable expression of Gal1 in HCC cell lines. Results and Discussion: Analysis of 48 HCC clinical samples revealed the significantly higher Gal1 expression in HCC late stage tumours compared to early stage. This overexpression was also observed in immunohistological staining of tumour tissues and elevated gal1 levels in patient blood serum samples. Overexpression corresponded to poorer disease-free survival, cirrhotic liver and venous invasion. By knocking down Gal1 expression in HCC cell lines BEL7402 and MHCC97L, functional experiment results showed reduced migration, invasion and anchorage independent growth. Similarly, when Gal1 was overexpressed in HCC cell line PLC/PRF/5, the migratory potential and the ability for anchorage independent growth increased. These results signify the importance of gal1 in regulating essential tumourigenic cell processes. Conclusion: Gal1 expression is significantly higher in HCC patient samples observed in the clinical setting and likewise in HCC cell lines. The oncogenic role of Gal1 has been implicated in HCC tumourigenesis by promoting cell migration, invasion and anchorage independent growth. No conflict of interest. 330 Mutant p53 promotes tumor progression by the endoplasmic reticulum UDPase ENTPD5 F. Vogiatzi1 , D.T. Brandt2 , J. Fuchs1 , K. Grikscheit2 , O. Timofeev1 , A. Nist3 , M. Mernberger1 , R. Grosse2 , T. Stiewe1 . 1 Institute of Molecular Oncology, Philipps-University, Marburg, Germany, 2 Institute of Pharmacology, PhilippsUniversity, Marburg, Germany, 3 Genomics Core Facility, Philipps-University, Marburg, Germany Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and often associated with progression from benign to invasive, metastatic stages. Mutations inactivate tumor suppression by p53 and endow the mutant protein with novel gain-of-function (GOF) activities that actively promote tumor progression, metastasis and therapy resistance. By comparative gene expression profiling of p53 mutated and p53-depleted cancer cells we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene. A comprehensive pan-cancer analysis of TCGA data revealed a highly significant correlation between GOF p53 mutations and elevated expression of ENTPD5. Mechanistically, regulation of ENTPD5 by mutant p53 is mediated by the histone H3 lysine 4 methyltransferase COMPASS complex. ENTPD5 has been shown to function in the endoplasmic reticulum as an UDPase to promote the N-glycosylation and folding of membrane proteins such as growth factor receptors and integrins. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals N-glycosylation in the endoplasmic reticulum as a novel mechanism underlying the progression of tumors with GOF p53 that could provide new possibilities for treating cancers driven by GOF p53 mutations. No conflict of interest. 331 TGFb-induced JMJD3 controls tumor cell microenvironment and myeloid cell polarization ´ 1 , A. Arias1 , A. Cascante1 , I. Cuartas1 , I. Huber Ruano1 , C. Raventos J. Seoane1 . 1 Vall d’Hebron Institute of Oncology VHIO, Translational research program, Barcelona, Spain Background: TGFb is a pleiotropic cytokine involved in many different cellular processes including proliferation, differentiation, adhesion, motility and apoptosis. Moreover, TGFb feeds the oncogenic process by inducing cancer initiation, cell self-renewal, angiogenesis, metastasis and immunosuppression.

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Both genetic and epigenetic modifications have been linked to cancer. Far from being innocuous, these epigenetic modifications are essential for maintaining transcriptional programs and determining cell fate and identity. JMJD3 is a histone 3 lysine 27 (H3K27me3) demethylase that plays a role in cellular differentiation during embryonic development and regulates cancer cell growth by modulating H3K27 methylation levels at site-specific promoters or enhancers. Previous work from our lab showed that JMJD3 expression was upregulated upon TGFb treatment in different cell types. Thus, we speculated that some of the TGFb functions might occur by modifications of the epigenetic landscape through JMJD3, and we decided to evaluate the impact of such modulation. Material and Methods: A549, H1666 and Lacun3 lung cancer cell lines were used for gene expression, chromatin immunoprecipitation, ELISAs and functional in vitro assays. Immune system effects were studied in human macrophages derived from monocytes isolated from PBMCs from healthy donors. Subcutaneous and lung orthotopic in vivo models were also used for studying tumor growth and immune system characterization. Results and Discussion: In order to determine the effects of TGFb activity on the epigenetic landscape, we performed microarrays and demonstrated that JMJD3 is specifically up-regulated through the Smad pathway upon TGFb treatment. By analyzing microarray data from cells expressing shJMJD3 and performing ChIP experiments, we have shown that JMJD3 regulates the expression of a group of genes that encode for cytokines that profoundly affect the tumor microenvirontment by modulating the immune system. In vitro functional assays have shown that JMJD3 affects tumor cell invasion abilities and myeloid cell polarization. In vivo orthotopic experiments in mice using A549 cells expressing an inducible shJMJD3 showed that JMJD3 down-regulation leads to a striking decrease in tumor size and to fewer metastases caused by the polarization of myeloid cells towards a more antitumoral phenotype. Conclusion: Our work demonstrates that TGFb-dependent JMJD3 regulation controls tumor growth by affecting tumor cell migrating abilities and, more importantly, myeloid cell polarization. The benefit of targeting JMJD3 and TGFb in combination arises as a potential therapeutic strategy for lung adenocarcinoma. No conflict of interest. 332 Specific EMT-inducers signature associates with oncogenic events in breast tumour progression C. Moyret-Lalle1 , E. Ruiz2 , C. Bardel3 , I. Treilleux4 , S. Courtois-Cox5 , A. Puisieux1 . 1 Universite´ Claude Bernard Lyon 1, Cancer Research Center of Lyon and LabEx DEVweCAN, Lyon, France, 2 Universite´ Claude Bernard Lyon 1, Cancer Research Center of Lyon, Lyon, France, 3 Universite´ Claude ´ ´ Bernard Lyon 1, LBBE, Lyon, France, 4 Centre Leon Berard, Pathology ´ ´ Berard, LYRIC, Lyon, France department, Lyon, France, 5 Centre Leon Background: Mammary epithelial cancer cells are able to reactivate an embryonic program, the Epithelial-to-Mesenchymal Transition (EMT), to acquire motility, renewal and de-differentiation capacities. EMT drives a profound genetic reprograming including reactivation of embryonic transcription factors (ETFs) that may cooperate with specific mitogenic stresses during tumorigenesis. Our goal is to determine whether specific expression networks between ETFs and mitogenic stresses exist in breast tumours, representing a driving force towards transformation in breast tumorigenesis. Material and Methods: We performed oncogenic cooperation assays in immortalized human mammary epithelial cells (HMEC–hTert) by using an ETF expression library in combination with various mitogenic stresses (activated b-catenin, overexpression of EGFR, deletion of PTEN and p53). In softagar colony assays, these mitogenic stresses are not sufficient to transform cells and form colonies. In contrast, the combination of ETFs and mitogenic stresses led to transformation of mammary human cells. In order to determine whether the ETFs signature was specific to the mitogenic insult, EMT-inducer expression was analysed in a large number of colonies. We have used a Chisquare and arborescences analyses to determine how expression of ETFs was interconnected in different mitogenic insult conditions. Results: We found that expression of Goosecoid and Zeb1 was specifically enriched in colonies generated when PTEN or p53 was silenced. Twist1 and Twist2 expression was selected following b-catenin activation whereas random ETF combinations were found to be expressed in colonies expressing a high level of EGFR. The impact of PTEN loss associated Goosecoid/Zeb1 signature was evaluated in vivo in a large cohort of TNBC (Triple Negative Breast Cancer). Goosecoid protein expression was significantly associated with nuclear PTEN exclusion. We have identified specific ETF signatures, depending on mitogenic insult during tumour progression. Therefore, their impact of EMT in mammary transformation might depend on mitogenic alterations already present in cells. Conclusions: A better understanding of how ETFs cooperate with mitogenic insults might allow us to propose new combined therapeutics. No conflict of interest.

333 New tools for the study of intratumour heterogeneity in cancer L. Gonzalez-Silva1 , L. Quevedo Palacio1 , T. Moreno Rodriguez1 , C. Revilla Gomez1 , R. Rad2,3,4 , D. Saur2,3,4 , I. Varela1 . 1 Instituto de Biomedicina y Biotecnolog´ıa de Cantabria CSIC-UC-Sodercan- Universidad de Cantabria, Departamento de Biolog´ıa Molecular, Santander, Spain, 2 Department ¨ of Internal Medicine II- Klinikum rechts der Isar- Technische Universitat Munchen¨ Munchen, ¨ Germany, 3 German Cancer Consortium DKTK4 Heidelberg, Germany, German Cancer Research Center, DKFZ, Heidelberg, Germany Introduction: Intratumour heterogeneity has been observed in multiple cancers and has been postulated as a critical aspect for tumour metastasis and treatment resistance. Therefore, a further characterization of its role in cancer progression and metastasis has become essential. The use of cell lineage tracking systems, combined with the use of genetically modified mouse models, could be of special importance to understand this process. A genetically engineered allele, called confetti, has been used for cell lineage tracking based in the random and inheritable expression of different fluorescent markers in Cre expressing cells. Nevertheless, the proteins selected for this construct present quite a lot of spectra overlapping that difficult the correct identification of the four different proteins and the analysis of potential protein combinations, which seriously limits the number of potential outcomes. Similarly, virus- and transposon-mediated alternatives have been developed. However, in these alternatives there is no control over the number of copies integrated or the genomic position for this integration, which precludes a clear interpretation of the results. In this project, we present the choice of an optimal combination of fluorescent proteins and the identification of new incompatible lox sites that has allowed the development of a new allele for cell lineage genetic tracking that overcomes these limitations and constitutes a great tool for the study of the dynamics of intratumour heterogeneity in cancer progression. Material and Methods: We have cloned more than ten different fluorescent proteins in a mammalian expression vector to analyse their spectrometric characteristics by confocal microscopy and cytometry. Additionally, we have developed a new expression construct that permits the identification of new incompatible recombination sites by CRE and DRE recombinases. Results and Discussion: We have found an efficient combination of five different fluorescent proteins that can be distinguished uniquely by cytometry and confocal microscopy. This, combined with the identification of new incompatible loxP sites, has allowed us to create a new allele for cell lineage tracking. Additionally, we have some preliminary results that suggest that a similar strategy can be used dependent on DRE instead of CRE recombinase, which opens new opportunities to combine this strategy with new mouse cancer models. Conclusion: The development of new tools of genetic tracking could help in the study of the role and dynamics of intratumour heterogeneity in cancer progression. Our results demonstrate that it is possible to implement an effective strategy of genetic tracking based in fluorescent proteins that could suppose a great advantage for the study of this process in cancer mouse models. No conflict of interest. 334 Combination therapy to target ERBB receptors in HER2 low breast cancer M. Berdiel-Acer1 , E. Sofyali1 , S. Burmester1 , R. Will2 , U. Korf1 , S. Wiemann1 . 1 DKFZ, Molecular Genome Analysis, Heidelberg, Germany, 2 DKFZ, Genomics and proteomics core facility, Heidelberg, Germany Introduction: Breast cancer is the most common cancer in women worldwide and a highly heterogeneous disease. 20−30% of breast tumor shows overexpression of HER2 receptor and could be targeted with specific drugs such as trastuzumab. A large number of patients show low/moderate levels of HER2 along with others ERBB receptors. Targeting EGFR or ERBB3 with powerful drugs could be one approach to increase survival of these patients. Material and Methods: Cell lines from different breast cancer subtypes were selected and levels of ERBB receptors were checked at RNA and protein levels (qRT-PCR and western blot respectively). siRNA (Dharmacon) for the receptors was transfected using Lipofectamine® RNAimax (Invitrogen). Therapeutic monoclonal antibodies targeting ERBB receptors were trastuzumab and pertuzumab (ERBB2), cetuximab (EGFR), lumretuzumab (ERBB3) and the EGFR tyrosine kinase inhibitor, erlotinib (Roche). 4-hydroxytamoxifen (SigmaAldrich) was also used to target the estrogen receptor. MCF7 TamR and BT474 TrasR resistant cell lines to tamoxifen and trastuzumab respectively, were included. MDA-MB-231 cell line overexpressing ERBB3 under an inducible promoter was designed to better characterize lumretuzumab mechanism of action. Cell viability was measured using Cell Titer Glo Luminescent assay (Promega). Results: Total levels of ERBB receptors varied among different cell lines depending on their subtype, ERBB3 and ERBB4 being specially higher expressed in T47D and MCF7 (luminal A). Their specific downregulation by siRNA, decreased viability of T47D around 50%, but without affecting

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 MCF7. Similar results were obtained when treatment with either cetuximab, erlotinib or lumretuzumab. In T47D cells, blockade of downstream signalling pathway of ERBB3 (mainly ERK and Akt) in presence of its main ligand (HRG1), was observed when administered together with lumretuzumab. On the contrary, lumretuzumab only blocked phosphorylation of ERBB3 but not ERK/Akt when delayed addition of HRG1, suggesting ERBB4 receptor as an alternative activator. Specific upregulation of ERBB receptors was detected in both resistant cell lines. For BT474 TrasR, when exposed to a drug combination of trastuzumab with either lumretuzumab or cetuximab, stronger effects were achieved (around 40% decrease in viability). The isogenic MDA-MB-231 cell line expressing ERBB3 under doxycycline addition, reached similar expression values to those in T47D cell line, increasing its proliferation rate when ERBB3 expressed. Conclusion: Although treatment of HER2 overexpressing breast tumors has been successful, targeting other ERBB members in a HER2 moderate/low scenario seems to be a promising approach when combining therapies; even in resistant cell lines. Thus, better characterization of the ERBB network may help to pave the way for a personalized treatment of HER2 low breast cancer. No conflict of interest. 336 Molecular mechanisms involved in the immunomodulation induced by LIF in cancer ´ 1, A. Sala Hojman1 , A. Soto1 , I. Huber-Ruano1 , V. Chigan¸cas1 , C. Raventos M. Vidal-Jorge2 , E. Mart´ınez-Saez ´ 3 , J. Sahuquillo2 , C. Espejo4 , J. Seoane1 . 1 Vall d’Hebron Institute of Oncology VHIO, Translational Research Program, Barcelona, Spain, 2 Vall d’Hebron Institute of Research- VHIR, Neurosurgery Department, Barcelona, Spain, 3 Vall d’Hebron University Hospital, Pathology Department, Barcelona, Spain, 4 Vall d’Hebron Institute of Research- VHIR, Neurology-Neuroimmunology Department, Barcelona, Spain Background: LIF is a pleiotropic cytokine that can act as an immunomodulatory factor in different biological processes, such as embryo implantation, organ transplantation tolerance and multiple sclerosis. LIF can act as an oncogenic factor and is highly expressed in certain tumor types. We hypothesized that tumors expressing high levels of LIF may promote an immune tolerant microenvironment that will preclude the anti-tumor immune response. Material and Methods: We studied the effect of LIF blockade on the immune system in the context of cancer using syngeneic mouse models and patientderived xenografts from GBM patients. We analyzed tumor-associated myeloid cells (TAMs), regulatory T cells (Tregs), effector T cells and NK cells populations. We corroborated the results obtained in our models using monocytes isolated from peripheral blood mononuclear cells from healthy human donors, and human organotypic cultures from GBM patients. We analyzed the TCGA database to extrapolate our results to a large cohort of patients. Results: We have observed that in tumors expressing LIF, the blockade of LIF inhibited tumor growth and caused tumor regression. The anti-tumor effect of LIF neutralization was mediated by the polarization of TAMs. LIF decreased the expression of M2-like markers such as MRC1, CD163 and CCL22 in human healthy monocytes and in TAMs from mouse syngeneic (non-small cell lung cancer, ovarian cancer and colorectal cancer) models, patient derived xenograft models, and organotypic cultures from glioblastoma. In turn, LIF neutralization decreased regulatory T cells and induced effector T and NK cell tumor infiltration. A positive correlation between M2-like TAMs and LIF expression was observed in glioblastoma, and patients with high levels of these factors presented worse prognosis. Conclusion: LIF protects the tumor from the immune system from the host through the re-education of TAMs. LIF blockade down-regulates CCL22 secretion by TAMs preventing Treg infiltration, and leading to the increase of effector T and NK cells within the tumor mass. Our results identify LIF as a promising therapeutic target and establish the translational potential of anti-LIF agents. No conflict of interest. 337 A relay of IL-1R and CXCR2 signals in the tumour microenvironment confer tolerance to MAPK antagonism in melanoma H. Young1 , E. Rowling1 , M. Smith1 , M. Bugatti2 , W. Vermi2 , N. Luheshi3 , C. Wellbrock1 , A. Hurlstone1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom, 2 University of Brescia, Department of Molecular and Translational Medicine, Brescia, Italy, 3 MedImmune Ltd, Division of Oncology, Cambridge, United Kingdom Introduction: Inflammation in the tumour microenvironment is now firmly established as a major contributor to tumour development and progression. More recently, microenvironment-derived inflammatory signals have been shown to modify the response of melanoma cells to targeted therapy and thereby contribute to treatment resistance. Materials and Methods: We have used in vitro co-culture systems and in vivo mouse models to investigate the crosstalk between macrophages, fibroblasts and melanoma cells in the melanoma microenvironment, in particular in response to therapy.

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Results and Discussion: We find evidence of a complex relay of paracrine signals between macrophages, fibroblasts and melanoma cells with melanoma cells inducing macrophages to secrete high levels of interleukin 1b (IL-1b). We establish that IL-1 signalling in the tumour microenvironment, which is elevated upon treatment with MAPK signalling inhibitors, is a major determinant of tolerance to inhibition of MAPK signalling. However, the effect of IL-1b on melanoma cells is largely indirect and mediated through additional paracrine signals, notably ligands of CXCR2, which we propose to be generated by fibroblasts that respond to IL-1b. Moreover we find that tolerance to MAPK signalling inhibition in melanoma cells via CXCR2 signalling is not through reactivation of ERK but rather through activation of the transcription factor NF-úB and induction of Bcl-2, which promotes melanoma cell survival despite reduced levels of MAPK signalling. Significantly, inhibiting IL-1 or CXCR2 signalling in vivo enhanced the efficacy of MAPK signalling inhibitors. Conclusion: Our work illustrates that a complex network of signals relayed between multiple cell types within melanoma tumours is critical in promoting resilience to MAPK antagonism. We therefore propose that targeting this network, for example by inhibiting IL-1 or CXCR2 signalling, will improve responses to current targeted therapies. No conflict of interest. 338 Widespread epigenetic activation of DNp73 in small cell lung cancer causes vulnerability to Tip60−p400 inhibition A. Nist1,2 , A.M. Krampitz1 , K. Schlereth1 , M. Mernberger1 , M.C. Moßner1 , T. Muley3 , R. Dammann4 , T. Stiewe1,2 . 1 Molecular Oncology, PhilippsUniversity Marburg, Marburg, Germany, 2 Genomics Core Facility, Philipps-University Marburg, Marburg, Germany, 3 Thoraxklinik, University Hospital Heidelberg, Heidelberg, Germany, 4 Institute for Genetics, Justus-Liebig University Giessen, Giessen, Germany Lung cancer is the leading cause of cancer-associated mortality worldwide with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) being the most common subtypes. Although newly diagnosed SCLC is exquisitely sensitive to first-line chemotherapy, the disease eventually progresses rapidly in virtually all patients resulting in a 5-year survival of less than 10%. There is therefore an urgent need to develop novel therapeutic strategies. Unfortunately, SCLC are genetically driven by inactivating mutations in the tumor suppressor genes p53 and RB1 and not by druggable oncogenic drivers. Using targeted genome sequencing of SCLC patients we identified a few cases with structural rearrangements in the p73 transcription factor gene that delete the exons encoding the N-terminal p73 transactivation domain. More frequently, in approximately half of all SCLC cell lines and primary tumor samples, we observed loss of DNA methylation in an intronic CpG island of the p73 gene, which thereby gains promoter activity and drives expression of N-terminally truncated DNp73 proteins missing the transactivation domain. In both cases the resulting transactivation-deficient DNp73 proteins sequester and inactivate any tumor suppressive full-length p73 proteins. Given that DNp73, in contrast to transactivating full-length p73, represses transcription we analyzed the underlying mechanism by RNAi screening. We identified multiple components of the chromatin-modifying Tip60−p400 complex as required for transcriptional repression by DNp73. In a parallel RNAi-based viability screen, we found the Tip60–p400 complex to be also essential for survival of SCLC cells − in particular those with elevated DNp73 expression. Together these findings indicate that SCLC cells, due to either genetic or epigenetic alterations in the p73 gene, become dependent on DNp73-mediated gene repression via the Tip60–p400 complex which confers a vulnerability that could be exploited for cancer therapy. No conflict of interest. 340 Role of the HIV matrix protein p17 in EBV-driven lymphomagenesis K. Mastorci1 , D.A. Fae` 1 , C. Giagulli2 , A. Carbone1 , P. De Paoli3 , A. Caruso2 , R. Dolcetti4 . 1 National Cancer Institute- Aviano PN- Italy, Translational Research, Aviano, Italy, 2 University of Brescia Medical School- BresciaItaly, Molecular and Translational Medicine, Brescia, Italy, 3 National Cancer Institute- Aviano PN- Italy, Scientific Direction, Aviano, Italy, 4 University of Queensland Diamantina Institute- Brisbane- Australia, Translational Research, Brisbane, Australia Background: Although it is well established that HIV+ individuals are at risk to develop Hodgkin (HL) or Non-Hodgkin lymphoma (NHL), the pathogenic relationship between HIV and the oncogenic Epstein Barr Virus (EBV) remains unclear. Our recent study showed a bidirectional interplay involving the EBVdriven up-regulation of CXCR2 and the HIV matrix protein p17-dependent upregulation of the EBV LMP1 oncogene through CXCR2 in B cells. However, it remains unclear whether p17 may promote the expression of a broader spectrum of EBV latency proteins and/or trigger EBV reactivation, thereby enhancing the likelihood of malignant evolution of EBV+ B cells in the HIV+ setting.

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Material and Methods: EBV-infected primary B-cells from healthy donors, LCL, HBL-1 and Mutu I lymphoma cell lines were treated with recombinant p17 proteins, including variants derived from HIV+ patients with lymphoma (LyPNB), and analysed by qRT-PCR or immunoblotting for the genes and proteins of interest. EBV DNA in culture supernatants was quantified by qRTPCR. LyPNB1 variant derived from a NHL EBV-; LyPNB2 from a diffuse large B-cell lymphoma EBV+/LMP-1+; LyPNB7 from a Burkitt lymphoma EBV+/LMP-1−. Results: p17, and particularly its natural variant S75X, up-regulates both LMP1 and EBNA2 proteins in Mutu I cells basally expressing only EBNA1, thus promoting a shift towards a broader latency pattern with a greater transforming potential. Furthermore, recombinant p17 proteins activate EBV lytic cycle as shown by upregulation of the viral lytic switch inducers ZEBRA and BRLF1. The completeness of p17-induced lytic cycle was verified by the enhanced expression of late lytic proteins and by quantification of virionic EBV DNA in culture supernatants. EBV lytic cycle may be triggered by B-cell receptor activation (BCR) and by a differentiation shift driven by the plasma cell differentiation factor XBP1. Thus, immunoblotting analysis showed that p17 proteins, particularly S75X, are able to up-regulate the expression of BCR-related kinases in Mutu I cells, consistently with the activation of BCR signalling. p17 variants are also able to up-regulate both the un-spliced and the spliced/active forms of XBP1 mRNA and protein, consistently with an activation of this transcription factor. Moreover, p17, and especially S75X, enhanced the expression of ATF6 and IRE, which function to up-regulate and activate XBP1. Notably, the LyPNB2 p17 variant also induced a strong activation of both these pathways, effects closely related with a stronger induction of EBV lytic replication as compared to the LyPNB1 and LyPNB7 p17 variants. Conclusions: These results consolidate the new paradigm in which HIV may be regarded as source of microenvironmental factors, i.e. p17, which may directly contribute to the development of malignant lymphomas in HIV+ patients, particularly those associated with EBV infection. No conflict of interest. 341 Leukemic cell death induced by the Notch-1 fragment-derived peptide evokes the immunogenic inflammation A. Kuniyasu1 , M. Makise1 . 1 Sojo University, Molecular Cell Pharmacology, Kumamoto City, Japan Background: Non-apoptotic cell death such as a necrosis is an important physiological process. We previously found that a 25mer hybrid peptide (termed Tat-RAM13), which conjugates the Notch-1 intracellular fragment RAM-13 peptide with a cell-penetrating HIV-1 Tat peptide, induces a caspaseindependent cell death in multiple human leukemic cell lines. Morphological features of the cell death resemble necrosis. To elucidate the immunogenic property of the dead cells for inflammation, we investigated the dead cell engulfment by macrophages and extracellular releases of high mobility group box 1 (HMGB1) as a mediator of Inflammation. Materials and Methods: All leukemia cell lines were maintained in 10% FCS, RPMI-1640 medium. Human normal T-cells were separated by Lympholyte Media from the healthy peripheral blood. All peptides were synthesized by Fmoc chemistry using the automatic peptide synthesizer. Jurkat-T cells were incubated with the peptides and/or compounds for 24 h, or by heating at 60o C for 10 min. Cell viability was estimated with WST8 reagent. The cells were fluorescently labeled with carboxyfluorescein succinimidyl ester and then added to thioglycolate-stimulated mouse macrophages-coated chamber slide glass. Engulfed cells were counted under the fluorescent microscope. Nontreated or dead cells were spun down. Supernatants and pelleted (cells) were subjected to western blotting analysis using anti-HMGB1 polyclonal antibody. Results and Discussion: We first examined whether thioglycollate-elicited murine macrophages phagocytose the carboxyfluorescein-labeled dead Jurkat-T cells. Macrophages readily engulfed vincristine-induced apoptotic cells, but Tat-RAM13-treated dead cells engulfing by macrophages were extremely few as well as heat (60ºC)-treated necrotic cells. Next, we investigated that the dead cells secrete the inflammatory HMGB1 protein. Western blot analysis revealed that HMGB1 was rapidly released from both heat- and peptide-treated Jurkat-T cells. In contrast, vincristine-treated dying cells faintly secret it. Conclusion: These data suggest that the Tat-RAM13-induced cell death shares similarity to necrosis in morphological and immunogenic features. The functional evaluation of secreted HMGB1 from the peptide-treaded leukemic cells is ongoing. No conflict of interest.

342 The AMPK-related kinase NUAK1 is a target for treatment of colorectal cancer J. Port1 , N. Muthalagu1 , M. Raja1 , T. Monteverde1 , M. Mezna2 , F. Ceteci3 , G. Murray4 , O. Sansom3 , S. Zanivan3 , D. Murphy1 . 1 University of Glasgow, Institute of Cancer Sciences, Glasgow, United Kingdom, 2 CRUK Beatson Institute, Drug Discovery Unit, Glasgow, United Kingdom, 3 CRUK Beatson Institute, n/a, Glasgow, United Kingdom, 4 University of Aberdeen, Pathology, Aberdeen, United Kingdom Background: MYC deregulation is widespread in human cancers and may represent a requisite node of oncogenic signaling, making MYC an extremely attractive candidate for selective treatment of cancer. As a transcription factor however, targeting MYC directly is challenging. We have therefor used a synthetic lethal strategy to identify kinases that are selectively required to maintain tumour cell viability when MYC is overexpressed. In a study published in 2012, we showed that AMPK and the related kinase ARK5 (NUAK1) are both required to sustain viability of MYC overexpressing cells, largely due to their roles in maintaining energetic homeostasis (Liu, Ulbrich et al. Nature 483, 7391). Materials and Methods: We have now taken a genetic approach to determine the requirement for NUAK1 for development and maintenance of colorectal cancer, a cancer that is typified by high levels of MYC overexpression. Results: Genetic deletion of NUAK1 at the onset of tumourigenesis prevents the emergence of colorectal tumours while RNAi-mediated depletion of NUAK1 in pre-existing tumours drives profound tumour shrinkage. Conclusions: Our results suggest that NUAK1 is a novel target for treatment of colorectal cancer warranting the development of NUAK1 selective small molecule inhibitors. No conflict of interest. 343 Metabolic profile of osteolysis in bone metastases S. Avnet1 , S. Lemma1 , M. Sboarina2 , P. Porporato2 , N. Zini3 , P. Sonveaux2 , N. Baldini4 . 1 Rizzoli Orthopaedic Insitute, Orthopaedic Pathophysiology and Regenerative Medicine Unit, Bologna, Italy, 2 Institute of Experimental and Clinical Research IREC- Universite´ catholique de Louvain Medical School, Pole of Pharmacology, Brussels, Belgium, 3 Institute of Molecular Genetics, CNR National Research Council of Italy, Bologna, Italy, 4 University of Bologna, Department of Biomedical and Neuromotor Sciences, Bologna, Italy Introduction: Bone metastasis (BM) is a dismal complication of cancer, occurring frequently in patients with advanced breast carcinoma. During metastatic progression, carcinoma cells harness osteoclast (OC) activity, promoting osteolysis. To adapt to hypoxia and/or to support proliferation, carcinoma cells adopt primarily glycolysis for energy production, therefore releasing lactic acid in the microenvironment through monocarboxylate transporter 4 (MCT4). Stressed by tumor cells, osteoblasts (OB) can also switch to glycolysis, further promoting lactate release. Here, we hypothesized that extracellular lactate that is released by cells of the tumor microenvironment is uptaken by OC through MCT1 to fuel oxydative phosphorylation (OXPHOS), thereby promoting OC formation and activity. Materials and Methods: We used human primary cultures of OC and OB, wild type breast carcinoma MDA-MB-231 (MDA) cells and MDA clone prone to form BM (bmMDA). OC differentiation and activity were analyzed by TRAP/nuclei staining, and osteolyse assay. Metabolic analyses were performed by electron microscope analysis, immunofluorescence for mitochondrial mass, Seahorse XF96 and CMA600 analyzers, JC-1 staining and ATP assay. MCTs expression was evaluated by RT-qPCR and western Blot. Result and Discussion: We found an extensive mitochondriogenesis along the differentiation process. In mature osteoclasts, mitochondria were included in a complex tubular network, increased in size, and rich of cristae at the ultrastructural level. We also found that fully differentiated OC rely on OXPHOS as the main source for energy production, whereas monocytic precursors have a higher glycolytic rate. The increased of OXPHOS in OC is coupled with an increased expression of MCT1, whereas OB, MDA and bmMDA express high levels of MCT4 and do not express MCT1. We also found a dose-dependent uptake of 14C-lactate by OC. Finally, exposure to sodium lactate promoted OXPHOS in OC. Our data demonstrate that lactate released by tumor cells and OB into the BM microenvironment can be uptaken by OC through MCT1 to fuel mitochondria metabolism. However, the inhibition of OXPHOS by the treatment of OC with not toxic dosage of rotenone resulted in an increased degradation of type I collagen, meaning that bone resorption relays on a glycolytic metabolism. Conclusions: Our findings provide evidence that the differentiation of osteoclasts requires energy that is mainly derived from mitochondrial oxidative metabolism and that breast carcinoma cells can fuel the OXPHOS metabolism of OC through the release of lactate. However, the peripheral cellular activity of bone matrix degradation is supported by glycolysis and further studies are needed to validate MCT1 as a target in osteolytic BM. A better understanding of energy metabolism of osteolysis holds the potential for future therapeutic interventions for BM. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 344 Coordinated erasure and adding up epigenetic marks define transcriptional programs during microglia reprogramming B. Kaminska1 , M. Maleszewska1 , A. Steranka1 , M. Smiech1 , B. Kaza1 , M. Dabrowski2 . 1 The Nencki Institute of Experimental Biology, Neurobiology Center- Laboratory of Molecular Neurobiology, Warsaw, Poland, 2 The Nencki Institute of Experimental Biology, Neurobiology Center- Laboratory of Bioinformatics, Warsaw, Poland Background: Microenvironment-specific signal activate macrophages and polarize to a specific phenotype by epigenetic and transcriptional mechanisms. Microglia, brain specific myeloid cells, accumulate in gliomas and acquire the proinvasive and immunosuppressive phenotype supporting tumor progression. It is poorly understood how microglia are reprogrammed in a responses to challenges and how signals are converted into sustained patterns of gene expression in brain pathologies. Material and Method: Transcriptome analysis of microglial cultures exposed to glioma (GCM) or lipopolysaccharide (LPS) shows activation of distinct signaling and metabolic pathways resulting in different patterns of gene expression. We assess DNA methylation and selected activating and repressive of histone modifications in glioma-driven and inflammatory activation of microglia, to determine their roles in protumorigenic reprogramming. Epigenetic enzyme inhibitors were used to assess the role of histone modifications in phenotype acquisition. Results and Discussion: We demonstrate unmethylated DNA and active histone marks at the promoter regions of glioma- and inflammation-specific genes in unstimulated microglia. Increase of HDAC expression/activity specifically in glioma-activated microglia erases active histone marks. At later times genes regulated in stimulus-dependent manner acquire repressive histone marks suggesting phenotype consolidation. Microglia pre-exposed to glioma attain “epigenetic memory” and have considerably reduced inflammatory responses after inflammatory activation. HDAC inhibitors block glioma-driven activation and restore ability to launch effective inflammatory responses. Conclusion: Our study provides evidence how reversible epigenetic mechanisms stably reprogram brain immune cells, which shapes antitumor responses in gliomas. Acknowledgement: Studies were supported by a grant 2012/04/A/NZ3/00630 from the National Science Center. No conflict of interest. 345 Targeting Ewing Sarcoma cells and the tumor microenvironment with OMTX003 anti-endoglin monoclonal antibodies P. Puerto Camacho1 , A.T. Amaral1 , J.L. Ordonez ˜ 1 , M.J. Robles2 , 1 1 ´ , S. Dom´ınguez3 , C. Jordan ´ Alvarez ´ Perez ´ , M. Biscuola2 , M. Lopez M. Fabre3 , E. Alava2 . 1 Hospital Universitario Virgen del Roc´ıo, Instituto de Biomedicina de Sevilla IBiS, Seville, Spain, 2 Hospital Universitario Virgen del ´ Roc´ıo, Anatomia Patologica, Seville, Spain, 3 Oncomatryx Biopharma S.L., Oncomatryx Biopharma S.L., Derio, Spain Background: Ewing Sarcoma (ES) is a bone/soft tissue neoplasia affecting mainly children and young adults. ES is the second most common primary bone tumor after osteosarcoma in this age group. ES are heavily angiogenic and can show strong vascular mimicry. Endoglin (ENG) is a surface marker with an important role in the establishment of neo-angiogenesis and vascular mimicry. ENG is shedded from the cell surface by Matrix Metalloproteinase 14 (MMP-14) in its soluble form (sEDG) to the extracellular compartment. Materials and Methods: mRNA levels were evaluated by q-RT-PCR. Protein and subcellular location were studied by western blotting, immunofluorescence and flow cytometry in a set of cancer/non-tumoral cell lines. sENG in cell line supernatants were determined by ELISA. DNA promoter methylation study was performed in 9 ES cell lines and mesenchymal stem cells. Cytotoxicity assays with new human Monoclonal Antibodies (MAb) targeting Mouse ENG (mENG OMTX003) and Human ENG (hENG OMTX003) were performed by MTT assay and caspase 3 activation was evaluated by DEVDase activity. Xenograft models (n = 30) with two ES cell lines were developed to study the in vivo tumor binding of the MAbs. A dose range study (DRS) was performed in healthy mice (n = 15) exposed to 3 doses of hENG and mENG OMTX003 for 3 weeks. Results: Whole protein levels correlated with the mRNA levels, and the subcellular location of ENG was primarily at the cell membrane. Only one cell line (1/9) lacks expression of ENG, and in fact, this is the only cell line with hyper-methylated ENG promoter. The ratio between sENG/ENG in cell membrane showed a positive correlation with the levels of MMP14, consistent with a role of this protease in ENG shedding. Specific in vivo binding of hENG OMTX003 MAb after administration in xenografted mice of 2 ES cell lines (ENG++and ENG+/-) was observed in the ENG++ tumors. Impaired tubular formation during exposure to OMTX003 was observed in endothelial cells. Regarding the DRS, histopathological evaluation of several organs revealed no significant drug related toxicity after treatment with hENG OMTX003 up to 10 mg/kg and mENG OMTX003 up to 5 mg/kg.

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Conclusions: ENG is directly implicated in angiogenesis promoted by endothelial cells in the tumor microenvironment, and is also expressed in ES tumor cells. In vitro, OMTX003 inhibition of hENG reduces proliferation and impairs tubular formation of endothelial cells. In vivo, OMTX003 MAbs present specific binding to corresponding mENG/hENG and no toxicity was observed up to 10 mg/kg dose. Efficacy of OMTX003 MAbs as anti-tumoral agents in mono/combined regimens is being currently assessed. Conflict of interest: Ownership: Oncomatryx Biopharma. Corporatesponsored Research: Oncomatryx Biopharma. 346 N-acetylglucosaminyltransferase 5 (Mgat5) mediated N-glycosylation of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) enhances EGFR signaling of cell invasion and metastasis in head and neck cancer M.H. Wu1 , W.F. Chiang2 , T.M. Cheng1 . 1 Taipei Medical University, Graduate Institute of Translational Medicine, Taipei, Taiwan, 2 Chi-Mei Medical Center, Oral and Maxillofacial Section, Tainan, Taiwan Introduction: Distinct glycosylation of membrane proteins could be a biomarker for cancer cells and is regulated by the coordinated actions of glycosyltransferase and glycosidase. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycophosphoinositol (GPI)-anchor glycoprotein and has been reported as a biomarker for cancers. However, the pathological role of glycosylation of CEACAM6 is poorly understood in head and neck squamous cell carcinoma (HNSCC). Material and Methods: We have developed an anti-CEACAM6 single domain antibody (sdAb) from an immune llama library which was used for immunohistochemical staining and for blockage of CEACAM6 functions. The N-glycosylation of CEACAM6 was inhibited by swainsonine, a complex N-glycosylation inhibitor, and Mgat5 (N-acetylglucosaminyltransferase 5) shRNA. Cell migration and invasion ability was measured by Boyden chamber assay. EGF (epidermal growth factor) induced intracellular signaling, EGFR (EGF receptor) clustering, EGFR intracellular trafficking were analyzed using Western blotting and immunofluorescence. Results: Using the anti-CEACAM6 sdAb, we found fully glycosylated CEACAM6 was a tumor marker of oral squamous cell carcinoma (OSCC) and was associated with the poor prognosis of early-stage OSCC patients. CEACAM6 promoted OSCC cell migration, invasion and metastasis through modulating cytoskeleton rearrangement. Blocking the complex N-glycosylation of CEACAM6 using swainsonine and Mgat5 shRNA attenuates CEACAM6 induced cell motility. Furthermore, the N-glycosylation of CEACAM6 enhanced EGFR clustering, phosphorylation and intracellular signaling. Finally, targeting CEACAM6 with the anti-CEACAM6 sdAb inhibited migration, invasion and EGFR signaling in CEACAM6-overexpressing cells. Conclusions: The Mgat5 mediated N-glycosylation of CEACAM6 is crucial for EGFR signaling of invasion and metastasis and targeting CEACAM6 with the anti-CEACAM6 sdAb could be a feasible therapy for HNSCC. No conflict of interest. 347 YAP, Cdc42EP3 and septins modulate mechano-transduction and the emergence of cancer-associated fibroblasts F. Calvo1 , R. Ranftl1 , S. Hooper2 , E. Sahai2 . 1 Institute of Cancer Research, Division of Cancer Biology − Tumour Microenvironment Team, London, United Kingdom, 2 The Francis Crick Institute, Cell Biology of the tumour microenvironment, London, United Kingdom Introduction: Cancer-associated fibroblasts (CAFs) are non-malignant cells prominently found in tumours that can favour tumour progression, dissemination and therapeutic resistance through remodelling of the extracellular matrix and signalling to cancer, endothelial and immune cells. As opposed to normal fibroblasts, CAFs present a pathologically activated status responsible for their tumour-promoting activities. Targeting CAFs represents an interesting alternative for therapeutic intervention, but a better understanding of the mechanisms governing their emergence, maintenance and tumour promoting abilities is still needed. Materials and Methods: We isolated fibroblasts from different stages of breast cancer progression and normal counterparts, and analysed their phenotype, function and gene expression. These analyses were performed in 3D physiologically relevant conditions that better mimic the in vivo setting. These conditions enhanced the phenotypic and functional differences between normal fibroblasts and CAFs, and permitted the identification of relevant mechanisms that in 2D conditions may be masked. We performed a global mRNA analysis of our set of fibroblasts to identify dysregulated genes and changes in coordinated gene expression programmes that were further explored using loss-of-function RNAi approaches. Relevant mechanisms were validated using in vivo models and clinical material. Results and Discussion: Our analyses revealed that enhanced mechanotransduction and activation of the transcriptional regulator YAP are signature features of CAFs. YAP regulates the expression of cytoskeletal genes that are needed for the maintenance of the CAF phenotype and for their

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ability to remodel the extracellular matrix and promote cancer cell invasion and angiogenesis. Matrix stiffening further enhances YAP activation, thus establishing a feed-forward self-reinforcing loop that helps to maintain the CAF phenotype. In addition, we identified Cdc42EP3, a previously uncharacterised Cdc42 effector, as a key regulator of CAFs. Cdc42EP3 links actin and septin fibres and is upregulated early in fibroblast activation. Cdc42EP3 induces a profound cytoskeletal rearrangement in CAFs that enables mechanotransduction and the emergence of pro-tumorigenic functions, which are also dependent on the presence of a septin fibrillary network. Conclusion: Our studies show for the first time how mechanical inputs in solid tumours modulate the aggressive behaviour of stromal fibroblasts via YAP. We demonstrate a critical role for coordination between the actin and the septin networks in CAFs that is required for mechano-transduction, force-mediating matrix remodelling, and promoting cancer cell invasion, angiogenesis and tumour growth. Finally, we describe the mechanism of action of Cdc42EP3 and define a novel molecular and biological role for septins in CAFs. No conflict of interest. 348 The contribution of the cancer initiating cell markers EpCAM and claudin7 to metastatic progression S. Heiler1 , M. Zoller ¨ 1 . 1 University Hospital Heidelberg, Tumor Cell Biology, Heidelberg, Germany Functional activity of EpCAM (EpC) in colorectal and pancreatic cancer progression depends on its association with palmitoylated claudin7 (cld7) which is excluded from tight junctions and is recruited to glycolipid-enriched membrane microdomains (GEM), where it cooperates with EpC and additional transmembrane and cytosolic molecules. How palmitoylated cld7 contributes establishing a metastatic phenotype is unknown. The loss of metastatic capacity of a rat pancreatic adenocarcinoma by a knockdown (kd) of cld7 or EpC is waved by the rescue of wt EpC, but not EpC with a mutation in the cld7 binding site (EpCmAG) or by cld7 with a mutation in the palmitoylation site (cld7mPalm). Cld7mPalm also does not rescue anchorage-independent growth, motility, invasiveness and apoptosis resistance. This is due to cld7, but not cld7mPalm associating with a3, b4, ezrin, uPAR and MMP14, which supports motility and invasion. Palmitoylated cld7 additionally is engaged in Pten repression, which allows Akt phosphorylation accounting for apoptosis resistance. Cld7 supports epithelial mesenchymal transition (EMT) gene expression and nuclear translocation of Snail, Sox2 and Notch. These activities of GEM-located palmitoylated cld7 are transferred into exosomes (TEX) such that exosomal palmitoylated cld7 promotes EMT in cld7kd cells. Notably, only palmitoylated cld7 associates with several components of the vesicle transport machinery required for exosome biogenesis and exosome release such that TEX release by cld7kd cells is strongly reduced and uptake by host cells and non-cancer-initiating cells is severely impaired. GEM-located palmitoylated cld7 acts as a cancer-initiating cell marker by associating with signaling molecules and EMT genes, which are transferred together with cld7 into TEX and account for the crosstalk with host and tumor cells. By the association with the vesicle transporter machinery GEM-located, palmitoylated cld7 actively contributes to TEX biogenesis. No conflict of interest. 349 The hypoxia marker CA-IX is prognostic in soft tissue sarcoma patients treated in the UK phase III VORTEX trial L. Forker1 , S. Sioletic2 , P. Shenjere3 , J. Irlam1 , H. Valentine1 , D. Hughes4 , A. Hughes5 , P. Gaunt6 , M. Robinson7 , C. West1 . 1 Institute of Cancer Sciences, University of Manchester, Manchester, United Kingdom, 2 Azienda Ospedaliero-Universitaria di Udine, Department of Pathology, Udine, Italy, 3 The Christie NHS Foundation Trust, Department of Histopathology, Manchester, United Kingdom, 4 Sheffield Teaching Hospitals NHS Foundation Trust, Department of Histopathology, Sheffield, United Kingdom, 5 Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom, 6 Cancer Research UK Clinical Trials Unit CRCTU, University of Birmingham, Birmingham, United Kingdom, 7 Academic Unit of Clinical Oncology, Weston Park Hospital, Sheffield, United Kingdom Introduction: Despite high distant relapse rates, there is no proven benefit for adjuvant chemotherapy in soft tissue sarcoma (STS). In a heterogeneous group of tumours, progress requires tailoring of therapy to individual features of tumour biology. Hypoxia is associated with metastasis and a reliable biomarker may identify around half of patients with localised, high grade STS at high risk of metastatic relapse for intensification of therapy. The surrogate markers HIF-1a, CA-IX and GLUT-1 have been associated with poor outcomes in STS. However, previous studies yielded conflicting results and a question remains as to which marker is superior. VORTEX provides an opportunity to explore this in a large cohort with robust outcome data treated with current surgical/radiotherapy techniques. Material and Methods: Between 2007 and 2013 216 patients with localised, resectable extremity STS were randomised to two adjuvant radiotherapy volumes in the phase III VORTEX trial. Tissue microarrays were constructed

from formalin fixed paraffin embedded tissue for patients who consented to the biobank (n = 206). Immunohistochemistry for HIF-1a, CA-IX and GLUT-1 was performed and the percentage of tumour cells stained estimated. Cores were scored twice by one scorer and independently by a sarcoma pathologist. Duplicate and independent scores correlated well (Spearman ø > 0.81). The pathology score was used if scores were discordant. Disease-free survival (DFS) was defined as time from randomisation to local recurrence, metastasis or death. Survival estimates were performed using Kaplan–Meier analysis; hazard ratios (HRs) and 95% confidence intervals (CI) were obtained using Cox regression analysis (univariate). Results and Discussion: Data were available for 165, 179 and 183 randomised patients for HIF-1a, GLUT-1 and CA-IX expression respectively. Staining was consistent with previous studies (HIF-1a nuclear, GLUT-1/CA-IX membranous). Patients with high CA-IX expression had worse DFS (HR 2.28, 95% CI 1.44–3.63, p < 0.001). HIF-1a expression was also associated with poor DFS (HR 1.76, 95% CI 1.01–3.08, p = 0.047). GLUT-1 expression was not prognostic. Hypoxia biomarkers can predict clinical benefit from hypoxia modification in multiple tumour types. Hypoxia influences malignant progression through promotion of invasion/cell motility and increasing genome instability with subsequent development of a metastatic phenotype. Therefore targeting hypoxia in STS may reduce metastatic relapse and CA-IX could be used as a predictive biomarker to select patients most likely to benefit. Conclusion: CA-IX shows the strongest association with prognosis in STS and may be the best current known marker to identify patients for hypoxia targeted therapy in a biomarker driven trial. No conflict of interest. 350 Monitoring the dynamics of clonal tumor evolution in vivo using secreted luciferases J.P. Charles1 , J. Fuchs1 , M. Hefter1 , J.B. Vischedyk1 , M. Kleint1 , F. Vogiatzi1 , A. Nist1 , O. Timofeev1 , M. Wanzel1 , T. Stiewe1 . 1 Center for Tumor Biology and Immunology Philipps University Marburg, Molecular Oncology, Marburg, Germany Tumors are heterogeneous cell populations that undergo clonal evolution during tumor progression, metastasis, and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumor cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukemia mouse models, fluorescent reporters are poorly suited for animals with solid tumors − the most common tumor types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA expressing tumor cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumor-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumor subclones in preclinical mouse models of tumor development, metastasis and therapy. In addition, we present recent advances on monitoring the impact of CRISPR-Cas9 induced gene mutations on drug responses with the help of secreted luciferases. No conflict of interest. 351 Cancer stem cell markers and exosomes ¨ 1 , U. Erb1 . 1 University Hospital Heidelberg, Tumor Cell S. Heiler1 , M. Zoller Biology, Heidelberg, Germany It is unknown whether there is a linkage between cancer stem cell (CSC) markers and metastasis/EMT promoting activities of CSC exosomes (TEX). The question was explored by a knockdown (kd) of the CSC markers CD44v6, Tspan8, EpCAM and cld7 in gastrointestinal cancer. Each kd was accompanied by loss of EMT and metastases formation, but TEX from CSCenriched populations sufficed repairing the defect of kd cells. As a kd of one CSC marker sufficed for a CSC breakdown we performed proteome and miRNA analyses of wt and kd TEX searching for joint features. The CSC markers are located in internalization prone glycolipid-enriched microdomains (GEM). GEM-located Tspan8 recruits a6b4, the cMET-CD44v6 complex is recruited upon ligand binding. Internalized cld7 becomes palmitoylated, which fosters GEM location and EpCAM association. In addition, Tspan8 and cld7 are engaged in TEX biogenesis. Tspan8 associates with molecular complexes promoting GEM internalization and scission. Tspan8 and palmitoylated cld7 associate with intracellular vesicle transporters. Proteome analysis and immunoprecipitation also confirmed GEM-located complex internalization including cytoskeletal linker and signaling molecules located at the inner membrane. By regulating cMET and Tspan8 transcription, CD44v6 favors a second level of connectivity. Furthermore, there is a strong overlap of miRNA recruited into TEX including several miRNA known to promote EMT and/or metastases. These miRNA are frequently not recovered or reduced in Tspan8kd, cld7kd and CD44v6kd TEX, which argues for a joint contribution

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 of these CSC marker in miRNA recruitment or in miRNA processing, which remains to be explored. Finally, Tspan8 and associated integrins play a major role in TEX targeting. CSC protein markers are gathered in GEM, become jointly internalized, are engaged in exosome biogenesis including loading and are recovered in TEX. Joint target modulation is promoted by associations between CSC markers and additional TEX components including miRNA. Thus, TEX-based diagnosis and therapy should appreciate the central role of CSC markers. No conflict of interest. 352 PKCtheta controls invasion of triple-negative breast cancer by direct phosphorylation of FAK L. Chadelle1 , V. Cadamuro1 , J. Liu1 , X. Wang1 , K. Belguise1 . 1 LBCMCPUniversite´ Paul Sabatier- CNRS UMR5088, LBCMCP- Universite´ Paul Sabatier- CNRS UMR5088, Toulouse, France Background: Triple-negative breast cancers (TNBC: negative for oestrogenreceptor, progesterone-receptor and human-epidermal-growth-factor receptor 2 [HER2]) account for 15−20% of breast cancers and are characterized by an extremely aggressive phenotype. Currently, there is no efficient targeted therapy that can prevent the metastatic dissemination of TNBC. The serine threonine kinase PKCQ has recently been identified as a potential new marker of TNBC and as a strong inducer of cancer invasion. However, how PKCQ controls tumour invasion in TNBC is still unclear. Material and Methods: To study PKCQ control of invasion, we worked on several breast cancer cell lines combining biochemical and imaging approaches. Results: Here our results show that PKCQ can promote invasion by activating the focal adhesion kinase (FAK) signalling that regulates the cycle of focal contact formation and disassembly. As a consequence of this activation, PKCQ enhances the dynamic of adhesion turnover, Rho GTPases activity and protrusions formation. We also have unravelled the molecular mechanisms by which PKCQ could control FAK: PKCQ interacts with and phosphorylates FAK at S890–892–893 thus leading to its opening and activation. By using non phosphorylatable mutants of FAK, we show that the identified phosphorylation sites are essential to the PKCQ controlled invasion. Conclusion: We show that PKCQ is controlling invasion by directly phosphorylating FAK at S890–892–893. We are currently testing the in vivo relevance of this mechanism by mouse metastasis assay. Next, we will investigate the possibility of a correlation between the presence of the identified phosphorylations on patient samples and the tumour’s aggressiveness. In the long term, this study could help develop new targeted therapy for TNBC such as PKCQ specific inhibitors that would limit metastasis formation. No conflict of interest. 353 Quantitative analysis of estrogen receptors alpha and beta expression in ovarian cancer patients by flow cytometry E. Dudko1 , T. Bogush1 , S. Kolomiytsev1 , O. Rjabinina1 , A. Tjulandina2 , V. Kirsanov3 , E. Bogush3 , S. Tjulandin2 , I. Mamichev1 . 1 N.N. Blokhin Russian Cancer Research Center, Laboratory of medical chemistry, Moscow, Russian Federation, 2 N.N. Blokhin Russian Cancer Research Center, Department of clinical pharmacology and chemotherapy, Moscow, Russian Federation, 3 N.N. Blokhin Russian Cancer Research Center, Department of surgery, Moscow, Russian Federation Introduction: The results of chemotherapy of ovarian cancer is not satisfactory enough and new approaches are needed. Perspective therapy strategy implying estrogen receptors (ER) as drug target is investigated. Usually only ER alpha are examined, but recent researches had shown that the other type of ER − ER beta − is a significant pathogenetic parameter of ovarian cancer as well. The purpose of the study is estimation expression and co-expression of both types of ER − ER alpha and beta − in ovarian cancer tissue. Materials and Methods: 53 serous ovarian cancer surgical biopsy specimens were analyzed by flow cytometry. Single-cell suspensions were incubated overnight with primary anti-ER alpha (SP1, Abcam) and anti-ER beta (14C8, Abcam) and then − for 1.5 h with secondary antibodies, conjugated with DyLight650 (ab98510 and ab98729, respectively). Cell fluorescence was analyzed with FlowJo 10.0.8 software and number of specifically fluorescent cells was calculated by using Kolmogorov–Smirnov statistical approach. Three levels of ER expression were analyzed: high − ER were revealed more than in 40% of the cells; low − in 15−40%; negative − less than in 15%. Data were treated by nonparametric methods, Spearman rank correlation coefficient (rs) was used for estimation of dependence between expression of ER alpha and beta. Results and Discussion: (1) ER beta was revealed in all the patients (pts) but ER alpha − in 75% only. (2) In the pts with ER positive tumors low ER alpha level was revealed in 1.5 times often in comparison to ER beta (57% vs 38%), but for high ER alpha level, opposite, it was 3 times rarely in comparison to ER beta (20% vs 62%). (3) In 19% of the pts co-expression high level of ER alpha and ER beta was revealed. (4) Moderate correlation

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between expression of ER alpha and ER beta was found (rs = 0.61; 95% CI 0.38−0.74). Conclusion: It was shown that (1) ovarian cancer tumors are actually ER positive ones with higher level and frequency of ER beta expression as compared to ER alpha; (2) expression of both markers was correlated each other and (3) in 1/5 of the tumors co-expression high level of ER alpha and ER beta was revealed. We suppose that controversial information about contribution of ER in pathogenesis of ovarian cancer is because one type of ER − ER alpha only − is estimated in the tumor routinely. We believe that quantitative determination both ER alpha and ER beta in the tumor let distinguish the pts with high level of ER expression (ER alpha, ER beta or both), who needs adjuvant antiestrogen therapy similarly with the ER alpha positive breast cancer pts. Acknowledgement: Supported by RFBR grants (15-04-06991-a, 16−34-01049mol-a) and President of Russian Federation grant MK-7709.2016.7. No conflict of interest. 354 Colorectal adenocarcinoma stem cells show distinct growth patterns depending on the culture environment A. Borys1 , P. Wołkow1 . 1 Jagiellonian University Medical College, Center for Medical Genomics OMICRON, Krakow, Poland Introduction: Disease models for basic cancer research rely predominantly on established cell lines and animal studies. Lgr5, an R-spondin receptor, and a specific marker of gastrointestinal tract stem cells, enabled in vitro culture and observation of all steps of their differentiation and self-organization from single cells to “mini-organs”. Organoid cultures are currently the state-of-theart model for studies of colon, esophagus and small intestine pathophysiology. We used this method to produce cancer colonoids from stem cells, isolated from LoVo cell line. LoVo cell line, established from Dukes’ type C, grade IV, colorectal adenocarcinoma, contains, according to the literature, a significant fraction of cancer stem cells (CSCs). Materials and Methods: The characteristics of CSC derived cultures were assessed in two types of growth environments: 2D and 3D (laminin-rich extracellular matrix). Lgr5+ CSCs were isolated from amplified LoVo cells with anti-Lgr5+ magnetic beads. Proportion of living and dead cells, were measured with fluorescence/luminescence assay at several time points. Developed colonoids were treated with demethylation agent 5-azacytidine and with monoclonal antibody Cetuximab. Results and Discussion: The results show that established LoVo cell line contains small but relevant fraction of stem cells (1.5−3%), which can be isolated and cultured. CSC growth pattern and cell morphology are grossly affected by 2D or 3D culture environment. Cells kept in 2D, form cell aggregates, very similar to general LoVo population with elongated cells extending toward the inter-cellular space. Cells cultured in 3D matrix grow as compact ‘colonoids’ with almost invisible cell junctions and very smooth edges. Three-dimensional growth conditions support cell survival − there is a significantly higher proportion of living cells and lower of dead cells than in 2D. Colon cancer ‘colonoids’ show distinct response patterns to cytotoxic drugs treatment. Cells treated with 5-azacytidine show higher rate of viable cells in three-dimensional culture than in 2D conditions. LoVo cells were demonstrated to exhibit the high level of EGFR protein and therefore should be very sensitive to monoclonal EGFR targeted antibody, e.g. Cetuximab. Our study shows that colon cancer ‘colonoids’ are even more affected by Cetuximab in 3D conditions than in traditional monolayer. Conclusion: CSCs derived from LoVo cell line present distinct growth patterns with varying culture environment. In 2D, they form a monolayer of spindleshaped cells, in 3D they generate round, spherical structures with smooth edges. As organoids should develop all cell types, present in vivo in human colon, we plan to investigate cell subtypes present in LoVo ‘colonoids’. Also, patterns of demethylation induced changes in gene expression are under evaluation by microarrays. No conflict of interest. 355 Approaches to elucidate cancer metabolism using zebrafish models M.C. Mione1 , A. Kalkbrenner2 , V. Gourain1 , M. Mayrhofer1 , S. Burkart1 , C. Muhle-Goll2 , B. Luy2 . 1 Karlsruhe Institute of Technology, Institute for Toxicology and Genetics, Eggenstein-Leopoldshafen, Germany, 2 Karlsruhe Institute of Technology, Institute of Organic Chemistry, Karlsruhe, Germany Introduction: Metabolic reprogramming is a hallmark of cancer, as proliferating cells require a large amount of nutrients, energy, and biosynthetic activity to produce the macromolecular components required by the growing tumour cells. This leads to a switch from oxidative metabolism to glycolysis (Warburg 1925; 1956) to utilize metabolites, such as lactate and glutamine, as building blocks for macromolecule biosynthesis. Previously, changes in metabolism were mostly attributed to opportunistic utilization of alternative biochemical pathways; however, the systematic analysis of cancer genomes and their expression profiling have revealed a surprising abundance of genetic

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alterations of key metabolic enzymes as early events in cancer. These are particularly interesting as enzymatic activities can be targeted with great specificity and success. To understand the consequences of altered enzymatic activities on cancer metabolism in vivo, analytical methods for quantifying metabolites, and system approaches to analyse the genetic and metabolome data in model systems are required. Materials and Methods: We have started to develop integrated maps of metabolic alterations at different stages of melanoma and glioma development using our zebrafish cancer models (Santoriello et al., 2010; Spitzner et al., 2014). We combined metabolome analysis (through NMR) and gene expression data (through RNA-Seq) at different stages of cancer development, in whole animals and dissected tumors. Results and Discussion: We identified metabolic reprogramming in the choline pathway leading to decreased activity of the trans-sulphuration pathway and S-adenosyl-methionine availability, and increased glutamine /acetate production in glioma due to extensive reprogramming of the TCA cycle. Conclusions: Changes in metabolites and enzyme expression leading to these metabolic reprogramming events were already detectable in transgenic larvae, thus suggesting that zebrafish cancer models may serve to study the evolution of metabolic reprogramming in cancer and provide an in vivo platform to screen for metabolic vulnerability in cancer. No conflict of interest. 356 Non-canonical Hedgehog/AMPK-mediated control of polyamine metabolism is required for medulloblastoma growth G. Canettieri1 , S. Coni1 , D.M. Laura1 , G. Sdruscia1 . 1 Sapienza University, Department of Molecular Medicine, Roma, Italy Introduction: Developmental Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs) and its aberrant activation is a leading cause of Medulloblastoma (SHH-MB, Hedgehog molecular subgroup). Treatment of SHH-MB patients with the FDA-approved Hh inhibitor Vismodegib (which targets the transducer Smoothened) has been disappointing because of the occurrence of resistance, attributed to Smo inactivating mutations or to activation of downstream effectors. Thus, it is now believed that the identification and targeting of novel downstream components represents a preferable option for this disease. Material and Methods: We used biochemical, cell biology and molecular biology approaches to characterize the molecular features of a novel mechanism involved in polyamine production upon Hh activation. The biological relevance of our findings has been evaluated in mice and patients with SHH MB. Results and Discussion: We show here that Hedgehog promotes polyamine biosynthesis in GCPs by engaging a non-canonical axis leading to ODC translation. This process is regulated by AMPK, which phosphorylates threonine 173 of CNBP in response to Hedgehog activation. Phosphorylated CNBP increases its association with Sufu, followed by CNBP stabilization, ODC translation and polyamine biosynthesis. Notably, CNBP, ODC and polyamines are hallmarks Hedgehog-dependent Medulloblastoma (SHH-MB) and genetic or pharmacological inhibition of this axis efficiently blocks Hedgehog-dependent proliferation of Medulloblastoma cells in preclinical settings. Conclusions: Together, these data illustrate a novel auxiliary mechanism of metabolic control by a morphogenic pathway with relevant implications in cancer. No conflict of interest. 357 The impact of Mycobacterium obuense on innate and adaptive immunity J. Crooks1 , S. Brown1 , C. Forss1 , A. Phythian-Adams1 , P. Cook1 , L. Rosa Brunet2 , A. MacDonald1 . 1 University of Manchester, MCCIR, Manchester, United Kingdom, 2 Immodulon Therapeutics Ltd, Immodulon Therapeutics Ltd, London, United Kingdom Background: IMM-101 is an immunomodulatory treatment, comprising of heat killed whole cell Mycobacterium obuense (NCTC13365), currently under clinical investigation in a variety of cancers, including pancreatic cancer. At present, available pancreatic cancer treatments only provide 5% survival rate over 5 years, with a median overall survival rate of 6−12 months, highlighting the need for new therapies. A phase II clinical trial (NCT01303172) has shown that treatment with the immunomodulator IMM-101 in parallel with gemcitabine increases median survival to 7.0 months in patients with metastatic pancreatic cancer, compared to 4.4 months following treatment with gemcitabine alone (Dalgleish, A.G. and The IMAGE I Trial investigators, 2015). In light of the promising results of this combination treatment, elucidation of how IMM101 induces its therapeutic effects is ongoing. Initial pre-clinical studies have suggested that antigen specific CD45RBlowCD44high cytotoxic CD8+ T cells are increased in IMM-101 treated animals and may play a key role.

Material and Methods: Dendritic Cell (DC) studies were conducted on IMM101 (or control) stimulated murine bone marrow derived GMCSF DCs or FLT-3 DCs, or human monocyte derived DCs. DC phenotypic activation was assessed by flow cytometry, and cytokine secretion assessed in culture supernatants by ELISA or cytokine bead array. CD4+ T cells used in murine coculture studies were isolated from the lymph nodes and spleens of transgenic OTII mice before being stained with CFSE to assess proliferation via flow cytometry. Results: We have investigated the impact of different concentrations of IMM101 on murine and human DC activation and function. GMCSF derived murine DCs displayed a dose dependent response to the immunomodulatory IMM101, with elevated expression of activation markers and increased secretion of IL-6, IL-12p40 and NO, compared to controls. This dose dependent response to IMM-101 was also replicated in murine FLT-3 generated DCs (pDCs, CD11b+ cDCs and CD24+ cDCs) and human monocyte derived DCs. Using murine DC:T cell co-cultures, we have demonstrated that IMM-101 influences the processing and presentation of antigen by DCs to CD4+ T cells. We have also shown that IMM-101 activated DCs instigate a T cell specific IFN-gamma, and a non-T cell specific IL-17, response following re-stimulation of draining lymph node cell preparations obtained 7 days after adoptive transfer of DCs into na¨ıve recipient mice. Conclusion: Our demonstration that IMM-101 activates primary DCs, both murine and human, and promotes IFN-gamma and IL-17 production in vivo helps build a better understanding of the fundamental mechanisms that may bridge innate and adaptive immune responses and may help elucidate the mode of action of this promising cancer treatment. No conflict of interest. 358 Novel methods for the isolation of tumor cells from human, mouse, and xenografted tumors D. Agorku1 , A. Langhammer1 , L. Willnow1 , K. Klingner2 , S. Tomiuk1 , 1 J. Kollet1 , S. Ruberg ¨ , J. Schuler ¨ 2 , A. Bosio1 , O. Hardt1 . 1 Miltenyi Biotec GmbH, Research and Development, Bergisch Gladbach, Germany, 2 Oncotest GmbH, In vivo Tumorbiology, Freiburg, Germany Solid tumors are vascularized and infiltrated by stromal cells such as leukocytes, endothelial cells and fibroblasts. The amount and composition of those non-tumor cells depends on various factors including tumor entity and stage, treatment history, status of the host organism and site of tumor growth. This results in a widely unpredictable contamination of non-tumor cells within tumor samples frequently hampering downstream applications like cultivation and molecular analysis. However, the direct isolation of target cells is difficult as there is a lack of markers exclusively expressed by tumor cells in many cases. To overcome these hurdles, we have developed methods allowing for the isolation of tumor cells from human, mouse, and xenografted tumors by specifically depleting non-tumor cells from the respective primary material by utilizing magnetic cell sorting. The combinations of antibodies specifically binding all non-tumor cells from human, mouse, and xenograft tumors have been identified by flow cytometrybased screening assays on multiple tissue samples as well as cell lines. The respective antibodies were coupled to superparamagnetic nanoparticles and optimized for efficient depletion utilizing MACS. Performance of nontumor cell depletion was assessed by flow cytometry and cell culture followed by immunocytochemistry of separated cells. Additionally, whole exome sequencing on bulk tumor and separated samples was performed to assess improvement in downstream analysis. Three different antibody cocktails were established for depletion of non-tumor cells from all common sources of tumor material. Using these optimized antibody combinations coupled to superparamagnetic nanoparticles allowed for the fast isolation of untouched tumor cells independent of tumor entity and origin. Typically, purities higher than 95% were achieved in less than 25 minutes. Even when starting with samples such as pleural effusions, containing only minor frequencies of tumor cells, purities of higher than 85% were reached by a single isolation step. By depletion of stromal cells, the downstream culture of tumor cells was significantly improved and standardized. In downstream analysis more SNPs were identified by next generation sequencing, and gene expression signatures of isolated tumor cells and cancer stem cells could be more reliably be interpreted upon removal of the bias caused by non-tumor cells. Taken together, we have developed easy and fast procedures to isolate tumor cells independent of tumor specific markers or tumor entity from human, mouse, and xenografted tumors. This allows for increased sensitivity, specificity and reproducibility in downstream analysis of tumor cells and subpopulations thereof. The “untouched” isolation of tumor cells allows for the subsequent sorting and analyses of minor tumor subpopulations, such as cancer stem cells. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 359 Dissecting the pro-tumoural role of the essential amino-acid transporter complex CD98/LAT1 Y. Cormerais1 , S. Giuliano1 , P.A. Massard1 , J. Durivault1 , E. Hitoshi2 , W. Michael3 , S. Parks1 , J. Pouyssegur1 . 1 Centre Scientifique de Monaco, Medical Biology, Monaco, Monaco, 2 J-Pharma- Co. Ltd., J-Pharma, Yokohama, Japan, 3 University of Colorado Denver- Anschutz Medical Campus, School of Pharmacy, Aurora, USA Background: The CD98/LAT1 complex is overexpressed in aggressive human cancers and is thereby described as a potential therapeutic target. This complex carries multiple protumoral activities with LAT1 (SLC7A5) promoting import of essential amino acids (EAA) and mTORC1 activity while CD98 (4F2hc) promotes b-integrin signalling. Considering this functional plurality, which member of the heterodimer is carrying the most prevalent pro-tumoural action? This notion is far from being clarified and remains controversial in the literature. Despite the fact that CD98 knockout cells have reduced AA transport, Ginsberg and colleagues reported that the pro-tumoural action in a teratocarcinoma model or growth promoting activities in T and B cells are mediated through b-integrin-CD98 signalling rather than the activity of LAT1. Considering the obligatory need for EAA in proliferating tissues and in particular tumours this is a surprising conclusion that we decided to investigate further. Material and Methods: We first created SLC7A5 (LAT1) or SLC3A2 (CD98) knockouts in the colorectal adenocarcinoma cell line LS174T using Zinc Finger Nucleases (ZFN). We monitored the impact of these ablations both in vitro (2D and 3D proliferation assay, western blot, transport activity) and in vivo (xenograft, western blot). Moreover, we confirmed our results both by genetic ablation and/or pharmacological inhibition in other cancer cell types such as lung (A549, H1975) and kidney (786-O and A498). Results and Discussion: Homozygous knockouts of each gene (CD98 or LAT1) demonstrated a clear interdependence of the two members of this complex. Each knockout respectively ablated 90% (CD98KO) and 100% (LAT1KO) of Na+-independent leucine transport activity. LAT1KO cells presented an AA stress response with ATF4 induction, mTORC1 inhibition and severe in vitro and in vivo tumour growth arrest. Rescuing a full functional expression of CD98 in LAT1KO cells had minimal impact. Surprisingly, CD98KO cells that maintain only 10% of the Na+-independent leucine transport rate (LAT1 transport) display a high mTORC1 activity and are capable of proliferating in vitro and in vivo at the same rate as the wild type cells. However CD98KO cells became extremely sensitive to a selective LAT1 inhibitor (JPH203) and genetic disruption of LAT1 (CD98/LAT1dKO). This finding demonstrates that the tumoural potential of CD98KO cells is due to residual LAT1 transport activity. Furthermore, specific pharmacological inhibition of LAT1 in 6 independent cancer cell lines strongly decreased mTORC1 activity and proliferation while CD98 expression was slightly increased. Conclusion: Our study clearly establishes that LAT1 transport activity is the key growth-limiting step of the CD98/LAT1 complex in multiple cancer types and advocates the pharmacology development of LAT1 transporter inhibitors as very promising anticancer target. No conflict of interest. 360 The role of E3 ubiquitin ligase Smurf2 in cancer M. Blank1 . 1 Bar Ilan University, Faculty of Medicine, Safed, Israel Introduction: Recent progress in cancer research has established a consequential link between impaired DNA damage response (DDR), altered gene expression, and compromised genome integrity. This “threesome” forms the main driving force in carcinogenesis, and evidently, this “carcinogenic triad” determines the therapeutic outcomes of anticancer therapies. Despite the importance of this triad in cancer biology, we know little about the regulatory mechanisms lying upstream and interlocking its components. This dearth of knowledge creates significant gaps in our understanding of the mechanisms leading to and operating in cancer, and impedes the design of novel, more efficient, therapeutic approaches for its treatment. Our recent studies suggested that Smurf2, an E3 ubiquitin ligase and recently discovered tumor suppressor, serves as the master regulator that spans through and integrates the components of the “carcinogenic triad” (Blank M et al., Nature Med 2012). Smurf2 (Smad specific ubiquitin protein ligase 2) is an evolutionarily conserved HECT-type E3 ubiquitin ligase. Initially, Smurf2 was discovered as a negative regulator of TGF-beta signaling, yet our and other studies showed that Smurf2 has other key functions, and intimately involved in carcinogenesis (Zou X et al., BBA 2015). Despite these proceedings, the mechanisms operating under the jurisdiction of Smurf2 and involved in the regulation of the components of the “carcinogenic triad” remain largely unknown. Materials and Method: In order to identify and characterize molecular mechanisms operating under Smurf2 auspices, we use a variety of biochemical, molecular and cell biology approaches, including mass spectrometry analysis.

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Results and Discussion: Through the probing of Smurf2 cellular interactome, we identified a number of novel, previously unknown molecular interactors of Smurf2. Currently, we are conducting studies to determine the functional significance of these interactions in the regulation of fundamental cellular processes operating in and regulating carcinogenesis and tumor cell sensitivity to anticancer therapies. Conclusion: Our data suggest that E3 ubiquitin ligase Smurf2 through its association with key cellular factors involved in chromatin structure transition processes, gene expression, DDR and genomic integrity maintenance is intrinsically involved in the regulation of pivotal cellular processes. No conflict of interest. 361 Genomic evolution and heterogeneity in 2,800 cancers D. Wedge1 , P. Spellman2 , P. Van Loo3 , PCAWG Working Group on Evolution and Heterogeneity. 1 Wellcome Trust Sanger Institute, Cancer Genome Project, Cambridge, United Kingdom, 2 Oregon Health and Science University, School of Medicine, Portland, USA, 3 Francis Crick Institute, Cancer Genomics, London, United Kingdom Introduction: Genetic intra-tumour heterogeneity (ITH) arises inevitably from the continual action of mutational processes. Selective pressure drives the expansion of individual cells, often leading to subclones that are detectable from bulk DNA sequencing. The occurrence of heterogeneity is of clinical importance, since different subclones have different proliferative potentials and responses to treatment. Here we present the first large-scale analysis of ITH using whole genome sequencing (WGS). Analysis of 2,800 tumours has yielded insights into the extent of heterogeneity in different tumour types, the order of acquisition of genetic aberrations, the evolution of mutational signatures, real-time estimates of landmark events in tumour development and the genetic drivers of subclonal expansion. Materials and Methods: The International Cancer Genome Consortium (ICGC) has obtained WGS of several thousand cancers, representing ~30 tumour types. The Pan Cancer Analysis of Whole Genomes (PCAWG) project has performed alignment and variant calling on 2,800 of these whole genomes and the PCAWG Working Group on Evolution and Heterogeneity, an international partnership of ~20 research groups, have carried out comprehensive analyses to identify the extent and nature of heterogeneity within the tumours and the mechanisms and processes that underlie tumour evolution. Results: Reconstruction of the subclonal architecture of over 2,000 tumours reveals variability in the extent of heterogeneity between tumour types. Separate analyses of different types of genetic aberration shows that some tumour types have more intra-tumour heterogeneity at the copy number level, while others acquire large numbers of single nucleotide variants subclonally. Chronological timing of whole genome duplications indicate that they commonly occur many years before diagnosis. Differences in mutational signatures of clonal and subclonal mutations indicate changes in the mutational processes operative on cancers at early and later stages of development. Within individual tumour types, some driver mutations are almost exclusively clonal, founding mutations, while others are very commonly subclonal. Subtypes may be defined from the agglomeration of numerous genomic features, indicative of the early determination of different pathways to cancer. Conclusion: Through a large-scale integrated analysis of whole genome profiles of over 2,000 cancers, we reveal differences in the evolutionary trajectories between tumour types and between individual tumours. No conflict of interest. 362 Modelling the tumour microenvironment and employing functional genomics identifies CREBBP as a novel tumour suppressor in triple negative breast cancers B. Peck1 , S. Maguire1 , E. Morrison1 , P. Wai1 , R. Natrajan1 . 1 Institute of Cancer Research, London, United Kingdom Recent next generation sequencing studies have comprehensively mapped the genetic landscape of breast cancer and revealed that only a small number of genes are recurrently mutated in more than 10% of unselected tumours (i.e. TP53, PIK3CA and GATA3), and that the vast majority of recurrent mutations occur at low frequencies. Although some have been shown to be drivers, i.e. confer a selective advantage, there are a myriad of significantly altered lower frequency mutations whose functional impact is unknown. We utilized a functional genomics approach silencing the 200 most frequently mutated genes in breast cancer in 3 dimensional (3D) spheroid cultures, using the MCF10a progression series cell line panel to identify novel loss-of-function mutations that affect breast cancer progression from non-malignant to highly invasive disease. Using cancer cell spheroids to more accurately recapitulate in vivo-like conditions, we identified 11 genes whose silencing with siRNA impacted growth in two or more cell lines in 3D. Furthermore, a second targeted validation screen showed that silencing of a cohort of these genes had limited

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effect under traditional 2D culture conditions. The silencing of the histone acetyltransferase CREBBP promoted growth in AT1, DCIS.com and CA1H cells in 3D while having no effect in 2D cultures. Investigation of The Cancer Genome Atlas (TCGA) showed that CREBBP was more frequently mutated in triple negative breast cancers (TNBCs). Moreover, at least a third of TNBCs also displayed haploinsufficient loss of CREBBP and this loss resulted in the upregulation of the pro-proliferative transcription factor FOXM1. Multivariate analysis of CREBBP expression showed that CREBBP loss was significantly associated with decreased relapse-free survival. In summary, using a functional genomics approach in 3D models we found that CREBBP silencing promotes growth in tumour spheroids. Furthermore, CREBBP activity is altered in a significant proportion of TNBCs and associated with decreased relapse survival. No conflict of interest.


Cell and Tumour Biology II 363 Mimicking disease progression features by modulation of the tumour microenvironment in stirred-tank culture systems M.F. Estrada1,2 , S.P. Rebelo1,2 , V.E. Santo1,2 , E.J. Davies3,4 , S. Abreu1,2 , M.T. Pinto5 , W. Sommergruber6 , P.M. Alves1,2 , E. Anderson6 , C. Brito1,2 . 1 ´ IBET- Instituto de Biologia Experimental e Tecnologica, Animal Cell Technology Unit, Oeiras, Portugal, 2 ITQB-UNL, Oeiras, Portugal, 3 Bioscience Oncology iMed AstraZeneca, R&D Oncology iMed, Cambridge, United Kingdom, 4 European Cancer Stem Cell Institute, Cardiff, United Kingdom, 5 Institute of Molecular Pathology and Immunology- University of Porto IPATIMUP, In vivo CAM Assays Unit, Porto, Portugal, 6 Boehringer Ingelheim RCV, Oncology, Wien, Austria Background: Tumour microenvironment is composed of a network of fibroblasts, endothelial and immune cells embedded in the extracellular matrix under specific physicochemical conditions (e.g., hypoxia and acidic pH). Together, these components influence tumour progression and drug resistance through tumour–stroma crosstalk. Numerous 3D tumour cell models have been established in recent years aiming to reflect the high complexity of the tumour microenvironment and to mimic tumour progression. However, most of these models which are generated in culture systems that do not allow long-term culture, continuous monitoring and often use bioactive scaffolds. Materials and Methods: Our strategy is based on the combination of stirred-tank culture systems and alginate microencapsulation of tumour aggregates alone (mono-culture) or together with stromal cells (fibroblasts and monocytes − co-culture). Breast and lung tumour cell aggregates were cultured for up to 20 days under specific physico-chemical conditions. Results and Discussion: Microencapsulation of tumor spheroids with fibroblasts and/or monocytes allowed the establishment of distinct epithelial and stromal compartments. MCF7, an oestrogen Receptor (ER)+ breast tumour cell line, established cell-cell contacts and polarised around small lumina in the interior of the aggregates, recapitulating the in vivo tissue organization. Over the culture period, fibroblasts secreted collagen into the stromal compartment and the presence of both fibroblasts and monocytes resulted in a pro-inflammatory environment. This was accompanied by a reduction of ER and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential only in co-cultures. These phenotypic alterations are typical of advanced stages of cancer. In contrast, the effects of fibroblasts were not as significant in NSCLC using H1650, H1437 and H157 suggesting that the effect of tumor–stroma cross-talk is cell line dependent. Moreover, treatment with fulvestrant, an ER antagonist, reduced cell concentration in mono-cultures but not in co-cultures although the number of proliferating cells was reduced in both cultures. Conclusions: In summary, we have developed a robust model system for long-term in vitro recapitulation of tumour–stroma crosstalk and monitoring of disease progression, applicable to several pathologies. This constitutes a new tool to study tumour–stroma crosstalk, disease progression and drug resistance mechanisms in vitro. Acknowledgements: We acknowledge Dr. Cathrin Brisken, Dr. Heiko van der Kuip and Dr. Moshe Oren for the supply of the cell lines. This research received support from the Innovative Medicines Initiative Joint Undertaking (grant agreement nº 115188), FCT (iNOVA4Health—UID/Multi/04462/2013). MFE is recipient of a PhD fellowship from FCT SFRH/BD/52208/2013. No conflict of interest.

364 Three-dimensional ECM-based cell culture models for cancer research K. Storch1 , E. Dickreuter1 , A. Vehlow2 , N. Cordes1 . 1 Oncoray − National Center for Radiation Research in Oncology TU Dresden, Medical Faculty Carl Gustav Carus, Dresden, Germany, 2 Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden − Rossendorf, Dresden, Germany Introduction: The need for three-dimensional (3D) cell culture models optimal reflecting in-vivo growth conditions is great and demanding. Taking into account that also 3D cell cultures will always be models, it would nonetheless considerable assist in identifying novel potential cancer targets, characterizing better schedules for conventional radiochemotherapy and understanding the prosurvival and resistance-mediating mechanisms of malignant tumors. Despite the fact that a large number of 3D cell cultures has emerged during the last decade, industry and researchers have missed to pay attention to validation. Validation meant as thorough characterization of cellular and noncellular features clearly demonstrating that the particular 3D cell culture of interest mimics fundamental and cancer-relevant properties. Here, we present a multifaceted characterization of a laminin-rich ECM (lrECM) based cell culture system for tumors of epithelial origin (carcinomas) with regards to e.g. cell morphology, transcriptome, signal transduction and DNA repair. Material and Methods: Human carcinoma cell lines of different origin (head and neck squamous cell carcinoma, lung carcinoma, colorectal and pancreatic carcinoma) were embedded into 3D lrECM (Matrigel). The cell morphology, clonogenic survival, number of residual DNA double strand breaks (DSB), expression and phosphorylation of a large panel of proteins and mRNA expression were analyzed and compared with corresponding tumor xenografts. Results: Our data demonstrate great similarities in cellular behavior and measured endpoints in carcinoma cells grown in a 3D lrECM and tumor xenografts. We show similar cell morphology, similar distribution of radiationinduced DNA DSB and comparable protein expression and phosphorylation levels in 3D grown cell cultures and xenografts. Conclusions: Based on its great similarity to in-vivo, our 3D lrECM cell culture model can easily and robustly be used for mechanistic studies, drug/radiation testing and identification of novel cancer targets. No conflict of interest. 365 The bone microenvironment as a master regulator of tumour cell dormancy in breast cancer − evidence from novel in vivo models P. Ottewell1 , H. Brown1 , G. Allocca1 , M.T. Haider1 , N. Brown1 , I. Holen1 . University of Sheffield, Oncology and Metabolism, Sheffield, United Kingdom 1

Introduction: Breast cancer bone metastases originate from dormant tumour cells residing in bone marrow niches that are insensitive to anti-proliferative drugs. Increased understanding of the mechanisms that regulate tumour cell dormancy and subsequent escape is required to develop novel therapies that target these key processes underpinning metastatic disease. We have developed the first in vivo models of tumour cell dormancy in bone and used these to investigate how escape from dormancy can be inhibited through therapeutic targeting of the bone microenvironment. Material and Methods: Luc-2+ve MDA-MB-231 breast cancer cells were labelled with lipophilic dyes (DID, DiL) allowing detection of non-proliferating (dormant) tumour cells in bone. Labelled cells were injected i.c. in mature (12-week old+) BALBc/nude mice and tumour growth tracked by in vivo imaging (IVIS). Animals with dormant cells in bone underwent ovariectomy (OVX)/sham or were exposed to low calcium diet, altering both bone turnover and microvascular structure of the metastatic niche. Therapeutic effects were investigated using bone-targeted agents (100ug/kg/week zoledronic acid (ZOL), 25 mg/kg/week Fc-OPG). Location and number of tumour cells in the long bones were quantified using 2-photon microscopy, effects on bone volume and structure by uCT and bone histomorphometry. Bone microvasculature identified using endomucin staining ex vivo was assessed using confocal microscopy. Results and Discussion: Tumour cells primarily located in highly vascularised trabecular areas of the metaphysis, in close proximity to osteoblasts and vessels. Injection of differentially labelled tumour cell populations in the same animal on separate days showed that tumour cells compete for access to a limited number of bone marrow niches. Individual tumour cells were present in the long bones of animals without evidence of overt tumours up to 6 weeks after tumour cell injection, demonstrating that our models are suitable for studies of dormant tumour cells. OVX triggered tumour growth in bone in 89% of animals compared with 11% in the sham group (P < 0.001). ZOL and FcOPG caused significant (p < 0.01) inhibition of OVX-induced tumour growth, indicating that escape from dormancy involves an osteoclastdriven mechanism. Bone microvasculature was also altered by OVX/ZOL reflecting the changes in bone structure and therefore potentially contributing to regulation of dormancy. Conclusion: Using novel models of tumour cell dormancy we provide evidence that modification of the bone microenvironment triggers tumour cell escape

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 from dormancy and progression in bone. We demonstrate that inhibition of bone and vascular remodelling post OVX reduces the development of bone metastasis in vivo, supporting the premise that targeting the bone microenvironment has potential to prevent breast cancer recurrence. No conflict of interest. 366 PI3KC2a, a new spindle associated protein involved in genomic instability and tumorigenesis M.C. De Santis1 , M. Martini1 , G. Federico1 , A. Ghigo1 , C.C. Campa1 , R. Chiarle2 , A. Sapino3 , E. Hirsch1 . 1 MBC − Molecular Biotechnology Center, Dipartimento di Biotecnologie Molecolari e Scienze per la Salute, Torino, Italy, 2 Harvard Medical School- Boston, Department of Pathology, USA, USA, 3 University of Turin, Department of Medical Sciences, Torino, Italy Background: PI3K signaling axis is one of the most frequently deregulated pathways in human cancer. Emerging evidences highlight the importance of class II enzymes in cell proliferation and survival. Material and Methods: To evaluate the oncogenic role of PI3KC2a in cancer, we targeted its expression in a breast cancer mouse model (neuT). Results: Heterozygous loss of PI3KC2a resulted in an initially delayed tumor onset followed by a faster growth rate in Pik3c2a+/-/neuT mice. In agreement with the delayed onset, Pik3c2a+/-/neuT E-MMETs (primary tumor cells derived from early stage of tumor development) displayed a reduced proliferative capacity and a delay in the progress from prophase to anaphase, accompanied by increased apoptosis. We found that Pik3c2a loss causes an aberrant microtubule (MT) spindle organization that, in turn, promotes genomic instability. In line with this, PI3KC2a is specifically enriched at the metaphase spindle and stabilize K-fibers during mitosis, acting as a scaffold protein for clathrin and transforming acidic coiled-coil protein 3 (TACC3). Truncated constructs of PI3KC2a were generated to identify the region of interaction with TACC3. Despite the aberrant MT organization, we demonstrate that tumors bypass the requirement of PI3KC2a through a common mechanism of progression. Multiple genes involved in the spindle-associated checkpoint, resulted alterated in fast growing (hare) compared to slow growing (turtles) tumors. The ability of tumors with low PI3KC2a to grow faster appeared to correlate with increased sensitivity to anti-microtubule agents like Paclitaxel. Conclusion: Taken together, these data demonstrate that PI3KC2a is a new spindle associated protein interacting with TACC3 and clathrin to stabilize MTs. The loss of PI3KC2a plays a crucial role in promoting genomic instability, altering chromosome congression/segregation during cell division. These findings will eventually validate PI3KC2a as a new prognostic tool thus allowing the development of a therapeutic option for breast cancer patients. No conflict of interest. 367 Angiogenic markers in hepatocellular carcinoma R. Albulescu1 , L.G. Necula1 , A.I. Neagu1 , V. Herlea1 , S.O. Dima2 , C. Tanase1 , I. Popescu2 . 1 Titu Maiorescu University- Bucharest- Romania, Medical Research Inst. “Nicolae Cajal”, Bucharest, Romania, 2 Fundeni Clinical Institute, Center of General Surgery and Liver Transplantation, Bucharest, Romania Introduction: Hepatocellular carcinoma (HCC), the most common primary malignancy of the liver is one of the most prevalent and lethal cancers. Neoangiogenesis contributes to metastasis and poor prognosis. Many studies investigated the relationship between VEGF and HCC development and progression. VEGF is up-regulated in most human tumors; in human specimens, increased expression of VEGF correlates with aggressive behavior of the tumors and poor prognosis. Several studies evaluated the serum level of VEGF and another angiogenic factors as potential prognostic factors for survival in patients with HCC. Materials and Methods: Tumoral and peritumoral samples from patients with HCC, selected based on prior identification of increased level of circulating angiogenic markers. All patients had recurrence at the time of survey. Immunohistochemistry was performed to investigate the expression of VEGF and CD34 proteins in HCC tissues on FFPE tissue samples. Dot-blot: 100 mg tissue was used for protein extraction and 400 mg of protein were incubated with the pre-coated Proteome Profiler™ Array − Human Angiogenesis Array Kit. Analysis of dot blot was performed with ImageJ 1.42 software. Results and Discussion: VEGF-positive expression was found in 70% of HCC patients. HCC tissues also showed positive staining for CD34. The results obtained from dot-blotting showed significant expression changes in tumor tissue compared to peritumoral tissue, for several proteins involved in angiogenesis. Among the proteins with increased expression in distinguishes basic FGF (basic fibroblast growth factor), IL-8 and VEGF − 4 times increased in tumor vs. peritumoral tissue. HCC is characterized by clinical heterogeneity and lack of efficient diagnostic markers and treatment strategies. Neovascularization represents a key step in the molecular mechanisms of HCC relapse and metastasis. VEGF and bFGF are involved in endothelial proliferation and migration, supporting angiogenesis and HCC progression/metastasis. Together with interleukin IL-8, VEGF and bFGF were

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associated with the transition from liver cirrhosis into HCC. VEGF is expressed in cirrhosis and during progression to malignancy, highlighting its major role in HCC development. Our study highlighted an increased expression of several angiogenic factors, such as bFGF, VEGF and IL-8, in tumoral tissue compared to peritumoral tissue, in HCC patients. Further studies are needed to validate these results. Conclusion: The results obtained by IHC and dot blot techniques highlighted the activation of several angiogenic factors, including VEGF, bFGF and IL-8 in HCC tumor tissues and these molecules may be potential targets for developing new therapeutical strategies in HCC through their effects on angiogenesis. Acknowledgement: The research presented in this paper was supported by EEA-JRP-Romania-Norway nr. 4SEE/2014. No conflict of interest. 369 New data on molecular phenotype of ascitic tumor cells in ovarian cancer patients T. Bogush1 , S. Kaliuzhny1 , E. Dudko1 , O. Rjabinina1 , N. Vichljantzeva1 , A. Tjulandina2 , E. Bogush3 , S. Tjulandin2 , M. Davydov4 , I. Mamichev1 . 1 N.N. Blokhin Russian Cancer Research Center, Laboratory of medical chemistry, Moscow, Russian Federation, 2 N.N. Blokhin Russian Cancer Research Center, Department of clinical pharmacology and chemotherapy, Moscow, Russian Federation, 3 N.N. Blokhin Russian Cancer Research Center, Department of surgery, Moscow, Russian Federation, 4 N.N. Blokhin Russian Cancer Research Center, Director of N.N. Blokhin Russian Cancer Research Center- Thoracic and abdominal surgery department, Moscow, Russian Federation Introduction: Recurrent stages III and IV of ovarian cancer with peritoneal dissemination and ascitis is the disease phase characterized by resistance to the most chemotherapy regimens used in the solid tumors. Authors supposed that one of the reasons of that are specific changes in the molecular phenotype of recurrent ascitic tumor cells which require chemotherapy different from the solid ovarian cancer treatment. To validate this hypothesis comparative study of some molecular markers was performed in solid and ascitic ovarian cancer cells. Material and Methods: Surgical specimens of serous ovarian adenocarcinoma and ascitis (14 samples of III stage of the disease) were analyzed by flow cytometry. Single-cell suspensions were incubated for 1.5 h with primary antiCD45 (HI30, Biolegend), anti-cytokeratin (AE1/AE3, DAKO) and anti-vimentin (SP20, BIOCARE) antibodies and then − for 1.5 h with anti-rabbit and antimouse secondary antibodies, conjugated with DyLight650 (ab98510) and DyLight488 (ab98637) respectively. Plots and quadrant regions were made up in WinMDI 2.9 software for double stained cells allocation. Results and Discussion: 1. It was established that ascitic cells presented common leucocytic marker CD45 and mesenchymal marker vimentin additionally to epithelial marker cytokeratin. The cells with “cytokeratin+/ CD45+/vimentin+” phenotype accounted for the most of ascitic tumor cells. 2. Cytokeratin positive cells in solid tumor specimens had no CD45 marker and expressed vimentin in some tumors only in 10−20% of the cells. 3. Hence, besides inhibition of anoikis (the specific form of epithelial cell death induced in fluid by the lack of contacts with matrix) reccurent ascitic ovarian cancer cells are characterized by (1) emperipolesis (the intracellular migration of leucocytes without damage of cancer cells and CD45 expression) and (2) phenotype of epithelial-to-mesenchymal transition with expression of vimentin. Conclusion: For the first time it was established that solid and recurrent ascitic ovarian cancer have clinically significant molecular differences, namely simultaneous expression of cytokeratin and non-epithelial markers CD45 and vimentin in the ascitic tumor cells. This observation confirms authors’ notions about needs for principally new approaches to therapy of recurrent ascitic form of the disease. Acknowledgement: Supported by RFBR grants (15-04-06991-a, 16−34-01049mol-a) and President of Russian Federation grant MK-7709.2016.7. No conflict of interest. 370 NEAT1, a long non-coding RNA, controls cell survival and is up-regulated in breast cancer Z.A. Almnaseer1 , M. Mourtada-Maarabouni1 . 1 Keele University, School of Life Sciences, Newcastle-Under-Lyme, United Kingdom Background: Nuclear Enriched Abundant Transcript 1 (NEAT1) is a nuclear long-non coding RNA transcribed from the familial tumour syndrome multiple endocrine neoplasia (MEN) type 1 locus on chromosome 11. NEAT1 is reported to be overexpressed in prostate cancer and a direct transcriptional target of hypoxia-inducible factor in breast cancer cells. The aims of this study were to determine: i) the effects of silencing NEAT1 on breast cancer cell survival, ii) the effects of NEAT1 silencing on the expression of two genes located on the same chromosome, iii) the levels of NEAT1 in breast cancer samples.

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Materials and Methods: MCF7 and MDA-MB 231 cells were transfected with NEAT1 antisense oligonucleotides (ASO), controls received scrambled oligonucleotide. In some experiments, cells were exposed to ultraviolet-C (UV-C) light post-transfection to induce apoptosis, and then culture viability and apoptosis were assessed. Commercial Breast Cancer cDNA Arrays were used to evaluate the levels of NEAT1 in breast cancer samples. NEAT1 expression was evaluated by qRT-PCR TaqMan® analysis, using relative standard curve method. Results: In MCF7 and MDA-MB-231 cells, siRNA-mediated silencing of NEAT1 reduced basal survival. NEAT1 silencing enhanced UV-induced cell death and this response was associated with a significant increase in the expression levels of Estrogen-Related Receptor Alpha (ESRRA) and BAD (BCL2-Associated Agonist of Cell Death). NEAT1 levels were found to be significantly increased in breast cancer samples. Conclusion: Overall, the results suggest that NEAT1 regulates cell survival and the expression of neighbouring genes in ER+ and TNBC cells. The substantial increase in NEAT1 expression levels in breast cancer tissues suggests that NEAT1 may function as an oncogene. No conflict of interest. 371 A novel role for p21-activated kinase 4 (PAK4) in endocrine resistance and disease progression 1 A. Santiago-Gomez ´ , I. Dragoni2 , R. NicAmhlaoibh2 , E. Trivier2 , J.M. Gee3 , A.H. Sims4 , S.J. Howell5 , R.B. Clarke6 . 1 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom, 2 Cancer Research Technology, Cancer Research Technology, London, United Kingdom, 3 Cardiff University, Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff, United Kingdom, 4 University of Edinburgh Cancer Research Centre, Applied Bioinformatics of Cancer Group, Edinburgh, United Kingdom, 5 The Christie NHS Foundation Trust, Medical Oncology, Manchester, United Kingdom, 6 The University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom

Background: Endocrine therapies are beneficial but two thirds of ER+ breast cancer patients will have de novo or acquired resistance to the therapy. One plausible explanation underlying the failure of endocrine therapies is the existence of a small cell subpopulation within the tumour known as Cancer Stem-like Cells (CSC), which we reported to accumulate after anti-estrogen therapy (Simoes et al, Cell Rep, 2015). p21-activated kinases (PAK) are a conserved family of serine/threonine kinases located at the intersection of several signalling pathways that are commonly deregulated in human cancers. Hence, PAKs are key regulators of several cellular processes such as survival, growth and estrogen signalling, among others. Material and Methods: ER+ models for acquired resistance to endocrine therapies and ER+ breast cancer metastatic patient-derived samples (pleural effusions or ascites) were used 1) to detect PAK1/4 expression; and 2) to test the effect of PAK1/4 inhibition on breast CSC activity using the mammosphereforming assay. Results and Discussion: We found that both PAK1 and 4 are overexpressed in Tamoxifen- and Fulvestrant-resistant, but not Estrogen-deprived MCF7 cells. PAK1/4 inhibition using a novel and specific compound abrogates CSC self-renewal in endocrine-resistant cells by the mammosphere-forming assay (>95% in TAMR and 80% in FULVR vs.vehicle). Similarly, PAK4 silencing using siRNA also impairs breast CSC activity in Tamoxifen-resistant cells, confirming that PAK4 is essential for maintaining CSC features. PAK1/4 expression in metastatic-patient derived samples (n = 18) is unrelated to breast cancer subtype, and there is a significant correlation of PAK4 mRNA expression to CSC activity (r = 0.756, p = 0.00714). Kaplan–Meier analysis using published microarray datasets of pre-treatment tumours from ER+ patients who subsequently received adjuvant Tamoxifen (n = 669) showed that elevated PAK4 gene expression was significantly associated with distant metastasis (cut-point p < 0.05). Furthermore, high PAK4 levels were associated with reduced overall survival in an independent cohort of untreated ER+ breast cancer patients (n = 343, p < 0.05), confirming that PAK4 predicts for resistance to Tamoxifen and poor prognosis in ER+ tumours. Examination of sequential samples of 2 ER+ metastatic patients taken at different time points during treatment showed that both PAK1/4 levels and CSC activity increase alongside with disease progression. Targeting of PAK1/4 using a specific inhibitory compound in ER+ metastatic primary samples (n = 6) impaired breast CSC activity up to 50%. Conclusion: PAK4 is as potential indicator of poor prognosis in ER+ breast cancer patients, is responsible for the acquisition of resistance to endocrine therapy and represents a possible target molecule whose inhibition could overcome resistance to anti-estrogen therapies. No conflict of interest.

372 Neuropilin 2 regulates lung disseminated tumor cells escape from dormancy and progression into metastasis P. Bragado1 , R. Alonso1 , M. Mancino1 , P. Fernandez-Nogueira1 , G. Fuster1 , P. Gascon1 . 1 Institut d’Investigacions Biomediques August Pi i Sunyer IDIBAPS, Oncology, Barcelona, Spain Background: Despite considerable advances in the treatment of cancer, about 50% of patients will still eventually develop metastatic disease which remains the main cause of cancer-related death. Disseminated tumour cells (DTCs) are the precursors of metastasis and understanding their biology is one of the most important challenges for the future of cancer research. Our previous studies have shown that TGFb2 through binding to TGFbR3 regulates DTCs fate through the induction of p38aMAPK and SMAD1/5. However, these DTCs dormancy may be reversed when micro-environmental conditions shift to support DTCs/micro-metastasis expansion. Nerve fibers and neuroendocrine mediators are present in organs that serve as key targets for breast and head and neck cancer metastasis, including the lymph nodes, lung, and bone. Our laboratory and others have shown that the nervous system is functionally relevant for tumour progression. We hypothesize that these neural mediators create a favourable microenvironment and regulate DTCs escape from dormancy and metastasis formation. Materials and Methods: We have use bioinformatics tools and patient databases to identify neural genes differentially expressed among breast cancer subtypes. We have used different Head and neck (HNSSC) and breast cancer (BCC) models including HEp3 (HNSCC) and MDAMB453 (BCC). We have also lung DTCs derived cell lines from both models. In addition from the HNSCC model we also have a dormant cell line derived from bone marrow DTCs (BM-D). For in vivo studies, we will use spontaneous metastasis models from orthotopic human xenografts in nude mice. In addition, we will also use the chorioallantoic membrane (CAM) chicken embryo system for rapid screening of in vivo phenotypes. Results: Using bioinformatics tools we have found that the semaphorins receptor Neuropilin 2 (NRP2), a co-receptor for TGFb ligands, is overexpressed in basal and HER2+ breast cancer patient samples and its expression in primary tumors correlate with worst prognosis. In basal breast cancer cell lines, NRP2 expression correlates with lower levels of DEC2 and p27 dormancy genes. Furthermore, we have found that NRP2 is downregulated in dormant cells and overexpressed in proliferative lung DTCs derived cell lines that express low levels of P-p38, DEC2 and p27 dormancy markers. Our results show that in lung DTCs derived cell lines, NRP2 inhibits TGFbR3 expression favoring TGFb1 signaling and promoting DTCs growth. In agreement with this, breast cancer lung metastasis express higher levels of NRP2 than quiescence lung DTCs in vivo, suggesting that NRP2 might play a role in lung DTCs proliferation. Conclusion: Therefore, we conclude that NRP2 can regulate breast cancer lung DTCs progression from a dormant state to a proliferative state and promote metastasis formation. No conflict of interest.

373 Targeting mismatch repair deficiency in endometrial cancer R. Begum1 , S. Martin1 . 1 Barts Cancer Institute, Molecular Oncology, London, United Kingdom Introduction: Endometrial cancer is the fourth most common cancer in the UK among women and deficiency of the DNA Mismatch repair (MMR) pathway is present in 30% of cases. This pathway is accountable for the repair of base-base mismatches and insertion/deletions loops that occur during DNA replication. MMR-deficient tumours are resistant to standard chemotherapeutic agents used in endometrial cancer, including carboplatin. Recently, the concept of synthetic lethality has been exploited as a means of identifying novel therapeutic agents in cancer. Materials and Methods: To this end, we have performed a synthetic lethal compound screen with a library of 1120 FDA-approved compounds, in a panel of MMR deficient and proficient endometrial cancer cells. The screen identified a number of ‘hits compounds’ which selectively kill the MMR deficient cells, and not the MMR proficient cells. The drug ‘hits’ were validated by generating dose-response curves with increasing concentrations of the drugs, comparing cell viability in the MMR proficient and deficient cells. Results and Discussion: Hit compounds from the drug screen were validated in a panel of endometrial cancer cell lines and the drug, pentamidine isethionate showed selective killing of the MMR deficient cell lines. However, the drug did not show selectivity in MMR-isogenic paired cell lines where the MMR gene was re-expressed in the cells. This suggested that the synthetically lethality was due to secondary mutations commonly found in MMR deficient cells. Immunoblotting of proteins commonly lost in MMR deficient cells showed reduction or total loss of ATM/phosphorylated-ATM in MMR deficient cells, which suggested a defect in the DNA damage response in these cells. Further investigation showed that upon treatment with pentamidine, there is a dramatic increase of yH2AX foci in MMR deficient cells, a marker of double strand

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 breaks. This indicated that defects in the DNA damage response in MMR deficient cells, induce sensitivity to pentamidine treatment. Conclusion: We have identified a drug, pentamidine isethionate, that is selective in MMR deficient endometrial cancer cells. Our analysis suggests that this selectivity may be due to common secondary mutations involved in the DNA damage response found in MMR deficient cells. Pentamidine isethionate may potentially be a novel therapeutic for the treatment of MMR deficient endometrial cancer. No conflict of interest. 374 Apobec3B expression in drug resistant MCF-7 breast cancer cell lines O. Onguru1 , S. Yalcin2 , C. Rosemblit3 , P. Zhang4 , S. Kilic5 , U. Gunduz6 . 1 Gulhane Military Medical Academy, Department of Pathology, Ankara, Turkey, 2 Ahi Evran University, Department of Food Engineering, Kırsehir, Turkey, 3 University of Pennsylvania Perelman School of Medicine, Department of Surgery, Philadelphia, PA, USA, 4 University of Pennsylvania Medical Center, Department of Pathology and Laboratory Medicine, Philadelphia, PA, USA, 5 Gulhane Military Medical Academy, Department of Public Health, Ankara, Turkey, 6 Middle East Technical University, Department of Biology, Ankara, Turkey Introduction: Apobec3B belongs to a protein family of cytidine deaminases that can insert mutations in DNA and RNA as a result of their ability to deaminate cytidine to uridine. It has been shown that Apobec3B-catalysed deamination provides a chronic source of DNA damage in breast cancers. We investigated Apobec3B expression in doxorubicin (MCF-7/Dox), etoposide (MCF-7/Eto), paclitaxel (MCF-7/Pac) and docetaxel resistant (MCF-7/Doc) MCF-7 breast cancer cell lines and tried to reveal its relation with drug resistance. Material and Method: Drug resistant sublines to doxorubicin, etoposide, paclitaxel and docetaxel, that were developed from sensitive MCF-7 cells (MCF-7/S) were used. We used immunocytochemistry, western blot (WB) analysis and RNA in situ hybridization technology (RNAscope ® ) for Apobec3B expression and compared its expression levels in drug sensitive and resistant MCF-7 cell lines. After RNAscope staining, slides were scanned and saved as digital images using scanner (Aperio ® ) and software. Quantitative scoring utilizing the number of punctate dots present within each cell boundary was performed. The parameters including positive cell percentage and signal intensity per positive cell were recorded and analyzed. BT-474 and MDAMB231 breast cancer cell lines, which were reported to have higher RNA expression for Apobec3B in a previous study used as a positive control for immunocytochemistry. Results and Discussion: In doxorubicin and etoposide resistant MCF-7 cell lines, APOBEC3B expression was approximately five-fold increased (23% and 24% respectively) with higher signal intensity (1.92 and 1.44 signal/ cell, respectively) compared to MCF-7/S (5%, 1.00 signal/ cell) with statistical significance (p < 0.001 for MCF-7/S vs MCF-7/Eto and p = 0.002 for MCF-7/S vs MCF-7/Dox). The increase of APOBEC3B expression in Docataxel resitant (7%, 1.05) and Paclitaxel resistant MCF-7 cell lines (8%, 1.05) was minimal. In immunocytochemistry, almost all of cells in MCF-7, BT-474 and MDA-MB231 cell lines showed cytoplasmic positivity with strong dot like staining in rare cells. In the evaluation of WB analysis, a single band for MCF7, two bands for SK-BR-3 and BT474 were observed. Conclusion: APOBEC3B RNA expression was increased in some population of tumor cells of drug resistant cell lines. APOBEC3B may be related to drug resistance in breast cancer for some drugs including Doxorubicin and Etoposide, subjecting some tumor cells to frequent mutation. No conflict of interest. 375 Sequential treatment of HCC cells with PI3K/Akt/mTOR pathway inhibitors prior to sorafenib attenuates cancer stem cell population D.C. Kahraman1 , T. Kahraman1 , R.C. Atalay2 . 1 Bilkent University, Molecular Biology and Genetics, Ankara, Turkey, 2 METU, Informatics Institute, Ankara, Turkey Introduction: Cancer stem cells are known to be responsible for drug resistance, metastasis and tumor relapse. Liver cancer stem cells are derived from damaged and transformed hepatic progenitor cells (HPC) during precancerous cirrhosis stage. Therefore, identifying novel inhibitors against CSCs in liver cancer is essential. It is well known that PI3K/Akt/mTOR pathway has an active role in hepatocellular carcinoma (HCC), and inhibitors of this pathway are promising chemotherapeutic agents in HCC. Our current aim is to study the bioactivities of small molecule kinase inhibitors against liver CSCs (LCSCs) acting through PI3K/Akt/mTOR pathway in comparison with controls such as Notch pathway inhibitors and DNA intercalators and HCC specific inhibitor sorafenib used in clinics. Material and Method: Huh7 and Hep3B cells were treated with the selected inhibitors grouped according to their mode of action: ZSTK474, PI-103, PI3Ki-a,

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PI3Ki-b, AKTi-2, AKTi-1,2, LY294002, Wortmannin, Rapamycin (Rapamune® ), NVP-BEZ235 (Dactolisib® ), Campthotecin, Doxorubicin (Adriamycin® ), DAPT and sorafenib (Nexavar® ) for 72 hours. Cells that could survive the drug treatment were collected and stained with liver cancer stem cell markers (CD133 and EpCAM), and then analyzed by flow cytometry. Small molecule inhibitors that were able to attenuate CD133+/EpCAM+ cell population were tested for their potential additive or synergistic effects in vitro on Huh7 and Hep3B cells and in vivo using hollow fibers in nude mice. Results and Discussion: In parallel with the literature, treatment of HCC cells with sorafenib, and DNA intercalators resulted in enrichment of CD133+/EpCAM+ cells. PI3K/Akt pathway inhibitors lead either to the enrichment of CD133+/EpCAM+ cells (ZSTK474, NVP-BEZ235) or did not have any effect (Akti-1,2, Akti-2, PI3Ki-a, PI3Ki-b) on subpopulations. However, mTOR inhibitor-Rapamycin, PI3K inhibitor-LY294002 and Notch inhibitor-DAPT significantly reduced the CD133+/EpCAM+ double positive cell population. Furthermore, combination studies revealed that treatment of cells with these inhibitors before, but not after, sorafenib treatment decreased the double positive side-population as opposed to sorafenib treatment alone. Conclusion: Our findings indicated that PI3K/Akt/mTOR pathway inhibitors may alter tumor composition in favor of either cancer stem cells (CD133+/EpCAM+) or non-stem cancer cells (CD133-/EpCAM-). Therefore, we claim that combinatorial chemotherapy in cancer may not always be in favor of the patient survival due to the side-population enrichment. Combined chemotherapy methods must be analyzed in detail and revised accordingly, since our data showed that the composition and the timing of the combined treatment with sorafenib can be in favor of the patient survival as well as a risk for tumor relapse. No conflict of interest. 376 Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumours 1 ´ , B.M. Simoes ˜ 1 , A. Denis1 , R. Eyre1 , K. Spence1 , A. Santiago-Gomez A. Sarmiento-Castro1 , I. Tanaka1 , D. Howat2 , S. Howell1 , R. Clarke1 . 1 Institute of Cancer Sciences- The University of Manchester, Breast Biology, Manchester, United Kingdom, 2 Evgen Pharma, Liverpool Science Park, Liverpool, United Kingdom

Background: Sulforadex (SFX) is a novel therapeutic comprising synthetic sulforaphane (SFN) stabilised within a-cyclodextrin. SFN has been studied as an anti-cancer compound for many years however its development has hitherto been hampered due to its inherent instability. Breast cancer stem-like cells (CSCs) have been identified in all molecular subtypes and are believed to be the drivers of breast cancer metastasis. The majority of breast cancers express the estrogen receptor (ER) but these hormone-responsive breast cancers frequently develop resistance to hormonal therapies (e.g. tamoxifen). We have established that CSCs in ER+ breast cancer lack ER expression, representing a potential mechanism of therapeutic resistance (Simoes et al, Cell Reports, 2015). Material and Methods: We investigated SFX effects on breast CSC activity using mammosphere formation and aldehyde dehydrogenase (ALDH) activity assays in patient samples and patient-derived xenograft (PDX) tumours. Cells from primary (n = 12) and metastatic (n = 15) samples were treated with SFX (5 mM) or vehicle control. Using a 14 day in vivo ‘window’ treatment, early (HBCx34) and metastatic (BB3RC31) ER+ PDX tumours were treated with SFX (300 mg/kg/day) alone or in combination with tamoxifen (10 mg/kg/day). Results and Discussion: SFX induced a reduction greater than 50% in the mammosphere formation efficiency (MFE) of both primary (Control: 0.52%±0.06 vs treated: 0.19±0.02, p < 0.001) and metastatic (Control: 0.93%±0.07 vs treated: 0.43±0.04, p < 0.001) patient cells. Importantly, SFX also abrogated breast CSC activity of HBCx34 and BB3RC31 PDX tumours. We observed that SFX decreases ALDH+ cells (HBCx34 Control: 6.3%±0.4 vs treated: 3%±0.6, p < 0.01; BB3RC31 Control: 3%±0.6 vs treated: 1%±0.2, p < 0.05) and MFE (HBCx34 Control: 0.64%±0.09 vs treated: 0.35%±0.03, p < 0.01; BB3RC31 Control: 0.89%±0.06 vs treated: 0.78%±0.04). SFX targets surviving CSCs identified by ALDH positivity and mammosphere-initiating capacity following tamoxifen treatment in vivo in ER+ PDX tumours. Combination treatment of tamoxifen and SFX reduced ALDH+ cells (HBCx34 TAM: 10%±0.4 vs TAM+SFX: 4.2%±0.4, p < 0.01; BB3RC31 TAM: 11.3%±1.1 vs TAM+SFX: 6.9±0.6, p < 0.05) and MFE (HBCx34 TAM: 0.81%±0.07 vs TAM+SFX: 0.34%±0.02, p < 0.01; BB3RC31 TAM: 0.47%±0.06 vs TAM+SFX: 0.27±0.04, p < 0.05). In on-going PDX experiments, which comprise endocrine therapy in combination with SFX for a period of 8 weeks, we will test the effects of the combination on tumour regression. Mechanistically, SFX potently inhibited the canonical Wnt pathway in MCF-7 cells and their endocrine-resistant derivatives and we are currently exploring SFX activity on other CSC regulatory pathways. Conclusions: Our data demonstrate the potential of SFX for clinically meaningful improvements to endocrine therapy in ER+ breast cancer by reversing CSC mediated resistance. No conflict of interest.

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377 Myc-dependent cell cycle progression through the activation of CDK1 and phosphorylation of p27 L. Garcia-Gutierrez1 , G. Bretones1 , I. Arechaga2 , D. Santamaria3 , M. Barbacid3 , J. Leon1 . 1 Instituto de Biomedicina y Biotecnologia ˜ de Cantabria-CSIC-UC, Departamento de Senalizacion Molecular y Celular, Santander, Spain, 2 Instituto de Biomedicina y Biotecnologia de Cantabria-CSIC-UC, Departamento de Microbiologia y Genomica, Santander, Spain, 3 Centro Nacional de Investigaciones Oncologicas, Grupo de Oncologia Experimental, Madrid, Spain Background: An important feature that defines a cancer cell is the lost of the cell cycle regulation. Myc is known to be a potent stimulator of cell proliferation. The enforced Myc expression in quiescent cells is sufficient to mediate cell cycle entry and for this purpose, p27 levels need to be reduced within the cell. There is a correlation between high levels of Myc expression with low levels of p27 in many human tumors. The best characterized pathway leading to p27 downregulation involves phosphorylation at Thr187 of p27. pT187-p27 is the target for SCFSKP2-mediated ubiquitination and degradation. The only kinase known so far to mediate this phosphorylation is the CyclinE/CDK2 complex. We have previously shown that Myc induces the expression of SKP2 as well as the pT187p27. Material and Method: We used the Kp27MER cell line, a K562 derivative cell line carrying a ZnSO4-inducible p27 construct and the MycER protein which can be activated by tamoxifen, and four mouse embryonic fibroblast derived cell lines: CDK2−/−, Cyclin E−/−, TKO (CDK2−/−; CDK4−/−; CDK6−/−) and CDK1lox/lox. Myc overexpressing cell lines were generated by lentiviral transduction. RT-qPCR and western blot to study gene expression. In vitro kinase assays to study pT187-p27 and phosphorylation detected by western blot using phospho-specific antibodies. Results and Discussion: Activation of Myc in Kp27MER cells leads to an increase of pT187-p27 and Cyclin A. Besides, it increases the in vitro kinase activity of CDK1 and CDK2. CDK1 complexes from cells overexpressing p27 were able to phosphorylate p27 in vitro upon Myc activation, but CDK2 complexes were not. Cyclin B/CDK1 was reported to phosphorylate p27 in vitro, but the involvement of Cyclin A is unknown. As cyclin A is induced by Myc in our model, we asked for the role of Cyclin A/CDK1 in p27 phosphorylation. In vitro kinase assays with immunoprecipitated Cyclin A complexes showed the same p27 phosphorylation pattern as CDK1 complexes did. Myc overexpression increased proliferation rates of MEFs lacking CDK2 or cyclin E, and decreases p27 expression. CDK1 and Cyclin A complexes from CDK2−/− MEFs showed increased pT187p27 levels when Myc was overexpressed. Similarly, CDK1 and CyclinA complexes from Cyclin E−/− MEFs showed increased pT187-p27 when Myc was overexpressed. In TKO MEFs, overexpression of Myc leaded to increased CDK1 kinase activity. CDK1 complexes from TKO MEFs did not phosphorylate p27 in vitro while CDK1 complexes of TKO MEF-Myc cells induced it. Cyclin B complexes from CDK1lox/lox MEFs phosphorylated p27 in vitro and Myc increased it, while knocking out CDK1 totally abolished it. Conclusion: Myc promotes p27 degradation by (a) inducing the expression of SKP2 as we previulsy reported, and (b) inducing its phosphorylation at Thr187, which is mediated not only by CyclinE/CDK2, but also by CyclinA-B/CDK1. No conflict of interest. 378 The role of High-Mobility Group Box 1 protein in mesenchymal– epithelial signalling in the tumour microenvironment A. Evans1 , S. Sharma2 , E. Hemers3 . 1 Liverpool John Moores University, Pharmacy and Bimolecular Sciences, Liverpool, United Kingdom, 2 Liverpool John Moores University, Pharmacy & Biomolecular Sciences, Liverpool, United Kingdom, 3 Liverpool John Moores University, Natural Sciences, Liverpool, United Kingdom Introduction: Glucose depravation, hypoxia, and acidosis are characteristic features of the central core most solid tumours. Myofibroblasts are stromal cells present in many such solid tumours including those of the colon, and they are known to be involved in all stages of tumour progression. HMGB1 is a nuclear protein that plays an important role in nucleosome stabilisation and gene transcription. HMGB1 is also released from immune cells and is involved in the inflammatory process. This work reports a novel finding that the microenvironmental condition of glucose depravation is responsible for the active release of HMGB1 from different types of cancer cell lines (HT-29, MCF-7 and A549) under normoxic conditions. Materials and Method: Human colonic myofibroblasts cells CCD18COCo, and cancer cells, colon HT-29, lung A549 and breast MCF7 were obtained from the ATCC and bladder cancer cells EJ138 from the ECACC. The human recombinant HMGB1, anti-HMGB1 antibody, anti-RAGE primary antibody and anti-TLR4 antibody were purchased from R&D systems. MEK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) were purchased from Cell Signalling Technology (USA). Medium was collected from HT-29, MCF-7, EJ138 and A549 cells after 48 h treatment with various conditioned or fresh medium and analysed by western blotting for the presence of HMGB1. The MTT assay was

used measure proliferation of myofibroblasts after treatment with conditioned medium with or without an MEK1/2 inhibitor or a PI3K inhibitor. Migration of CCD18Co cells in response to medium previously conditioned on HT-29 cells for 20 h was studied using 8 mm pore Boyden chamber inserts in 24-well plate system (BD Bioscience). In addition, invasion assays of myofibroblast cells were performed using 8 mm pore matrigel matrix Biocoat® inserts according to the manufacturer’s instruction (Becton Dickinson). Results and Discussion: Recombinant HMGB1 (10 ng/ml) was shown to trigger proliferation in myofibroblasts cells via activation of PI3K and MEK1/2. Conditioned medium collected from glucose deprived HT-29 colon cancer cells was shown to stimulate migration and invasion of colonic myofibroblasts, and these processes were significantly inhibited by immunoneutralising antibodies to HMGB1, RAGE and TLR4, along with specific inhibitors of PI3K and MEK1/2. Conclusion: Together these data suggests that HMGB1 released from the cancer cells under glucose depravation is involved in stimulating colonic myofibroblast migration and invasion and that this was through activation of RAGE and TLR4, resulting in activation of the MAPK and PI3K signalling pathways. Thus, it is proposed that HMGB1 may be released by cancer cells in areas of low glucose in solid tumours with the resulting activation of myofibroblasts. Therefore, HMGB1 may be considered as potential therapeutic target to inhibit solid tumour growth. No conflict of interest. 379 Glioblastoma cancer stem cells: Adaptive or stable phenotype? A. Dirkse1 , A. Golebiewska1 , N.H.C. Brons2 , T. Buder3,4 , A. Deutsch3 , S. Leite5 , N. Sauvageot5 , S. Senn5 , C. Herold-Mende6 , S. Niclou1,7 . 1 Luxembourg Institute of Health LIH, Oncology- Norlux Neuro-Oncology Laboratory, Luxembourg, Luxembourg, 2 Luxembourg Institute of Health LIH, Core Facility Flow Cytometry, Luxembourg, Luxembourg, 3 ¨ Dresden, Zentrum fur Technische Universitat ¨ Informationsdienste und ¨ Technik und Hochleistungsrechnen ZIH, Dresden, Germany, 4 Hochschule fur ¨ Informatik / Mathematik, Dresden, Germany, Wirtschaft Dresden, Fakultat 5 Luxembourg Institute of Health LIH, Centre of Competence for Methodology and Statistics CCMS, Strassen, Luxembourg, 6 University of Heidelberg, Division of Experimental Neurosurgery- Department of Neurosurgery, Heidelberg, Germany, 7 University of Bergen, KG Jebsen Brain Tumour Research Center- Department of Biomedicine, Bergen, Norway Introduction: Glioblastoma (GBM) is a highly malignant brain tumor where no curative treatment is available. According to the cancer stem cell (CSC) hypothesis GBMs rely on a small subpopulation of cancer cells with stemlike properties responsible for tumor progression and recurrence. Recent experimental data from GBM and other cancers however suggest that CSCs cannot be defined by specific marker expression and may in fact not be a stable entity but a population of cells adapting to a changing microenvironment. We have previously shown that glioma CSCs do not represent a genetically homogenous population. Material and Method: Here we examined inter- and intra-tumoral heterogeneity based on stem cell-associated marker expression profiles. Tumor cell subpopulations were classified based on their expression of four chosen cell membrane markers (CD133, CD15, A2B5 and CD44) using multicolor flow cytometry. 16 subpopulations were separated and analyzed for their self-renewal capacity and their ability to reform the original heterogeneous cell population. Mathematical modelling was used to calculate state transitions between phenotypes and predict the adaptive response of CSCs. Results and Discussion: Similar to GBM biopsies, we observed markers to be heterogeneously expressed in glioma stem-like cells and primary cultures. All analyzed tumor cell subpopulations were able to proliferate and carried stem-cell properties including self-renewal potential. Moreover all subpopulations were able to adapt their marker profiles to give rise to the original subpopulations. Interestingly, mathematical modeling revealed a different propensity in reforming the original heterogeneity between subpopulations over time, which was independent of their proliferation index. Conclusion: Our results suggest that glioma CSCs do not represent a stable entity and that intra-tumoral heterogeneity in GBM at least partially results from a high adaptation capacity. This implies that glioma treatment approaches should take into account the potential of cancer cells to change their state. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 380 Relevance of TERT promoter mutations in poorly differentiated and anaplastic thyroid carcinomas. Association with RAS, BRAF or PIK3CA activating mutations N. Feas-Rodr´ ´ ıguez1 , R. Munoz ˜ 1 , T. Alvarez Gago2 , J.J. Mateos Otero2 , J.M. Cameselle Tejeiro3 , G. Garcia-Rostan4 . 1 Institute of Molecular Biology and Genetics IBGM, Group Pathobiology of Cancer: Inter-- intra-tumor heterogeneity and Molecular Targets, Valladolid, Spain, 2 School of Medicine − Valladolid University, Department of Pathology, Valladolid, Spain, 3 Hospital Cl´ınico Universitario Santiago Compostela − School of Medicine, Department of Pathology, Santiago de Compostela, Spain, 4 Institute of Molecular Biology and Genetics IBGM, Group Pathobiology of Cancer: Inter-- intra-tumor heterogeneity and Molecular Targets, Valladolid, Spain Poorly Differentiated Thyroid Carcinomas (PDCs) and Anaplastic Thyroid Carcinomas (ATCs) are ORPHAN CANCERS, derived from the follicular epithelium of the thyroid gland. Rare, late stage, advanced, metastatic iodine refractory thyroid tumor histotypes with a dismal prognosis for which do not exist efficacious therapies. Recently, it has been postulated that mutations in the promoter region of the TERT gene, which encodes the catalytic subunit of telomerase, may exert a pivotal role in thyroid tumor dedifferentiation, progression and behavior. In the study, whose preliminary results we are submitting we aim to determine the prevalence of TERT promoter mutations in 80 PDCs and 80 ATCs. We seek to determine its clonal/subclonal nature, its presence/absence in better differentiated components within the same tumor, its segregation with metastatic cells to local or distant sites, its association or not with established oncogenic events in PDCs and ATCs (e.g. mutations in BRAF, RAS and PIK3CA). The analysis, by means of PCR and direct sequencing, of 31 and 36 samples from 16 PDC and 18 ATC cases respectively has shown that TERT promoter mutations are highly prevalent in aggressive thyroid tumor histotypes [62.5% PDCs; 72% ATCs]. The overall frequency of TERT C228T (−124 C>T) and TERT C250T (−146 C>T) mutations is 56% (19/34 cases). TERT C228T mutants are far more common than TERT C250T mutants [31.25% versus 12.5% in PDCs and 55.5% versus 11% in ATCs]. Four cases [3 PDCs and 1 ATC] display mutations at other sites within the promoter region, some of them already described in COSMIC. Among PDCs and ATCs TERT mutations seem to be a clonal event, being present in all the different areas analyzed within a particular tumor. Moreover, TERT mutants segregate with tumor dedifferentiation. In all of the cases in which a better differentiated component within the same tumor was screened the mutation was present in all components. Sixteen cases (16/34 − 47%) have previously been characterized for the presence/absence of BRAF, RAS and PIK3CA mutations and ten of them bear the C228T or C250T mutation (10/16 − 62.5%). Two cases (2/10 − 20% − 1PDC + 1ATC) are concurrently mutated at RAS, 3 cases (3/10 − 30% − 1PDC + 2ATC) at BRAF and 1 case (1/10 − 10% − 1ATC) at PIK3CA. The preliminary results of the project indicate that TERT promoter mutations are highly prevalent in aggressive, orphan thyroid cancers. The data so far indicate that TERT mutants are clonal, segregate with tumor dedifferentiation and occasionally coexist with other genetic events that have been claimed by different groups to be crucial in PDC and ATC development and behavior. On the whole, the findings of the project, that we expect to have completed by the time of the Congress, may significantly impact on the rationale of future, tailored gene targeted therapies applied to patients with PDC and ATC. No conflict of interest. 381 Role of AXL in invasion and drug resistance of breast and colon cancer cells W.M. Abdel-Rahman1 , N.A. Al-khayyal2 , V.A. Nair3 , M.S. Ayad2 . 1 University of Sharjah, Medical Lab Sciences, Sharjah, U.A.E., 2 University of Sharjah, College of Medicine, Sharjah, U.A.E., 3 University of Sharjah, SIMR, Sharjah, U.A.E. Introduction: AXL is a receptor tyrosine kinase, part of the TAM family (Tyro3, AXL and Merk). It regulates wide array of processes including cell proliferation, migration, invasion and Epithelial to Mesenchymal Transition (EMT). It was found to be over-expressed in some cancer types such as breast and colorectal cancer. Since EMT is controlled by many transcription factors, one of which is the p53 tumor suppressor gene, our aim here was to characterize the expression of AXL in relationship to p53 status and its role in EMT in breast and colorectal cancer. Material and Methods: We used 14 cell lines including 3 isogenic pairs with different p53 status. Breast cancer cell lines were MCF7, 1001 (p53 mutant clone of MCF7), CAL-51, MDA-MB-231, MDA-MB-361, ZR-75-1, T47D and BT-549. Colon cancer cell lines were HCT116, HCT116.p53 mutant line, RKO, RKO.p53 knokout line and SW480. HeLa cervical cancer cell line was used as a positive control for EMT. AXL expression was examined by Western blotting, followed by AXL siRNA silencing and collagen invasion assay. Cell viability analysis was performed after exposure to chemotherapeutic agent using sulforhodamine B (SRB) assay.

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Results and Discussion: AXL showed positive expression in the p53 mutant breast cancer cell lines while the matching p53 wild type cells were negative. In colon cancer cell lines, wild type p53 cells showed weak expression, whereas the mutant/knockout ones showed much higher expression compared to their parental cells. siRNA silencing of AXL in 1001 (p53 mutant cell line) was >95% efficient. The AXL-silenced cells were less invasive in the standard invasion assay. AXL silencing did not show significant effect on breast cancer cell sensitivity to Doxorubicin or colon cancer cell sensitivity to 5FU or irinotecan. However, AXL expressing cells developed more invasive potentials after exposure to chemotherapy compared to the AXL-silenced cells. Conclusion: We showed differential expression of a potential EMT candidate AXL in breast and colorectal cancers and a positive correlation with the p53 status. We have demonstrated a functional role of AXL in tumor invasion particularly after exposure to chemotherapy. These results could have clinical applications in cancer treatment and prognosis. No conflict of interest. 382 Leptin sustains hypoxic phenotype through modulation of mitochondrial homeostasis in prostate cancer cells M. Bologna1 , A. Calgani2 , C. Vicentini1 , D. Biondi1 , A. Ciafarone1 , P. Muzi1 , A. Angelucci2 . 1 University of L’Aquila, Dept. Life- Health- Environmental Sciences, L’Aquila, Italy, 2 University of L’Aquila, Dept. Clinical and Biotechnological Sciences, L’Aquila, Italy Background: Leptin is a cytokine produced by the adipose tissue in response to food intake; its serum levels are chronically high in obese subjects. In different carcinomas, obesity and high serum levels of leptin are risk factors for advanced tumor stage and poor prognosis. It has been proposed that adipose tissue, through the release of leptin, can regulate whole-body energy homeostasis. Leptin is a well-known modulator of energy metabolism both at central and peripheral sites. Physiological action of leptin in modulating the metabolic adaptation of different peripheral tissues supports the hypothesis that it could also exert a direct effect on cancer cells. Materials and Methods: We investigated leptin signaling in prostate cancer cell lines PC3 and LNCaP, in normoxic and hypoxic conditions. Results: Leptin treatment stimulated proliferation of prostate cancer cells in hypoxia and anoxia. Leptin effects were independent from oxidative phosphorylation and were associated with mitochondrial homeostasis, stabilization of mitochondrial membrane potential and of ROS production. In normoxia, leptin determined a reduction in mitochondrial respiration with a concomitant upregulation of UCP2 and of uncoupled respiration. In hypoxia leptin treatment sustained an additional stabilization of HIF-1a and the upregulation of the lactate exporter MCT4, determining the maintenance of a high lactate production rate. Furthermore, leptin counteracted the down modulation of SIRT1 induced by hypoxia, and persistent high levels of SIRT1 were involved in HIF-1a stabilization. Elevated expression levels of UCP2 and MCT4 were also found significantly associated with serum leptin in cancer tissues from prostate cancer patients. Conclusions: Leptin action, through the modulation of cancer metabolism, may explain, at least partially, the association between obesity and prostate cancer. Leptin can sustain cancer progression through the direct modulation of cancer cell energetics. This effect could be particularly important in hypoxic environment and when mitochondrial respiration is impaired. The proposed mechanism, involving new targetable intermediates, such as MCTs and SIRT1, offers new opportunities in diagnosis stratification and treatment for prostate carcinoma patients with high serum leptin levels. No conflict of interest. 383 The impact of monocarboxylate transporter expression on metabolic function in prostate cancer cells L. Hutchinson1 , A. Boyers1 , A. Chadwick2 , I. Stratford1 . 1 The University of Manchester, Manchester Pharmacy School, Manchester, United Kingdom, 2 The University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom Background: The tumor microenvironment is subjected to variations in oxygen tension which results in hypoxic regions. Hypoxia in tumors is associated with radioresistance and a metastatic phenotype. Hypoxic conditions cause tumor cells to favor anaerobic metabolism, however this switch to glycolysis is also found in tumor cells under aerobic conditions, known as the ‘Warburg effect’. An increased reliance on glycolysis results in large quantities of lactate, which needs to be transported out of tumor cells to maintain the intracellular pH. The monocarboxylate transporters, MCT1 and MCT4, regulate the transport of lactate in tumor cells and here we report the effect of overexpression or knockdown of these transporters on metabolism, growth and response to therapy in prostate cancer cell lines. Materials and Methods: Human prostate cancer cell lines were stably transfected, using lentiviral vectors, to show overexpression of MCT1 (DU145, LNCaP and PC3), overexpression of MCT4 (LNCaP), or knockdown of MCT4 (DU145 and PC3). Using these stable cell lines, the effect of overexpression or

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silencing of MCT1 or MCT4 was assessed in vitro. A lactate assay was used to investigate the effect on intra- and extracellular lactate under varying oxygen tensions. Effect on cell cycle progression was assessed using flow cytometry and toxicity of docetaxel was determined using MTT and SRB assays. Finally, a metabolome siRNA screen was carried out to determine if MCT4 expression was related to sensitivity to toxicity resulting from knockdown of a range of metabolic proteins. Results: Expression levels of MCT1 and MCT4 were confirmed by western blot and immunofluorescence. Silencing of MCT4 in DU145 and PC3 cell lines increased intracellular lactate under hypoxic conditions. Knockdown of MCT4 resulted in sensitization to docetaxel treatment under both normoxic and hypoxic conditions. Overexpression of MCT1 in PC3 cells caused cell cycle arrest in the G2/M phase in both normoxia and hypoxia. The metabolome screen indicated that, in wild type LNCaP cells, down-regulation of the protein product of the genes encoding 2,4-Dehydrocholesterol Reductase or Pyruvate Dehydrogenase Phosphatase Catalytic Subunit 2 was synthetically lethal. Overexpression of MCT4 resulted in protection against these lethal effects. Conclusions: Altering the expression of MCT1 and MCT4 in human prostate cancer cells had a variety of effects on cell function. Overexpression of MCT1 caused cell cycle arrest in PC3 cells which may impact on radiosensitivity as cells are most vulnerable to radiation treatment when in the G2/M phase. The identification of two proteins that cause toxicity when silenced in LNCaP wild-type cells, but not LNCaP cells showing overexpression of MCT4, is an interesting lead for further investigation. No conflict of interest. 385 The heterogeneity of circulating lung tumor cells from non-small cell lung cancer patients S.L. Kong1 , S.J. Tan2 , T.K.H. Lim3 , H.M. Poh1 , T.Z.X. Yeo2 , X. Liu1 , Y.W. Chua3 , A.A. Bhagat4 , W.T. Lim5 , A.M. Hillmer1 . 1 Genome Institute of Singapore- A*STAR, Cancer therapeutics and stratified oncology, Biopolis Street, Singapore, 2 Clearbridge Accelerator Pte Ltd, Science Park Drive, Singapore, 3 Singapore General Hospital, Department of Pathology, Outram Road, Singapore, 4 Clearbridge BioMedics Pte Ltd, Science Park Drive, Singapore, 5 National Cancer Center, Division of Medical Oncology, Hospital Drive, Singapore Background: The heterogeneity and plasticity of individual tumor subclones potentially drive development of resistance under selection pressure from anticancer therapy. Molecular analyses of single circulating tumor cells (CTCs) may allow us to understand the critical pathways that mediate the bloodborne dissemination of cancer, where bulk analyses of primary or metastatic tumors fail due to tumour heterogeneity. Material and Methods: In this study, we evaluated the heterogeneity of single CTCs isolated from non-small cell lung cancer (NSCLC) patients recruited from National Cancer Centre Singapore. A total of 7.5 ml EDTA blood was processed on the Clearbridge BioMedics ClearCell® FX system. Single CTCs were enumerated using a novel single cell isolation machine, DropCell that operates based on hydrodynamic focusing and negative antibody selection. Whole genome amplification was performed on the enumerated single cells followed by amplicon-based deep sequencing on a custom-designed lung cancer gene panel. Results: A total of 23 CD45-negative single cells were isolated from 7 NSCLC patients. As verified by the Sanger sequencing, we found similar EGFR driver mutation in the matched primary tumor and 5 single CTCs samples. A more comprehensive mutation profiling of the single CD45-negative cells and matched primary tumor is being processed and validated. Conclusions: This study demonstrates the possibility of single cell CTC isolation and targeted amplicon sequencing approach in characterizing the mutation profiles of NSCLC CTCs. This allows us to evaluate the degree of heterogeneity in CTCs and the matched primary tumor. No conflict of interest. 386 Induction of senescence in primary glioblastoma cells by serum and TGFb R. Kumar1 , A. Gont1 , T. Perkins2 , I. Lorimer1 . 1 Ottawa Hospital Research Institute, Cancer Therapeutics, Ottawa, Canada, 2 Ottawa Hospital Research Institute, Regenerative Medicine Program, Ottawa, Canada Introduction: Glioblastoma is the most common type of adult primary brain tumour and has a median survival after diagnosis of a little more than a year. Glioblastomas have a high frequency of mutations in the Telomerase reverse transcriptase (TERT) promoter and Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) locus that are expected to render them resistant to both replicative and oncogene-induced senescence. While traditional glioblastoma cell lines cultured in serum have demonstrated the ability to undergo premature senescence, the ability of primary glioblastoma cells to do so has not been studied in detail. Materials and Methods: Primary glioblastoma (PriGO) cells were harvested from human patients with glioblastoma and cultured in serum free media

under hypoxic conditions (5% oxygen). Senescence was determined primarily by the senescence-associated b galactosidase (SA-b-Gal) assay. Markers of senescence Promyelocytic Leukemia (PML) bodies and p21 were detected by immunofluorescence and immunoblotting respectively. Global gene expression profiling was determined using Affymetrix microarray human gene chips. Thrombospondin expression was determined by enzyme-linked immunosorbent assay (ELISA). Ras pathway inhibition was achieved with the MEK inhibitor U0126. Results and Discussion: Exposure of PriGO8A primary glioblastoma cells to media with 10% serum induced a senescence-like phenotype characterized by increased SA-B-Gal activity, PML bodies and p21 and morphological changes characteristic of senescence. This occurred in the absence of any increase in gH2AX foci or senescence-associated secretory phenotype (SASP). Microarray expression analysis showed that serum exposure increased the expression of genes associated with the Transforming growth factor beta (TGFb) pathway. Treatment of PriGO8A cells with TGFb was sufficient to induce senescence in these cells. The response of PriGO8A cells to serum was dependent on basal expression of the TGFb activator protein thrombospondin. Primary glioblastoma cells from three additional patients showed a variable ability to undergo senescence in response to serum. Cells that lacked basal thrombospondin expression did not undergo senescence in response to serum, but did in response to TGFb. Cells that had low level constitutive TGFb pathway activation driven by Ras pathway activation only underwent senescence in response to TGFb when Ras pathway activity was blocked. Conclusion: TGFb is a key mediator of premature senescence in primary glioblastoma cells, its activity dependent on the TGFb activator protein thrombospondin as well as Ras pathway activation. Primary glioblastoma cells therefore retain a functional senescence program which can potentially be exploited therapeutically. No conflict of interest. 387 Yeast as a model system to screen purine derivatives against human CDK1 and CDK2 kinases T. Mayi1 , M.E. Huang2 , L. Vernis2 , M. Legraverend3 , G. Faye2 . 1 Institut ˆ 110–112- Centre Universitaire- 91405 Orsay- France, INSERM Curie- Bat. ˆ 110–112- Centre UniversitaireU612, Orsay, France, 2 Institut Curie- Bat. ˆ 91405 Orsay- France, CNRS UMR3348, Orsay, France, 3 Institut Curie- Bat. 110–112- Centre Universitaire- 91405 Orsay- France, CNRS UMR176, Orsay, France Background: Cyclin-dependent kinases (Cdk) play crucial roles in cell cycle progression. Aberrant activation of Cdk1 has been observed in a number of primary tumors and Cdk2 is deregulated in various malignancies. The therapeutic value of targeting Cdk1 and Cdk2 has been explored in a number of experimental systems. Material and Methods: Taking advantage of the fact that deletion of the yeast CDC28 gene is functionally complemented by human CDK1 or CDK2, in vivo screen system have been set up to evaluate the inhibitory potency of purine derivatives against these two human Cdks. Three isogenic strains highly sensitive to small molecules and harboring genes CDK1, CDK2 or CDC28, have been constructed under the control of the CDC28 promoter. In a proof of principle assay, the inhibitory effect of 82 purine derivatives on the growth rate of these strains were determined. Results: Thirty-three of 82 purine derivatives were revealed to be able to inhibit the Cdk1- or Cdk2-harboring strains but not the Cdc28-harboring strain, suggesting a specific inhibitory effect on human Cdks. Conclusions: Our data demonstrate that the yeast-based assay is an efficient system to identify potential specific inhibitors that should be preferentially selected for further investigation in cancer cultured human cell lines. No conflict of interest. 388 The vacuolar ATPase subunit E controls apoptosis and is up-regulated in breast cancer S.N. Mohammed1 , M. Mourtada-Maarabouni1 . 1 Keele University, School of Life Sciences, Newcastle-Under-Lyme, United Kingdom Background: Vacuolar H+ATPase (V-ATPase) is a multi-subunit proton pump involved in the acidification of a wide variety of organelles. Dysfunction of the V-ATPase has been associated with several diseases, including cancer. Increased V-ATPase activity is reported to be critical for invasion of highly metastatic breast cancer cells and its expression at the plasma membrane correlates with the invasive characteristics of various malignant cells. Functional expression cloning identified V-ATPase subunit E (ATP6V1E) as a potential apoptosis regulatory gene. The aims of this study were to examine the effects of modulating ATP6V1E expression on the survival of breast cancer cells. In addition, we have examined the expression level of ATP6V1E in breast cancer samples. Materials and Methods: MCF7 and MDA-MB-231 cells were transfected either with a mammalian expression plasmid encoding ATP6V1E transcript, control cells received empty vector. Culture growth, cell viability, apoptosis,

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 and long term survival were assessed. The level of ATP6V1E transcripts in breast cancer cDNA samples, from commercial Breast Cancer cDNA Arrays, was determined by qRT-PCR TaqMan® analysis. Results: Over-expression of ATP6V1E levels in both cell lines resulted in an increase in basal apoptosis level and a reduction in short and long term survival. ATP6V1E expression was found to be significantly down-regulated in breast cancer samples. Conclusion: The present data suggest that the ATP6V1E regulates apoptosis and cell survival in breast cancer cells. The pro-apoptotic effect exerted by a single subunit of V-ATPase is a novel observation. The reduced expression of ATP6V1E in breast cancer samples suggests that a decrease in the expression levels may be significant in oncogenesis. No conflict of interest. 390 Distinct HER2 distribution and homo-dimerization patterns on subpopulations of breast cancer cells − correlative light- and electron microscopy in liquid for cancer stem cell characterization D. Peckys1 , N. De Jonge2 . 1 Center for Integrative Physiology and Molecular Medicine- Saarland University, Molecular Biophysics, Homburg, Germany, 2 INM − Leibniz Institute for New Materials- Saarbrucken¨ Germany, Innovative Electron Microscopy Group, Saarbruecken, Germany Background: A recognized key problem in cancer biology and clinical oncology is the inherent heterogeneity of tumors, caused by epigenetic, genetic, and metabolic influences. However, most of today’s molecular knowledge of cancer cells − especially at the proteome level − is derived from analytical methods that generate population average data. These kind of data bear the risk that of hiding important but sporadic cellular events, and rare cells, such as cancer stem cells. Many molecular targets of the new biological generation of anti-cancer drugs reside in the plasma membrane. Among these are the members of the epidermal growth factor receptor family (HER1−3), which are overexpressed in a variety of cancer types and are important for cancer cell proliferation. Activated states of these receptors, mostly their association into dimers, trigger intercellular signaling pathways for cell migration, proliferation, and other phenotypes. Insights about their functioning are mostly based on population average data, since it has not been possible to examine these receptors at the single cell level in categorized cancer subpopulations for endogenous membrane expression densities with sufficient spatial resolution until recently. Material and Methods: We here present a new correlative light- and electron microscopy methodology capable of imaging thousands of endogenously expressed membrane proteins in the plasma membrane of many single cells. In addition, the approach allows studying endogenously expressed proteins in their native environment, i.e. the hydrated and intact plasma membrane. We used this methodology to examine the distribution and homo-dimerization of the orphan receptor HER2 in HER2 overexpressing breast cancer cells, categorized in phenotypic distinct subpopulations. This was achieved by applying a specific HER2 labeling and a characterization of four distinct phenotypes within the cancer cell population, including breast cancer stem cells (CD44+/CD24-/low). Results: Selected cells representative for each subpopulation were imaged with a spatial resolution of 3 nm while keeping the cells in liquid. Automated image processing of hundreds of images provided detailed information about the distribution pattern of individual HER2 molecules and their homodimerization status. A statistical analysis based on calculating the pair correlation function from hundred thousands of individual HER2 positions revealed remarkable differences in functionality between cellular regions, and between subpopulations of cells, which has possible relevance for studying cancer metastasis and drug response. Conclusions: Correlative light- and electron microscopy in liquid revealed different HER2 distribution patterns and homo-dimerization states on breast cancer stem cells, and other phenotypic distinct cell subpopulations, compared to bulk cancer cells. No conflict of interest.

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compound, decreased GBM cell line cell viability. Flow cytometry results revealed that isochaihulactone dose-dependently triggered G2/M phase arrest and apoptosis. Our in vivo study showed that isochaihulactone suppressed tumors substantially in a GBM xenograft model as well as DDIT3 and NAG-1 overexpression. Results: In this study, we discovered isochaihulactone which can regulate ER homeostasis in GBM cell lines and increase ER apoptosis-related DDIT3 expression independently of chaperone protein GRP78 expression. In the mean time, isochaihulactone dose-dependently triggered G2/M phase arrest and apoptosis and we also found Isochaihulactone induced DDIT3 overexpression to modulate NAG-1 same as our in vivo study result. Conclusion: Overall, isochaihulactone caused glioblastoma cell line apoptosis by disrupting ER homeostasis through an increase in DDIT3 and NAG-1 expression. We propose ER stress as a potential target for GBM therapy in the future. No conflict of interest. 392 Grb2 facilitates EGF-dependent GEP100-Arf6 pathway activation leading to lung cancer invasion and metastasis T. Menju1 , K. Hijiya1 , H. Motoyama1 , A. Aoyama1 , F. Chen1 , T. Sato1 , M. Sonobe1 , S. Feller2 , H. Sabe3 , H. Date1 . 1 Kyoto University- Graduate School of Medicine, Thoracic Surgery, Kyoto, Japan, 2 Martin-Lutter University, Institute of Molecular Biology, Halle-Wittenberg, Germany, 3 Hokkaido University- Graduate School of Medicine, Molecular Biology, Sapporo, Japan Introduction: Invasive and metastatic activities are the most challenging hallmark of cancer in clinical settings. Our previous reports have shown that GEP100 activates Arf6 by its binding to activated EGFR leading to epithelio–mesenchymal transition (EMT) and cancer progression. They also have revealed the phosphorylated tyrosines in the C-terminal EGFR necessary for the binding to GEP100, are well known to be Grb2-binding sites as well. Here we have examined the enhancing effect of Grb2 on the binding of GEP100 with EGFR and the Arf6 activation. Furthermore, the clinical significance of co-expression of Grb2 and GEP100 was analysed. Materials and Methods: GST-tagged proteins including PH domain of GEP100 or SH2/SH3 domain of Grb2 inserted into pGEX vector were purified and used for the mutual binding assays. A549 cells with HA-tagged Grb2 or HA alone expression were stimulated with EGF, and the lysates were applied for the immunoprecipitation assay against GEP100. Then, Arf6 activities of these cells were analyzed by the pulldown assay with GST-tagged GGA protein. Furthermore the in vitro invasive activities of those cells were measured by Matrigel invasion assays. Grb2 and GEP100 of the tumor cells were immunostained in resected human lung adenocarcinoma specimens. These expression data of the two molecules integrated with their clinicopathological factors and EMT status information previously published were examined with regard to their invasive and metastatic activities. Results: Grb2 and GEP100 were physically associated through the PH domain of GEP100 and both the SH2 and N-terminal SH3 domain of Grb2, not its C-terminal SH3 domain. Exogenously aberrant expression of Grb2 in A549 lung cancer cells enhanced the association between activated EGFR and GEP100, consequently, Arf6 activation, and in vitro invasive activity, according to the expression level of Grb2. Additionally, A549 cells with HAGrb2 overexpression showed stronger EMT activation in response to EGF stimulation than those with control vector Among 239 lung adenocarcinoma specimens on tissue microarrays, 131 (55%) and 65 (27%) cases of patients were positive for Grb2 and GEP100, respectively. Tumors with double-positive for Grb2 and GEP100 (45 cases) showed significantly more aggressive EMT status (p = 0.0116) and higher node-metastatic potential (p = 0.0082, nodepositive/negative; 12/33 to 7/77) than the double-negative one (84 cases). Conclusion: Grb2 enhances the binding of GEP100 to EGFR leading to Arf6 activation with EMT and promotes lung cancer invasion and metastasis via GEP100-Arf6 pathway. No conflict of interest.

391 Isochaihulactone-induced NAG-1 overexpression mediated by endoplasmic reticulum stress-independent DDIT3 leads to glioblastoma multiforme cell apoptosis

393 N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents

S.F. Tsai1 , H.J. Harn2 . 1 China Medical University, Center for Neuropsychiatry, Taichung, Taiwan, 2 China Medical University and Hospital, Department of Pathology, Taichung, Taiwan

C. Mazzoccoli1 , V. Ruggieri1 , T. Tataranni1 , F. Agriesti1 , I. Laurenzana1 , A. Fratello2 , N. Capitanio2 , C. Piccoli1,2 . 1 Laboratory of Pre-clinical and Translational Research- IRCCS-CROB- Referral Cancer Center of BasilicataRionero in Vulture (PZ), Italy, 2 Department of Clinical and Experimental Medicine- University of Foggia- Foggia, Italy

Background: The endoplasmic reticulum (ER) is a major site of cellular protein production homeostasis regulation, which plays a major role in cancer. Glioblastoma multiforme (GBM) is the most malignant brain tumor characterized, which always always activate the unfolded protein response under the ER stress condition. Material and Methods: We first use western blot to verified ER stress related protein level in each GBM cell lines compared to normal astrocyte. After that, the MTT assay showed isochaihulactone, which we found a nature

Background: Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to originate from undifferentiated neural crest cells. N-acetylaspartate (NAA) is the second most abundant metabolite present in the central nervous system (CNS) and its levels are changed in a wide array of CNS disorders. Decreased levels of NAA, associated with loss of neurons or mitochondrial dysfunction, are found in neuroblastoma tumor.

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Material and Method: SH-SY5Y neuroblastoma cells were treated with increasing doses of NAA (2, 4, 8 and 16 mM) for 72 h and cell viability was assessed by MTS assay. The gene expression profile induced by 4mM NAA treatment in SH-SY5Y cells was examined using the ILLUMINA array technology. Apoptotic and differentiating effects of 4mM NAA treatment for 72 h were evaluated by flow cytometric analysis. The levels of proteins involved in the apoptotic pathway and differentiation were measured by Western Blotting. Results: NAA treatment in SH-SY5Y neuroblastoma cells has elicited morphological and neuronal differentiating effects evident with the neurite outgrowth and increased expression of specific differentiating markers: Microtubule-associated protein 2 (MAP2) and Tyrosine Hydroxylase (TH). Exposure of cells to NAA has induced activation of apoptotic pathway, associated to decreased levels of Bcl-xl and Survivin and increased levels of p53, p21 and p27 proteins. Moreover, NAA-treated SH-SY5Y cells have proved to be more sensitive to the chemotherapy drugs, Cisplatin and 5-Fluorouracil, when compared to the untreated control. Conclusions: To our knowledge, this is the first study to demonstrate the neuronal differentiating effects of NAA treatment in neuroblastoma cells and its ability to enhance sensitivity to antineoplastic drugs. No conflict of interest. 394 Glucose deprivation as new therapeutic approach to target pancreatic cancer cell metabolism T. Tataranni1 , F. Agriesti1 , V. Ruggieri1 , C. Mazzoccoli1 , I. Laurenzana1 , R. Scrima2 , N. Capitanio2 , C. Piccoli1,2 . 1 IRCCS CROB, Laboratory of Pre-Clinical and Translational Research, Rionero in Vulture Pz, Italy, 2 University of Foggia, Department of Clinical and Experimental Medicine, Foggia, Italy Background: Pancreatic cancer (PC) is the fourth leading cause of cancer related deaths and available therapeutic strategies, based on conventional chemotherapy, result in a progressive resistance to treatment. As PC cells metabolism significantly diverges from normal cells, targeting cellular metabolism may represent a new therapeutic strategy. In this study, we characterized the metabolic profile of two PC cell lines and investigated their response to glucose deprivation. Material and Methods: BxPC3 and PANC1 cell lines were cultured in control medium (CM) or in glucose deprived medium (GDM) for 24 h. O2 consumption was measured by a Clark-type electrode. The specific activities of respiratory chain complexes as well as the concentration of NADH and NAD+ were assayed spectrophotometrically. MTS assay assessed cell viability, apoptosis and ROS were detected by flow-cytometry following Annexin-V assay and DCFH-DA staining, respectively. Results: BxPC3 and PANC1, cultured in CM, underwent a comparative metabolic characterization. BxPC3 displayed a lower efficiency of oxidative phosphorylation system (p < 0.01 vs PANC1) together with a lower activity of each complex of the mitochondrial respiratory chain and a reduced cellular content of the co-factor NAD (p < 0.05), compared to PANC1. Interestingly, GDM culture significantly reduced cell viability in both cell lines compared to CM with BxPC3 showing a marked sensitivity to energy deprivation compared to PANC1 (p < 0.001). GDM culture partially restored mitochondrial respiration in BxPC3 (p = 0.005 vs CM), leading to a remarkable cytotoxic effect only in BxPC3, as demonstrated by a significant ROS production (p < 0.01 vs CM) and a sustained apoptosis induction (p < 0.01 vs CM). Conclusions: Our results reveal that BxPC3 and PANC1 display distinctive metabolic properties and the reduction of the glucose intake significantly affects survival of BxPC3, cell line characterized by a low oxidative metabolism. Defining the metabolic feature may represent, therefore, an additional important tool for targeting pancreatic cancer and developing new alternative therapeutic strategies to overcome chemo-resistance and improve prognosis and survival of patients affected. No conflict of interest. 395 Hypoxia-induced angiogenesis, EMT and stem cell characteristics using next-generation sequencing in lung cancer

conditions. For transcriptome analysis, mRNA of BEAS-2B and A549 cell lines were analyzed using next-generation sequencing (Hiseq2500 system). For further validation, angiogenesis markers were analyzed by western blotting. EMT was assessed with western blotting, wound healing assay and Matrigel invasion assay, and stem cell characteristics were assessed with RT-PCR, immunostaining, soft agar colony formation assay, sphere formation assay and in vivo mice tumor model. Next-generation sequencing revealed significant changes in the expression of angiogenesis, EMT and stem cell markers after hypoxic stress. Among the angiogenesis markers, VEGF and HIF-2a were increased. EMT markers related in hypoxia showed decrease in E-cadherin and increase in fibronectin, vimentin, N-cadherin, a-SMA, Snail, Slug, ZEB1 and ZEB2. Stem cell markers such as CXCR4, Oct4 and Nanog were increased at least one lung cancer cell line in hypoxic condition compared with in normoxic condition. Functional assays for EMT and stemness acquisition indicated that hypoxic stress increased wound healing, Matrigel invasion, sphere formation and in vivo mice tumor formation. These results suggest that hypoxia induces angiogenesis markers expression which is associated with EMT and stemness acquisition in lung cancer. Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2014R1A2A1A11052422). No conflict of interest. 396 Induction of cellular senescence and hair follicle stem cell dysfunction upon p16INK4a expression in the skin N. Azazmeh1 , R. Amiel-Tokarsky1 , A. Helman1 , I. Ben-Porath1 . 1 Institute for Medical Research − Israel Canada − The Hebrew University-Hadassah school of medicine, Developmental Biology and Cancer Research, Jerusalem, Israel Introduction: Cellular senescence is a physiologic stress response program, in which cells cease to proliferate and undergo dramatic alterations in morphology, metabolic activity and gene expression. Senescence is mediated mainly by the p14ARF/p53 and p16INK4a/Rb tumor suppressor pathways, which are interconnected, yet their distinct contributions to the different components of the senescence program are not fully understood. Senescence is involved in tumor suppression, aging, multiple pathologies and normal embryonic development, however, many fundamental aspects of its beneficial and detrimental roles in tissue remodeling and physiology remain poorly elucidated. Materials and Methods: We have developed mice that allow inducible activation of p16INK4a, a senescence activator, in the epidermis of the skin. Using this model, we set out to study the effects of p16INK4a activation on senescence induction, on skin structure and hair follicle stem cell function. Results: We found that p16INK4a activation led to cell cycle arrest and senescence formation within the epidermis. Although expressed only in a subset of cells, p16INK4a caused pronounced epidermal alterations, including an increase in thickness and keratinocyte enlargement, and dysfunction of hair follicle stem cells. Upon long-term induction, we found that p16INK4a activation led to expansion of differentiated epidermal layers, alopecia, and dermal structure modification that were distinct from those observed during normal aging. p16INK4a activity induced substantial changes in wound closure, and in overall epidermal proliferation rates. Conclusion: Our findings indicate that p16INK4a activity can induce dramatic structural and functional changes in the epidermis, most likely acting through both cell autonomous and non-autonomous mechanisms. Our data shed light on one of the most important cellular genes and mechanisms regulating aging and cancer. No conflict of interest. 397 Loss of c-KIT in thyroid cancer cells: A functional study to investigate its role in tumor differentiation and progression

N. Kang1 , S.J. Kim2 , Y.K. Kim2 . 1 The Cancer Research Institute, College of Medicine- The Catholic University of Korea, Seoul, South Korea, 2 Department of Internal Medicine- Seoul St. Mary’s Hospital, College of Medicine- The Catholic University of Korea, Seoul, South Korea

F. Lessi1 , S. Franceschi1 , E. Tantillo1 , F. Panebianco2 , M. Menicagli1 , M. La Ferla1 , P. Aretini1 , G. Bevilacqua1 , I. Marchetti3 , C.M. Mazzanti1 . 1 Fondazione Pisana per la Scienza Onlus, Genomic Section, Pisa, Italy, 2 Pathology- University of Pittsburgh, Division of Molecular Genomic, Pittsburgh, USA, 3 University of Pisa and Pisa University Hospital, Section of Cytopathology, Pisa, Italy

Hypoxia, a major phenomenon in solid tumors, can promote the metastatic potential of tumor cells which is associated with chemoresistance and poor prognosis. It was reported that various angiogenesis factors including VEGF and HIF, were associated in cancer development and progression by hypoxia. In addition, both epithelial–mesenchymal transition (EMT) and cancer stem cells play an important role in malignant progression in many human tumors. We investigated the effect of hypoxic stress on the angiogenesis, EMT and stemness acquisition in lung cancer. Normal lung cell (BEAS-2B) and lung cancer cell lines (A549, H292, H226 and H460) were incubated in either normoxic or hypoxic (below 1% O2)

Background: Thyroid carcinoma is the most common malignancy of the endocrine system, representing approximately 90% of cases. Among these, papillary thyroid carcinoma (PTC) is the most frequent histologic type, comprising 70% to 85% of all thyroid malignancies. Nowadays, thyroid fineneedle aspiration (FNA) cytology is the best available test in the evaluation of a thyroid nodule. The role of c-KIT (member of receptor tyrosine kinase subclass III) in human tumours still remains to be explained. There have been reports that c-KIT is highly expressed in some tumours but on the other hand, its expression is lost in breast cancer and melanoma. In thyroid, c-KIT expression in malignant tumours decrease rather than in the benign nodules, suggesting a

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 negative involvement in tumoral progression. We performed a functional study on K1 human thyroid carcinoma cell line to confirm c-KIT role in thyrocyte differentiated phenotype rather than cell division. Material and Methods: We analysed the expression levels of PAX8 mRNA (marker of thyrocytes differentiation) with Real Time PCR on 69 FNA samples (39 benign samples and 30 malignant ones) on which c-KIT mRNA expression levels were observed in a previous our paper (Tomei et al. 2012). On the basis of these results, c-KIT was overexpressed in K1 PTC cell line. We evaluated the effects of this overexpression by proliferation assay (WST1); then we performed western blot, real time-PCR and immunofluorescence assay with c-KIT and PAX8 antibodies. Indeed we evaluated the morphological aspect of the transfected cells reviewed by senior cytopathologist. Results: PAX8 expression level was significantly higher in the benign group compared to the malignant group, inversely to c-KIT expression. In fact c-KIT expression (previous results) significantly correlated with the results of PAX8 expression. About the functional study, we observed that with the overexpression of c-KIT in thyroid cancer cells we have an increase of the expression levels of PAX8, that is a marker of differentiation for thyrocytes. Then the levels of proliferation decrease in the K1 cancer cell line statistically significative (P < 0.0001). Moreover we observed that the morphological aspect of the tumoral cells after the transfection of c-KIT changed, resembling normal cells. Conclusions: All our results may lead to confirm that c-KIT is involved in the differentiation in thyroid tissues and is lost in thyroid tumoral progression. Thereafter, studies about a possible role for c-KIT in the treatment of PTC, could be useful. No conflict of interest. 398 Characterization of the role of ASB6 in head and neck cancer initiating cells C.K. Chen1 , P.F. Su2 , J.F. Lo1,2,3 . 1 Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan, 2 Department of Dentistry, Taipei Veterans General Hospital, Taipei, Taiwan, 3 Department of Dentistry, National Yang-Ming University, Taipei, Taiwan Background: Head and neck squamous cell carcinomas (HNSCC) are clinically with characteristics of poor prognosis and recurrence. Previously, our study demonstrates that Areca nut extracts treatment not only enhances carcinogenesis but also up regulates mRNA expression of Ankyrin repeat and SOCS box containing 6 (ASB6). ASB6, decoded from ASB6 gene, is a 418-amino acid-long peptide consisting of 6 ankyrin repeats and a C-terminal SOCS box domain. Others show that ASB6 inhibits insulin receptor by interacting to APS adapter protein and Elongin BC complex. In addition, we have identified the existence of cancer stem cells (CSCs; also termed as cancer initiating cells (CICs)) which are important for tumor growth, metastasis and drug resistance in HNSCC. Herein, we want to characterize the role of ASB6 on mediating CICs of HNSCC (HN-CICs) during head and neck cancer tumorigenesis. Material and Method: We used shRNA to knockdown or lentivirus to overexpress expression of ASB6 in HNSCC cells to examine the expression profile of stemness markers and Epithelial–mesenchymal transition proteins in vitro. The phenotypic effects including invasion and migration capability of abovementioned cells were analyzed. Further, sphere formation ability, a hallmark of cellular stemness, of the manipulated HNSCC cells was examined. Results: First, we found that expression of ASB6 protein was increased in more proliferative HNSCC (SASVO3), enriched NH-CICs and metastatic cells (SASM5). Furthermore, we observed up-regulation of abovementioned ASB6 protein in the cell membrane preparation in OECM1 and SASM5 cells. Secondly, shRNAi was applied to knockdown expression of ASB6 in SASM5 cell and we observed that protein level of ASB6 was decreased. By flow cytometry, we observed that expression of stemness marker such as CD44 and ALDH was both down regulated. Same findings were also displayed in SAS and OECM1 sphere cells. Immunoblot analyses also demonstrated down regulation of Oct4, Nanog, and glucose transporter 3 in SAS and OECM1 sphere cells with shRNAi of ASB6. For malignancy analyses, we observed that reduction of migratory ability and anchorage independent growth of SAS and OECM1 sphere cells with shRNAi of ASB6. Finally, SAS and OECM1 cells over-expressing ASB6 with HA-tag were generated and found that CD44 is up-regulation. Therefore, gain-of-function of ASB6 to mediate cancer stemness and malignancy in OSCC is continuously under investigated. ASB6 might mediate stemness by inhibit insulin receptor and enhance glucose consumption. It is of importance to uncover how ASB6 regulates stemness and metastasis and the molecular mechanism. Conclusion: Together, we will characterize the role of ASB6 to mediate tumorigenesis and stemness of HN-CICs and metastatic capability in HNSCC. Targeting on HN-CICs through ASB6 can be potential therapeutic for future HNSCC treatment. No conflict of interest.

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399 ATRX expression and ALT phenotype in uterine leiomyomas 1 2 T. Ahvenainen1 , N. Makinen ¨ , R. Butzow ¨ , P. Vahteristo1 . 1 University of Helsinki, Research Programs Unit- Genome-Scale Biology and MedicumDepartment of Medical and Clinical Genetics, Helsinki, Finland, 2 University of Helsinki and Helsinki University Hospital, Medicum- Department of Pathology and Laboratory of Helsinki University Hospital, Helsinki, Finland

Introduction: Uterine leiomyomas (ULMs) are the most common gynecologic tumors. Majority of the tumors are classified as conventional ULMs, whereas approximately 10% of the lesions represent several histopathological variant subtypes. These variants include mitotically active ULMs, ULMs with bizarre nuclei and cellular ULMs. Uterine leiomyosarcoma (ULMS), which is considered the malignant counterpart of ULM, is aggressive smooth muscle cancer. Although the pathogenesis of ULMS is poorly understood, it is suggested that a small fraction of tumors may arise from currently uncharacterized ULM precursor. We have recently shown that alpha thalassemia/mental retardation syndrome X-linked (ATRX) is frequently mutated in ULMSs, commonly accompanied with alternative lengthening of telomeres (ALT) phenotype. In addition, aberrant ATRX function is has been associated, with death-domain associated protein (DAXX), with ALT. Materials and Methods: Primary study material consisted of tissue microarrays (TMAs) with 93 formalin-fixed paraffin-embedded (FFPE) samples representing various ULM subtypes and 63 conventional ULMs. Immunohistochemical (IHC) staining of ATRX and DAXX was performed to all samples, which were further scored by a pathologist specialized in gynecological pathology. Telomere-specific fluorescence in situ hybridization was used to determine ALT phenotype in all TMA samples. The results were independently analyzed by two investigators. Both aberrant ATRX expression and suspected ALT phenotype were validated from whole paraffin tissue sections. Results and Discussion: Loss of ATRX expression was found in 3% (3/93) of ULM variants. All 63 conventional ULMs showed normal ATRX expression. Aberrant DAXX expression was not observed in variant or conventional ULMs. We are currently determining ALT phenotype status for all samples, with few aberrant samples already identified. Conclusion: It is important to investigate whether a particular ULM phenotype may develop to ULMS. If these lesions could be identified to a particular ULM subgroup with known molecular markers, it would provide an effective way to detect leiomyomas with malignant potential. In return, this would have clinical significance assisting the diagnostics and development of therapies. Here we show that a fraction of variants have an aberrant ATRX expression and represent an ALT phenotype, and thus for this part their pathogenesis resemble that of ULMS. No conflict of interest.

400 Activation of CXCR4 by aberrant promoter demethylation in lung cancer N. Kang1 , S.J. Kim2 , Y.K. Kim2 . 1 The Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul, South Korea, 2 Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea As a cancer stem cell marker, CXCR4 has been known to be closely associated with cell survival, stemness acquisition. Previous studies reported that the level of CXCR4 is increased after hypoxic condition in several types of cancer. However, the mechanism of the increased CXCR4 expression has not been well understood. We investigated whether aberrant promoter demethylation could induce CXCR4 activation by using hypoxic stress in lung cancer. Human normal lung cell (BEAS-2B) and lung cancer cell lines (A549, H292, H226 and H460) were incubated under hypoxic condition. Transcriptome and methylation analysis using Next-generation sequencing were performed by Hiseq2500 system. For further validation, CXCR4 expression was analyzed by RT-PCR and western blotting. To determine whether CXCR4 is reactivated, cell lines were treated with a DNA methyltransferase inhibitor (AZA). Hypoxiainduced DNA demethylation was identified by methylation-specific PCR and bisulfite sequencing. Stem cell characteristics were assessed by sphere formation assay and in vivo mice tumor model. NGS results revealed that CXCR4 expression was increased after hypoxic condition, whereas CXCR4 methylation was reduced. CXCR4 was activated by either in hypoxic condition and by treatment with AZA. MSP showed decreased CXCR4 promoter methylation in hypoxic condition compared with normoxic condition. Functional stem cell assay indicated that hypoxic stress increased sphere formation and in vivo mice tumor formation. These results suggest that hypoxia induces stem cell characteristics which are related with CXCR4 reactivation by promoter demethylation. Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2014R1A2A1A11052422). No conflict of interest.

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401 Neuroendocrine differentiation of prostate cancer cells grown in nude mice in presence of bone marrow stromal cells in androgen deprivation conditions G.L. Gravina1 , A. Mancini1 , A. Colapietro1 , A. Vetuschi2 , S. Pompili2 , R. Sferra2 , S. Delle Monache3 , C. Festuccia1 . 1 University of L’Aquila, Department of Biotechnological and Applied Clinical Sciences, L’Aquila, Italy, 2 University of L’Aquila, Department of Biotechnological and Applied Clinical Sciences- Laboratory of Human Anatomy, L’Aquila, Italy, 3 University of L’Aquila, Department of Biotechnological and Applied Clinical Sciences − Laboratory of General Pathology, L’Aquila, Italy Background: Prostate cancer (PCa) is usually characterized by an excellent prognosis largely due to little biological aggressiveness and the power of hormonal deprivation (ADT) therapy. Nevertheless, a significant percentage of patients develops a progressive and a castration-resistant disease (CRPC). PCa that has metastasized to bone undergoes critical interactions with bone marrow stromal cells (BMSc), ultimately promoting tumor survival and growth. This is associated both to cancer stem cell (CSC) proliferation and neuroendocrine (NE) differentiation as function of hormone status. Methods: A mixture (1:100) of 22rv1 PCa cells and murine BMSc was xenografted in intact or castrated male nude mice. Parallely, PC3, PCb2, VCaP and C4-2B (bone derived PCa) or 22rv1 and DU145 (primary and brain derived PCa) were tested for apoptosis and NE differentiation in vitro in co cultures with BMSc or in presence of conditioned media from BMSc and PC3 cells were engrafted in the tibia. Western blots, elisa assays for TGF-b1, neuron-specific enolase (NSE) and chromogranin A (chromo A) levels were tested. Results: The inoculation of BMSc alone was not able to growth in subcutaneous engraftment. Although 22rv1 cells are a model for primary CRPC, these cells maintain sensibility to androgens. Tumors originated from 22rv1/BMS cell mixture are larger than tumors originated from 22rv1 alone. This difference was higher when tumors were grown in castrated nude mice. Due to the significance of TGF-b in cytostasis and bone metastasis, the role of TGF-b1 was investigated in the context of PCa-BMCs interactions. 22rv1 cells basally express NSE and chromo A. The expression of these NE markers was, however, increased in tumor grown in castrated nude mice. The presence of BMSs increases angiogenesis and mesenchymal cell recruitment. In vitro, we shown that NE phenotype, acquired in co-culture conditions between 22rv1 and BMSc or culturing 22rv1 cells with exogenous TGF-b1, was associated to increased pro-inflammatory cytokine expression and monocyte/macrophage recruitment. However, bone derived PC3, PCb2, VCaP and C4-2B metastatic cells showed reduced NE differentiation in presence of BMS or TGF-b1. Conversely, intratibial PC3 engraftment showed an increased expression of CSC markers. Androgen insensitive CSC, borne in the highly hypoxic bone marrow niche, or NE cells are generated and this allows to overcome ADT or the difficulties of growing up in a hostile environment such as bone. Therefore, NE and CSC differentiation represent alternative mechanisms to evade PCa apoptosis in the bone. Conclusions: The use of drug targeting CSC recruitment (TGF-b1 blocking antibodies or TGFbR inhibitors) or the growth of NE cells (somatostatin antagonists) might be used for the treatment of bone metastatic PCa. No conflict of interest. 402 K-Ras modulated microRNAs induce tumorigenesis and drug resistance in NSCLC L. Shi1 , M. Garofalo1 . 1 Cancer Research UK Manchester Institute, Transriptional Networks in Lung Cancer, Manchester, United Kingdom Background: The KRAS oncogene regulates gene expression through multiple molecular mechanisms, and its dysregulation culminates in tumour progression. MicroRNAs (miRNAs) are small non-coding RNAs that have been established as main players in tumorigenesis. Material and Methods: To investigate whether KRAS was able to directly modulate microRNA expression we overexpressed both KRAS WT and KRAS G12D in NSCLC cells. Results: Two microRNAs, miR-30c and miR-21, were significantly modulated by both. We show that miR-30c and miR-21 overexpression induces cell proliferation, cell cycle progression and migration/invasion via inhibiting important tumour suppressor genes, such as NF1 and RASA1. In clinical specimens of NSCLC, miR-30c and miR-21 inversely correlated with the respective targets. Conclusions: In summary, our study defines that miR-30c and miR-21 silencing may be beneficial for future therapeutic applications in NSCLC. No conflict of interest. 403 TGF-b induced stemness acquisition using next-generation sequencing in lung cancer S.J. Kim1 , N. Kang2 , Y.K. Kim1 . 1 Department of Internal Medicine- Seoul St. Mary’s Hospital, College of Medicine- The Catholic University of Korea, Seoul, South Korea, 2 The Cancer Research Institute, College of MedicineThe Catholic University of Korea, Seoul, South Korea Transforming growth factor-b (TGF-b) is known to inhibit cell growth in benign cells but promotes tumor invasion and metastasis by inducing an epithelial–

mesenchymal transition (EMT). EMT is a differentiation switch through which epithelial cells differentiate into mesenchymal cells. It occurs in the process of tissue morphogenesis during development, wound repair and cancer progression in adult tissues. EMT is often associated with acquisition of stemlike characteristics. In this study, we investigated whether EMT induced by TGF-b could acquire stem-like characteristics in lung cancer. Human normal epithelial (BEAS-2B) and cancer (A549, H292, H226 and H460) cell lines were incubated with 10 ng/ml of TGF-b for 3 days. Transcriptome and methylation analysis of BEAS-2B and A549 cells treated with TGF-b were performed by using next-generation sequencing (Hiseq2500 system). Western blotting was performed to analyze the expression of epithelial marker (E-cadherin) and mesenchymal markers (fibronectin, vimentin, N-cadherin and a-SMA). RT-PCR was performed to analyze the expression of variable stem cell markers (CD44, CD133, CXCR4, ABCG2, CD117, ALDH1A1, EpCAM, CD90, Oct4, Nanog, SOX2, SSEA4, and CD166). Wound healing assay, Matrigel invasion assay and sphere formation assay were used to assess functional characteristics of EMT and stemness acquisition. Next-generation sequencing revealed significant changes in the expression of stem cell markers, CD44, ALDH1A1 and CD90 in both BEAS-2B and A549 cells. The changes in the expression of EMT and stem cell markers induced by TGF-b were variable according to lung cell lines. Except for H460 cell line, lung cell lines showed at least one or more increased stem cell markers expression with TGF-b. Functional analysis revealed increased wound healing, Matrigel invasion and sphere formation after TGF-b treatment. TGF-b induced EMT was associated with acquisition of stem-like characteristics. Various expression patterns of stem cell marker were observed according to different lung cancer cell lines. Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2014R1A2A1A11052422). No conflict of interest. 404 Acriflavine inhibits the epithelial-to-mesenchymal transition in vitro in liver and pancreatic cancer cells: restoring the phenotype and drug sensitivity and reducing ATF4 mediated stress response A. Bulle1 , J. Dekervel1 , P. Windmolders1 , L. Lambrechts2 , E. Van Cutsem3 , C. Verslype3 , J. Van Pelt1 . 1 KU Leuven, Hepatology, Leuven, Belgium, 2 KU Leuven, VIB − Oncology, Leuven, Belgium, 3 KU Leuven, Digestive Oncology, Leuven, Belgium Background: Epithelial-to-mesenchymal transition (EMT) is considered an important driving mechanism behind aggressive cancer phenotype. The EMT program is largely dependent on the cell’s microenvironment. Acriflavine (ACF) is a heteroaromatic dye with antibacterial and antiviral effects. Recently, ACF was suggested as anticancer agent and further blocks the hypoxia-inducible factor (HIF) pathway, an important driver of cancer aggressiveness. How ACF works in cancer is however unknown. Aim: Identification of the working mechanism, molecular pathways and signaling of ACF in EMT cancer cells. Methods: Three in vitro models were developed of EMT induction: (1) Panc-1: human pancreatic cancer cells stimulated with TGF-b1, (2) severe hypoxia: Panc-1 with CoCl2 and (3) Drug resistance against sorafenib in human liver cancer HepG2. Cell models were characterized on morphology, functional (invasion and migration), protein and transcriptome analysis and for drug sensitivity (IC50). Cells were treated with EMT inducers in combination with ACF. Results: – ACF inhibits EMT induction by TGF-b1, hypoxia or acquired drug resistance. Cell-cell contact was lost together with acquisition of a spindleshaped morphology in all models. Cells acquired enhanced invasive capacities. Gene and protein expression studies of EMT markers confirmed downregulation of CDH1 and upregulation of SNAI1. Co-treatment with ACF the Panc-1 cells partially regained their epithelial characteristics, including closer cell-cell contact. Drug resistant HepG2S1 cells underwent marked changes in morphology, invasive capacity and gene and protein expression compared to parental HepG2 cells, compatible with pronounced EMT. ACF strongly inhibited the invasive potential of stimulated Panc-1 cells and HepG2S1 cells with upregulation of epithelial markers and downregulation of mesenchymal markers. – Acriflavine reactivates interferon signaling inhibited by TGF-b1. Based on the transcriptome data of the TGF-b1 model, both the iRegulon tool and GSEA identified the mediators of interferon signaling as key transcription factors in the working mechanism of ACF. – ACF treatment of unstimulated Panc-1 and HepG2S1 cells leads to strong inhibition of the unfolded protein response (UPR). GSEA and the iRegulon tool pointed towards either ATF4, ATF3 or CEBPB as main transcription factors involved in genes down-regulated upon ACF treatment. – Acriflavine restores drug sensitivity. Treating resistant HepG2S1 cells with an on itself non-toxic concentration of ACF leads to a significant enhancement of sorafenib sensitivity.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Conclusions: There is a general molecular mechanism by which Acriflavine blocks EMT-aggressive cancer cells, independent of the way the cells were stimulated. We could identify as most prominent pathways: interferon signaling and UPR. No conflict of interest. 405 Depletion of g-glutamylcyclotransferase inhibits cancer cell growth via cellular senescence caused by CDK inhibitor induction K. Matsumura1 , N. Susumu1 , I. Hiromi1 , A. Eishi2 , K. Susumu3 , K. Akihiro3 , Y. Tatsuhiro1 . 1 Kyoto Pharmaceutical University, Clinical Oncology, Kyoto, Japan, 2 Kyoto Pharmaceutical University, Clinical and Translational Physiology, Kyoto, Japan, 3 Shiga University of Medical Science, Urology, Shiga, Japan Background: Chromosome 7 open reading frame 24 (C7orf24) was originally identified by a proteomic analysis on clinical samples as a highly expressed protein in various types of cancer, and later shown to be a gglutamylcyclotransferase (GGCT), a major enzyme involved in glutathione metabolism. As it has been shown that higher levels of GGCT expression are associated with poorer clinical outcomes and knockdown of GGCT inhibits cancer cell growth in vitro and in vivo, GGCT is considered as a promising candidate for a novel therapeutic target. However, the cellular events caused by GGCT depletion have not been fully characterized, and the mechanisms underlying the growth inhibition of cancer cells remain elusive. Material and Methods: GGCT was depleted using siRNAs in MCF7, MDA-MB-231, PC3, A172, Hela, and LNCaP cells. Induction of cellular senescence was evaluated with senescence-associated b-galactosidase (SA-b-Gal) staining. Expression levels of p21WAF1/CIP1 and p16INK4A were assessed by qRT-PCR and Western blotting. Effects of double knockdown of p21WAF1/CIP1 or p16INK4A together with GGCT on cell cycle regulation and cell growth were assessed by flow cytometry, and trypan blue dye exclusion test. Results: GGCT knockdown induced G0/G1 cell cycle arrest and suppressed proliferation, without evident induction of cell death by 4 days post siRNA transfection, despite undergoing morphological changes into flat and enlarged shapes. Thereafter, from 6 to 7 days post transfection, the ratio of dead cells drastically increased, without activation of apoptotic pathway. We found that GGCT knockdown induces significant cellular senescence in the multiple cancer cell lines. Cyclin dependent kinase (CDK) inhibitor p21WAF1/CIP1 and/or p16INK4A were upregulated in all cell lines tested. We identified p21WAF1/CIP1 as a significantly upregulated gene in GGCT-depleted MCF7 breast cancer cells. Simultaneous knockdown of p21WAF1/CIP1 significantly decreased induction of cellular senescence and rescued subsequent growth inhibition, demonstrating that the cellular events are dependent on the p21WAF1/CIP1 upregulation. In contrast, in GGCT silenced MDA-MB-231 breast cancer cells, p16INK4A instead of p21WAF1/CIP1 was induced and played a regulatory role in induction of senescence, suggesting cellular context dependent induction of CDK inhibitors. Conclusions: Our findings demonstrate that induction of cellular senescence mediated by the upregulation of CDK inhibitors is a major event underlying the anti-proliferative effect of GGCT depletion, highlighting the potential of GGCT blockade as a therapeutic strategy to target a key enzyme in cancer cells involved in amino acid metabolism to induce cellular senescence. No conflict of interest.

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induces apoptosis, while sunitinib and CQ alone do not. Knocking down ATG5 and ATG7 as well as lysosome-associated membrane protein 2 (LAMP2) by lentiviral delivery of shRNA confirmed the results obtained with pharmacological autophagy inhibitors. Silencing of these ATG genes and of LAMP2 leads to slightly reduced clonogenic potential, however addition of sunitinib potentiates this effect. Cell death is not observed under control conditions, but treatment with sunitinib leads to apoptosis in LAMP2 knockdown cells. Additionally, preliminary data in vivo indicates that the combination treatment is more effective than the single treatments. Conclusion: Our data clearly indicate that autophagy inhibition in combination with sunitinib might be beneficial for the treatment of pNETs. No conflict of interest. 407 Histone deacetylases-1 promotes urothelial cell migration and invasion by modulating p63/Rho-kinase-1/pMLC2 signalling K. Srivastava1 , V. Smith1 , C. Breen1 , K. McCloskey1 . 1 Queen’s University Belfast, Center For Cancer Research and Cell Biology, Belfast, United Kingdom Background: Upregulation of histone deacetylases (HDAC) and loss of the transcription factor p63 are associated with poor prognosis in high grade urothelial tumours. The current study focused on epigenetic modification of p63 and its downstream effects during the progression of invasive urothelial carcinomas. Material and Methods: Primary and immortalised human urothelial cells (HUC, SV-HUC), and bladder cancer cell lines (T24, HT1376) were cultured either on human foreskin fibroblast-embedded collagen-I to establish 3Dorganotypic rafts of urothelium or as 2D monolayers. Scratch wound assay was used to study the rate of cell migration in aforementioned cells before harvesting total RNA and protein to study the differential expression of HDAC1,2,3, p63, p21, Rho-kinase1 (ROCK1) and myosin light chain-2 (MLC2). Results: The 3D-organotypic urothelium rafts exhibited distinct biology with invasive incidents observed only with T24 cells. In contrast, non-invasive HT1376 cells had a significantly higher number of tumour formations. These observations were consistent with the rate of T24 cell migration which was enhanced compared to the other cell lines. qPCR and Western blotting demonstrated enhanced expression of HDAC1 and HDAC3 with depleted levels of p63, ROCK1, pMLC2-Ser19, total MLC2 and p21 in both cancer cell lines compared to normal cells (N = 3, p < 0.05). These results coincided with the loss of stress fibre formations in the cancer cell lines, visualised by immunofluorescence detection of F-actin, indicating that cytoskeletal reorganization may be pivotal for cell migration. Exposure to HDAC inhibitors (vorinostat (pan) and entinostat (HDAC1, 3)) restored p63 mRNA and protein expression (N = 3, p < 0.05) in the cancer cells and attenuated the rate of cell migration in the invasive T24 cells. Transient knockdown of HDAC1 in T24 cells mimicked the results with HDAC inhibitors by restoring the mRNA/protein levels of p63, ROCK1, pMLC2-Ser19 and p21 (N = 3, p < 0.05), while successive depletion of p63 abolished the effects of HDAC1 knockdown. Conclusions: Upregulation of HDAC1 promotes cell migration and invasion by modulating p63-ROCK1/pMLC2 signalling-evoked cytoskeletal remodelling in bladder cancer cells. HDAC1 inhibitors restored p63 expression while mitigating cell migration, highlighting their therapeutic potential in bladder cancer. No conflict of interest.

406 Targeting autophagy in pancreatic neuroendocrine tumors T. Wiedmer1 , M.P. Tschan1 , A. Perren1 , I. Marinoni1 . 1 Institute of Pathology, Department of Experimental Pathology, Bern, Switzerland Introduction: Pancreatic neuroendocrine tumor (pNET) patients often display primary or secondary resistance to sunitinib, an anti-angiogenic multi-receptor tyrosine kinase inhibitor, which is approved for the treatment of late stage pNETs. Sunitinib targets mainly endothelial cells and pericytes. However, subsets of pNETs show expression of target receptors not only in the vasculature but also in the tumor cells. Additionally, sunitinib accumulates in lysosomes. Cellular stress caused by sunitinib treatment might increase autophagy in pNETs through a direct mechanism in the cancer cells or through induction of hypoxia in the microenvironment. Since autophagy can promote cell survival in unfavorable environments or upon treatment, we hypothesize that autophagy inhibition might increase therapy efficacy of sunitinib. Material and Methods: Two different pNET cell lines are used for in vitro experiments. In the in vivo experiment Rip1Tag2 mice are treated daily for three weeks with the autophagy inhibitor chloroquine (CQ), sunitinib or their combination. Results: In vitro we found that sunitinib significantly induces autophagy. Combining sunitinib with CQ significantly reduces cell viability when compared to sunitinib treatment alone. Importantly, combining sunitinib and autophagy inhibition reduces the recovery upon removal of the treatment by 50%, whereas the single treatment does not show any differences in colony numbers as compared to control treated cells. The combination treatment

408 Development of ex vivo phenotypic assays to enable precision medicine in the context of high grade serous ovarian cancer L. Nelson1 , A. Tighe1 , A. Clamp2 , R. Edmondson3 , G. Jayson3 , S. Taylor1 . 1 University of Manchester, MCP, Manchester, United Kingdom, 2 Christie Hospital, Medical Oncology, Manchester, United Kingdom, 3 University of Manchester, Insitute of Cancer Sciences, Manchester, United Kingdom Introduction: Precision medicine in the context of cancer chemotherapy requires gleaning sufficient information about a patient’s tumour to then decide which drug regimens to adopt. These decisions are typically based on the mutated oncogenic and/or tumour suppressor pathways driving the tumour. However, not all cancers are driven by actionable mutations, especially high grade serous ovarian cancer (HGSOC) where beyond p53 mutation, common driver mutations are rare. Moreover, not all drivers are betrayed by easilyidentifiable mutations. For example, while ~50% of HGSOCs are predicted to have homologous recombination defects, and thus benefit from PARP inhibitors, only ~22% harbour lesions at the BRCA1/2 loci. We therefore aim to establish an array of ex vivo phenotypic assays to measure the robustness of various cell cycle processes, including mitotic checkpoints, p53 responses and DNA damage repair pathways. As a first step, we set out to establish procedures that permit facile isolation of primary ovarian cancer cells, plus culture conditions suitable for high resolution cell cycle analysis. Methods: Under the auspices of the Manchester Cancer Research Centre Biobank, anonymised ascites from chemotherapy naive patients were collected

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then processed on the same day. Cells were isolated by centrifugation and subjected to an enzymatic disaggregation process. Tumour and stromal cells were then separated using magnetic beads, plated into OCMI media (Ince et al (2015) Nat Comm), then cultured under low oxygen conditions. Following passage, populations were analysed by flow cytometry, immunofluorescence microscopy and TP53 genotyping. Both populations were then analysed by time-lapse microscopy approaches to analyse cell cycle parameters. Results and Discussion: Flow cytometry and immunofluorescence microscopy revealed a tumour cell population positive for EPCAM and CA125 and a stromal population positive for CD105, CD44 and Vimentin. Sequencing TP53 transcripts identified mutations in the tumour population but not the stromal fraction, confirming the veracity of the magnetic bead separation. Time-lapse microscopy and serial passage showed that the tumour and stromal fractions had extensive proliferative potential, thus confirming that the OCMI/low oxygen combination facilitates propagation of primary ovarian cancer cells suitable for high resolution cell cycle analysis. No conflict of interest. 409 Neural crest derived progenitors give rise to tumor stroma and contribute to aggressiveness in high-risk neuroblastoma P. Linares-Clemente1 , D. Aguilar-Morante1 , R. Pardal1 . 1 Instituto de Biomedicina de Sevilla IBiS, Medical Physiology and Biophysics. Univ. of Seville., Seville, Spain Background: Pediatric tumors arise during embryonic development within growing tissues full of stem/progenitor cells. Given this scenario, the existence of non-transformed stem cells included within the aberrant tumoral niche, with a potential role in tumor biology, is an intriguing and unstudied possibility. Material and Methods: We established stem cell cultures from tumor samples of Neuroblastoma (NB) patients. We characterized the cells and determined their tumorigenicity by xenotransplanting them in SCID mice. Results and Discussion: We describe the presence and function of nontransformed neural crest-derived progenitor cells in high-risk NB tumors. These cells are able to differentiate into neural crest typical mesectodermal derivatives, such as smooth muscle actin-positive (aSMA+) myofibroblasts, giving rise to tumor stroma and promoting proliferation and tumor aggressiveness in a mouse model of the disease. Furthermore, an analysis of gene expression profiles in high-risk NB reveals a neural crest stem cell (NCSC) gene signature that is associated to stromal phenotype, expression of ACTA2 (encoding for aSMA) and high probability of relapse. Therefore, we propose NCSC gene expression signature and aSMA expression as new prognostic criteria to improve stratification of high-risk NB tumors. Conclusions: High-risk NB tumors might contain a population of nontransformed NCSCs that would be able to undergo mesectodermal differentiation, contribute to angiogenesis, and therefore promote aggressiveness and tumor growth. Our results might facilitate the design of new therapies against a subset of high-risk NBs, by targeting NCSCs and their contribution to stroma-dependent processes such as immune response, cell migration or angiogenesis. Acknowledgement: This research has been supported by ‘Asociacion ´ Espanola ˜ contra el Cancer’ ´ (AECC), Spanish Ministry of Science (SAF), and European Union (ERC). No conflict of interest. 410 ALDH1 expression and activity increase during tumor evolution in sarcoma cancer stem cell populations L. Martinez-Cruzado1 , J. Tornin1 , L. Santos1 , A. Rodriguez1 , R. Rodriguez1 . Hospital Universitario Central de Asturias, IUOPA, Oviedo, Spain

analysis. Gene expression profiling of CSC-related genes was analyzed using The Human Cancer Stem Cells RT2 Profiler PCR Array (SABiosciences). ALDH activity was assayed by the Aldefluor® assay (StemCell Technologies). In vitro transformation properties were analyzed in soft agar colony formation assays. In vivo tumor formation ability was analyzed after the inoculation of different subpopulations into inmunodeficient mice in limiting dilution assays. All animal research protocols were approved by the Animal Research Ethical Committee of the University of Oviedo. Results and Discussion: In vivo tumor formation assays showed that the tumorsphere cultures from xenograft-derived cells, but not from the cell-oforigin models, were enriched in CSCs, evidencing the emergence of bona fide CSCs subpopulations during tumor progression. Relevant CSC-related factors, like ALDH1 and SOX2, were increasingly upregulated in CSC subpopulations during tumor progression and importantly, the increased levels and activity of ALDH1 in these subpopulations was associated to enhanced tumorigenicity. Conclusion: Our findings indicate that, besides being used as a CSC marker, ALDH1 could also be useful to track the malignant potential of CSC subpopulations during sarcoma evolution. No conflict of interest. 411 Notch3–EGFR crosstalk in triple negative breast cancers (TNBC): new therapeutic possibilities G. Diluvio1 , F. Del Gaudio2 , G. Franciosa1 , M.V. Giuli1 , M.P. Felli3 , D. Bellavia1 , I. Screpanti1 , S. Checquolo4 . 1 “Sapienza” University of Rome, Medicina Molecolare, Rome, Italy, 2 Karolinska Institute, Department of Cell and Molecular Biology, Stockolm, Sweden, 3 “Sapienza” University of Rome, Experimental Medicine, Rome, Italy, 4 “Sapienza” University of Latin, Medico-Surgical Sciences and Biotechnology, Latin, Italy Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease that does not express therapeutically targetable ER, PR or HER2 receptors making this aggressive subtype difficult to treat. Several therapies are being developed that target specific biomarkers of TNBC, including several tyrosine kinase inhibitors (TKIs) targeting EGFR, since EGFR expression is approximately seen in 60% of TNBCs. Unfortunately, TKIs have shown only limited clinical activity against breast cancers, often due to the existence of compensatory pathways that confer resistance to EGFR inhibition, such as Notch receptor signalling. Indeed, increasing evidence shows that both Notch and EGFR pathways are used by TNBC cells to escape EGFRtargeted inhibition. In the present study we investigated in more details the mechanism(s) by which Notch and EGFR crosstalk occurs in TNBC, focusing our attention at the level of cell membrane rafts compartment, where EGFR localized in TNBC TKIs resistant cells. Material and Method: Cell coltures and drugs treatments; Western blot analysis; Biochemical rafts isolation; Immunofluorescence assay; FACS analysis; RT-PCR/qRT-PCR; siRNA silencing; Proliferation assay. Results: We firstly demonstrated that both EGFR and Notch3 receptor colocalized in the rafts compartment of some TNBC resistant cells. Interestingly, when we silenced Notch3, as well as lipid rafts were depleted of cholesterol using lovastatin, TKI resistant cells were sensitized to gefitinib drug, suggesting a close relationship between EGFR and Notch3 receptor starting at the cell surface membrane, where lipid rafts may provide a platform to facilitate the activation of downstream pathways in the absence of EGFR kinase activity. Conclusions: Our data provide the molecular basis for improved screening for the expression of these oncogenes enabling patients to optimize their personal treatment options and predict potential treatment resistance. No conflict of interest.

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Background: Tumors evolve from the initial tumorigenic events to increasingly aggressive behaviors in a process usually driven by cancer stem cells (CSCs) subpopulations. Mesenchymal stromal/stem cells (MSCs) may act as the cell-of-origin for sarcomas, and CSCs presenting MSC features have been identified in many types of sarcomas. Accordingly, we have previously developed sarcoma models using human MSCs transformed with relevant oncogenic events (cell-of-origin models). The process of emergence/evolution of CSC subpopulations from the cell-of-origin in sarcomagenesis has been barely addressed. Here, we have compared the tumorigenic properties of bulk cultures and CSC-enriched subpopulations both in the sarcoma cell-of-origin models and in their corresponding tumor xenograft-derived cells to analyze the evolution of CSCs subpopulations during tumor progression. Material and Methods: We used a panel of transformed bone marrowderived human MSCs able to initiate sarcomas [spindle cell sarcoma (MSC5H-GFP and MSC-5H-O cells) and myxoid liposarcoma (MSC-4H-FC and MSC-5H-FC cells)]. In addition we used several cell lines derived from xenograft tumors generated by these cell-of-origin models (T-4H-FC#1, T-4HFC#3, T5H-GFP#1, T5H-FC#1 and T5H-O). CSC subpopulations from these cell types were enriched by their ability to grow as self-renewed floating spheres (tumorspheres). The expression of different proteins was analyzed by Western Blotting, immunofluorescence staining or immunohistochemical

412 Phenotypic and miRNA transcriptomic evaluation of cellular tumour spheroid as model of breast cancer stem cell studies W.Y. Ho1 , S.K. Yeap2 . 1 The University of Nottingham Malaysia Campus, Biomedical Sciences, Semenyih, Malaysia, 2 Universiti Putra Malaysia, Institute of Bioscience, Serdang, Malaysia Cellular tumour spheroids derived from breast cancer cell lines MCF-7 and MDA-MB-231 have been widely utilised as a model for breast cancer stem cell (CSC). In most studies, breast CSC phenotype is distinguished or characterised via expression of CD44/CD24, ALDH1 and/or stem cell markers such as Sox-2 and Nanog. However, the suitability and reliability of using these spheroids as a paradigm for breast CSC remains elusive. In this study, we cultivated spheroids from MCF-7 and MDA-MB-231 using serumdepleted media for enrichment of CSC. We then evaluated CSC properties in the spheroids via a few parameters including morphological assessment, drug resistance, spheroid formation efficiency, expression of stem cell-related markers as well as miRNA profiling. Cellular characterisation showed cell renewal capacity and elevated resistance against tamoxifen in both types of spheroids. CD44+CD24− expression and ALDH1 activity were elevated. Cells dissociated from the spheroids were also capable of forming secondary spheroids, with MCF-7 developing more compact spheres as compared to MDA-MB-231. While exhibiting similar trend in cellular characteristics, miRNA

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 expression profile was different between the two cell lines. Only 20 miRNAs were regulated similarly in these spheroids. Stem cell related pathways were found to be differentially regulated between the two types of spheroids as shown by gene ontology prediction of functional annotations based on their miRNA expressions. To illustrate a few, insulin signalling pathway was found to be dysregulated in MCF-7 spheroids but not in MDA-MB-231 spheroids while the latter was predicted to regulate Wnt and Notch signalling pathways distinctively. From these findings, we conclude that breast cancer spheroids from MCF-7 and MDA-MB-231 could serve as reliable models for CSC related studies. Additionally, miRNA profiling of the spheroids demonstrated differential regulation of CSC related pathways between the different breast cancer cell lines. This also highlights the need for prudent selection of cell type in future research of targeted CSC therapies in varied subtypes of breast cancer. No conflict of interest. 413 The human L-asparaginase ASPG is cytotoxic for leukemic cells S. Belviso1 , M. Menniti2 , N. Perrotti2 , R. Iuliano1 . 1 Universita’ degli Studi “Magna Græcia” di Catanzaro, Medicina Sperimentale e Clinica, Catanzaro, Italy, 2 Universita’ degli Studi “Magna Græcia” di Catanzaro, Scienze della Salute, Catanzaro, Italy Background: L-Asparagine is an essential amino acid for the growth of tumor cells, in particular leukemic cells, that do not express a sufficient amount of L-asparagine synthetase. Thus, bacterial asparaginases, which are enzymes that hydrolyze L-asparagine into L-aspartate and ammonia, were used from early seventies of the past century in the treatment of acute lymphoblastic leukemia. Despite their therapeutic efficacy, bacterial asparaginases show some side effects caused by immune reactions. In this study, we investigated the antitumor potential of human ASPG, also named 60 kDa Lysophospholipase, a protein with asparaginase activity that might become suitable for replacing bacterial enzymes used in the treatment of leukemias. Material and Method: Recombinant bacterial vectors, expressing GSTtagged ASPG and GST-tagged inactive ASPG-T19A mutant, were generated. Expression of recombinant proteins was induced by isopropyl b-D-1thiogalactopyranoside in BL21 E.Coli and proteins were then extracted and purified by glutathione sepharose beads. L-Asparaginase activity was measured by Nesslerization method. Human leukemia K562 and NALM-6 cell lines and Human Dermal Fibroblasts-adult cells (HDFa) were used as cellular models. Cytotoxicity was determined by tripan blue exclusion method and Cell Counting Kit-8 assay. Results: We detected a significant L-asparaginase activity, with a time dependent ammonia release, in purified GST-ASPG but not in GST-ASPGT19A. The kinetic data were fitted with Hill equation from which we estimated a Hill slope of 6.9 and a S0.5 of 13 mM. Cancer cell lines were treated with serial dilutions of GST-ASPG or GST-ASPG T19A (from 100 nM to 0.01 nM) for 24 hours and then cytotoxicity was measured and compared with GST treated cells. GST-ASPG but not GST-ASPG T19A decreased cell viability in a dose dependent manner in the cancer cell lines tested (IC50 in K562 = 6.30 nM, IC50 in NALM-6 = 1.34 nM). GST-ASPG, at the same concentrations used for cancer cells, did not affect cell viability of HDFa cells. Conclusion: GST-ASPG significantly decreases the viability of leukemia cell lines by its asparaginase activity and it may be suitable for replacing bacterial enzymes approved for anticancer therapy. No conflict of interest. 414 Runt-related transcription factor 2 (Runx2) is responsible for galectin-3 overexpression in human thyroid carcinoma E. Kaptan1 , S. Sancar Bas1 , A. Sancakli1 , H. Gumushan Aktas2 , S. Bolkent1 . 1 Istanbul University Faculty of Science, Department of Biology, Istanbul, Turkey, 2 Harran University Art and Science Faculty, Department of Biology, Sanliurfa, Turkey Background: Runx2, which is a transcription factor regulating gene expression during bone formation, is overexpressed in various types of cancer to activate metastasis to bone. Its overexpression has been also shown in thyroid cancers. Runx2 promotes metastatic ability of cancer cells by directly activating matrix metalloproteinase (MMP) genes in particular. Galectin-3 (Gal-3) extensively expressed in normal and transformed cells and it is responsible for many cellular processes. Gal-3 overexpression in various cancer cells strongly correlates with metastatic potential. Additionally, galectin-3 overexpression has been revealed in the thyroid cancers having high malignant character and, proposed as a powerful marker for its diagnosis. In this study is aimed to investigate whether there is any relationship between runx2 transcription factor and regulation of galectin-3 expression in different human thyroid carcinoma cell lines. Material and Methods: To show effects of runx2 transcription factor on gal-3 expression, we developed Runx2 knockdown model in the thyroid carcinoma cell lines; anaplastic 8505C and 8305C and, papillary TPC-1 by using siRNA transfection. We analyzed gal-3 protein expression levels in the runx2-silenced

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cell lines using western blotting. Also we evaluated protein expression levels of MMP-2 and 9 recognizing gal-3 as a substrate in the same cells. Results: We detected runx2 and gal-3 protein expressions in the all cell lines that we used. The protein expression level of gal-3 was downregulated in runx2-silenced 8305C and TPC-1 cell lines. However, changings in gal-3 protein expression level in runx2 silenced 8505C cell line were not evident. Researchers pointed out that runx2 regulated gal-3 expression in human pituitary tumors and glioma cells. In this investigation, we revealed that regulation of gal-3 expression is strongly associated with runx2 transcription factor in human thyroid carcinoma. Conclusion: Human MMPs recognizes human galectin-3 as a substrate and cleaves it at the specific amino acid site. This cleavage of galectin-3 by metalloproteinases strongly improves its binding interaction to laminin and endothelial cells. These results show that galectin-3 contributes to cell aggregation leading to cancer cell metastasis. Therefore, regulation of galectin-3 expression through Runx2 seems like one of the key regulatory mechanism for malignant potential of thyroid carcinoma. No conflict of interest. 415 Notch3 sustains CXCR4 expression in acute T cell lymphoblastic leukemia progression F. Ferrandino1 , G. Bernardini1 , P. Grazioli1 , A.F. Campese1 , D. Bellavia1 , S. Checquolo2 , I. Screpanti1 , M.P. Felli3 . 1 “La Sapienza” University of Roma, Department of Molecular Medicine, Roma, Italy, 2 “La Sapienza” University of Roma, Department of Medico-Surgical Sciences and Biotechnology, Latina, Italy, 3 “La Sapienza” University of Roma, Department of Experimental Medicine, Roma, Italy Introduction: Acute T cell lymphoblastic-leukemia (T-ALL) is the most common of childhood cancers. The majority of T-ALL cases are driven by Notch deregulated signaling, that activates different downstream pathways essential for TALL cell proliferation and leukemia-initiating cell (LIC) activity. The oncogenic function of Notch3 in T-ALL was demonstrated by a murine model of our laboratory, characterized by enforced expression of the Notch3 receptor active form (N3-IC) in immature thymocytes (N3-ICtg). Aberrant proliferation and maturation at the preT/T transition phase and constitutive activation of preTCR and NF-kB signalings were observed in N3-ICtg mice. Multiple signals from stroma sustain T cell differentiation programs in the thymic niche. Cooperative signaling among the preTCR, CXCR4 and Notch are required at b selection for the continued differentiation from Double Negative (DN) to Double Positive (DP) thymocytes. The stromal cell derived factor SDF-1 (CXCL12) and its receptor CXCR4 promote survival of DN thymocytes and regulate the migration during the DN/DP transition. The CXCR4/SDF-1 axis has been recently suggested to play a role in the pathogenesis of T-ALL. Material and Methods: CXCR4 and Notch3 cell-surface evalution was performed by FACS analysis with freshly isolated DP T cells obtained from thymus, blood, spleen and bone marrow of N3-ICtg and wt mice. Statistical analysis of the data was performed. Biochemical analysis of the modulatory effect of Notch3 overexpression in TALL1, a human leukemic CD3+/CD4+/CD8+ cell line, was performed by FACS, RealTime-PCR and Western Blot. Results and Discussion: In Notch3-ICtg mice, DP-gated thymocytes display an increased level of CXCR4 expression per cell with respect to wt. Furthermore, most of the DP thymocytes highly co-express Notch3 and CXCR4. Abnormally represented DP T cells at different ages in lymphoid organs of N3-ICtg show a combined Notch3/CXCR4 expression suggesting a crosstalk of the two receptors, possibly modulating their leukemogenicity. Through inhibition of Notch signaling (GSI treatment) or by silencing Notch3 gene expression (shRNA) we could modulate CXCR4 cell-surface expression in TALL1 (CD4+/CD8+/CD3+ leukemic cell line). Conclusion: Overall, our results are suggestive of Notch3 deregulated pathway as a molecular mechanism that may modulate DP T cells egress from thymus, in early steps of T-ALL development, by forcing CXCR4 cell-surface expression through a beta-arrestin-mediated mechanism. No conflict of interest. 416 Identification of novel cancer biomarkers of prognostic value using specific gene regulatory networks D. Chudasama1 , V. Bo2 , M. Hall3 , V. Anikin4 , G. Pados5 , A. Tucker2 , E. Karteris6 . 1 Brunel Univesity London, Life Sciences, Uxbridge, United Kingdom, 2 Brunel Univesity London, Computer Sciences, Uxbridge, United Kingdom, 3 Mount Vernon Hospital, Mount Vernon Cancer Centre, Northwood, United Kingdom, 4 Royal Brompton & Harefield NHS Foundation Trust, Division of Cardiothoracic Surgery, Harefield, United Kingdom, 5 University of Thessaloniki, Medical School, Thessaloniki, Greece, 6 Brunel University London, Life Sciences, Uxbridge, United Kingdom Background: Ovarian cancer is the second leading cause of death among gynaecologic malignancies in the world, with the majority of patients experiencing a recurrence. Microarray analyses of ovarian cancer patients over

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the past years have led to the discovery of a number of “molecular signatures” associated with this cancer. However, adoption of these multi-gene signatures in the clinical environment for diagnostic or prognostic testing still remains controversial. Moreover, there are limitations including data mining, statistical analyses and clinical sample acquisition. Material and Methods: Using microarray data of multiple cancer studies and a combination of glasso and bayesian networks, we first derived a uniquenetwork for ovarian cancer and then identified the genes that are uniquely involved in this mechanism. We have validated the expression of the genes derived in ovarian cancer and controls and expanded the observations in lung and breast cancer using qRT-PCR. In order to examine putative prognostic biomarkers in ovarian cancer we have used the OvMark software. Results: Unique networks identified 4 novel markers for ovarian cancer: small proline-rich protein 1A (SPRR1A), follistatin like 1 (FSTL1), collagen type XII alpha 1 (COL12A1) and RAD51 associated protein 1 (RAD51AP1). Using clinical samples, we demonstrate that RAD51AP1 and FSTL1 are significantly overexpressed in ovarian cancer patients compared to controls. Interestingly, OvMark predicted poorer overall survival (OS) of ovarian cancer patients with high expression of both RAD51AP1 and FSTL1. We expanded on these observations, assessing the relative expression of these four genes in lung and breast cancers. RAD51AP1, COL12A1 and SPRR1A were significantly overexpressed in lung when compared to ovarian cancer, and FSTL1 was significantly overexpressed in ovarian compared to lung cancer and breast cancer. Conclusions: Collectively, our data described the generation of specific biomarkers that could potentially support current clinical practice and improve long term outcomes. No conflict of interest. 417 Somatic MED12 exon 1 nonsense mutation in T-cell acute lymphoblastic leukemia escapes nonsense-mediated mRNA decay and prevents protein nuclear localization K. Kampj ¨ arvi ¨ 1 , T. Heikkinen1 , S. Keskitalo2 , P. Von Nandelstadh3 , 1 V. Rantanen4 , E. Pitkanen ¨ , K. Lehti5 , S. Mustjoki6 , M. Varjosalo2 , P. Vahteristo1 . 1 University of Helsinki, Research Programs Unit- GenomeScale Biology and Medicum- Department of Medical and Clinical Genetics, Helsinki, Finland, 2 University of Helsinki, Institute of Biotechnology, Helsinki, Finland, 3 University of Helsinki, Research Programs Unit- Genome-Scale Biology and Medicum- Department of Pathology, Helsinki, Finland, 4 University of Helsinki, Research Programs Unit- Genome-Scale Biology and Institute of Biomedicine, Helsinki, Finland, 5 University of Helsinki, Research Programs Unit- Genome-Scale Biology and Finnish Cancer Institute, Helsinki, Finland, 6 University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, Hematology Research Unit Helsinki- Department of Hematology- Department of Clinical Chemistry, Helsinki, Finland Introduction: Mediator complex subunit 12 (MED12) is a component of the Mediator complex that regulates RNA pol II-dependent transcription. Somatic mutations in the 5 end of X chromosomal MED12 gene are highly frequent in hormone-dependent female tumors: uterine leiomyomas and breast fibroadenomas and phyllodes tumors. All observed mutations have been missense mutations or frame retaining insertions and deletions in exons 1 and 2. Interestingly, similar mutations have been observed with lower frequencies also in chronic lymphocytic leukemia (CLL). Somatic nonsense mutation in MED12 5 end was recently observed in a patient with T-cell acute lymphoblastic leukemia (T-ALL). The mutation (c.97G>T) affects last amino acid of exon 1, E33, which is a mutational hotspot in CLL but not in female tumors with recurrent MED12 mutations. Materials and Methods: Analysis of patient sample indicated the mutation to be expressed on a transcriptional level. To analyze the functional effects of the mutation, we generated stable Flp-In 293 T-Rex cell lines expressing MED12 wild type and mutant derivatives E33X, E33K (CLL hotspot), and G44D (uterine leiomyoma hotspot). Western blotting and immunofluorescence was used to analyze the expression and localization of the derivatives, while effects on protein-protein interactions were analyzed with proteome-wide mass spectrometry. Results and Discussion: Western blot analysis and peptide identification mass spectrometry showed that E33X transcript is able to escape nonsense mediated mRNA decay and that alternative translation initiation site is used for translation of an N-terminally truncated protein product. Nuclear localization signal (NLS) was observed to exist on this untranslated region as the localization of MED12 E33X derivative to nucleus was prevented by the mutation. In silico prediction tools and further validation with specific NLS-modified MED12 constructs confirmed the domain to be located at highly conserved region in the N-terminus. Compared to the protein-protein interactions presented by wild type MED12, both missense mutations (E33K and G44D) led to diminished association between MED12 and other kinase module components (MED13, CDK8/19 and Cyclin C). Instead, almost complete loss of interactions between MED12 and all other Mediator subunits were observed with E33X and NLS-modifying derivatives. BioID mass

spectrometry analysis of transient interactions showed reduced associations of E33X and NLS derivatives also with importin-a proteins and other components of nuclear pore complex. Conclusions: E33X containing MED12 mRNA is able to escape nonsense mediated decay and encodes an N-terminally truncated protein. Lack of this NLS-containing region alters interactions with importin molecules and prevents protein’s transportation to the nucleus. No conflict of interest. 419 Expression profiling of tumour budding cells in colorectal cancer suggests an EMT-like phenotype and molecular subtype switching L. De Smedt1 , S. Palmans1 , O. Govaere1 , J. Wouters1 , D. Barras2 , J. Dekervel3 , S. Tejpar4 , D. Lambrechts5,6 , X. Sagaert1,7 . 1 KU Leuven, Translational Cell and Tissue Research, Leuven, Belgium, 2 Swiss Institute for Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland, 3 UZ Leuven, Hepatology, Leuven, Belgium, 4 UZ Leuven, Digestive Oncology, Leuven, Belgium, 5 KU Leuven, Translational Genetics, Leuven, Belgium, 6 VIB, Vesalius Research Center, Leuven, Belgium, 7 UZ Leuven, Pathology, Leuven, Belgium Introduction: Tumour budding is referred to as the presence of single cells or small clusters of up to five tumour cells at the invasive margin of colorectal carcinoma and is considered as an additional prognostic marker besides TNMstaging. It is generally hypothesized, but not yet proven, that the feature of tumour budding is the histologic representation of epithelial–mesenchymal transition (EMT). In the present study, we aim to investigate the molecular signature of tumour budding cells and to compare it to the corresponding tumour bulk signature. Materials and Methods: Tumour bulk and budding cells were laser capture microdissected from eight fresh-frozen (FF) colorectal cancer samples and processed for RNA-sequencing. Since little RNA was obtained from budding cells, a special low-input (~1 ng) mRNA library preparation protocol was used. Gene expression profiles of tumour budding cells as compared to tumour bulk were investigated for established EMT signatures, consensus molecular subtype (CMS), gene set enrichment and pathway analysis. Results: A total of 296 genes were differentially expressed with a FDR 4 folds), but also enhanced cancer metastasis in orthotopic injection model (>3 folds). ZO-1 in CAFs seems play a pivotal role in the coupled migration of metastasis processes. Knockdown of ZO-1 in CAFs resulted in dramatically cutback the interaction between cancer cells and CAFs. Conclusions: These results propose that CAFs play imperative roles in the initiation stage of cancer metastasis via coupled migration and the direction of movement. It also implies that the interaction between cancer cells and CAFs might be a novel treatment strategy in cancer metastasis by targeting the direct contact components between cancer cells and CAFs. No conflict of interest.

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424 Glioma cell-of-origin and tumor cell plasticity L. Rousso-Noori1 , O. Pilo Kerman1 , S. Talmor1 , P. Magod1 , D. Friedmann-Morvinski1 . 1 Tel Aviv University, Biochemistry and Molecular Biology, Tel Aviv, Israel Background: Glioblastoma (GBM) is the most common and malignant type of primary brain tumor. It represents one of the deadliest human cancers, with an average survival at diagnosis of about one year. This poor prognosis is due to therapeutic resistance and tumor recurrence after surgical removal. One of the most important hallmarks of GBM is tumor heterogeneity. Intra-tumor heterogeneity is becoming the focus of research interest in the past few years, and tumor cell plasticity as a new source of cancer stem cells (CSC) has added another level of complexity to this phenomenon. Material and Method: Using a CRE-inducible lentiviral based mouse GBM model we were able to show that gliomas can originate from differentiated cells in the central nervous system [1]. In this study, primary cortical neurons and astrocytes obtained from SynapsinI-Cre or GFAP-Cre transgenic mice, respectively, were transformed using HRAS-shp53 lentivirus and reprogrammed to a cancer stem-like state. Differential gene expression analysis and transcriptional network analysis was performed on RNAseq data from these populations of cells. Results: Differential gene expression analysis identified a common set of genes up-regulated in dedifferentiated transformed neurons and astrocytes compared to their parental mature neurons and astrocytes. Pathway enrichment analysis on these differentially regulated genes revealed focal adhesion pathway to be significantly up-regulated. To assess the functional role of the up-regulated genes during dedifferentiation, we used lentiviruses expressing shRNAs to silence these genes in transformed neurons and astrocytes. Inhibition of focal adhesion up-regulated genes blocked the formation of neurospheres, affected their proliferative capacity and compromised the plasticity of these cells. Conclusion: We believe that tumor cell plasticity is an important player in GBM heterogeneity and hence inhibition of key players in dedifferentiation of glioma cells can be a therapeutic approach to treat GBM. Reference(s) [1] Friedmann-Morvinski D., Bushong EA, Ke E, Soda Y, Marumoto T, Singer O, Ellisman MH, Verma IM. Science. 2012 Nov 23; 338(6110): 1080−4. No conflict of interest. 425 Mechanisms of p53-mediated chemosensitivity of VMY on medulloblastoma cells A. Naeem1 , V. Yenugonda1 , O. Rodriguez1 , M.L. Avantaggiati1 , B. Rood2 , S. Karam3 , C. Albanese4 . 1 Georgetown University Medical CenterWashington DC, Oncology, Washington, DC, USA, 2 Children National Medical Center, Clinical Neuro-Oncology, Washington, DC, USA, 3 University of Colorado, Radiation Oncology, Denver, CO, USA, 4 Georgetown university Medical Center- Washington DC, Oncology and Pathology, Washington, DC, USA The p53 pathway is considered a key determinant of anti-tumor responses in many tumors; however, its roles in the regulation of cell survival, chemo-sensitivity and chemo-resistance in medulloblastoma (MB), a primitive neuroectodermal tumor, are much less well defined. Importantly, we have recently reported that the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both Doxorubicin and the experimental drug VMY. Enhanced late stage-apoptosis was implicated in this increased MB cell death. In the present study, the role of p53 (through its inhibition by Pifithrin-a) in the cytotoxic activity of VMY was evaluated through expression analysis of D556 medulloblastoma cells. Using Ingenuity Pathway Analysis, we identified G-protein-coupled receptors (GPCR) clusters that control both calcium homeostasis and neuronal apoptosis. In addition, induction of key Forkhead transcription factor genes and the repression of target genes including MDM2, GDF 15 (growth differentiation factor 15) and NRG (neuregulin), were also found to be uniquely regulated by p53 suppression in the presence of VMY. These data provide novel mechanistic information regarding the role of p53 in neuroectodermal tumor chemoresistance and provide new approaches for enhancing clinical outcomes in MB. No conflict of interest. 426 LOX and LOXL2 in pancreatic cancer microenvironment K. Gardian1 , M. Durlik1 . 1 Mossakowski Medical Research Centre, Department of Surgical Research & Transplantology, Warsaw, Poland Background: Pancreatic cancer remains the most aggressive malignancy of all human cancers. Patients have an extremely poor prognosis, with less than 5% surviving for 5 years and a median survival period of 6 months. Pancreatic cancer is characterized by strong desmoplastic reaction, which consist a barrier in providing therapeutic agents to cancer cells. Main contributors for fibrotic stroma are stellate cells − myofibroblasts that can also influence the

cancer cell motility. Lysyl oxidases are enzymes that catalyses the crosslinking of collagens and elastin in the ECM. What is more they have also been associated with creating metastasis. LOXL2 has been reported to participate in creating metastatic niche in hepatocellular carcinoma while LOX influence creating pre-metastatic bone lesions. The aim of this study was to evaluate role of LOXL2 and LOX in pancreatic cancer − creating fibrotic stroma and influence possibility of metastasis. Material and Methods: Tumor tissue samples and lymph nodes were obtained from 40 patients, who underwent macroscopically curative resection. Patients had not received any preoperative radio- or chemotherapy. Tissue specimens were analysed with immunohistochemistry using antibodies against LOX, LOXL2 and SMAa (in tumor tissue). Results: Pancreatic cancer cells are the source of LOXL2 and LOX, stellate cells express LOXL2. Expression of both enzymes was cytoplasmatic. In lymph nodes LOX expression was noticeable but not LOXL2. Comparison with clinical features showed that high expression of LOXL2 and LOX was associated with shorter overall survival and lymph node metastases. Conclusion: Lysis oxidases influence progression of pancreatic cancer. It seems that their role is to rebuild tumor stroma and in this way they increase cancer cell motility. Further research is necessary to establish how LOX influence creating lymph nodes metastases. No conflict of interest. 427 Endometrial cancer stem cells: tumorospheres characterization and cancer stem cells markers M.J. Carvalho1 , M. Laranjo2 , T. Costa3 , N. Almeida3 , M. Abrantes2 , J. Casalta-Lopes3 , A. Paiva4 , M.F. Botelho2 , C. Oliveira5 . 1 University Hospital Centre of Coimbra, Gynecology A Service, Coimbra, Portugal, 2 Faculty of Medicine of University of Coimbra, Institute of Biophysics/BiomathematicsCIMAGO- CNC.IBILI, Coimbra, Portugal, 3 Faculty of Medicine of University of Coimbra, Institute of Biophysics/Biomathematics, Coimbra, Portugal, 4 Portuguese Institute of Blood and Transplantation, Coimbra, Portugal, 5 Faculty of Medicine of University of Coimbra, CIMAGO, Coimbra, Portugal Introduction: The cancer stem cell (CSC) are a population with properties of tumour initiation, of resistance to therapy and with metastatic potential. The aim of this study was to characterize endometrial tumorospheres in vitro, particularly relative to CSC markers. Material and Methods: Endometrial cancer cell line ECC1 was submitted to sphere forming protocol. The first spheres generation (ES1) was cultured in adherent conditions (G1). This procedure was repeated to obtain successive generations of spheres (ES1, ES2 and ES3) and spheres-derived adherent cells (G1, G2 and G3). Sphere forming capacity evaluated the number of spheres originated per plated cell number. The self-renewal capacity was evaluated by dissociation and accessing spheres cell potential to form new ones. Adherent populations doubling time was evaluated. Fifth day spheres were photographed to access projection area. The cloning capacity was evaluated for all the populations, plating the cells in adherent conditions and colony assessment after 12 days. The expression of CD133, CD24 and CD44 were evaluated by flow cytometry and the expression of aldehyde dehydrogenase (ALDH) was evaluated by western blot. Results and Discussion: The three sphere populations and the three derived adherent populations were obtained from ECC-1 cell line ECC-1. The spheresforming capacity of G1, G2 and G3 ranged from 2.22% to 2.54%, and selfrenewal was higher in ES3 than ES1, which also showed lower projection area, emphasizing stemness. The cloning efficiency was lower in spheres than in ECC-1 (p < 0.001), however, regain of adhesion highlights spheres plasticity. ECC-1, G1, G2 and G3 showed similar doubling time. The mean fluorescence intensity (MFI) for CD133, was higher in spheres than ECC-1, with significance in ES3 (p = 0.048). There were no differences in CD24 between cells populations. CD44 was higher in spheres than ECC-1, with significance in ES1 (p = 0.006). ALDH expression, was significantly higher in ES1 (1.41±0.25, p = 0.014), ES2 (1.86±0.46, p = 0.001) and ES3 (2.10±0.46, p = 0.004) compared with ECC-1. The expression for G1 (1.27±0.26), G2 (1.36±0.23) and G3 (1.27±0.35) was not significantly different from ECC-1. Herewith, spheres populations showed enhanced expression of CSC markers and its expression decrease with differentiation into adherent conditions. Conclusions: Endometrial spheres presented self-renewal, differentiation and enhanced expression of CSC markers, namely CD133, CD44 and ALDH compared with parental cell line and derived adherent populations. The spheres protocol selectively isolated a population with CSC properties. Acknowledgement: Funding: Portuguese Foundation for Science and Technology (SFRH/SINTD/60068/2009; PEst-C/SAU/UI3282/2013 and UID/ NEU/04539/2013 − COMPETE-FEDER) and CIMAGO. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 428 Engineering glioblastoma microenvironment in a chip to study cell response J.M. Ayuso1 , R. Monge1 , G. Llamazares1 , M. Virumbrales-Munoz1 , A. Lacueva1 , M. Olave1 , M. Doblare1 , I. Ochoa1 , L. Fernandez1 . 1 Aragon Institute for Engineering Research, Mechanical Engineering, Zaragoza, Spain Introduction: Glioblastoma (GBM) is the most common of primary brain tumours. Nowadays, the survival mean time in patients who received medical treatment, based on local radiotherapy and chemotherapy with temozolomide (TMZ), is 12−14 months. Despite all research, development of more effective treatments remains elusive. This tumour is characterized by its extremely invasive behaviour, and is distinguished from other brain tumours by necrosis foci with a highly cellularized surrounding area. Recent evidences suggest these hypercellularized regions, known as pseudopalisades, are forming migrating cell waves; which expand across the brain led by nutrient and oxygen gradients. This hypothesis explains the poor efficiency of conventional anti-tumorigenic treatments in GBM, which are targeting proliferative cells. However, development of new drugs against this tumour invasion remains challenging due to the lack of systems capable to recreate this complex tumour microenvironment. In this context, microfluidics arises as a powerful tool to recreate these complex scenarios. Thereby, we present a microfluidic system to study GBM behaviour under the physiologic microenvironment. Materials and Methods: The presented microfluidic system includes a central microchamber to locate GBM embedded into a 3D collagen hydrogel, mimicking the Extracellular matrix. Flanking this central microchamber there are two lateral microchannels which remained hydrogel free, mimicking blood vessels and allowing medium perfusion. Due to the system design, we are able to monitor oxygen and glucose profiles across the central microchamber. Moreover, we are able to study cell behaviour under nutrient and oxygen gradient conditions by controlling medium perfusion on lateral microchannels. Results and Discussion: When cultured within the microdevices, GBM cell metabolism created an oxygen gradient along the system. Under these circumstances, GBM cells invaded the tissue towards the oxygen source, creating a migratory front which behaved similar to the pseudopalisades found on patients. Interestingly, if GBM cells were cultured under a homogeneous hypoxia, no directional migration was observed. When these migrating GBM cells reached nutrient-enriched regions, they became more aggressive and proliferation was dramatically increased. As a result, cells invaded the lateral microchannel. We could find a perfect agreement between our results and the analysis of patient histological slices. Conclusion: In this work, we present a microfluidic system capable of mimicking the specific GBM microenvironment. Specifically, the presented system allowed the culture of GBM cells under hypoxia/nutrient starvation conditions. The results show GBM cells within the presented microfluidic device generated a migrating pseudopalisade similar to what is expected on GBM patients. No conflict of interest. 429 Tumour suppression by a protein hydroxylase A. Zayer1 , H. Smith2 , M. Pillai3 , R. Sekirnik4 , J. Zak5 , X. Lu5 , C. Schofield4 , M. Bix6 , P. Ratcliffe7 , M. Coleman2 . 1 University of Cambridge, University of Cambridge, Cambridge, United Kingdom, 2 University of Birmingham, Institute of Cancer and Genomic Sciences, Birmingham, United Kingdom, 3 St Jude Childrens Hospital, Department of Immunology, Memphis, USA, 4 University of Oxford, Chemistry Research Laboratories, Oxford, United Kingdom, 5 University of Oxford, Ludwig Institute for Cancer Research, Oxford, United Kingdom, 6 Chiba University, Dept. of Immunology, Chiba, Japan, 7 University of Oxford, Nuffield Department of Medicine, Oxford, United Kingdom Introduction: Hydroxylation is an emerging modification catalysed by a family of ‘2-oxoglutarate (2OG)-dependent oxygenases’ that includes the Collagen and HIF hydroxylases, TET 5hmC demethylases, and JmjC histone demethylases. These enzymes are widely implicated in cancer due to roles in hypoxia signalling, extracellular matrix formation, and epigenetics, and because of their inhibition by oncometabolites. Although other sub-families of 2OG-oxygenases exist they are less well characterised and their role in cancer remains largely unexplored. We are defining the biochemical targets and biological functions of JmjC protein hydroxylases, a poorly characterised family of 2OG-oxygenases related to the JmjC histone demethylases, and have discovered that these enzymes regulate fundamental biological processes that are commonly deregulated in cancer. Myc-Induced Nuclear Antigen (MINA) is a JmjC protein hydroxylase that we have shown promotes ribosomal 60S subunit abundance through modification of Rpl27a. Consistent with a role in supporting growth and ribosome biogenesis, MINA is overexpressed in several tumour types. However, our recent work suggests MINA also has the capacity for tumour suppressor activity. Here we describe our investigation of the MINA protein hydroxylase and its tumour suppressor role.

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Results: Anchorage-independent growth assays indicate that reconstitution of MINA into knockout K-Ras-transformed cells and MINA mutant human tumour epithelial cell line suppresses transformation. These suppressor affects are recapitulated in xenograft tumour growth assays and in cancer models using transgenic MINA knockout mice. Consistent with the possibility of MINA inactivation in human tumours, cancer sequencing databases include nonsense, frameshift, splice site and missense mutations across a variety of tumour types. Importantly, missense mutations are clustered in ‘hotspots’ within functionally important domains and can inhibit hydroxylase activity. Conclusion: Our work demonstrates that MINA is a novel class of tumour suppressor, and indicates that further investigation into the roles of JmjC protein hydroxylases in cancer is warranted. No conflict of interest. 430 Biomimetic microfluidic platform for 2D3D co-culture cancer models M. Virumbrales-Munoz1 , J.M. Ayuso1 , R. Monge1 , G. Llamazares1 , M. Olave1 , M. Doblare1 , L. Fernandez1 , I. Ochoa1 . 1 Aragon Institute for Engineering Research, Mechanical Engineering, Zaragoza, Spain Background: Nowadays, cancer is seen as a multifactor process in which cells interact with other cell types and the extracellular matrix. Tumour microenvironment is crucial in cancer development and prognosis, especially in the metastasis process, which is responsible for 90% of cancer-related deaths. Particularly, endothelium interaction with the tumour has long been investigated, since one of the key steps in metastasis is cell intravasation. Nonetheless, the exact mechanisms of tumour-endothelium cross-talk are not fully known. Thus, there is nowadays an urging need to develop cancer models which accurately reproduce tumour microenvironment. In this context, microfluidics provides unique features to create robust, high-throughput and straightforward biomimetic cell-culture platforms. We developed a versatile and easy-to use microfluidic device for co-culture assays. The system can be operated without any external equipment thanks to a passive pumping mechanism. The resulting model combines two and three-dimensional cultures of different cell types. Materials and Methods: The presented microfluidic device comprises three independent arrays of microwells. The wells present on each array are open from the top, and connected by a microchannel. Cancer cells were embedded in a collagen matrix and seeded in a three-dimensional environment inside the three microchannels. As a result, all microwells were filled, creating a flat surface. Next, a two-dimensional endothelium was established on top of the microdevice. Hence, both cultures remain connected through the hydrogel but are distinctively patterned. The integrity of the endothelium was validated via immunofluorescence, and drug diffusion processes were carried out in the described setup. Finally, immunotherapeutic drugs were assayed in our model. Results and Discussion: The described microdevice was found biocompatible for cancer and primary cell lines. Furthermore, the endothelium was found to be flat, healthy and highly interconnected, as demonstrated by the cell interactions via VE-cadherin. Besides, the endothelium was responsive to inflammatory molecules: the addition of TNF-a resulted in the appearance of pores in the endothelium. Finally, drug diffusion was studied for nanoparticles and other soluble molecules. Our experiments show how the developed device can be used for an easy and deep study on diffusion of nanoparticles and molecules through endothelium. Conclusion: We developed a robust, high-throughput tool to perform coculture assays. This microdevice was successfully validated with a cancer co-culture with an endothelium. No conflict of interest. 431 CD133+ subpopulation was identified in miR-135a induced early transformation process of HPV-infected cells to cervical cancer W. Zhu1 , D.C.K. Yuen1 , R.T.K. Pang1 , W.S.B. Yeung1 . 1 The University of Hong Kong, Obstetrics and Gynaecology, Hong Kong, Hong Kong Introduction: Cervical cancer carcinogenesis initiates from the persistent infection of high-risk type human papilloma virus (HPV). Under long-term HPV infection, normal cervical epithelia cells firstly enter into a precancerous stage, and then gradually transform into cancer cells. HPV onco-proteins E6 and E7 interrupt normal cell cycle and lead to transformation. MicroRNAs are non-coding negative regulators of gene expression. Abnormal microRNA expression associates to the cervical cancer transformation. CD133+ cancer stem cells are involved in tumor formation in various tumors including cervical cancer. Previously, microRNA-135a (miR-135a) was found to be highly expressed in cervical cancer lesions. Stable force-expression of miR-135a transformed HPV E6/E7 immortalized cells into tumor cells with CD133+ subpopulation that is tumorigenic in nude mice. However, whether miR-135a induces CD133+ subpopulation formation in early transformation process is unclear. Here, we mimic the cervical cancer multiple step transformation

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process by serial transient miR-135a force-expression in a precancerous cell model and examine in vitro tumor forming ability of its CD133+ subpopulation. Material and Method: NC104-E6/E7 is normal cervix epithelia cell-line immortalized by HPV E6 and E7. The transformation process was done by serial miR-135a transient force-expressions. Functional assays examined the tumorigenic properties after each miR-135a force-expression and compared to the control force-expression counterpart. Correspondingly, the CD133+ and CD133− subpopulations are isolated by CD133 antibody coated microbeads and their in vitro tumor sphere forming abilities are examined. Results and Discussion: After 3 successive miR-135a force-expressions, NC104-E6/E7-3t-135a cells exhibited higher invasiveness. Other malignant properties such as proliferation and migration were only slightly changed compared to the control group. The expression of E-cadherin was decreased accompanied by an increase of N-cadherin during the miR-135a mediated transformation process, indicating that the epithelial–mesenchymal transition (EMT) has occurred. CD133+ cells were found after 3 successive miR-135a force expressions. The CD133+ cells isolated from the NC104-E6/E7-3t-135a cells exhibited higher tumor spheres forming ability; the number and size of tumor spheres formed were larger than their CD133− counterparts. CD133+ subpopulation from the two control cell lines, NC104-E6/E7 and NC104-E6/E73t-ctrl cells failed to form tumor spheres. Conclusion: MiR-135a mediated cervical cancer malignant transformation is a multi-step process. Force expressing miR-135a favored the EMT process of precancerous cell model, which might be responsible for the stemness acquisition of cancer stem cells. This transformation process induced formation of CD133+ subpopulation. No conflict of interest. 432 DigiWest multiplex protein profiling of extracellular vesicles to decipher cancer metastasis and to identify biomarkers G. Erdmann1 , C. Sachse1 , M. Templin2 , J. Gross3 . 1 NMI TT GmbH, Pharmaservices, Berlin, Germany, 2 NMI, Protein Profiling & Assay ¨ Development, Reutlingen, Germany, 3 University Medical Center Gottingen, Haematology and Oncology, Goettingen, Germany Background: Improved diagnostic tests for cancer treatment are crucial in the clinic as well as during drug development. Technically, liquid biopsies are highly desirable, which has raised interest in CTCs and secreted extracellular vesicles. Most cell types secrete extracellular vesicles (EVs), a heterogeneous population of membrane particles, named according to their size and origin, as microvesicles (MVs) (250–1000 nm) or exosomes (50–100 nm). They have been described as a specific form of inter/extracellular communication. For example, tumor cell-derived EVs prime metastatic sites for incoming tumor stem cells (Peinado et al. 2012). In addition, a variety of signaling molecules are secreted on EVs from their sending cells, e.g. Wnt proteins, which allows them to induce Wnt signaling activity on target cells (Gross et al. 2012) and even to enhance migration and invasiveness of tumor cells (Menck et al. 2013). Hence, interrogating signaling molecules transmitted via tumor derived EVs may reveal biomarkers that might be more conclusive than circulating nucleic acids. Thus, analyzing proteins incorporated on EVs may offer important diagnostic information prior to and during treatment. Our aim is to analyze proteins traveling on extracellular vesicles and determine their utility as biomarkers. Methods: Extracellular vesicles were purified from different breast cancer cell lines as well as plasma from cancer patients by a differential centrifugation, separating exosomes and microvesicles by ultracentrifugation. To simultaneously detect a wide range of proteins traveling on the EVs, a novel protein profiling platform, DigiWest, was utilized. This method combines sizebased separation by gel electrophoresis with the Luminex bead system into a highly multiplexed “digital Western blot”. Using this approach, >90 proteins were analyzed for their presence on extracellular vesicles from as little as 10 mg of total protein. Results: Using the DigiWest assay on extracellular vesicles, we were able to identify and quantify known specific marker proteins for exosomes as well as microvesicles, respectively. Overall, we identified proteins specific for one type of vesicles or the other, but also found proteins equally present in both sample types. Furthermore, we identified a number of signaling proteins and receptors on both extracellular vesicles fractions that we further analyze for their robustness and relevance to breast cancer. Conclusion: Monitoring signaling molecules on extracellular vesicles using the DigiWest multiplex protein profiling technology is a highly promising approach for elucidation of the biological function of extracellular vesicles during tumor metastasis as well as for identification of specific biomarker candidates. Conflict of interest: Other Substantive Relationships: NMI TT Pharmaservices offers DigiWest as a service (for research use only).

433 Fasting in combination with curcumin induces a fatal energy “black out” in tumor cells L. Raffaghello1 , G. Bianchi1 , N. Bertola1 , A. Amaro2 , G. Angelini2 , L. Emionite3 , S. Ravera4 , U. Pfeffer2 . 1 G.Gaslini Children’s Hospital, Laboratory of Oncology, Genova, Italy, 2 IRCCS AOU San Martino − IST Istituto Nazionale per la Ricerca sul Cancro, Molecular Pathology, Genoa, Italy, 3 IRCCS AOU San Martino − IST Istituto Nazionale per la Ricerca sul Cancro, Animal Facility, Genoa, Italy, 4 University of Genoa, Department of Pharmacy, Genoa, Italy Introduction: The dietary polyphenol curcumin (CUR) has been described to exert anti-tumoral effects without toxicity. At present, 126 clinical studies using CUR or its derivatives are registered at clinicaltrials.gov. CUR has recently been defined as a caloric restriction mimetic (CRM), as it mimics biochemical and functional effects of calorie restriction (CR). In mice, two day cycles of fasting with only water access (short term starvation: STS) represent a peculiar example of CR that has been shown to protect normal cells but not tumor cells against cytotoxicity of chemotherapy and to render many tumors more susceptible to chemotherapy through an anti-Warburg effect. Preliminary clinical data indicate that in cancer patients fasting is feasible, not associated with major toxicity and able to reduce several side effects associated to chemotherapy. Aim of this study is to investigate the effects and mechanisms of fasting in combination with CUR on tumor metabolism. Material and Method: We tested the cytotoxicity of different concentrations of CUR +/− STS (1% FBS + 0.5 g/L glucose) on murine and human cell lines of melanoma, neuroblastoma, breast, colon cancer, and chronic lymphatic leukemia by Trypan Blue and Annexin V staining. Reactive oxygen species production was measured by 2 ,7 -dichlorodihydrofluorescein diacetate staining. Protein expression and activity of the key glycolytic enzymes, respiratory chain complexes as well as autophagy and proliferative pathways (PI3K/AKT/mTOR) were studied by Western blot and spectrophotometric assays. Results and Discussion: CUR reduces cell proliferation and induces apoptosis of various tumor cell lines, without affecting ROS production. It exerts an anti-Warburg effect by decreasing the activity of the main glycolytic enzymes. CUR inhibits ATP synthase and reduces O2 consumption leading to a dramatic drop of ATP production. STS shows similar anti-proliferative and apoptotic effects. STS-treated tumor cells exhibit a significant induction of respiratory chain complexes I, III, IV associated to a striking uncoupling between induced O2 consumption and reduced ATP synthesis. This latter event leads to a significant increase of ROS generation and apoptosis. The highest tumor cytotoxicity was observed for the combination of STS and CUR that additively reduces glycolysis and oxidative phosphorylation leading to a complete impairment of energy charge and consequent cell death. Differences between cell lines based on their basal respiratory activity are observed. The efficacy of STS+CUR in vivo is currently being tested. Conclusion: Fasting combined with CUR causes a strong suppression of tumor cell viability by inducing a fatal energy impairment. Thus, this association represents a promising strategy for the treatment of human cancer. No conflict of interest. 434 Crizotinib overcomes microenvironment mediated drug resistance in Ph positive leukemia O. Regev1 , N. Kidan1 , H. Khamisie1 , J. Mahajna1 . 1 Galilee Technology Center- Migal, Cancer Drug Discovery Program, Kiryat Shmona, Israel Background: Abl Kinase Inhibitors (AKIs) exhibiting good clinical efficacy in Ph+ leukemia. However, in one-third of patients, first-line TKI treatment will eventually become ineffective, which attributable to either non-mutational or kinase mutations in BCR/ABL. Conditions within the Bone Marrow (BM) niche contribute to reduced drug sensitivity of cancer cells residing in the BM including Ph+ leukemia. Crizotinib, a kinase inhibitor designed to target c-ALK for the treatment of a subtype of lung cancer, exhibits activity also against the ABL-kinase. Aim: Our aim is to understand BM microenvironment role in Ph+ leukemia AKI drug resistance and investigated the therapeutic potential of Crizotinib for the treatment of advanced and therapy of resistant disease. Methods: To study BM microenvironment mediated drug resistance in Ph+ leukemia; we exposed CML cells to condition media (CM) collected from mesenchymal cells. Cell proliferation was monitored by XTT assay and apoptosis inducing function of AKIs was assayed by following PARP cleavage. In addition we followed levels of STAT proteins. Results: CM collected from mesenchymal cells confers Imatinib drug resistance to CML cells. However, CM collected from ovarian or murine skin melanoma cells were not efficient in conferring drug resistance to Imatinib. Moreover, no significant change in CM mediated Imatinib drug resistance when CM were filtered with 0.4 or 0.2 micron. Exposer of CML cells to CM abolished ability of Imatinib to induce PARP cleavage. In contrasts, Crizotinib overcomes CM-mediated drug resistance. Levels of variety of soluble factors in the CM were monitored using a cytokine assay kit and revealed the presence

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 of significant amount of mIL-3. Furthermore, we showed that exposure to CM caused a significant increase in levels of pSTATs. Conclusion: These findings indicate that soluble factors found in CM contributes to Imatinib drug resistance in CML. Additionally, Crizotinib was active in overcoming CM-mediated drug resistance. No conflict of interest. 435 Impact of Aspirin on factors associated with breast cancer lymph node metastasis S. Khan1 , K. Gilligan1 , K. O’Brien1 , B. Moloney1 , I.S. Miller2 , E. Ramphul1 , T.I. Barron3 , K. Bennett2 , A.T. Byrne2 , M.J. Kerin1 , R.M. Dwyer1 . 1 Lambe Institute for Translational Research, Surgery, Galway, Ireland, 2 Royal College of Surgeons Ireland, Physiology & Medical Physics Dept, Dublin, Ireland, 3 Trinity College Dublin, School of Medicine, Dublin, Ireland Introduction: Long-term aspirin use in breast cancer patients has been associated with reduced spread to lymph nodes, potentially mediated through VEGF-C/-D. VEGF-C is a pro-angiogenic factor regulated down-steam of cyclooxygenase-2 (COX-2), which is a target of Aspirin. This study aimed to investigate circulating VEGF-C in breast cancer patients compared to healthy controls. The impact of Aspirin on VEGF-C secretion and angiogenic factors In Vitro, and on tumour recurrence In Vivo was also determined. Materials and Methods: Circulating levels of VEGF-C were measured by ELISA in serum from breast cancer patients (n = 58) and healthy controls (n = 20). Primary Normal stromal cells were derived from fresh tissue samples harvested in theatre with informed consent. Human Mesenchymal Stem Cells (MSCs) were isolated from the iliac crest of healthy volunteers. Cells were cultured either individually or with HCC-1954 breast cancer epithelial cells. Cells were exposed to Aspirin, conditioned media harvested, and secreted VEGF-C quantified. A tubule formation assay was performed to determine the impact of Aspirin on angiogenesis. Pro-angiogenic protein expression was investigated using an array. In vivo, breast tumour bearing mice were treated with Aspirin, tumours resected and disease recurrence monitored using bioluminescent and photoaccoustic (PA) imaging. Results and Discussion: Circulating VEGF-C levels were found to be elevated in breast cancer patients (5438±1270 pg/ml) compared to controls (3560±1477 pg/ml, p > 0.01). MSCs secreted highest levels of VEGF-C (305±35 pg/ml), followed by normal stromal cells (153±21 pg/ml), with no detectable levels secreted by HCC-1954 cells. Aspirin exposure resulted in loss of VEGF-C secretion from co-cultured cell populations, and reduced ability to form tubules. Decreased levels of angiogenesis related proteins were also observed following treatment with Aspirin, with the greatest decrease seen in Urokinase Plasminogen Activator (uPA, −42%). Preliminary In Vivo results revealed reduced tumour recurrence in mice treated with Aspirin. Conclusion: The data presented here highlights elevated circulating VEGF-C in breast cancer patients. Further, Aspirin was shown to have a significant impact on VEGF-C and uPA. Initial results also revealed that Aspirin may impact breast cancer recurrence in vivo. Acknowledgement: Funding: Irish Cancer Society BREAST-PREDICT (CCRC13GAL). No conflict of interest. 436 RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation E.J. Arenas Lahuerta1 , R. Gomis Cabre´ 1 , M. Morales1 . 1 IRB Barcelona, Oncology, Barcelona, Spain Despite a recent decrease, breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. In Estrogen Receptor Negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Therefore, a better understanding of the biology of breast cancer lung metastasis is required. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we show that RARRES3 is a metastatic suppressor gene in breast cancer. Using the ER− MDA-MB-231 breast cancer cell line model and lung metastatic derivatives, we functionally validated that RARRES3 loss of expression confers a selective advantage for the colonization of the lung in vivo (xenografts and syngeneic models). RARRES3 silencing engages metastasis initiating capabilities by facilitating extravasation and adhesion of the tumor cells to the lung. Furthermore, RARRES3 phospholipase A1/A2 activity contributes to tumor cell differentiation, thereby blocking lung metastasis demonstrated in 3D organotypic cultures and by a re-initiation assay in vivo. Therefore, our results show that genes selected for metastasis contribute to the different steps of this process and represent the random accumulation of traits that provide the necessary advantage for adaptation to the microenvironment of a different organ. RARRES3 restrains the lung metastatic capacity of breast cancer cells and more important, RARRES3 levels in the primary tumor are clinically relevant as may predict risk of relapse.

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The contribution of RARRES3 to differentiation over self-renewal suggests that reduced RARRES3 expression could be also predictive of therapy-resistant tumors, identifying patients possibly requiring new therapies designed to target breast cancer-initiating cells. Therefore, screening for compounds that activate RARRES3 may contribute to the development of new differentiation-inducing strategies to target therapy-resistant breast tumors. Main point: Functionally validate RARRES3 as a lung metastatic tumor suppressor gene in breast cancer in vitro, in vivo, and the most important, the clinical relevance in a human cohort. No conflict of interest. 437 Role of integrated TGF-b superfamily signaling in melanoma progression E. Tuncer1 , D. Zingg1 , P. Cheng2 , A. Antunes1 , J. Haeusel1 , C.X. Deng3 , I. Kleiter4 , L. Sommer1 . 1 Institute of Anatomy, University of Zurich, Zurich, Switzerland, 2 Department of Dermatology, University Hospital Zurich, ¨ Zurich, ¨ Switzerland, 3 National Institutes of Health, Genetics of Development and Diseases Branch, Bethesda- MD 20892, USA, 4 Department of Neurology, St. Josef-Hospital Ruhr-University, Bochum, Germany In various cancer types including melanoma, signalling by TGF-b superfamily has been implicated in both suppressing tumour growth and promoting metastasis. Several members of the TGF-b superfamily are expressed in melanoma and have been shown to affect tumour cell proliferation or invasiveness in a context-dependent manner. However, the role of the integrated TGF-b signalling in melanoma progression in vivo remains to be determined. Here, we show that conditional deletion of the TGF-b mediator Smad4, which abrogates all canonical TGF-b family signalling, abolishes tumourigenesis in a transgenic mouse model of melanoma. Intriguingly, this phenotype was brought about by reduced proliferation of tumour cells, thus pointing to the existence of pro-proliferative TGF-b family factors. After screening several TGF-b superfamily ligands, we identified BMP7 as a crucial ligand for melanoma cell proliferation. Moreover, BMP7 was sufficient to override the cytostatic and pro-invasive effects of TGF-b and Nodal. We next asked whether melanoma tumourigenesis could be increased by SMAD signalling activation. For that, we conditionally depleted Smad7 in a transgenic melanoma mouse model. Interestingly, Smad7 depletion resulted in massive metastasis formation in vivo. In agreement, cutaneous melanoma patients with low SMAD7 expression display reduced overall survival. Taken together, our results suggest that TGF-b signalling promotes melanoma growth, and in the context of reduced SMAD7 expression induces metastasis formation. No conflict of interest. 438 Tissue transglutaminase correlates with disease progression and the acquisition of epithelial–mesenchymal transition in colorectal cancer cells O. Ayinde1 , Z. Wang1 , C. Tselepsis2 , M. Griffin1 . 1 Aston University, School of life and health science, Birmingham, United Kingdom, 2 University of Birmingham, Institute of Cancer and Genomic Sciences, Birmingham, United Kingdom Background: Tissue transglutaminase (TG2) is a multifunctional and ubiquitous enzyme. Clinical studies indicate TG2 is elevated in various cancer cell types, however conflicting reports posit both pro and anti-tumour role for the enzyme. Previous studies using murine model showed TG2 regulates colorectal cancer cell (CRC) progression, however the role of TG2 in the human disease remains unclear. This study employs human CRCs at different disease stage to investigate the role of TG2 in tumour progression. Methods: TG2 expression was manipulated using Lentiviral particles carrying wild type TG2 or shRNA for TG2 in human CRCs RKO, SW480 and SW620. Correlation of TG2 expression with epithelial–mesenchymal transition (EMT), CRC chemo-resistance to 5 Fluorouracil (5-FU) and cancer stem cell (CSC) formation were characterized using Western blotting, XTT assay, and spheroid formation for CSCs. Results: Results show TG2 was sufficiently expressed in the metastatic cell line SW620 when compared to primary cell lines SW480 and RKO. SW620 cells exhibited higher resistance to 5FU treatment, while knockdown of TG2 using shRNA in these cells reduced cell resistance to 5FU. SW620 demonstrated EMT markers while SW480 and RKO expressed predominantly epithelial markers. TG2 over expression and TG2 shRNA treated cells showed increased and decreased expression of mesenchymal and epithelia markers respectively when compared to their corresponding parental cell types. TG2 knockdown resulted in the reduced ability of SW620 cells to form spheroids enriched with cancer stem cell phenotype in culture. Conclusions: This study has shown that TG2 correlates with in vitro tumour progression in human CRCs and that TG2 is involved in EMT, drug resistance and CSC formation. Therefore TG2 could serve as a potential biomarker for CRC tumour progression with therapeutic significance. No conflict of interest.

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439 Targeting COPZ1 in thyroid tumour cells M.C. Anania1 , E. Cetti1 , D. Lecis2 , L. Cleris3 , M. Mazzoni1 , S. Pagliardini1 , G. Manenti4 , A. Greco1 . 1 Fondazione IRCCS Istituto Nazionale Tumori, Dep. of Experimental Oncology and Molecular Medicine- Molecular Mechanisms Unit, Milan, Italy, 2 Fondazione IRCCS Istituto Nazionale Tumori, Dep. of Experimental Oncology and Molecular Medicine- Molecular Mechanisms of Cell Cycle Control Unit, Milan, Italy, 3 Fondazione IRCCS Istituto Nazionale Tumori, Dep. of Experimental Oncology and Molecular MedicineChemoprevention Unit, Milan, Italy, 4 Fondazione IRCCS Istituto Nazionale Tumori, Dep. of Predictive and Preventive Medicine- Genetic Epidemiology and Pharmacogenomics Unit, Milan, Italy Background: The incidence of thyroid carcinoma (TC) is rapidly increasing. Although generally associated with good prognosis, a fraction of TC is not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. To identify novel therapeutic targets for TC, we performed a functional screening of a siRNA library in tumour (BCPAP) and normal (NThy-ori 3−1) thyroid cell lines, and we have identified a set of genes whose silencing interferes with the growth of tumour cells, while sparing the normal ones (non oncogene addiction paradigm). Among these, we found COPZ1 (coatomer protein complex z1) gene, which encodes a subunit of the heptameric coatomer protein complex I, involved in intracellular vesicular traffic, autophagy and lipid homeostasis. Further studies showed that COPZ1 represents a common vulnerability for thyroid tumour cells, as its inhibition reduced the viability of several thyroid tumour cell lines, regardless the histotype or oncogenic lesion. Here we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death, and explored its use as therapeutic target for thyroid tumours. Material and Methods: We analyzed the effect of COPZ1 depletion on the growth of TPC-1 (papillary) and 8505C (anaplastic) tumour thyroid cell lines, performing cell viability and colony forming assays. The activation of apoptosis was investigated analyzing by WB the cleavage of Caspase 3 and PARP. The inhibition of autophagy was assessed investigating by WB the accumulation of LC3II protein, and by IF the presence of autophagosomal puncta (in cells stably expressing the autophagosome marker GFP-LC3). To evaluate the in vivo effect of COPZ1 silencing, nude mice were xenografted with 8505C cells. At day 17 after injection, tumours were treated twice a week with siCOPZ1 oligos (mixed to MaxSuppressor™) and monitored for 34 days. Results: In both the papillary and anaplastic cell lines, we observed that COPZ1 depletion is associated with decrease of cell viability, abortive autophagy and apoptosis induction. Then, we evaluated the in vivo effect of COPZ1 silencing. Preliminary experiments using mouse xenograft models showed that COPZ1 silencing significantly reduced tumour growth, thus demonstrating that COPZ1 depletion impairs the growth of thyroid tumour cells also in an in vivo experimental setting. Conclusions: We found that COPZ1 gene plays a role in the biology of thyroid cancer, being essential for sustaining the oncogenic phenotype. This paves the way for designing novel therapeutic approaches based on COPZ1 inhibition, useful for thyroid tumour patients who do not respond to standard therapies and rapidly progress to malignant forms. Notably, being effective on tumour but not normal cells, these novel therapeutic approaches may have limited side effects. No conflict of interest. 440 miR-214 in stroma cells and tumor progression D. Dettori1 , F. Orso1 , E. Penna1 , L. Salmena2 , P.P. Pandolfi3 , M. Caselle4 , D. Taverna1 . 1 Molecular Biotechnology Center, Dept. Molecular Biotechnology and Health Sciences, Turin, Italy, 2 MSB 4211 1 King’s College Circle, Department of Pharmacology & Toxicology, Toronto, Canada, 3 Beth Israel Deaconess Medical Center Harvard Medical School, Department of Pathology, Boston, USA, 4 University of Physics, Dep. of Physics, Turin, Italy Background: Malignant melanoma is one of the most aggressive and fatal human cancers. In the past years a substantial number of works have demonstrated the role of miRNAs in melanoma pathogenesis. Our laboratory has shown that miR-214 is highly expressed in invasive human melanomas and it contributes to melanoma spread by controlling tumor cell migration and invasion in vitro and, more importantly, tumor cell extravasation and metastasis formation in mice. Mechanistically, we showed that this occurs via the regulation of specific direct and indirect target genes in tumor cells, for instance TFAP2, ITGA3, ALCAM and miR-148b. Moreover we have evidences indicating that various stroma cells express high levels of miR-214 and that stroma cells can influence melanoma cell migration. We generated or obtained mice overexpressing or depleted for miR-214 and we are now investigating the role of stroma cells in tumor progression in these mice. Materials and Methods: We injected B16.F10 melanoma cells (syngenic) subcutaneously into the right flank of genetically modified mice (miR-214over and miR-214ko) and evaluated tumor volumes and micro or macro metastases. In parallel we quantified the number of circulating tumor cells by taking

blood samples from these mice and expanding the cell content in culture. B16.F10 cells were also injected in the tail vein of these mice to evaluate the number of pulmonary nodules. From our miR-214 modified mice we isolated and characterized stroma cells (i.e. MEFs) and used them for co-culture experiments with melanoma cells. Results: Our results underline that miR-214 can play a role in the stroma and tumor cell communication in vitro and in vivo. Detailed experiments will be presented at the meeting. Conclusion: By using miR-214 modified mice we aim at the identification of miR-214 functions in tumor and tumor-associated stroma cells. No conflict of interest. 442 Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein S. D’Aguanno1 , D. Trisciuoglio1 , M. Desideri1 , V. Farini1 , M. Di Martile1 , T. De Luca1 , M.G. Tupone1 , A. Urbani2 , D. Del Bufalo1 . 1 Regina Elena National Cancer Institute, Research- Advanced Diagnostics and Technological Innovation Department, Rome, Italy, 2 University of Rome Tor Vergata, Department of Experimental Medicine and Surgery, Rome, Italy Background: Bcl-2, the most studied member of the bcl-2 family proteins, plays a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2 interacting factors should provide a critical advance in understanding the different functions of bcl-2. Material and Methods: Bcl-2 immunocomplexes (IM), obtained from H1299 human lung adenocarcinoma cells overexpressing FLAG-bcl-2 protein were separated by SDS-PAGE gel and analyzed by mass spectrometry for protein identification. Identified proteins were analyzed by Ingenuity Pathway Analysis (IPA). In vitro validation of bcl-2 binding to its novel putative interacting protein, SLIRP, was performed by co-immunoprecipitation (co-IP) experiments in both lung H1299 and breast MDA-MB-231 human carcinoma cell lines overexpressing FLAG-bcl-2. Cellular proteins co-localization was investigated by immunofluorescence. Apoptosis was evaluated by cytofluorimetric analysis of propidium iodide and annexin V-binding assays. Cellular oxidative stress was evaluated by cytofluorimetric determination of intracellular reactive oxygen species. Mitochondrial genes expression was assessed by quantitative Realtime PCR. Results: Proteins obtained by bcl-2 IM were identified by mass spectrometry and, analyzing them by bioinformatics tools, we evidenced that a significant part was associated to mitochondrial function. This evidence prompted us to validate the identified interacting proteins starting from SLIRP (SRA stem-loop interacting RNA-binding protein), a protein mainly associated to mitochondria and involved in maintaining mitochondrial mRNA homeostasis. Co-IP of SLIRP with bcl-2 was strengthened in FLAG-bcl-2 IM obtained from both H1299 and MDA-MB-231 cell lines overexpressing FLAG-bcl-2 by using antibodies against both FLAG epitope and endogenous SLIRP. As expected, SLIRP subcellular localization was mitochondrial in both H1299 and MDA-MB-231 cell lines. Interestingly, partial co-localization of bcl-2 and SLIRP was observed in the transfectants overexpressing FLAG-bcl-2. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein, thus contributing to regulate mRNA levels of mitochondrial SLIRP targets. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding. Conclusion: In this work, we investigated the bcl-2 interactome in human lung adenocarcinoma cells by single-step affinity purification coupled to mass spectrometry protein identification. This approach allowed to evidence, for the first time, a novel mitochondrial associated function related to bcl-2. No conflict of interest. 444 Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage S. Alsafadi1 , A. Houy1 , A. Battistella1 , T. Popova1 , M. Wassef2 , E. Henry3 , F. Tirode1 , A. Constantinou4 , S. Piperno-Neumann5 , S. Roman-Roman3 , M. Dutertre6 , M.H. Stern1 . 1 Institut Curie- PSL Research University, INSERM U830, Paris, France, 2 Institut Curie- PSL Research University, CNRS UMR 3215/INSERM U934, Paris, France, 3 Institut Curie- PSL Research University, Translational Research Department, Paris, France, 4 IGH-Institute of Human Genetics, CNRS UPR 1142, Montpellier, France, 5 Institut CuriePSL Research University, Department of Medical Oncology, Paris, France, 6 Institut Curie- PSL Research University, CNRS UMR 3348, Orsay, France Background: Discovery of recurrent missense mutations in splicing factors in cancers revealed the importance of the spliceosome pathway as a direct actor in carcinogenesis and questioned functional roles and molecular mechanisms of these mutations. SF3B1 (Splicing Factor 3B Subunit 1A) encodes for a core component of the U2 snRNP complex of the spliceosome and is found mutated in 20% of uveal melanomas (UM), tumors characterized by an overall low mutation burden.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Materials and Methods: We conducted an in-depth RNA-Seq analyses of 74 primary UM, mutated or not for SF3B1. To validate the output of our analyses, we established an SF3B1(MUT)-specific splice assay in human cell models and combined bioinformatics with site-directed mutagenesis in order to investigate the SF3B1 potential branchpoints. Results: We identified 1491 differential junctions in SF3B1(MUT) tumors, of which 79% corresponded to usage of alternative 3 splice sites. Modeling the differential junctions in SF3B1(WT) and SF3B1(MUT) cells allowed us to determine that the deregulated splice pattern is strictly dependent on SF3B1(MUT) status and on the proximal sequence of the 3’ss. SF3B1(WT) knockdown or overexpression did not reproduce the SF3B1(MUT) splice pattern, qualifying SF3B1(MUT) as change-of-function mutants. Mutagenesis of predicted branchpoints revealed that the SF3B1(MUT)-promoted splice pattern is a direct result of alternative branchpoint usage. Conclusion: This study provided a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and revealed a role for alternative branchpoints in disease. No conflict of interest. 446 The pseudokinases KSR link metabolism to the MAPK/ERK pathway A. Verlande1 , D. Poteˇ ˇ sil2 , M. Krafˇc´ıkova´ 3 , Z. Zdrahal ´ 2 , L. Trant´ırek3 , S. Uldrijan1 . 1 St. Anne’s University Hospital Brno − International Clinical Research Center, Center of Biomolecular and Cellular Engineering, Brno, Czech Republic, 2 Masaryk University − Central European Institute of Technology, Proteomics, Brno, Czech Republic, 3 Masaryk University − Central European Institute of Technology, Structural Biology, Brno, Czech Republic Background: Melanomas can be divided with respect to the activating mutations that stimulate the MAPK/ERK pathway into two major groups: BRAFV600E and NRAS mutant melanomas, found mutated in 45% and 20% of all melanomas respectively. While BRAFV600E mutant melanoma cells rely on the mutant BRAF protein to activate MEK1/2, NRAS mutant melanoma cells recruit CRAF to the plasma membrane for activation. The MAPK/ERK pathway, as other cancer-producing pathways, has already been linked with the promotion of the metabolic switch in cancer. Cells from some tumors often display an altered metabolic profile compared to their normal counterparts (glucose avidity, its catabolism through aerobic glycolysis and lesser use of the oxidative phosphorylation). In this study, we investigate if modulation of metabolic pathways has an effect on the MAPK/ERK pathway in melanoma. Material and Methods: BRAFV600E mutant melanoma cell lines (A375, MelHo, RVH421) and NRAS mutant melanoma cell lines (IPC298, MelJuso, SKMel30) were used in this study. All cell lines were cultivated in RPMI-1640 containing 2 g/L of glucose. We induced metabolic modulation with glycolysis inhibitors (2-deoxy-D-glucose (2DG), 5-thio-D-glucose, 6-aminonicotinamide) and complex I of the electron transport chain inhibitors (Rotenone, Metformin (+ AMPK activator)). We determined the phosphorylation pattern of CRAF protein kinase by mass spectrometry after exposure to 2DG and compared it with the one that developed upon no treatment with 2DG. We then selected several phospho-sites for further investigation and mutated the targeted sites to non-phosphorylatable residues by site-directed mutagenesis. CRAF kinase assays were performed with immunoprecipitated CRAF (endogenous or overexpressed) and analyzed by western blotting. Results: We show that the MAPK/ERK pathway is hyperactivated after various metabolic stresses in BRAFV600E and NRAS mutant melanoma cells. In the latter subtype, CRAF has an increased kinase activity and can bypass an inhibitory signal as its inactivation by the PKA kinase. We used a combination of proteomics and biochemistry to investigate the possible phosphorylation sites responsible for the hyperactivation of CRAF protein kinase after exposure to the metabolic drugs and identified threonine 269 as a critical phosphosite. Furthermore, our findings demonstrate that the hyperactivation of CRAF after metabolic changes is a RAS-independent mechanism and that the pseudokinases KSR play a major role in this process. Conclusion: Our results indicate that modulating metabolic pathways impacts the MAPK/ERK signaling in melanoma and that the KSR proteins establish the link between them. Understanding the precise regulation of the MAPK/ERK pathway is crucial as it remains the most therapeutic targeted signaling pathway for the treatment of melanoma. Acknowledgement: This work was supported by the EU FP7 project ICRCERA-HumanBridge, reg. No. 316345. No conflict of interest.

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447 Targeting breast cancer stem cells by inhibiting Wnt pathway: Combination of Pd (II) complex and niclosamide E. Ulukaya1 , M. Erkisa2 , S. Aydinlik2 , B. Cevatemre2 , F. Ari2 , V. Yilmaz3 . 1 Uludag University/Faculty of Medicine, Clinical Biochemistry, Bursa, Turkey, 2 Uludag University/Faculty of Science, Molecular biology, Bursa, Turkey, 3 Uludag University/Faculty of Science, Chemistry, Bursa, Turkey Introduction: Cancer stem cells (CSC), which are rare in tumor mass, are responsible for the development of tumor cell heterogeneity, a key feature for resistance to cancer chemotherapeutics. Wnt signaling pathways are reactivated in CSC and these pathways play essential role in self-renewal and proliferation of these cells. Therefore, we studied the cytotoxic effect of a palladium (Pd II) complex ({Pd(sac)(terpy)(sac).4H2 O}) on breast cancer stem cells in association with the DNA damage and oxidative stress. This complex was synthesized at the chemistry department of Uludag University. This complex was also combined with niclosamide, a Wnt pathway inhibitor, with the hope of enhancing the cytotoxic effect of the complex. Material and Methods: To determine cytotoxic and apoptotic effects of Pd (II) complex by inhibiting the wnt signaling pathway, breast cancer stem cells (CD44+/CD24− cells) were first propagated from MCF-7 cell line (parental) and then mammosphere formation was induced. Characteristic cell surface markers (CD44+/CD24−) were determined by flow cytometry to confirm that the mammospheres contain some CSCs. The cytotoxic activitiy of treatment groups (Niclosamide (1.25 mM) and Pd(II) complex (12.5, 25, and 50 mM) alone and their combination) on MCF-7-derived spheres (mammospheres) was investigated via the ATP viability assay. Caspase-cleaved cytokeratin 18, which is a marker for apoptosis and total cytokeratin 18 were used to determine the cell death mechanism (apoptosis with M30 ELISA or necrosis with M65 ELISA). The results were confirmed with Hoechst dye 43332/PI staining for nuclear morphology and cell membrane integrity as well as annexin-V-FITC. For the mechanism of apoptosis, the caspase 3/7 activity, and mitochondria membrane potential, DNA damage (gH2AX assay) and oxidative stress (ROS) level were measured by flow cytometry. Results: The combination of Pd(II) complex and niclosamide caused greater decrease in cell viability, compared to Pd(II) complex alone. This combination also increased the M30 levels in mammospheres at relatively higher doses (25 and 50 mM), suggesting relative resistance in mammospheres against the the same dose of the complex. Pyknotic nuclei, a well known marker for apoptosis, were observed after treatment with both Pd(II) complex and in combination with the inhibitor. In addition, the combination increased the level of caspase 3/7 (+) cells, Annexin-V (+) cells, and the depolarization of mitochondria membrane in mammospheres. Despite increasing levels of gH2AX, this combination interestingly reduced ROS levels in mammospheres. Conclusion: Signalling pathways are potential targets for CSC-targeted therapy and cancer drug discovery. Based on these results, combination of Pd (II) complex with niclosamide can be regarded as a novel and effective approach for the treatment of breast cancer. No conflict of interest. 448 Dysregulated cell signalling as an oncogenic basis for the development of clear cell sarcoma of kidney (CCSK) N. McDonagh1 , E. O’Meara1 , M. Alsulami2 , E. Dillon2 , G. Cagney2 , M.J. O’Sullivan1 . 1 Our Lady’s Children’s Hospital, National Children’s Research Centre, Dublin, Ireland, 2 University College Dublin, Conway Insitiute, Dublin, Ireland We previously characterized the chromosomal translocation t(10;17)(q22;p13) in CCSK to show an in-frame chimeric transcript YWHAE-NUTM2 encoding the fusion protein 14-3-3e-NUTM2. A similar fusion was identified in high-grade endometrial stromal sarcoma, lending credence to this as an oncogenic driver event. We are studying expression of 14-3-3e-NUTM2 in a cellular context to understand CCSK cell biology. Cell line generation: As no primary CCSK cell line exists, we created a cell line model. Full length YWHAE-NUTM2 fusion sequence was amplified from a CCSK tumour and cloned into pcDNA3HA and phrGFPIIN. HEK293 and BJhTERT were stably transfected with pLVX Tight Puro containing (HA-/GFP-14-3-3e-NUTM2) and pLVX Tet-On Advanced. Selected cells were induced to express the fusion using doxycycline. Subcellular fractionation: After treatment, cells were harvested using a high sucrose buffer to isolate the cytoplasmic fraction followed by a high salt buffer for the nuclear fraction. KinexKam850 Microarray 50 mg protein from doxycycline-/mock-treated cells was applied to the microarray which was analysed for protein binding to the 850 antibodies arrayed. Immunoprecipitation (IP) IP was performed using whole cell/nuclear/cytoplasmic lysates of doxycycline-/mock-treated cells. Lysates were incubated overnight with Sigma HA-conjugate beads/Dynabeads cross-linked (using DMP) to Genetex 14-3-3e antibody. While 14-3-3e is cytoplasmic, we observed HA-14-3-3e-NUTM2 in nuclear and cytoplasmic fractions, suggesting the NUTM2 fusion sequence facilitates nuclear translocation. This altered localisation could lead to novel protein

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interactions. Whole cell, nuclear and cytoplasmic lysates doxycycline-/mocktreated, were applied to the microarray. From the heat-map, a short list of proteins which were up- or down-regulated after treatment was generated. These include oncogenes, apoptotic mediators, heat shock and MAPK proteins. Validation of several findings was carried out by western blot including anti-apoptotic Mcl-1 and Bcl-xL. In contrast to other 14-3-3 family members, 14-3-3e preferentially heterodimerises. We investigated interaction of the fusion protein with 14-3-3e. IP of HA-14-3-3e-NUTM2 showed no pull-down of WT14-3-3e by the fusion or vice versa suggesting no interaction. Initial analysis of mass-spectrometry (MS) data on proteins pulled down by HA14-3-3e-NUTM2 and 14-3-3e suggests the fusion interacts with multiple 14-3-3 family members amongst other proteins. We created a cell line model to study the effect of induced expression of 14-3-3e-NUMT2 and find it, in contrast to 14-3-3e, is localised to both nucleus and cytoplasm. By antibody array and IP-MS Analysis, we are detailing protein expression differences and interactions resulting from 14-3-3e-NUTM2 expression to unravel the resultant dysregulated cell biology underpinning CCSK. No conflict of interest. 449 Adhesion signalling in melanoma biology Z. Miskolczi1 , M. Smith2 , C. Wellbrock3 . 1 The University of Manchester, The Faculty of Life Sciences, Manchester, United Kingdom, 2 The Universoty of Manchester, Faculty of Life Sciences, Manchester, United Kingdom, 3 The University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom Introduction: Melanoma is a type of skin cancer originating from the pigment producing cells, melanocytes. It is the deadliest skin cancer, requiring a better understanding of the drivers of the disease. The lineage specific transcription factor MITF is the master regulator of melanocyte development and is important in melanoma. MITF regulates genes involved in survival, migration and fate-decisions and acts in a level dependent manner. This project aims to dissect the role of MITF and how extracellular-matrix (ECM) signalling could influence its function and regulate melanoma cell behaviour. Since it is well know, that tumour tissue tends to be stiffer than the healthy tissue, we focused on how matrix stiffness could make the cells more aggressive by modulating MITF expression and/or function. Material and Method: We performed a microarray analysis of high and low MITF expressing cells embedded in 3D collagen or on tissue culture plates. In addition, we plated melanoma cells on collagen hydrolgels with defined stiffness’s (50 kPa, 8 kPa, 2 kPa, 0.2 kPa) and performed gene expression analysis for MITF and differentiation genes. We assessed changes in cellcycle and invasion as well as in main focal adhesion regulators via western blot. Results and Discussion: We identified distinct gene signatures depending on the cell line and cellular environment indicating several complex signalling networks. MITF itself was differentially expressed when cells were embedded in collagen or plated on plastic. MITF and pigmentation genes were downregulated in cells in collagen indicating a de-differentiation process. MITF levels, proliferation rate and survival also changed dependent on matrix stiffness. On lower stiffness gels, cells are less differentiated, less proliferative and more prone to apoptosis; whereas on stiffer gels, melanoma cells display a more differentiated and proliferative phenotype. Transition from a soft gel to a stiff gel causes hyper activation of SRC/FAK signalling resulting in increased MAPK signalling and survival advantage. This could allow cancer cells to proliferate faster as the tumour microenvironment gets stiffer, or survive at novel, stiffer secondary sites. Conclusion: Our data suggest that the stiffness of the extracellular matrix could influence melanoma cell biology, and this could be relevant in the context of melanoma progression. No conflict of interest. 450 The ERBB network facilitates KRAS-driven lung tumorigenesis S. Neidler1 , A. Hedley2 , B. Kruspig1 , T. Monteverde1 , K. Hewit2 , B. Neiswandt3 , A. Rosenwald4 , E. Shanks2 , C. Dick1 , D. Murphy1 . 1 University of Glasgow, Institute of Cancer Sciences, Glasgow, United Kingdom, 2 CRUK Beatson Institute, Beatson Institute for Cancer Research, Glasgow, United ¨ Rudolf Virchow Center, Wurzburg, ¨ Kingdom, 3 University of Wurzburg, ¨ Institute of Pathology, Wurzburg, ¨ Germany, 4 University of Wurzburg, Germany Small molecule inhibitors of EGFR have improved patient response rates in NSCLC driven by EGFR mutation. Mutation of KRas, downstream of EGFR, is associated with resistance to EGFR-targeted therapy. EGFR belongs to a family of 4 related ERBB/HER kinases and broad specificity multi-ERBB inhibitors have recently become available but to date have not been tested in KRas-mutant cancers, owing to the failure of EGFR-selective molecules in this context.

Using an inducible genetically engineered mouse model (GEMM) of KRas/Myc-driven lung Adenocarcinoma, we have identified a highly reproducible intra-tumoural transition that occurs sporadically in a subset of early stage lung tumours. The transition manifests as a pronounced shift in discrete tumour sub-populations from phosphor-Erk negative to p-Erk positive status that coincides with the loss of cell polarity and reduced (tumour) tissue organisation. We used laser-capture micro-dissection coupled with RNA-SEQ analysis to profile the gene expression changes associated with this transition and discovered a surprising upregulation of promiscuous EGFR/HER/ERBB-family ligands, Epiregulin and Amphiregulin, along with increased WNT pathway activation, and increased glycolytic enzyme expression. Treatment with a dual EGFR/ERBB2 inhibitor, Neratinib, reduced proliferation of several human NSCLC cell lines expressing G12 mutant KRas proteins and suppressed formation of lung tumours in our GEMM model, revealing an unexpected requirement for the ERBB network in supporting KRas-driven lung cancer. Our data suggest that broad-specificity ERBB inhibitors may have therapeutic value in the treatment of KRas-mutant lung cancer. No conflict of interest. 451 Hypoxic conditions regulate the molecular content, release and uptake rates of extracellular vesicles produced by colorectal cancer cells A. Line¯ 1 , E. Zandberga1 , A. Abols1 , C. Bajo Santos1 , R. Toleikiene1 , 1 P. Zayakin1 , D. Pupola ¯ , K. Shvirksts2 , M. Grube2 , U. Riekstina3 . 1 Latvian Biomedical Research and Study Centre, Cancer Biomarker and Immunotherapy group, Riga, Latvia, 2 University of Latvia, Institute of Microbiology and Biotechnology, Riga, Latvia, 3 University of Latvia, Faculty of Medicine, Riga, Latvia Introduction: Extracellular vesicles (EVs) are a heterogeneous group of membrane-contained vesicles released in the extracellular space and biofluids by a variety of normal and cancerous cells. EVs can transfer various proteins, lipids and nucleic acids from a cell of origin to recipient cells, where they can trigger diverse physiological and pathological responses. In the current study we explored the effects of hypoxia on the release, uptake and molecular content of EVs produced by colorectal cancer cells. Materials and Methods: Human colorectal cancer cell lines SW480 and SW620 that are originally derived from primary and metastatic tumour from a single patient were cultured under hypoxic or normoxic conditions. Exosomeenriched EV fractions were isolated from cell culture medium by using sequential centrifugation, filtration and size-exclusion chromatography steps. EVs were visualised by electron microscopy, quantified by NanoSight and characterised by Western blot analysis and FT-IR spectroscopy. For uptake experiments, EVs were labelled with Exo-APC, incubated with the cells and their uptake rate was analysed by FC. Small RNA libraries were constructed from hypoxic and normoxic cells and their EVs and sequenced using Ion Proton platform. Results and Discussion: Nanoparticle tracking analysis showed that the isolated EVs ranged in size from 30 to 200 nm consistent with the size of exosomes. The release rate differed between SW480 and SW620 cells and the hypoxic conditions significantly enhanced the release of EVs by SW620 cells and altered their size distribution profile. FT-IR spectroscopy revealed that hypoxia substantially altered the carbohydrate content in EVs released by both cell lines. The vesicles were positive for CD9, CD63, CD81 and Alix and negative for GM130. Importantly, they expressed hypoxia-inducible CAIX that potentially might allow isolation of hypoxic EVs from biofluids. Under normoxic conditions, the cells preferentially internalised normoxic EVs, while hypoxic conditions enhanced the uptake of hypoxic EVs. Preliminary RNASeq data analysis revealed a set of miRNAs that were induced by hypoxia in the cells. However only a few of these miRNAs were also found to be upregulated in hypoxic EVs, while a subset of miRNAs that were not significantly upregulated in the cells were found to be upregulated in the EVs. Conclusions: These data show that hypoxia affects the release and uptake rates of EVs and may serve as a signal for sorting specific miRNA and carbohydrate cargo into EVs. This suggests that EVs mediate hypoxia signalling among cancer cells and hypoxia may represent a natural EV targeting mechanism. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 452 Chloroquine enhances the antiangiogenic effect of doxorubicin on the chorioallantoic membrane (CAM) in vivo E. Armutak-Ikitimur1 , E. Gurel-Gurevin2 , H.T. Kiyan3 , O.B.B. Esener1 , S. Aydinlik4 , A. Uvez5 , K. Dimas6 , E. Ulukaya7 . 1 Istanbul University, Histology and Embryology, Istanbul, Turkey, 2 Istanbul University, Biology, Istanbul, Turkey, 3 Anadolu University, Pharmacognosy, Eskisehir, Turkey, 4 Uludag University, Biology, Bursa, Turkey, 5 Istanbul University, Health Sciences, Istanbul, Turkey, 6 Thessaly University, Pharmacology, Larissa, Greece, 7 Uludag University/Faculty of Medicine, Clinical Biochemistry, Bursa, Turkey Introduction: Angiogenesis is the process through growth of blood vessels from the existing vasculature. Anti-angiogenesis agents, such as bevacizumab, could increase tumor hypoxia thus upregulating autophagy activation of tumor cells which is considered to play cytoprotective role in most cases. Studies focused on the relationship between autophagy and anti-angiogenic therapies are rare. In our preliminary studies, we noticed the antiangiogenic activity of doxorubicin. Therefore, inhibition of autophagy by using pharmacological inhibitors such as chloroquine may stand an effective strategy to overcome chemotherapy or anti-angiogenesis therapy resistance. For this purpose this study was carried out to evaluate the in vivo antiangiogenic effects of the combination of doxorubicin and chloroquine as well as the effects of their single use. Methods: Doxorubicin (0.25 mg/pellet), Chloroquine (1.5 mg/pellet) and Standarts (Cortisone and (±)-Thalidomide, Prednisone) (5 mg/pellet) were dissolved in a 2.5% (w/v) agarose solution. For ease of application pellets of these solutions (10 ml) were prepared and applied drop wise on circular stainless steel supports of 5 mm diameter and cooled to room temperature for solidification and applied on to the chick chorioallantoic membrane (CAM). The angiogenic activities of doxorubicin and chloroquine alone and their combination were evaluated in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Cytotoxic effect of DXR (0.1−1mM), CQ (0.25−32mM) and their combination was investigated by employing ATP assay in Human umbilical vein endothelial cells (HUVECs). Results: The CAM treated with combination of doxorubicin (0.25 mg/pellet) and chloroquine (1.5 mg/pellet) showed significantly very high anti-angiogenic effect (score 1.5±0.6) whereas doxorubicin (0.25 mg/pellet) showed high antiangiogenic effect (score 1.2±0.3) and chloroquine (1.5 mg/pellet) showed no effect alone compared to the positive controls cortisone (score 1.2±0.2), prednisone (score 0.9±0.7) and (±)-thalidomide (score 0.9±0.2) tested at 5 mg/pellet. Furthermore, both Doxorubicin and chloroquine alone and in combination showed neither membrane toxicity nor irritation at tested concentrations. According to the ATP viability assay, combination of doxorubicin and Chloroquine caused a significant decrease in cell viability compared with the effect of doxorubicin alone in a dose-dependent manner. Conclusion: These results suggested that Chloroquine has enhanced the antiangiogenic activity of Doxorubicin on the CAM at the tested concentrations. No conflict of interest. 453 Protein kinase CK2 regulates prostate cancer cell migration and proliferation through phosphorylation of PRH/HHEX E. Marcolino de Assis Junior1 , Y. Hasan Siddiqui1 , R.M. Kershaw2 , E.H. Humphreys2 , S. Chaudhri3 , P.S. Jayaraman2 , K. Gaston1 . 1 University of Bristol, School of Biochemistry, Bristol, United Kingdom, 2 University of Birmingham, Division of Immunity and Infection- School of Medicine, Birmingham, United Kingdom, 3 Queen Elizabeth Hospital, Pathology, Birmingham, United Kingdom Background: The transcription factor PRH/HHEX (Proline Rich Homeodomain/Haematopoietically-Expressed Homeobox) controls cell proliferation, differentiation and migration. The activity of this protein is decreased in some tumours and in some subtypes of leukaemia. Our previous work showed that in haematopoietic cells Protein Kinase CK2 phosphorylates PRH resulting in the inhibition of PRH DNA binding activity and the misregulation of PRH target genes. CK2 activity is known to be elevated in prostate cancer and in many other disease states. Materials and Methods: PRH and phosphorylated PRH (pPRH) expression levels in prostate cell lines and prostate tissue samples (normal prostate, benign prostatic hyperplasia, and prostatic adenocarcinoma) were determined using western blotting and immunohistochemistry, respectively. PRH was knocked down in normal immortalised prostate epithelial cells using shRNA. PRH and CK2 were over-expressed in these cells and in prostate cancer cell lines using transient transfection. Cell migration/invasion and cell proliferation were examined using Transwell assays and MTT assays, respectively. CK2 was inhibited using TBB and DMAT. Results and Discussion: PRH and pPRH are present in normal immortalised prostate epithelial cells and in normal prostate. Moreover, nuclear pPRH is elevated in benign prostatic hyperplasia, prostatic adenocarcinoma, and prostate cancer cell lines. PRH knockdown increases the motility of normal prostate epithelial cells whereas PRH over-expression inhibits prostate cancer

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cell migration and prevents the invasion of an extracellular matrix by these cells. Significantly, CK2 over-expression blocks the repression of prostate cancer cell migration and invasion by PRH, but does not block the effects of a mutated PRH protein that cannot be phosphorylated by CK2. PRH knockdown in normal immortalised prostate cells also results in increased cell proliferation and an increase in the population of cells capable of colony formation in Matrigel. Inhibition of CK2 reduces PRH phosphorylation and reduces prostate cell proliferation. However, the effects of CK2 inhibition on cell proliferation are abrogated in PRH knockdown cells. Conclusions: In both normal and transformed prostate epithelial cells PRH expression limits cell proliferation and cell migration/invasion. Increased phosphorylation of PRH in prostate cancer cells by CK2 increases cell proliferation and cell migration/invasion. Importantly, the effects of CK2 inhibitors on the proliferation of prostate cells are mediated by PRH. Further clinical studies are required to evaluate the value of PRH and pPRH as potential biomarkers that could identify patients suitable for treatment with CK2 inhibitors. No conflict of interest. 454 Tumor heterogeneity testing in gastrointestinal stromal tumors using droplet digital PCR ´ 2 , P. Szepe ´ 3 , L. Plank3 . 1 Comenius Z. Lasabova´ 1 , K. Jaˇsek1 , M. Grendar University in Bratislava- Jessenius Faculty of Medicine in Martin, Biomedical Center Martin- Division Oncology, Martin, Slovak Republic, 2 Comenius University in Bratislava- Jessenius Faculty of Medicine in Martin, Biomedical Center Martin, Martin, Slovak Republic, 3 Comenius University in BratislavaJessenius Faculty of Medicine and University Hospital in Martin, Department of Pathological Anatomy, Martin, Slovak Republic Introduction: Gastrointestinal stromal tumors (GISTs) are characterized by KIT/PDGFRa mutations. Around 10−15% of all diagnosed GISTs are without mutations in these two genes and are referred to as wild-type (WT) GISTs. However, using different molecular methods, preferably Sanger sequencing and allele-specific approaches, the BRAF V600E mutation was reported in 3−6% cases and represents an alternative molecular pathway in the early tumorigenesis in WT GISTs. The aim of this study was to confirm and quantify the presence of the BRAF mutation using sensitive droplet digital PCR (ddPCR) in GISTs samples V600E positive by Sanger sequencing and/or here implemented allele-specific (AS) PCR. Material and Methods: Formalin-fixed paraffin-embedded (FFPE) tissue sections were collected as a part of the National GIST Registry from 705 patients with GIST in years 2004–2014 and were characterized by immunohistochemistry. Mutation analysis of KIT, PDGFRA and BRAF was performed by Sanger sequencing and AS PCR. DNA was extracted from 9 FFPE tissue section previously tested positive for V600E by AS-PCR. DNA extracted from the V600E harboring RKO cell line was used as a positive control for the assessment of analytical sensitivity. Amplifications were carried out in a reaction volume of 20 ml with ddPCR Mut Assay BRAF on the QX200 Droplet Digital PCR System (Bio-Rad). The analytical sensitivity was evaluated using the R library chemCal by calibrating the regression line. The fractional abundance representing the amount of mutated DNA in the background of all tested DNA was calculated by the QuantaSoft software. Results and Discussion: The analytical sensitivity of the ddPCR was assessed in serial dilutions of mutated DNA (from 70 ng to 0.0007 ng) into 70 ng of the wild-type DNA in quadruplicates. The mutation could be detected even at 0.0007 ng with the fractional abundance of 0.00417%. The limit of detection (LoD) was established as 3.4293 copies/ml. This represents the sensitivity of 0.0162%. After extrapolation to 100 ng of input DNA, we were able to detect the V600E mutation in all AS-PCR positive samples with the fractional abundance from 1.9% to 0.06%. The sample containing 1.9% mutated copies was positive by Sanger sequencing, however, the sample containing 0.06% mutated copies was not. We were able to obtain a very high analytical sensitivity to study the tumor heterogeneity and all WT GISTs in our cohort should be tested by this method. Conclusion: The results of our study show that the identification of V600E by very sensitive ddPCR has potential value for identifying patients that could benefit from treatment with BRAF inhibitors. Acknowledgement: This work was supported by Biomedical Center Martin (ITMS 26220220187) and APVV-14-0273. No conflict of interest. 455 Chemotherapy resistance in breast cancer: A role for senescent fibroblasts D.W. Perkins1 , L. O’Leary1 , C.M. Isacke1 . 1 Institute of Cancer Research ICR London, Breast Cancer Research, London, United Kingdom Background: Despite significant advances in endocrine therapies and targeted agents, chemotherapy remains the mainstay treatment for metastatic breast cancer. Unfortunately tumours frequently develop resistance during repeated cycles of treatment, termed chemoresistance. There are two mechanisms by which chemoresistance can arise: tumour autonomous, whereby

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tumour cells become resistant by changing their internal signalling pathways; and non-autonomous which involve interaction and paracrine signalling within the tumour stroma to elicit a protective effect on the tumour cells. Whilst tumour autonomous resistance mechanisms are well studied, comparatively less is known about how non-autonomous resistance arises. Fibroblasts are a key component of the tumour stroma in many cancers. There is now evidence that genotoxic chemotherapy agents cause DNA damage in stromal fibroblasts causing them to become senescent and alter their cocktail of secreted factors. This senescence associated secretory phenotype (SASP) has been shown to have many pro-tumorigenic effects. Together this suggests that chemoresistance, after repeated cycles of treatment, is contributed to by changes in stromal signals from senescent fibroblasts. Understanding what changes occur in the SASP, and which signalling pathways are activated in the tumour cells has the potential to enable development of adjuvant drugs to administer alongside chemotherapy to slow the onset of chemoresistance, increase the efficacy of the treatment and reduce the negative side effects felt by patients. Methods: Within my PhD project I am developing in vitro co-culture assays to provide a model of fibroblast interaction with tumour cells. To better mimic the in vivo interaction between the two cell types I am employing 3D spheroid culture systems. Results: I have demonstrated that the fibroblast component of tumour spheroids elicits marked growth and phenotype alterations of the tumour cells. Further, my data indicates that chemotherapy treatment induces protumorigenic fibroblast SASPs and could promote chemoresistance. Conclusions: Chemotherapy induces senescence in benign fibroblasts which has pro-tumorigenic effects. These studies will be expanded into screens to identify the components which relate fibroblast signalling to resistance mechanisms in tumour cells. These studies are clinically relevant because they have the potential to identify novel strategies for blocking stromal mediated non-autonomous chemoresistance in tumour cells. No conflict of interest. 456 Chemotherapy and inflammation show a NFúB- and STAT3-dependent pro-tumorigenic potential L. Pezze` 1 , F. Alessandrini1 , Y. Ciribilli1 . 1 Centre for Integrative BiologyCIBIO- University of Trento, Laboratory of Molecular Cancer Genetics, Trento, Italy Introduction: Tumor microenvironment (TME) plays a critical role in cancer progression and response to therapies. Therapeutic intervention strategies for cancer treatment can induce inflammation, resulting in changes in the TME. To investigate the response of cancer cells to chemotherapy in the context of an inflammatory microenvironment, we studied the effects driven by the chemotherapeutic drug Doxorubicin (Doxo), able to stabilize p53 and the inflammatory cytokine TNFa, inducing NFúB. Using expression microarrays, we previously demonstrated that the Doxo+TNFa combined treatment determined a strong up-regulation of migration-related genes as well as increased motility in breast cancer cells (MCF7). Based on these results we investigated the signaling pathways underlying the Doxo+TNFa synergy, specifically the involvement of p53, NFúB and STAT3, that is one of the master regulators in response to cancer-related inflammation. Methods: We obtained p53, p65 and STAT3 knock-out MCF7 and MDAMB-231 cells (breast cancer-derived) and p53 KO U2OS cells (osteosarcomaderived) by using the CRISPR/Cas9 technology. We also pharmacologically inhibited NFúB and STAT3 pathways using respectively BAY11–7082 and Stattic compounds. Mammary acini were obtained with human nontransformed mammary cells MCF10A cells grown in matrigel and tubeformation assay was performed with Human Umbilical Vein Endothelial Cells (HUVEC). Migratory potential was assessed by wound healing assay. Results: We confirmed the synergistic regulation (both up- and downregulation) of a group of 10 different genes upon Doxo+TNFa treatment in different cell line models coming from different cancer types (MCF7 and MDA-MB-231-breast, A549-lung, U2OS-osteosarcoma). Moreover, we demonstrated that this effect was only partially p53-depenedent but strongly Doxo-dependent using p53 mutated MDA-MB-231 cells or knocking-out p53 in MCF7 or U2OS p53 wild type cells. Using BAY11–7082 we were able to demonstrate an NFúB-dependent regulation of most of the genes analyzed. Furthermore, in order to demonstrate a STAT3-dependent regulation of upregulated genes upon combined treatment, we pharmacologically inhibited or knocked-out STAT3 in MCF7, MDA-MB-231 and U2OS cells. Results demonstrated a STAT3-dependent regulation for some of the selected genes and STAT3 inhibition resulted also in a reduced migration potential of cancer cells, particularly in U2OS. We also demonstrated that the combined Doxo+TNFa treatment was not only able to disrupt the 3D architecture of mammary acini observed with MCF10A cells grown within matrigel, but also to stimulate the tube-forming potential of HUVEC cells. Conclusions: We propose that the combined treatment with Doxo+TNFa can lead to the activation of specific gene expression programs that may impact on cancer phenotypes and potentially modify the efficacy of cancer therapy. No conflict of interest.

457 The role of tumour heterogeneity in melanoma progression E. Rowling1 , A. Chapman1 , B. Telfer2 , A. Hurlstone1 , C. Wellbrock1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom, 2 University of Manchester, School of Pharmacy and Pharmaceutical Science, Manchester, United Kingdom Introduction: Tumours usually display a high degree of genotypic and phenotypic heterogeneity. In melanoma, cell subpopulations have been characterized that differ in gene expression profiles, proliferation rates, and invasiveness. The transcription factor MITF (MIcrophthalmia Transcription Factor) plays a crucial role in these phenotypes with high expression correlating with a proliferative phenotype and low expression with an invasive phenotype, and histological analyses have shown that MITF expression is heterogeneous within tumours. We have previously demonstrated the importance of these MITF high and low phenotypes and how they can communicate reciprocally and cooperate. Particularly in melanoma invasion, poorly invasive high MITF expressing cells can co-invade with highly invasive low MITF expressing cells, a process termed ‘co-operative invasion’. Our study also showed the importance of extra-cellular matrix (ECM) deposition in cooperative invasion, specifically Fibronectin (FN), which is essential for effective cell invasion. Further preliminary work looked at FN and MITF heterogeneity in the metastatic cascade and found circulating tumour cells were primarily detected in mice bearing MITFlow and FNhigh tumours. Here we have investigated the communication between FNhigh with FNlow cells during various steps involved in the metastatic cascade to further elucidate the mechanisms involved. Materials and Methods: A pair of melanoma cell lines 501mel (MITFhigh, FNlow) and WM266-4 cells (MITFlow, FNhigh) were chosen for the study along with WM266-4 shFN cells, in which FN is depleted via RNAi. In vitro assays with 501mel cells alone or combined with wild type WM266-4 or WM266-4 shFN cells evaluated the effects of heterogeneity on spheroid formation, adhesion, anoikis and the cell cycle. Exogenous FN was also used as a tool to confirm the involvement of the ECM molecule. In vivo studies looking at the effects of the different cell types alone and in combination on metastasis were completed. Results and Discussion: In anoikis assays FNhigh WM266-4 cells showed less cell death than FNlow 501mel cells and FN depleted WM266-4. However, in heterogeneous populations consisting of 501mel cells and WM266-4 cells, cell death was reduced. Moreover, in heterogeneous populations the cell numbers of 501mel cells increased compared to 501mel cells alone. Similar observations were made in melanoma spheroids, where the presence of FN expressing cells increased aggregation and reduced apoptosis. Heterogeneous populations also showed increased invasion through matrigel and adhesion to endothelial cells. Conclusion: We have found that cells harboring differing phenotypes can work co-operatively and communicate to survive and proliferate in a non-adherent environment and that the ECM molecule Fibronectin plays a key role in this communication. No conflict of interest. 458 Low primary intra-tumoural heterogeneity versus high genomic disparity between primary and distant sites in NSCLC B. Polzer1,2 , N. Wendler1 , R. Fahrioglu-Yamaci1 , F. Elsner1 , B. Cucuruz1 , M. Lindner3 , H.S. Hofmann4 , B. Passlick5 , C.A. Klein1,2 . 1 Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany, 2 Project group “Personalized Tumor Therapy”, Fraunhofer Institute for Toxicology and Experimental Medicine, Regensburg, ¨ Germany, 3 Department of Surgery, Asklepios Fachkliniken Munchen-Gauting, Gauting, Germany, 4 Department of Thoracic Surgery, University of 5 Regensburg, Regensburg, Germany, Department of Thoracic Surgery, University Medical Center Freiburg, Freiburg, Germany Introduction: Deep sequencing of primary tumours and metastases revealed striking clonal heterogeneity between and within individual patients and hinted at an early branching in the genetic evolution of primary and metastatic tumour cells. Moreover, disseminated cancer cells (DCCs) can be detected in bone marrow (BM) or lymph nodes (LN) of patients with smallest tumours, suggesting that systemic spread is an early event during progression in many types of cancer. Thus, local and systemic disease most likely progress in parallel. Therefore, we asked whether detection and molecular analysis of DCCs in BM and LN of patients with early stage non-small cell lung cancer (NSCLC) could provide information on mechanisms of metastasis that may eventually become clinically useful. Material and Methods: We collected LN and BM samples of 119 patients with operable NSCLC. Presence of DCCs was detected by staining against the epithelial cell adhesion molecule (EpCAM, CD326) for LN and cytokeratins 8/18/19 for BM samples. The patients were followed for a median of 42 months (range 1–139 months) and detection of DCCs was correlated with overall survival by univariate and multivariate analysis. Additionally, single DCCs

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 were isolated and DNA amplified by Ampli1TM whole genome amplification. Genomic profiles of isolated DCCs from LN and BM were analyzed by single cell comparative genomic hybridization and compared to profiles of various laser-microdissected areas of primary NSCLC tumours. Results: DCCs in LN or BM were detected in 41.2% of 119 patients and were associated with significantly reduced overall survival (logrank test, P = 0.014). Moreover, we found that especially patients showing both haematogenous and lymphatic spread at the time of surgery showed poor outcome (logrank test, P < 0.001). Interestingly, primary tumour areas and single DCCs from LN showed significantly more chromosomal gains or losses than DCCs from BM (Kruskal Wallis test, P < 0.001). Additionally, we frequently detected a clear clonal relationship between multiple analysed primary tumour areas of the same patient, indicating low intra-tumour heterogeneity in primary NSCLC. Similarly, individual LN DCCs displayed many shared aberrations in the majority of patients. However, comparing the genomic profiles of the dominant clones isolated from primary tumour and LN-DCCs we identified striking differences. Conclusions: In summary, intra-tumoural heterogeneity for genomic alterations is much lower than the disparity between primary cancer cells and DCCs isolated from distant sites. This result questions the approach to rely on the genetic profile of primary tumour cells as surrogate marker for systemic therapy. Therefore, detection and molecular characterization of DCCs could provide valuable information as prognostic and predictive biomarker in NSCLC. No conflict of interest. 459 The activities of cell surface NADH-oxidases, pyruvate kinase and lactate dehydrogenase: Signatures for cancer cells in precarious nutrient conditions A.M. Otto1 , P. Biechl1 , H. Fernandes1 , M. Gkiouli1 , J. Hintermair1 , J. Hutterer1 . 1 Munich Technical University, Institute of Medical Engineering IMETUM, Garching, Germany Background: While it is accepted that tumor metabolism changes with malignancy and a variable microenvironment, there is a lack of quantitative biochemical characterization of metabolites and enzyme activities under varying growth conditions. Such information is of particular relevance for the implementation of clinical metabolic imaging. It is thus the aim of this study to determine the activities of two glycolytic enzymes, i.e. pyruvate kinase and lactate dehydrogenase (LDH), as well as of a cell surface NADH oxidase (NOX) in two cell lines of different malignant potential growing under limiting nutrient conditions. Material and Methods: MCF-7 and the more malignant MDA-MB231 cells were cultured as monolayers in DMEM (with 2% FCS) containing various combinations of glucose, glutamine and lactate. Cell number, the activities of pyruvate kinase and lactate dehydrogenase (LDH) were measured, and NADH and NAD+ levels determined. A WST-1 (“proliferation”) kit was used to assay cell surface NADH oxidase (NOX) activity. Results: Growth of MCF-7 cells is more sensitive to glucose in the 1−5 mM range than that of MDA-MB231 and depends also on the glutamine concentrations. Very different patterns of metabolite-dependency were observed for cell surface NOX activity, which is suppressed by 1 mM glutamine (as opposed to 0.1 mM) and enhanced by added lactate (25 mM) in both cell lines. This NOX activity has been reported to be fed by cytosolic NADH, and indeed, 2−3-fold increases in NADH concentrations were observed with lactate. In both cell lines, NOX activity was also rapidly enhanced upon inhibiting the mitochondrial NADH oxidase by rotenone, which leads to “excess” cytosolic NADH. NOX may also contribute to the reoxidation of NADH to NAD+, which is required for glycolysis, and, moreover, due to the transfer of hydrogen through the plasma membrane, to the maintenance of cytosolic alkalinity. In MCF-7 cells pyruvate kinase activity was highest with a 2.5 mM glucose/0.1 mM glutamine combination, while LDH activity was about 2−3-fold lower, meaning that pyruvate diverges also in to other metabolic pathways. In contrast, pyruvate kinase activity was markedly lower in MDAMB231 than in MCF-7 cells and was equal to LDH activity in limiting nutrient conditions. This suggests that lactate production is the main fate of glycolytic pyruvate in the more malignant cells. Conclusions: Cell surface NOX activity along with the potential activities of pyruvate kinase and LDH are measurable signatures with which the metabolic phenotype of cells with different states of malignancy could be distinguished. These biochemical parameters will serve as references for the evaluation of data from metabolic profiling and imaging. Acknowledgement: This project is supported by the “Wilhelm Sander-Stiftung”, Munich, Germany. No conflict of interest.

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460 Investigation of the possible relationship of cancer stem cell subpopulation and metastatic potential of an oral squamous cell carcinoma cell line N. Martins Lopes1 , R. Alves da Silva Alavarce2 , R. Carneiro Ortiz1 , T.J. Dion´ısio1 , G. Pompermaier Garlet1 , E. Graner3 , V. Soares Lara2 , C. Oliveira Rodini1 . 1 School of Dentistry of Bauru − University of Sao Paulo, Biological Sciences, Bauru, Brazil, 2 School of Dentistry of Bauru − University of Sao Paulo, Department of Stomatology, Bauru, Brazil, 3 School of Dentistry of Piracicaba − Unicamp, Department of Oral Diagnosis, Piracicaba, Brazil Background: The presence of metastasis in cervical lymph nodes is the most significant prognostic factor for the oral squamous cell carcinoma (OSCC), one of the most common malignant neoplasms of the head and neck. Studies have associated the mechanisms of tumor progression, recurrence and metastasis with the presence and maintenance of cancer stem cells (CSC), which correspond to the most migratory and highly metastatic cellular subpopulation when compared to other cell subpopulations within the tumor. The purpose of the present study was to validate an in vitro experimental model to investigate the participation of CSC on OSCC invasion and metastasis. Parental (SCC-9) and metastatic (SCC-9 LN1) OSCC cell lines obtained after in vivo tumorigenesis assays were characterized regarding the presence and proportion of CSC subpopulation as well as related CSC markers. Material and Methods: CSC phenotype within the whole tumor population was evaluated based on the capacity to form tumor colonies, mainly holoclones, and spheres. qPCR was also conducted to verify the differential expression levels of CD44, BMI-1 and ALDH-1 transcripts on both OSCC cell lines using human palate epithelial cells (HPEC) generated by primary culture as controls. Results: The quantification of tumor holoclones and spheres were greater in the metastatic compared to parental cell line (fold 2 and >2, respectively). Transcripts related to the CSC phenotype were overexpressed in both tumor cell lines when compared to normal oral keratinocytes. Specifically, the metastatic cell line SCC-9 LN1 showed higher expression of CD44, BMI-1 and ALDH-1 (fold >2) when compared to parental cell line. Conclusion: These preliminary results confirm the presence of a CSC subpopulation in both tumor cell lines. Additionally, the metastatic potential of SCC-9 LN1 cells may be a consequence of their higher CSC subpopulation phenotype compared to parental SCC-9. Complementary studies are being conducted in order to also evaluate the higher epithelial–mesenchymal transition (EMT) phenotype of the metastatic SCC-9 LN1 cell line, since the process of tumor invasion and metastasis is related to both CSC and EMT phenotypes. No conflict of interest. 461 Investigating the molecular significance of aberrant HER2, HER3 and downstream partner signalling in castrate resistant prostate cancer M. Aalsamraae1 . 1 Newcastle University, Northern Institute for Cancer Research, Newcastle, United Kingdom Introduction: Prostate cancer (PC) is the most common male cancer in the UK with a man’s lifetime risk of developing PC being 1 in 8 (Prostate Cancer UK). The androgen receptor (AR) has a crucial role in the proliferation and growth of the prostate gland. Increased expression of HER2 and HER3 is suggested to play an important role in castrate resistant prostate cancer (CRPC), however it not clear how HER2 and HER3 regulates AR in this advanced stage of PC. This study aims to interrogate the mechanism of HER2/HER3 activation of the AR in CRPC, utilizing our in-house developed androgen receptor inhibitor resistant cell line models, including a Casodex-Resistant cell line, an EnzalutamideResistant cell line and an ARN509-Resistant cell line. Methods: 1. QPCR: To determine the mRNA level of HER2 and HER3 (+/-Heregulin). Data was normalised against the house keeping gene hprt1. 2. Western blot: used to detect the protein level of the HER2 and HER3 (+/− Heregulin) a-tubulin was used as loading control protein. 3. Luciferase assay: The LNCaP-7B7 cell line contains a chromosomally integrated PSA-luciferase promoter, thus enabling the analysis of AR activity on the transcription of its downstream targets such as PSA. 4. Microarray: used to determine the gene expression on a genomic scale. The microarray was used to determine the genes profile of LNCaP resistant to anti-androgen and LNCaP sensitive to anti-androgen drugs. Results: The preliminary data showed an increase in expression of HER2/3 in castrate resistant cells compared with LNCaP cells at both the protein and mRNA levels. We have also demonstrated through luciferase assays that heregulin-stimulated activation of the AR can be abrogated through both pan-HER inhibition and AKT inhibition. Consequently, we have performed a gene microarray comparing parental LNCaP cells with our in-house produced Enzalutamide-resistant LNCaP cell line +/− heregulin stimulation, this resulted in identification of gene expression signature of Enzalutamide resistance. Conclusion: Increased expression of HER2/3 in drug-resistant LNCaP cells compared with LNCaP cells at both proteins and mRNA levels was observed.

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Both pan-HER inhibitor and AKT inhibitor abrogate the increased AR activity seen on stimulation with Heregulin. We now have 4 target genes that characteristic of Enzalutamide resistance that could represent either future biomarkers of drug-resistance or potential therapeutic targets in advanced disease. No conflict of interest. 463 Spheroids versus monolayers: are the cells metabolically the same? M. Gkiouli1 , A. Otto1 , J. Hintermair1 . 1 Munich Technical University, Institute of Medical Engineering IMETUM, Garching, Germany Background: Monolayer cultures as tumor models do not mimic well an in vivo tumor. Therefore, 3D cultures are being increasingly used as in vitro models that mimic tumor conditions in a more physiological manner with respect to diffusional limitations of oxygen, nutrients and cellular waste products as well as pH gradients. To be able to evaluate what kind of differences could occur in the metabolic activities when tumor cells are cultured as 3D- as opposed to 2D-cultures, two different breast carcinoma cell lines were analysed for their metabolic responses to limiting nutrient levels and variable pH. Material and Methods: MCF-7 and the more malignant MDA-MB231 cells were cultivated as monolayer and as spheroid cultures with variable extracellular pH and tumor-relevant glucose and glutamine levels. Cell number was determined by nuclei counting. Metabolic activity was measured with a water soluble metabolic tetrazolium assay (WST-1, “proliferation assay”), which reflects the activity of a cell surface membrane NADH-oxidase that is dependent on intracellular NADH. Moreover, lactate production and glucose consumption were determined in cell culture supernatants. Results: The results show that in general tumor cell growth and metabolic activity do not correlate; they are dependent on the extracellular pH and on the concentration and the ratio of glucose / glutamine. In 3D cultures, NADHoxidase activity per cell number is increased up to 10-fold compared to 2D cultures. Moreover, MDA-MB231 cells have a higher NADH oxidase activity than MCF-7 cells. The amount of glucose converted to lactate is higher in 3D than in monolayer cultures, and it is higher in MDA-MB231 cells than in MCF-7 cells. Finally, with increasing acidity, in both cell lines growth as well as NADH-oxidase and glucose consumption become less dependent on the variable levels of nutrients, a common feature for both 2D and 3D cultures. Conclusions: There are differences in the metabolism of spheroids compared to their monolayer cultures, revealing the necessity for recapitulating in the 3D cultures metabolic parameters that have been established in 2D cultures, especially in the context of testing for anticancer activity, as well as for choosing cellular systems for metabolic profiling and imaging. Acknowledgment: This project is supported by the “Wilhelm Sander-Stiftung”, Munich, Germany. No conflict of interest. 465 Development and characterisation of a novel syngeneic, spontaneous breast cancer metastasis model U. Jungwirth1 , G. Qiong1 , S. Wantuch1 , C. Isacke1 . 1 The Institute of Cancer Research, Breast Cancer Now Research Centre, London, United Kingdom Background: The development of novel therapeutic strategies for targeting metastatic disease is restricted by our lack of knowledge of molecular mechanisms underlying the metastatic process. However, it is clear that successful metastatic colonisation involves a close crosstalk between tumour cells and the metastatic microenvironment. Unfortunately, there are no in vitro models that recapitulate all the different steps of the metastatic cascade and in vivo human xenograft and PDX models are transplanted into immunocompromised mice that lack an intact immune system. Material and Methods: In this study we developed two syngeneic, spontaneous Balb/c breast cancer metastasis models, namely D2A1-LuM1 and D2A1-LuM2, from the poorly spontaneous metastasising D2A1 cell line by serial in vivo passaging. We characterised the models in vivo, by immunohistochemistry and in vitro, using 2D and 3D cultures. Furthermore, we performed gene expression profiling to identify differentially modulated pathways. Results: Both D2A1-LuM1 and D2A1-LuM2 spontaneously metastasis from the fat pad of Balb/c mice giving rise to macrometastatic diease in the lungs. The metastatic potential of the two models is consistent with 2D colony growth and 3D tumour spheroid growth assays, showing that the D2A1-Lum1 clone is more aggressive than the D2A1-LuM2 clone. Gene expression profiling identified 218 (111 upregulated, 107 downregulated) differentially expressed genes (with a p-value of 0.001) in the metastatic clones compared to the parental D2A1 cells, including invasion and cell death pathways. Conclusions: Currently researchers rely on the 4T1 spontaneous breast cancer metastasis model. Consequently, there is a need for additional syngeneic models. The metastatic D2A1 clones described here, together with the recently described EO771.LMB metastatic clone, provide important

independent syngeneic models for investigating the molecular and cellular mechanisms underpinning metastatic breast cancer progression. No conflict of interest. 466 A kinome-scale synthetic lethality screen reveals an essentiality of CDK13 for 19q12 amplified cancer cells R. Stark1 , W. Krek1 . 1 Swiss Federal Institute of Technology, Institute of Molecular Health Sciences, Zurich, ¨ Switzerland Introduction: Amplification of the chromosomal locus 19q12 is a common feature among multiple tumor types including stomach, liver, lung, breast and ovarian cancer. The most frequent malignant tumors with 19q12 amplifications are high-grade serous ovarian carcinomas for which therapy and cure options are very limited due to chemoresistance. 19q12 encompasses multiple genes among which two genes, CCNE1 and URI1, have been associated with oncogenic function.CCNE1 encodes Cyclin E which controls G1/S phase transition in complex with CDK2 during cell cycle; URI1 is an unconventional member of the prefoldin family of molecular chaperones (Gstaiger et al, 2003; Theurillat et al., 2011). To identify genes critical for the survival of 19q12 amplified tumors, we performed a kinome-focused shRNA screen in 19q12 amplifed and non-amplifed ovarian cancer cells. Identified kinases could serve as potential therapeutic targets in the treatment of 19q12 amplified cancers. Methods: Using a lentivirally-delivered shRNA library, we performed parallel pooled shRNA screens in 6 ovarian cancer cell lines (3 with 19q12 amplification and 3 lacking the amplicon). Cell line dependencies on 537 kinase-encoding genes were interrogated by 5 shRNAs per gene. The proliferation effect of each shRNA in each cell line was assessed by transducing a population of 2.7 mio cells with one shRNA virus per cell and determining the relative enrichment or depletion of each of the 2684 shRNAs after 12 population doublings using Next Generation Sequencing of the individual hairpin sequences. Results: After quality control, count normalization and differential representation analyses (Robinson et al, 2010, Dai et al, 2014), Analytic Technique for Assessment of RNAi by Similarity (ATARiS) (Shao et al, 2013) was applied, which produces quantitative, gene-level phenotype values that provide an intuitive measure of the effect of gene suppression in each sample. The top 5 hits were validated in a biological context by performing clonogenic assays after applying RNAi with 2 distinct shRNAs. Hit validation was performed in all 6 cell lines and an extended panel of 19q12 non-amplified ovarian cancer and breast cancer cell lines. The number of remaining colonies was determined 14 days after application of the respective knockdowns. From the top 5 hits, CDK13 and CSNK1E (CK1e) were confirmed by subsequent validation studies. Conclusion: By screening the human kinome for potential therapeutic targets specifically required for 19q12 amplified ovarian cancer cell lines, we identified CDK13 and CK1e to be preferentially required for the survival of 19q12 amplified but not non-amplified cancer cells. Thus, CDK13 and CK1e may represent potential targets for therapy in 19q12 amplified cancer cells of diverse origins. No conflict of interest. 467 Mcl-1 dynamics influence mitotic slippage and death in mitosis O. Sloss1 , C. Topham1 , S. Taylor1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom Background: Microtubule-binding drugs are used to treat a variety of cancers, however the link between treatment and tumour cell death is still unclear. In cell culture these drugs induce a mitotic arrest, caused by spindle assembly checkpoint activation. Following a sustained arrest, cells can commit to mitotic death or mitotic slippage (mitotic exit without division). Whereas mitotic slippage is caused by declining Cyclin B1 levels by APC/C ubiquitination, the mitotic death signal is not well defined. Protein levels of anti-apoptotic factor Mcl-1 decline during a mitotic arrest, suggesting that Mcl-1 acts as a mitotic death timer. We investigated the effect of proteasome-mediated degradation of Mcl-1 on mitotic death as well as inhibition of several E3 ligase complexes previously shown to target Mcl-1 for degradation. Additionally, it has been proposed that apoptotic network factors could influence the rate of mitotic slippage. Therefore, we also analysed the effect of Mcl-1 levels on mitotic slippage. Materials and Methods: Two colorectal cell lines (RKO and DLD-1) were treated with a variety of small molecular inhibitors and siRNAs during a mitotic arrest in order to analyse the effect these methods had on Mcl-1 stability in a mitotic arrest. In parallel, time-lapse microscopy was utilised in order to analyse cell fate of populations of cells treated with microtubule-binding drugs in order to see if the changes in Mcl-1 levels could influence the rate of mitotic death and mitotic slippage. In addition, mutant forms of Mcl-1 believed to influence its degradation were over-expressed and similarly analysed. Results: Treatment of cells with a proteasome inhibitor significantly delayed mitotic death and this delay was dependent on increased Mcl-1 levels. Additionally we show that Mcl-1 is synthesised during mitosis and blocking protein synthesis with cycloheximide accelerates mitotic death. Inhibition of

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 E3 ligases APC/C-Cdc20, SCF-Fbw7 and MULE or expression of a lysineless mutant of Mcl-1 had no effect on the rate of mitotic death. Conversely, levels of Mcl-1 appeared to influence mitotic slippage at the same time as mitotic death. This effect appeared to be dependent on a D-box like motif of Mcl-1 recognised by the APC/C, suggesting binding competition between Mcl-1 and Cyclin B1 for the complex. Conclusions: These results show that Mcl-1 turnover is important for regulating the rate of mitotic death in response to microtubule-binding drugs. However, efforts to solidify a mechanism for Mcl-1 degradation argue against the importance of the cannonical ubiquitin-proteasome pathway. This study shows that Mcl-1 levels can influence the rate of mitotic slippage at the same time as mitotic death, thus suggesting future analysis regarding factors that contribute to either phenotype must be used with caution. Reference(s) Sloss et al (2016) Oncotarget. No conflict of interest. 468 Functional characterization of the interaction between the stem cell factor SOX2 and the cell cycle regulator P27 in normal and cancerous stem cells V. Moncho-Amor1 , L. Garros2 , C. Galichet1 , K. Rizzoti1 , M. Matheu2 , R. Lovell-Badge1 . 1 The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom, 2 IIS Biodonostia, Oncology Area, San Sebastian, Spain The discovery of a subpopulation of cancer cells with properties of stem cells (SCs) (cancer stem cells, CSCs) has implications for therapy because these cells are postulated to be responsible for the origin, progression and recurrence of cancers. SOX2 and P27 are essential during embryonic development and in the regulation of SCs in multiple tissues, such as the pituitary and brain. We have shown that P27 represses Sox2 expression in embryonic stem cells (ESCs) (Li et al., 2012). The physiological relevance of this interaction is demonstated in vivo as Sox2 haplo-insufficiency rescues aspects of the p27 null phenotype (Li et al., 2012). The SOX2−P27 interaction may therefore regulate the balance between quiescence and proliferation in SCs and be involved, if altered, in the transformation into CSCs. To demonstrate such a role, we are characterizing the function of SOX2−P27 in neural and glioma SCs, and also in pituitary adenomas displayed by p27 null mice. We show here that in differentiated neural SCs in vitro, p27 is recruited to the Sox2-SRR2 enhancer. This suggests, as demonstrated in ESCs, that it induces repression of Sox2, extending the relevance of the SOX2−P27 interaction to neural SCs. In line with this idea, there is an inverse correlation within them in glioma cells both in stem cell and differentiation conditions. In addition, P27 overexpression represses SOX2 levels in glioma cells. The intermediate lobe (IL) of the pituitary is populated by POMC (pro-opiomelanocortin) positive melanotrophs. Despite being differentiated, these also express low levels of SOX2. In p27 null ageing mice, tumors develop in IL; we observe that in pre-tumoral IL, melanotrophs express more SOX2 but less POMC, suggesting that malignancy is associated with incomplete or de-differentiation of melanotrophs. SOX2 dose appears crucial as we show here that reduction in SOX2 expression levels, such as in yellow submarine mutants (Kiernan et al., 2005), completely prevent IL tumor formation in p27 null; ysb double mutants. In parallel, we observe that ectopic blood vessels develops in IL, while proliferation increases both in melanotrophs and in pituitary stem cells (PitSCs), which express SOX2. In agreement with these data, more pituitary spheres (pituispheres) are generated from p27 null IL compared to control. To determine whether pituitary SCs or melanotrophs are the origin of the tumours that develop in p27 null animals, we are performing lineage-tracing experiments using SOX9CreERT2, which is active in the PitSCs (Rizzoti et al., 2013), and POMCreERT2, which targets melanotrophs. Preliminary results suggest that SC fate is modulated by proliferating melanotrophs in p27 null mice. In conclusion, these results give new insights into SOX2−P27 interactions and their relevance in both SCs and differentiated cells for initiation of tumorigenesis. No conflict of interest. 469 Gadd45: A new player in liver tumorigenesis U. Rosato1 , J.M. Salvador1 . 1 National Center for Biotechnology, Immunology and Oncology, Madrid, Spain The growth arrest and DNA damage-45 (Gadd45) family comprises 3 proteins, GADD45A, GADD45B and GADD45G, which control key cellular processes such as cell cycle arrest, proliferation and apoptosis. Gadd45b was found to be deregulated in Hepatocellular carcinomas (HCC) and its promoter methylated. Although several indirect evidences point out to Gadd45b as a suppressor of liver tumorigenesis, a study given a definitive answer to this question is still missing. To address the role of Gadd45b in hepatic carcinogenesis, we took advantage of the Diethylnitrosamine (DEN) model. This alkylating agent has been used extensively to induce hepatic tumors in mice. In this study, wild type and Gadd45b−/− mice were injected with DEN. After 9 months, mice were sacrificed and tumor development was assessed. Strikingly, a significant percentage of Gadd45b−/− mice did not show any sign of tumor development

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whereas 100% of wt mice developed tumors. In the early response, DEN induces a wave of cell death, where necrotic hepatocytes release. These events are crucial for tumor development at later stages. We found that Gadd45b mRNA is upregulated upon DEN injection and Gadd45b−/− mice display an impaired apoptotic response. This decreased apoptotic response following DEN injection results in a decrease proliferative phase. Our results indicate an unexpected tumor promotive role of Gadd45b in response to DEN injection. This role is probably exerted by targeting the Map kinase cascade, which in turn enhances the early and compensatory proliferative process that are crucial for the later stage tumor development. No conflict of interest. 470 Nandrolone affects cell growth and differentiation in hepatoma cells C. Piccoli1 , F. Agriesti2 , T. Tataranni2 , C. Mazzoccoli2 , V. Ruggieri2 , R. Scrima1 , O. Cela1 , C. Pomara1 , N. Capitanio1 . 1 University of Foggia, Clinical and Experimental Medicine, Foggia, Italy, 2 Laboratory of Pre-Clinical and Translational Research, Basilicata Cancer Centre, Rionero in Vulture PZ, Italy Background: Sexual hormones, estrogens and androgens, determine biological response in a tissue- and gender-specific manner and have a pivotal role in endocrine-mediated tumorigenesis. Androgen signalling, mediated by the androgen receptor (AR), is critical factor influencing growth of normal and malignant cancer cells. Hepatocellular carcinoma (HCC) may be modulated by both estrogens and androgens hormones during its initiation, progression and metastasis. The purpose of this study was to investigate the role of Nandrolone in regulating proliferation and differentiation of HCC. Material and Methods: Human hepatocellular carcinoma cell line HepG2 was treated with Nandrolone Vetranal, a synthetic androgen ligand, for 48 hs and its viability and proliferation was assessed by MTS and cell cycle analysis, respectively. The expression of protein involved in cell cycle regulation and differentiation markers were analysed by western blot and real time PCR. Endogenous respiration and respiratory chain complex activities were measured by high resolution respirometry and spectrophotometric teqniques respectively. Stemness surface markers expression was detected by flow cytometry. Results: Nandrolone treatment caused cell growth inhibition associated to a downregulation of cyclin D1 and an upregulation of the cyclin-dependent kinase inhibitors p21Waf1/Cip1 leading to cell cycle arrest in the G2 phase. Moreover, the activation of AKT signalling with a consequent phosphorylation of GSK-3beta and a significant reduction of mitochondrial respiratory activity were also observed, thus suggesting a role in the control of the metabolic reprogramming. Finally, a significant increase of the stemness markers like Nanog, Myc, Lin28, KLF4 and SOX2 was detected following Nandrolone treatment, also confirmed in a human pulp mesenchymal stem cells. Conclusions: Nandrolone shows a strong anti-proliferative effect in hepatocellular carcinoma cell line promoting cellular dedifferentiation. No conflict of interest. 471 ZEB1 induce chemoresistance through epithelial–mesenchymal transition in prostate cancer cells ´ 1. H.R. Contreras1 , O. Orellana-Serradell1 , D. Herrera1 , E.A. Castellon 1 University of Chile, Faculty of Medicine, Santiago, Chile Background: During the progression of prostate cancer (PCa) the tumor cells lose their intercellular connections and undergo important phenotypic changes known as epithelial–mesenchymal transition (EMT). In this process, ZEB1 plays a central role in the acquisition of mesenchymal phenotype, invasive ability and resistance to chemotherapy. The present work aims to determine the effect of knock down and overexpression of ZEB1 on proliferative, migratory and invasive capacities of PCa cell lines 22RV and DU145. Materials and Methods: Commercial PCa cell lines of low (22RV1, ATTC, Cat. CRL-2505) and high (DU145, ATCC, Cat. HTB-81) ZEB1 expressions were used. ZEB1 knock down and ZEB1 overexpressing cell lines were obtained by transduction with lentiviral vectors (GenTarget Inc). EMT markers were assessed by real time PCR and western blot. Cells were treated with Docetaxel (Tocris inc.) Cell proliferation was assayed by tetrazolium assay (MTT). Results: Cell lines with ZEB1 overexpression showed an increased cell proliferation and a low sensitivity to Docetaxel treatment. Also, ZEB1overexpressing cells showed high expression of mesenchymal genes (i.e. Vimentin) and low expression of epithelial genes (i.e. E-Cadherin). Conversely, ZEB1 knocked down cells showed a decreased proliferation rate and high sensitivity to Docetaxel treatment with an increase in E-cadherin expression and a decrease in Vimentin. These expression changes were associated to a phenotypic reversion from mesenchymal to epithelial type favoring cell growing, treatment resistance and cancer aggressiveness. Conclusions: ZEB1 is involved in regulating EMT and chemoresistance in PCa cells. Acknowledgement: Funding: Projects Fondecyt 1151214 (HRC) and 1140417 (EAC). No conflict of interest.

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473 Exploring the role of inflammation in ETV1-expressing prostate tumours V. Ubertini1 , P. Wang1 , H.S. Leong2 , K. Glass3 , E. Baena1 . 1 CRUK Manchester Institute, Prostate Oncobiology, Manchester, United Kingdom, 2 CRUK Manchester Institute, RNA Biology / Computational Biology, Manchester, United Kingdom, 3 Brigham and Women’s Hospital, Department of Medicine, Boston, USA Introduction: Prostate cancer (PCa) is the second most common cancer in the UK. The incidence of PCa is strictly related to age (>75 years old). At early stages the therapeutic options are curative. Patients who relapse following treatment of localized disease or with metastatic disease at diagnosis are primarily treated by androgen deprivation therapy (ADT). Most patients show a good initial response to ADT, but within 1−3years, PCa will become unresponsive and recur as castration-resistant prostate cancer (CRPC). ETS transcription factor gene fusions are one of the key PCa alterations identified in ~50% of human PCa. Among them, ETV1 is the second most frequently rearranged gene, after ERG. Clinical data revealed that high levels of ETV1 expression are often found in more advanced PCa, whereas ERG overexpression is mainly associated with localized PCa, being recently classified as independent PCa subtypes. Specifically, ETV1 is able to interfere with the lipid metabolism regulation as well as to induce a proinflammatory gene signature, pathways implicated in hormone unresponsive lethal disease. Material and Methods: Gene expression and chromatin-binding studies in human prostate cell lines (3D cultures) and genetically engineered mouse models (Tmprss2-ETV1) have been performed to clarify the transcriptional role of ETV1 in PCa initiation and progression. Cytokines array, multiplex IHC and flow cytometry assays have been established to address the roles of ETV1 in promoting inflammation. Results and Discussion: We demonstrated that ETV1 overexpression on prostate epithelial cells induce an inflammatory gene signature that could be responsible of PCa initiation and/or progression to the metastatic CRPC. Indeed, a big proportion of old Tmprss2-ETV1 mice showed areas of immune infiltration. ETV1-driven lipid metabolism (i.e. arachidonic acid (AA), sphingolipids) may promote a pro-tumorigenic microenvironment. Indeed, AA pathway is responsible for the production of prostaglandins, important mediators of inflammation and tumour immune evasion. We are currently assessing which downstream targets of ETV1 are essential for promoting the lipidic metabolic reprogramming and its relationship with co-expressed inflammatory genes as responsible for prostate epithelial cell transformation, as well as tumour growth. Conclusions: Our initial studies suggest ETV1 involved in a process of chronic inflammation that could facilitate prostate cancer initiation and progression. Noteworthy, several evidences associated prostatitis to the development of PCa, highlighting the importance of inflammation in this type of tumour. Targeting ETV1 or its downstream targets could be a valid therapeutic approach, in cooperation with existing treatments, for patients carrying ETV1 fusions or ETV1 overexpression. No conflict of interest. 474 Extracellular matrix anisotropy in breast cancer invasion and metastasis D. Park1 , R. Jenkins1 , A. Labernadie2 , E. Wershof3 , X. Trepat2 , P. Bates3 , L. Jones4 , E. Sahai1 . 1 Francis Crick Institute, Tumour Cell Biology- Lincoln’s Inn Fields Laboratory, London, United Kingdom, 2 Institute of Bioengineering of Catalonia, Integrative Cell and Tissue Dynamics, Barcelona, Spain, 3 Francis Crick Institute, Biomolecular Modelling Laboratory- Lincoln’s Inn Fields Laboratory, London, United Kingdom, 4 Barts Cancer Institute, Centre for Tumour Biology, London, United Kingdom Introduction: Increased extracellular matrix density and fibre anisotropy is associated with disease progression and poor outcome in several cancer types, including breast. Collagen and fibronectin are reorganised by fibroblasts from a reticular meshwork to aligned bundles running perpendicular to the tumour boundary, facilitating directed migration of tumour cells into the local tissue. Despite the obvious consequences of ECM alignment on invasion and metastasis, little is known about how fibroblasts control matrix anisotropy and orientation relative to the tumour border. Material and Methods: To unravel the molecular basis of this behaviour we have conducted RNA Seq analysis, siRNA and drug screens on a library of fibroblasts of different origin and disease states, that exhibit a range of ECM remodelling behaviours. Results: We find that pathways contributing to cell polarity, adhesion and contact inhibition of locomotion are required for the efficient generation of highly aligned anisotropic ECM. Aligned ECMs enhance the persistence and efficiency of tumour cell migration and drive a strand like pattern of invasion that sits between single cell and collective behaviours. We also explore tumour cell intrinsic requirements for migration along aligned ECM, focusing on a subtype of breast cancer (ILC) that displays strand-like behaviours at both primary and metastatic sites. We find that streams of ILC cells display highly

correlated velocities suggesting supra-cellular coordination in this type of migration. Results from a siRNA screen for the regulation of this behaviour are also presented. Conclusion: ECM fibre alignment is a complex emergent property of many cancer-associated fibroblasts that depends upon PDGFR, integrin and actomyosin function. Positive feedback can reinforce fibre alignment. As a result, small variations in fibre alignment at cancer cell–fibroblast interfaces can greatly alter whether ECM is aligned parallel or perpendicular to tumour boundaries with profound consequences for cancer cell invasion. No conflict of interest. 475 Functional dissection of the role of Myc in pancreatic tumourigenesis N. Muthalagu1 , J. Morton1 , W. Clark1 , A. Hedley1 , O. Sansom1 , D. Murphy1 . 1 Beatson Institute for Cancer reaserch, Beatson Institute for Cancer reaserch, Glasgow, United Kingdom Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths worldwide, with an estimated overall 5-year survival of less than 5%. A mutation in small GTPase Kras is found in up to 90% of all human PDACs, whereas CGH analyses of primary PDAC tumours reveals a high frequency of low level amplification of chromosome 8q24, which includes c-Myc locus. Recent studies using mouse model of PDAC (KPC) suggested crucial role for endogenous Myc in PDAC development. Here we aim to identify the mechanistic role of oncogene Myc in PDAC development. GEMM expressing KrasG12D has been previously reported to develop pancreatic intraepithelial neoplasms (PanINs) and rarely develop PDAC. We used mouse model expressing oncogenic Kras and transgene Myc under Rosa26 locus. To date our results show that c-Myc co-operates with KrasG12D and accelerates PDAC development, when activated in utero or in the adult pancreas. To understand the course of tumour evolution we made use of inducible cre (CreER) to activate both Myc and oncogenic Kras, this will provide an opportunity to understand how Myc co-operates with Kras in terms of tumour initiation. Our results suggest that 3days of oncogene activation drives widespread prolifereation and 14days is enough to drive PanIN1. We used Laser capture micro dissection coupled with RNA sequencing understand the gene expression changes in PanIN3 in comparison to PanIN1. Oncogene induced senescence is one of the major barrier preventing progression of Kras induced PanINs to PDAC. In our study, we show that superimposing Myc expression in mice with Kras induced PaniNs is enough to overcome oncogene induced senescence. No conflict of interest. 476 Multi-omics studies of intra-tumour heterogeneity in colorectal cancer ´ 4, G. Wa˛tor1 , M. Suski2 , A. Borys1 , J. Swirta3 , E. Chronowska1 , M. Barczynski P. Wołkow1 . 1 Jagiellonian University Medical College, Center for Medical Genomics − OMICRON, Krakow, Poland, 2 Jagiellonian University Medical College, Chair of Pharmacology, Krakow, Poland, 3 G. Narutowicz Municipal Specialist Hospital in Krakow, Clinical Department of General Surgery, Krakow, Poland, 4 Jagiellonian University Medical College, 3rd Chair of General Surgery, Krakow, Poland Introduction: Colorectal cancer is one of the most common cancers with growing incidence and low 5-year survival. The same clinical presentation can result from several different genetic mechanisms and patients can vary widely by their prognosis as well as response to treatment. Until now, very little is known about intra-tumour heterogeneity of colorectal cancer. Existing intratumour heterogeneity and lack of clear and well defined molecular targets makes it difficult to conduct efficient anti-tumour therapy. The use of complex omics approach should help to get insight into complexity (heterogeneity) of colorectal cancer genetic background and should open the way to translate this knowledge into clinical decisions in the future. Materials and Methods: Four early stage colorectal cancer primary tumours were subjected to this study. For transcriptomics analysis we isolated total RNA from 50–100 mg of tissue, with mRNA and small RNA (less than 200 bases) in separate fractions. Using gene expression microarrays, we analysed 3 tumours (3 tumour samples and corresponding healthy mucosa). Additionally, we performed profiling of tumour associated miRNA on 8 samples using pre-spotted qPCR plates. Raw data from IDAT files were analysed using Rsudio (beadarray, limma packages) whereas Cq value were analysed using GenEx software. For proteomic analysis we extracted and digested with trypsin another 4 samples. Resulting peptides were subjected to off-line strong cation exchange fractionation and analysed by LC-MS/MS system. For secondary analysis we used PANTHER Classification System. The patient’s voluntary informed consent was obtained before participation in the study, according to the recommendations of the Jagiellonian University Medical College Committee of Bioethics. Results and Discussion: Shotgun proteomics approach enabled to detect from 1086 to 1584 peptides with FDR below 2%. Microarray analysis showed

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 differences in gene composition in pathways such as gonadotropin releasing hormone receptor pathway or B cell activation pathway. Unexpectedly, we observed lack of MYC gene expression in the central region of the tumour comparing to the peripheral region and even at half the radius region. We also detected up-regulation of LFNG and TNFRSF10B genes, exclusively in the central region. Conclusion: We confirmed intratumour heterogeneity at the level of transcriptome. As we suspected, proteomics revealed huge differences in peptide composition between healthy mucosa and tumour samples while only minority changes between intratumour samples. We think that data integration analysis should reveal the biological nature of intratumour heterogeneity. No conflict of interest. 477 Human aldehyde dehydrogenase 7A1 (ALDH7A1) expression affects cancer cell proliferation, migration and cell protection against oxidative stress L. Elsalem1 , A. Fotopoulos2 , A. Papathanasiou2 , S. Allison1 , J. Moreb3 , Z. Cournia2 , K. Pors1 . 1 Institute of Cancer Therpeutics, Faculty of Life Sciences- University of Bradford, Bradford, United Kingdom, 2 Biomedical Research Foundation, Academy of Athens, Athens, Greece, 3 Division of Hematology/Oncology, Department of Medicine- University of Florida, Gainesville, USA Introduction: ALDH7A1 has important antioxidant role against oxidative stress caused by reactive oxygen species (ROS). Abnormally high expression of ALDH7A1 has been found in ovarian cancer, prostate cancer and matched bone metastasis, while its expression in NSCLC has been linked with cancer recurrence. Most of these studies concern the expression of ALDH7A1 but relatively little is known about its biological roles in cancer. Here, we report on the implication of ALDH7A1 expression in cell proliferation, migration and protection against ROS. We also report on novel chemical probes that can be used as starting points for further chemical tool discovery to study ALDH7A1 role in cancer. Materials and Methods: H1299, a NSCLC cell line that has low endogenous ALDH expression was stably transduced with ALDH7A1. The gene and protein expression was evaluated in H1299/7A1 and H1299/RFP cells (control cells with red florescent protein vector) using qRT-PCR and western blot, respectively. ALDH activity was measured with the ALDEFLUOR assay. The MTT assay was used to study cell proliferation, while cell migration was measured with the scratch assay. ROS generation was detected using carboxy-H2DCFDA and dsDNA breaks were measured using the expression of phosphorylated H2AX protein. The anti-proliferative effect of anticancer drugs was evaluated using the MTT assay. Five compounds with the highest binding affinity for ALDH7A1 from a virtual screening of 24,000 compounds were used to probe ALDH7A1 activity. Results and Discussion: ALDH7A1 was found to be highly expressed in H1299/7A1 at both the gene (85-fold) and protein (9-fold) levels compared to H1299/RFP cells. ALDH activity was 17-fold higher in H1299/7A1 than H1299/RFP. ALDH7A1 enhanced H1299 cell proliferation and migration. In addition, it significantly reduced the generation of ROS (>90% less). H1299/7A1 cells had significantly less phosphorylated H2AX expression (>70% less), indicating that ALDH7A1 was protecting cells against DNA damage caused by ROS. The effect of computationally designed compounds on ROS generation showed that ICT11501 resulted in significant more ROS in H1299/7A1 cells, suggesting inhibition of ALDH7A1 functional activity. No difference in the cell survival of both H1299 cells was found upon treatment with cytotoxic and molecularly targeted drugs. Conclusion: Our data revealed a possible role of ALDH7A1 in mediating cell proliferation and migration. To our knowledge, this is the first study describing an antioxidant role of ALDH7A1 in the context of cancer and points to its role in protecting cells from DNA damage caused by ROS. ICT11501 appears as a good starting point for medicinal chemistry to be performed, leading to more potent and selective ALDH7A1 inhibitors to be developed, which can be used as tool compounds to further explore the importance of ALDH7A1 in cancer. No conflict of interest. 478 Progesterone receptor antagonists are potential inhibitors of breast cancer stem cell activity D. Alferez1 , R. Eyre1 , K. Spence1 , W. Hayhurst2 , C. Chresta3 , H. Sacha1 , R. Clarke1 . 1 Institute of Cancer Sciences, Manchester Cancer Research Centre- The University of Manchester, Manchester, United Kingdom, 2 Manchester Medical School, University of Manchester, Manchester, United Kingdom, 3 Oncology iMed, Alderley Park- AstraZeneca, Manchester, United Kingdom 75% of Breast cancers (BCs) express estrogen receptor (ER) and progesterone receptor (PgR), and such patients receive anti-estrogens such as tamoxifen as endocrine therapy. However, resistance to therapies result in tumour recurrence and we recently reported that cancer stem cells (CSCs) play a role (Simoes et al., Cell Reports, 2015).

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Progesterone receptor (PgR) is established to regulate normal breast epithelial stem cells, suggesting PgR as a potential therapeutic target in breast CSCs. To investigate the effects on breast CSCs, we tested 2 non-steroidal PRAs; onapristone (Schering AG/Arno Ther.) and AZ4425 (AstraZeneca), in 2 ER/PR+ BC cell lines (MCF-7 & T47D) and 24 patient-derived samples (PDS) comprising early and metastatic BC. We assayed CSC activity using the mammosphere (MS) suspension assay in vitro and the limiting dilution assay in-vivo, and also preclinical anti-tumour activity in an ER+/PR+ patient derived xenograft (PDX) model. In addition, new predictive biomarkers to facilitate personalisation of anti-progestin therapies were investigated, such as accumulation of PgR into nuclear foci (PgRNF). In the presence of 10nM of progesterone, AZ4425 (100nM) inhibited mammosphere forming efficiency (MFE) in MCF-7 cells by 32% (p < 0.0001) and in T47D by 21% (p < 0.0001). Similar results were seen with onapristone (34% inhibition in MCF-7 (p < 0.002) and 34% in T47D cells (p < 0.0002). AZ4425 treatment significantly reduced MFEs in 6/10 early BC PDS and in 9/14 metastatic PDS. Onapristone also produced comparable reductions in MFEs, 5/10 in early and 7/14 in late BC PDS respectively. In vivo transplantation of T47D monolayer cells showed that AZ4425 pre-treatment for 72-hrs reduced tumour formation and CSC frequency, confirmed by ELDA analysis. A PDX tumour treated in vivo for 24 days showed greater anti-tumour potential in combination with tamoxifen than with AZ4425 alone. However, MFEs (CSC activity) measured ex-vivo were significantly reduced with AZ4425 alone or in combination (by 52% and 66% respectively). Pilot data on PgRNF as a biomarker suggest larger responses to PgR antagonists in PgRNF+ tumours. In conclusion, combination of anti-progestins AZ4425 with current antioestrogen therapies such as Tamoxifen may overcome resistance mechanisms by targeting breast CSCs that are resistant to endocrine therapy. Our pilot data support the potential use of PRNF as marker of PgR antagonism to potentially select patients. No conflict of interest. 479 The dual role of ALCAM in endometrial cancer L. Devis1 , N. Masia´ 1 , E. Mart´ınez1 , F. Brochard-Wyart2 , A. Gil-Moreno1 , S. Dufour3 , A. Santamaria1 , F. Alameda4 , J. Reventos ´ 1 , E. Colas ´ 1 . 1 Vall d’Hebron Research Institute, Biomedical Research Unit in Gynecology, Barcelona, Spain, 2 Centre National de la Recherche Scientifique-Institut Curie, Unite Mixte de Recherche 168, Paris, France, 3 Centre National de la Recherche Scientifique-Institut Curie, Unite Mixte de Recherche 144, Paris, France, 4 Hospital del Mar, Pathology Department, Barcelona, Spain Background: ALCAM is a transmembrane glycoprotein, from the Immunogloublin superfamily. Its main function is related to cell-cell adhesion through homotypic and heterotypic interactions. ALCAM has been associated to many cancers, however its expression in the tumor and its cell localization have generated great controversy to determine its prognostic value. In this study, we sought to evaluate the heterogeneous expression of ALCAM in endometrial cancer (EC) and its prognostic value; and to elucidate the molecular mechanisms associated to its differential expression in epitheliallike and mesenchymal-like cellular contexts. Methods: We constructed a tissue microarray (TMA) of 116 patients. For each patient, two differential regions of the tumor, superficial and the invasive front, were included. We performed immunohistochemistry of ALCAM and other EC related proteins: E-Cadherin, b-Catenin, ETV5, and P-ERK. For in vitro studies, we used two epithelial EC cell lines (Hec1a and Ishikawa) and a model of EC mesenchymal cells. Functional assays (micropipette assays, adhesion on different matrices, migration and invasion) and modulation of ALCAM were performed to understand the role of ALCAM in each cellular context. Results: Expression of membranous ALCAM (mALCAM) was heterogeneously expressed within the same tumor. We observed that its expression was significantly reduced in the invasion front compared to the superficial tumor in patients. Interestingly, correlation of mALCAM with other EC molecules was specific of location. In the superficial zone, mALCAM showed a significant correlation with the E-Cadherin/b-Catenin complex. This correlation was lost at the invasive front, where E-Cadherin was repressed. Interestingly, mALCAM was critical for the dialog between ETV5 transcription factor and ERK phosphorylation, only at the invasive front. To understand the molecular mechanisms that govern the dual function of ALCAM under epithelial and mesenchymal settings, we firstly used a set of stable endometrial cancer cell lines that mimic the superficial tumor. Inhibition of ALCAM in two models of epithelial cell lines, Ishikawa and Hec1a, impaired cell-cell adhesion, and decreased migration and invasion. Secondly, we used ETV5 overexpressing Hec1a cell lines as a model of mesenchymal EC cells. In this mesenchymal context, the ratio of membranous/cleaved ALCAM was significantly altered. Overexpression of full ALCAM at the cell membrane reversed the invasive capabilities promoted by ETV5 associated to cell-cell adhesion, migration; partially through the MAPK/ERK pathway. Conclusions: ALCAM is expressed heterogeneously in EC tumors. This study unveils a dual role for ALCAM, pointing to an association of ALCAM with cell

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adhesion molecules in the superficial tumor, and with mesenchymal inductors, such as ETV5, and MAPK/ERK pathway at the invasive front. No conflict of interest. 480 BRAF inhibition promotes BRAF mutant human melanoma cell survival under nutrient-deprived conditions through activation of mitochondrial metabolism T. Delgado-Goni1 , S. Wantuch2 , P. Workman3 , R. Marais4 , M.O. Leach1 , M. Beloueche-Babari1 . 1 Institute of Cancer Research, Radiotherapy and Imaging, London, United Kingdom, 2 Institute of Cancer Research, Breast Cancer Research, London, United Kingdom, 3 Institute of Cancer Research, Cancer Therapeutics Unit, London, United Kingdom, 4 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom Background: BRAF-MEK1/2 signaling inhibitors have shown remarkable clinical activity in BRAF-mutant melanoma. However, responses are not durable suggesting activation of alternative tumour survival mechanisms that allow resistance to therapy. This work investigates the re-programming of glucose metabolism following BRAF inhibition as an adaptive response, and its significance for cell survival in a nutrient depleted environment. Material and Methods: WM266.4 (BRAFV600D) cells were treated with the BRAF inhibitor vemurafenib (2mM, 24 h) in standard culture media containing 100% 5mM [1-13C]glucose. Cell extracts were analyzed by 13C NMR spectroscopy to assess alterations in glucose metabolism. To assess the significance of these alterations for cell survival and for their dependence on glycolysis, glutamine and TCA metabolism, cell counts were obtained from WM266.4 and SKMEL28 (BRAFV600E) cells grown in different nutrientdeprived conditions, with or without vemurafenib, for 48 h: 1mM glucose, 1mM glucose without glutamine and 1mM glucose without glutamine and pyruvate. Results: Vemurafenib induced a reduction in [3-13C] extracellular lactate in treated WM266.4 cells with respect to controls (62.9±13.1%; P = 0.01). A significant decrease in the intracellular [3-13C]lactate levels after treatment (53.1±18.6%, P = 0.01), concomitant with a significant increase in [1-13C]glucose (up to 286.9±105.5%, P = 0.04), indicated decreased glycolysis and glucose utilization after internalization. A significant elevation in [2-13C] and [3-13C]glutamate (to 185.2±42.6% and 171.4±38.9% of controls, respectively, P < 0.05) and [2-13C]glutamine (135.1±14.1, P = 0.01) in treated cells indicated an increased pyruvate carboxylase (PC) flux, consistent with an increased [2-13C]/[4-13C] glutamate ratio (from 0.10±0.04 to 0.30±0.04 (P = 0.02). Accordingly, the enzyme activity of PC showed a significant increase (159±67%, P = 0.04) under BRAF inhibition. WM266.4 and SKMEL28 cells exhibited a significant reduction in cell counts in low glucose media that was emphasised when glutamine was removed. However, the number of viable cells was significantly higher in treated samples, indicating that vemurafenib reduces the dependency of these cells on glucose (p = 0.03 and p = 0.01 respectively) and glutamine (p < 0.01) for proliferation and survival. When pyruvate was removed, the growth advantage under treatment was abolished for both lines, consistent with the dependency of treated cells on mitochondrial metabolism, with PC flux requiring pyruvate availability. Conclusion: BRAF inhibition with vemurafenib in BRAF mutant human melanoma cells alters glucose utilization and activates mitochondrial metabolism with consequences that enable improved survival under nutrientdeprived conditions. This metabolic shift may enable survival and the emergence of resistant clones following treatment. No conflict of interest. 481 Single cell analysis of intratumour heterogeneity in childhood acute lymphoblastic leukaemia V. Turati1 , H. Mike2 , J. Herrero1 , A. John1 , S. Richardson1 , B. Gaal1 , L. Mark3 , S.E. Jacobsen4 , T. Enver1 . 1 UCL Cancer Institute, Cancer Biology, London, United Kingdom, 2 Institute of Child Health, Genetics & Genomic Medicine, London, United Kingdom, 3 Fluidigm Corporation, Single-cell Biology, San Francisco, USA, 4 University of Oxford, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom Background: Tumours are best viewed as a matrix of genetic and epigenetic heterogeneity which presents a rich substrate for selection and clonal evolution in response to therapy. Childhood Acute Lymphoblastic Leukaemia (cALL) is a paradigmatic disease for which static snapshots of mutational diversity at diagnosis revealed genetically distinct subclones arranged in complex branching architectures and identified genetic variegation in leukaemia propagating cells. The molecular mechanisms driving relapse of cALL are still, however, largely unknown and whether genetic or epigenetic factors can cause therapeutic resistance remains to be investigated. Material and Methods: To obtain a “real-time” longitudinal analysis of subclonal dynamics through treatment we established an in vivo mouse model that allows a patient tumour to be independently exposed to treatment multiple times. This has allowed us to track the fate of genetic subclones at single cell resolution by means of multicolour FISH with five mutation markers, enabling us to distinguish between deterministic and stochastic mechanisms of

selection during therapy. Single cell whole genome sequencing was adopted to extend the analysis’ resolution. Secondary transplantation and limiting dilution assays were used to assess the leukaemia initiating ability of resistant cells, and small cell number RNAseq (SMART-seq) was adopted to transcriptionally profile the latter. Results: Treatment of sensitive ALLs results in a striking reduction in leukaemic burden, but the overall extent of genetic diversity is unaffected, suggesting that resistance in ALL is largely independent of genetic variegation. Nevertheless, limiting dilution secondary transplantation assays suggest that exposure to treatment affects the functional properties of the tumour, enriching for cells with leukaemia initiating potential. Trascriptome analysis reveals that treatment-exposed cells express features characteristic of more primitive haematopoietic cells. Upon chemotherapy withdrawal cells revert to a treatment-na¨ıve signature consistent with the selection during treatment of a population of cells with stem-like properties. To further investigate the full extent of genetic heterogeneity and its relation to these transcriptional features of resistance, we are currently complementing our genetic studies with higher resolution single cell whole genome sequencing of selected specimens. Conclusions: Overall this analysis provides novel insight into the contribution of genetic and transcriptional/epigenetic heterogeneity to resistance to cytotoxic chemotherapy. In the case of cALL phenotypic heterogeneity appears to play a larger role then genetic diversity, particularly with regards to cell cycle state and developmental stage. No conflict of interest. 483 sept4/Arts regulates stem cell apoptosis and skin regeneration Y. Fuchs1 , S. Brown2 , T. Gorenc2 , J. Rodriguez2 , E. Fuchs3 , H. Steller2 . 1 Technion − Israel Institute of Technology, Lokey Center for Life Sciences and Engineering and The Faculty of Biology, Haifa, Israel, 2 Rockefeller University/HHMI, Strang Laboratory of Apoptosis and Cancer Biology, New York, USA, 3 Rockefeller University/HHMI, Laboratory of Mammalian Cell Biology and Development, New York, USA Adult stem cells are essential for tissue homeostasis and wound repair. Their proliferative capacity must be tightly regulated to prevent the emergence of unwanted and potentially dangerous cells, such as cancer cells. We found that mice deficient for the proapoptotic Sept4/ARTS gene have elevated numbers of hair follicle stem cells (HFSCs) that are protected against apoptosis. Sept4/ARTS−/− mice display dramatic improvement in wound healing and regeneration of hair follicles. These phenotypes depend on HFSCs, as indicated by lineage tracing. Inactivation of XIAP, a direct target of ARTS, abrogated these phenotypes and impaired wound healing. Our results indicate that apoptosis plays an important role in regulating stem cell-dependent regeneration and suggest that this pathway may be a target for regenerative medicine. Reference(s) Fuchs et al., Science 2013. No conflict of interest. 484 Tumor progression marker for breast cancer − survivin gene (BIRC5) Y. Shlyakhtunou1 . 1 Vitebsk State Order of Peoples’ Friendship Medical University, Oncology, Vitebsk, Belarus Breast cancer is a leader in the structure of morbidity and mortality of the female population from malignant tumors. Distant metastases are the main cause of death of patients, a substrate for the development of which are circulating tumor cells (CTCs). However, only one-search ethics cells is not sufficient to form a complete picture of the nature and course of the tumor process in individual patients. Determination of the expression of tumor-genes responsible for the different processes of tumor progression allows a more complete picture. Such genes include the gene survivin (BIRC5) family of inhibitors of apoptosis (IAP). Objective: To study the expression of the gene survivin in the tumor tissue of breast carcinoma, as well as CTCs in the peripheral blood of patients with breast cancer. Material and Methods: Using real-time PCR was investigated expression of the gene survivin in 16 samples of primary invasive ductal carcinoma of the breast, three samples of benign tumors − fibroadenoma of the breast, as well as 26 samples of peripheral blood of patients with breast cancer at various stages of tumor and stage specific treatment, and 3 healthy people are controlled. After homogenization of frozen tumor samples was extracted RNA, followed by cDNA synthesis and determination of expression of survivin. Were isolated from peripheral blood using CTCs microspheres carrying antibodies to EpCAM, after several stages passed as tumor RNA extraction followed by standard stages real-time PCR. Results: In primary breast carcinoma was determined by high expression of survivin gene in all 16 samples with the average value (M±m) 1.40±0.49 (min 1.21; max 3.41). The highest figures were determined expression in tumors of medium and high grade (G II−III) with lymphovenous invasion

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 (LVSI +). In samples of benign tumor in 2 of 3 expression of survivin was not determined, and one was 0.015. In CTCs, isolated from peripheral blood of breast cancer patients, all 26 samples as determined by the gene expression of survivin with an average value (M±m) 0.90±0.19 (min 0.26; max 3.90). The level of expression of the control samples did not exceed 0.003. It should be noted that the maximum volume of expression obtained in samples of tumor patients with stage N +, and especially M1, on TNM classification. Any legitimate expression of survivin, depending on the size of the tumor had been received. In patients, receiving chemotherapy observed average expression of survivin gene, but never approached the indicators of control. Conclusions: Determination of expression of the gene survivin in primary tumor and the CTCs may be treated to one of the most promising markers of tumor progression and monitoring of breast cancer therapy. No conflict of interest. 485 WWP2 controls endothelial permeability by regulating the RAP1–RADIL signaling pathway F. Renzi1 , M.F. Baietti1 , L. Abbasi1 , K. Gevaert2 , A. Sablina1 . 1 VIB, center of human genetics, Leuven, Belgium, 2 VIB, center of human genetics, Gent, Belgium Background: The small GTPase RAP1 plays a crucial role in endothelial functioning, suggesting its potential contribution in the regulation of cancer cell extravasation. The RAP1 downstream effector RADIL is involved in the formation of endothelial cell junction and control of endothelial permeability. Our previous results revealed that monoubiquitination of the RAS-like GTPases alters their binding to specific downstream effectors, thus promoting particular signaling pathways. In this study, we investigate the role of reversible ubiquitination in regulation RAP1 signaling and function. Material and Methods: Mass spectrometry analyses identified WWP2 as potential interactor of RAP1. The interaction between RAP1 and WWP2 was further confirmed by reciprocal immunoprecipitations and co-localization analysis of the two proteins. We also assessed the effect of WWP2 expression on status of RAP1 ubiquitination. Endothelial permeability was measured by quantifying the amount of FITC-dextran passing through a monolayer of Human Umbilical Vein Endothelial Cells. Results: We found that RAP1 undergoes monoubiquitination within its effector binding domain. Our results implicated the E3 ubiquitin ligase WWP2 in control of RAP1 monoubiquitination. Importantly, depletion of WWP2 negatively affects the binding of RAP1 to its effector RADIL, resulting in increased endothelial permeability. Furthermore, increased endothelial permeability triggered by loss of WWP2 facilitates extravasation of cancer cells through a monolayer of endothelial cells. Altered endothelial permeability caused by WWP2 depletion could facilitate the spreading of metastatic cancer cells. Recently several anti-cancer approaches have been focused on the inhibition of E3 ligases. Specifically, it has been proposed that inhibition of WWP2 can suppress tumor growth by increasing levels of the tumor suppressor PTEN. However, our data indicate that suppression of WWP2 in stroma may increase metastatic spreading. Conclusion: WWP2-mediated RAP1 monoubiquitination plays a key role in the regulation of endothelial function and extravasation of cancer cells by promoting the interaction between RAP1 and its downstream effector RADIL. Understanding the mechanisms that affect tumor extravasation can lead to the development of specific therapies that aim to reduce the metastatic potential of cancer cells. No conflict of interest. 486 Expression of survivin gene (BIRC5) and ErbB-2 (Her-2/neu) in lymphocytes in breast cancer Y. Shlyakhtunou1 , V. Semenov2 . 1 Vitebsk State Order of Peoples’ Friendship Medical University, Oncology, Vitebsk, Belarus, 2 Vitebsk State Order of Peoples’ Friendship Medical University, Infectious Diseases, Vitebsk, Belarus It is now known that the presence of tumor infiltrating lymphocytes (TILs) is an indirect indication of the active anti-tumor immunity and combined with improved prognosis in patients, breast cancer (breast cancer) suffering from resectable cancer. According to the literature, patients with a high content of TILs holding one chemotherapy alone was associated with a 5-year disease-free survival rate of 91%. Adding Anita-Her-2 directional drug trastuzumab in this group did not improve outcomes. On the other hand, it was observed nonsignificant decrease in 5-year disease-free survival to 80%. The explanation of this phenomenon yet. Perhaps this is due to antigen-presenting mechanism of lymphocytes themselves. Objective: To study the expression of the gene survivin (BIRC5), and ErbB-2 (Her-2/neu) in lymphocytes infiltrating breast carcinoma tissue, and in peripheral blood lymphocytes of patients suffering from breast cancer. Materials and Methods: After homogenization of frozen tumor specimen isolated lymphocytes (CD45 +) by separation. In the same way the lymphocytes isolated from the peripheral blood. Using real-time PCR Gene

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expression was investigated BIRC5 ErbB-2 and lymphocytes (phenotype) 16 samples of primary invasive ductal breast carcinomas and in 26 samples of peripheral blood of patients suffering from breast cancer. Results: B lymphocytes isolated from all carcinoma samples was determined by gene expression BIRC5 and ErbB-2 with a mean value (M±ms) for survivin 2.00±1.29 (min 0.003; max 4.283) and 0.57±0.29 (min 0.024; max 1.459) for the Her-2/neu. B lymphocytes isolated from peripheral blood, in all the 26 samples was determined by the expression of ErbB-2 (Her-2/neu) with the average value (M±ms) 0.661±0.521 (min 0.259; max 1.405). In 19 samples, representing 73% of the expression of survivin gene was determined with the average value (M±ms) 0.248±0.171 (min 0.035; max 0.488). The 7 samples (27%) yielded negative results BIRC5 gene expression. Conclusion: The findings suggest that a sort of “tension” cellular immunity directly in the tumor tissue and the bloodstream. The wide range of indicators may suggest a different immune response of the organism to the tumor. This study indicates a possible predictive value of TILs as a biomarker that can identify a group of patients who are highly sensitive to chemotherapy and do not require the addition of trastuzumab. However, this hypothesis requires confirmation in randomized trials. No conflict of interest. 487 Identification and exploitation of collateral vulnerabilities to transcription and RNA processing inhibitors in tumor subtypes L. Frischknecht1 , Y. Christinat1 , C. Britschgi1 , W. Krek1 . 1 Molecular Health Sciences, Biology, Zurich, ¨ Switzerland Introduction: Genome instability is a general feature of cancer cells. Frequently, these alterations cause deletions of loci containing tumor suppressor genes and concurrently lead to hemizygous copy loss of flanking genes. The latter, when affecting genes acting in functionally essential processes, generates cancer specific vulnerabilities that could be exploited therapeutically. Material and Method: Bioinformatics analysis and data integration was performed on the Achilles, CCLE and TCGA databases (Broad Institute and NIH) with a focus on the URI1 onco-chaperone network. Drug response curves were performed on a panel of different representative cancer cell lines using PrestoBlue® cell viability reagent. CRISPR interference and activation was used to up- or down-regulate relevant genes to test causality in mediating collateral vulnerability. Results and Discussion: URI1 is a member of the prefoldin chaperone complexes with established oncogenic properties in multiple cancers. Analysis of the Achilles database (http://www.broadinstitute.org/achilles), containing information from loss-of-function screens in 216 cancer cell lines, revealed an URI1 onco-chaperone gene network comprised of four subnetworks involved in transcription, RNA processing, nuclear transport and protein degradation that demonstrated very similar survival properties as URI1. Further in silico analysis revealed that the dependency of cancer cells for their survival on genes in this new network correlated with copy number alterations of five of these genes as well as on the transcriptional burden of cancer cells as assessed by the copy number of a list of established driver oncogenes. Chemical inhibition of components of the individual subnetworks validated differential sensitivity of cancer cells as a function of hemizygous loss of network genes and oncogene copy number. The copy number changes of the five genes correlating with network sensitivity are driven by amplification or deletion of neighboring oncogenes and tumor suppressor genes respectively. The strongest correlation was observed with the copy number of POLR2E, a shared subunit of all three nuclear RNA polymerases and established chaperone target of URI1. POLR2E shows frequent hemizygous losses in melanoma, as a flanking gene of the tumor suppressor STK11. Melanoma cell lines having only one copy of POLR2E displayed increased sensitivity to RNA polymerase inhibition. Conclusion: Collateral hemizygous deletions of ‘housekeeping-genes’ that comprise the URI1 onco-chaperone network might be an interesting novel therapeutic target especially in cancers characterized by increased demand on the functions of the identified gene subnetworks. No conflict of interest. 488 ERK5 is required for pro-tumour macrophage activation E. Giurisato1 , W. Vermi2 , C. Tournier1 . 1 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom, 2 University of Brescia, Medicina Molecolare e Traslazionale, Brescia, Italy Background: A number of transcription factors have been implicated in the rearrangement of the transcriptional profile that sustains the reprogramming of tumour-associated macrophages (TAMs) to facilitate carcinoma development. In contrast, we have a very limited understanding of the signalling mechanisms that governs this process in response to tumour-derived signals. The extracellular-regulated protein kinase 5 (ERK5) is a unique mitogenactivated protein kinase (MAPK) which has previously been linked to the pathophysiology of cancer.

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Results: In this study, we have found that ERK5 is strongly expressed in pro-tumour macrophages present in various human carcinomas. Moreover, targeted deletion of the erk5 gene reduced M-CSF-induced proliferation of murine bone marrow mononuclear cells. This correlated with enhanced differentiation and increased expression of the cyclin-dependent kinase inhibitor p21. The absence of ERK5 also impaired the transcriptional activation of pro-tumour genes in differentiated macrophages. Accordingly, we found that erk5-deleted macrophages exhibited decreased expression of antiinflammatory mediators and trophic factors which subsequently halted tumour cells growth. Conclusions: Together these results indicate that ERK5 is a critical regulator of pro-tumour macrophage activity. Owing to the prevalence of TAMs in cancer and their unique influence upon disease progression and malignancy, macrophage-targeted manipulation via anti-ERK5 therapy represents a novel promising strategy in cancer immunotherapy. No conflict of interest. 489 A preliminary characterisation of de novo lipogenesis in pancreatic (Capan-1) and liver (HepG2) cancer cell lines N. Lambri1 , A. Snabaitis1 , A. Le Gresley1 , H. Modjtahedi1 , M. Stolinski1 . 1 Kingston University, Faculty of Science- Engineering and Computing, London, United Kingdom Background: Cancer cells demonstrate elevated levels of de novo lipogenesis (DNL), which represents a component of the reprogramming of tumour metabolism, driving uncontrolled cell growth. Inhibition of this pathway results in reduced proliferation rates, cell viability and tumour size. This preliminary study aims to investigate the effects of nutrient driven-responses on cell proliferation as well as the regulation of DNL, in pancreatic and liver cancerous cells. Methods: Capan-1 cells were grown in RPMI media containing 11, 5.5 and 2.75mM glucose and HepG2 cells grown in DMEM media containing 25, 5.5 and 2.75mM glucose under normoxic, full serum conditions for 5 days. Effects on cell growth were determined using the IncuCyte real-time imaging system. Samples of growth media were collected every 24 h for the measurement of glucose, glutamine and lactate concentrations using quantitative NMR (Bruker AV 600 NM). De novo lipogenesis was determined by growing cells with 50%, [U-13C6] glucose, under the conditions described above. Cells were collected every 24 hours over the 5 day period and levels of palmitate enrichment determined by gas chromatography–mass spectrometry (Agilent, 6890). Results: NMR data of cell culture media showed a rapid decrease in glucose and glutamine levels, and as a result of the high glycolytic flux present in cancer cells, high levels of lactate with both cell lines. Cell proliferation was shown to be dependent on glucose concentration. However, Capan-1 cells demonstrated reduced growth rates as well as an increase in cell death compared to HepG2 cells when supplied with similar levels of nutrients. Furthermore, HepG2 cells demonstrated different palmitate enrichment levels when cultured under 25, 5.5 and 2.75mM glucose (TTR enrichment values of 0.3543, 0.1987 and 0.1325 respectively; n = 1 at 48 hours). Capan-1 cell palmitate enrichment did not initially change (up to 72 hours), with an average TTR value of 0.3217. After 72 hours TTR palmitate enrichment values declined rapidly in Capan-1 cells when cultured with 5.5 and 2.75mM glucose. Conclusion: The data suggests that HepG2 cells may be capable of regulating DNL which may relate to reduced rates of cell death. Capan-1 cells seem to be unable to regulate DNL processes as enrichment levels of palmitate do not change under this experimental model. Further understanding of the regulation of DNL may provide novel therapeutic strategies that can be developed targeting cell survival. No conflict of interest. 490 Influence of stemness genes on metastatic capacity of cancer stem cells in prostate cancer E.A. Castellon ´ 1 , R. Valenzuela1 , F. Cifuentes1 , T. Thomson2 , H.R. Contreras1 . 1 University of Chile, Faculty of Medicine, Santiago, Chile, 2 CSIC, Institut de Biologia Molecular de Barcelona, Barcelona, Spain Introduction: Prostate cancer (PCa) is one the most important causes of men cancer mortality in the world. Recurrence rate and high resistance to hormonal treatments are major drawbacks. There is convincing evidence that cancer stem cells (CSCs) are responsible, at least in part, for this fact. Many signaling pathways and transcription factors are associated in keeping the stemness phenotype, promoting the survival of CSCs even after therapy. Knocking down stemness genes involved in these processes may reverse CSCs features making them more sensitive to treatments. The main objective of this research is to evaluate the effect of knocking down of stemness genes on metastatic capacity of CSCs in PCa. Materials and Method: Tumor explants were obtained from PCa patients undergoing radical prostatectomy. Protocols were approved by our institutional Ethical Committee (Hospital and Faculty). Cells were cultured under unattachment conditions favoring CSCs spheres formation. Stemness genes

SOX2, KLF4 and MYC were knocked down using specific shRNAs. The effect of this gene silencing on apoptosis, clonogenic and invasion were assayed. Also, the metastatic capacity and progression of SOX2-knocked down CSCs was evaluated in a orthotopic NOD/SCID mice model for human PCa. Results and Discussion: Tumorspheres were enriched in CSCs with stem phenotypic and genotypic features. Knocking down of SOX2, KLF4 and MYC reached more than 50% of inhibition. CSCs knocked down for those genes showed a increased apoptotic activity and decreased clonogenic and invasive capacity. SOX2-silenced CSCs decreased the growth tumor rate and completely inhibited metastasis in a orthotopic NOD/SCID model for PCa. The stemness genes evaluated are relevant in maintaining the stem functional signature of CSCs from PCa, promoting anti-apoptotic, clonogenic, invasive, tumorigenic and metastatic properties. Conclusion: SOX2 gene expression seems to have a determinant influence in metastatic progression of PCa and could be considered as a suitable therapeutic target. Acknowledgment: Funding: Projects Fondecyt 1140417 (EAC) and 1151214 (HRC). No conflict of interest. 492 Functional characterization of a tumor suppressor micro-RNA in ovarian cancer B. Majem1 , A. Parrilla1 , A. Soriano2 , A. Gil-Moreno3 , M.F. Segura2 , A. Santamaria1 . 1 Vall Hebron Research Institute VHIR- University Autonoma of Barcelona, Group of Biomedical Research in Gynecology- Cell cycle and cancer group, Barcelona, Spain, 2 Vall Hebron Research Institute VHIRUniversity Autonoma of Barcelona, Group of Translational Research in Child and Adolescent Cancer, Barcelona, Spain, 3 Vall Hebron University Hospital, Department of Gynecological Oncology, Barcelona, Spain Introduction: Ovarian cancer (OC) is the fifth cause of cancer in women and the leading cause of death among gynecological malignancies in developed countries. Since the accompanying symptoms are very unspecific, around 70% of the patients are diagnosed in advanced stages of the disease with a 5-years survival rate less than 25%. Therefore, identifying key players in the development of the tumor and developing novel and efficient therapeutic strategies is paramount. Aberrant expression of micro-RNAs (miRNA) in cancer suggests key functions of miRNAs in tumorigenesis. Our aim is to unravel the role of a micro-RNA in the initiation and progression of OC, deciphering also whether it directly regulates the response to standard chemotherapy. Material and Methods: miRNA expression levels were evaluated in human ovarian samples from benign ovarian diseases (BOD) and early and late serous OC tissues. Clinically relevant cell lines (A2780p, SKOV3, OAW42, OVCAR4) were used to explore the functional role of this miRNA in vitro. A list of putative mRNA target genes was obtained from the miRWalk database, and mRNA and protein levels of a panel, including the most relevant ones (n = 25), were analyzed by RTqPCR and Western Blotting, respectively. Direct binding of the miRNA to the downregulated genes was analyzed by a 3 UTR luciferase reporter assay. Finally, in vivo models were generated to prove the anti-tumorigenic role and the potential use of this miRNA as a therapeutic target. Results: In this study, the regulation and function of a miRNA that is significantly under-expressed (about 10-fold) in early and late OC, was evaluated. Overexpression of this miRNA resulted in a significant decrease in cell proliferation and marked increased apoptotic cell death in vitro, accompanied by an activation of the apoptotic pathway seen at the molecular level. In silico bioinformatics analysis of putative mRNA targets predicted several genes, amongst which a gene with a key role in the apoptotic cascade was analyzed and found to be downregulated at both mRNA and protein levels when overexpressing the corresponding miRNA. A 3 UTR luciferase reporter assay confirmed the direct binding of the studied miRNA to this gene. Importantly, we could reproduce the described phenotypic consequences of overexpressing this miRNA in vivo, suggesting that it acts as a tumor suppressor gene in OC. Conclusion: We have discovered a tumor suppressor miRNA that induces apoptosis in vitro and in vivo through directly inhibiting the expression of a key anti-apoptotic gene, indicating not only a potential pivotal role in the genesis and progression of OC, but also offering new therapeutic strategies to overcome the disease in the future. No conflict of interest. 493 Interrogating tumour–stroma interactions mediating escape from breast cancer metastatic dormancy L. O’Leary1 , A. Van Weverwijk1 , C. Isacke1 . 1 The Institute of Cancer Research, Breast Cancer Research, London, United Kingdom Introduction: In breast cancer patients the development of metastatic disease can occur many years after the removal of the primary tumour and systemic treatment. Indeed, >50% of these patients will have presented with a late

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 (>5 years) distant relapse, indicating that disseminated tumour cells can reside for prolonged periods of time as “dormant” single cells or non-productive micrometastasis in distant sites. Despite late relapse being a common occurrence in breast cancer, the mechanisms underlying the maintenance of the dormant state and the subsequent escape from dormancy remain poorly elucidated. This can be attributed to a lack of animal models, which can recapitulate the natural progression to dormancy. Material and Methods: In this study we have identified two human breast cancer cell lines which either in a spontaneous metastasis setting or when inoculated directly into the circulation, gives rise to dormant cells in the lungs of immunocompromised mice. In addition to this we have established an in vitro dormancy assay and an ex vivo lung slice approach in order to interrogate the stromal factors identified from the in vitro and in vivo models, that are required for the progression from a dormant state to a proliferative one. Results and Discussion: In both the in vitro and in vivo models we have demonstrated that these dormant cells can be stimulated to develop productive macrometastasis. Using an ex vivo lung slice approach, we were able to maintain viability of the lung slices for up to 21 days with the breast cancer cells remaining dormant as seen in vivo. This provides a platform to strictly manipulate stromal factors and monitor the emergence from dormancy in live tissue. Conclusion: Using these models we can interrogate the contribution of the microenvironment to the emergence from metastatic dormancy with an overall aim to identify and develop clinically relevant novel strategies to suppress or prevent the development of metastatic disease. No conflict of interest. 495 Anticancer effects of lipid encapsulated Bilberry J. Thornthwaite1 , S. Thibado2 , T. Ballard3 , B. Goodman4 . 1 Cancer Research Institute of West TN, Cancer Therapeutics, Henderson, USA, 2 Union University, Chemistry, Jackson, USA, 3 University of Tennessee, Biochemistry, Knoxville, USA, 4 Freed Hardeman University, Nursing, Henderson, USA Rapidly accumulating laboratory and clinical research evidence indicates that anthocyanins have anticancer activity and more intriguing, the evaluation of bilberry anthocyanins as chemo-preventive agents is progressing. These applications are collectively due to the anthocyanins up-regulating tumor suppressor genes, inducing apoptosis in cancer cells, repairing and protecting genomic DNA integrity, which is important in reducing age-associated oxidative stress, as well as improving neuronal and cognitive brain function. Bilberry anthocyanins have pronounced health effects, even though they have a low bioavailability. To increase the bioavailability, Bilberry was encapsulated in 8 nm diameter liposomal nanospheres, called NutraNanoSpheres (NNS), at a concentration of 2.5 mg/50 mL. These Bilberry NNS were used to study the apoptotic/cytotoxic effects on K562 cancer cells. Flow cytometric fluorescent quantification of the uptake of Propidium Iodide in a special cell viability formulation into dead K562 cells was used to determine the effects of Bilberry on the viability of K562 cells. The concentrations of Bilberry that showed the highest levels of percentage inhibition, relative to the control populations, were biphasic, showing 60−70% inhibition between 0.018–1.14 mg/ml (n = 6) and 60% inhibition at 80 mg/ml. The lowest % inhibition (30%) occurred at 40 mg/ml. The LD50 was determined to be 0.01–0.04 mg/ml of Bilberry per 105 K562 cells at 72 hr. of cell culture exposure. At 48 hr. incubation, the highest % inhibition was only 27%, suggesting a long-term apoptotic event being involved. These levels, which showed direct cytotoxic effects, were 10 times lower than what is required for the Bilberry that is not encapsulated. The 10 fold increase in bioavailability with the Bilberry NNS and its water solubility show the feasibility of using Bilberry NNS in cancer patient clinical trials. No conflict of interest. 496 A novel pre-clinical model for imaging cancer-associated inflammation K. Finegan1 , M. Babur1 , D. Forster2 , J. O’Connor3 , C. Tournier4 , K. Williams1 . 1 University of Manchester, Manchester Pharmacy School, Manchester, United Kingdom, 2 University of Manchester, Wolfson Molecular Imaging Centre, Manchester, United Kingdom, 3 University of Manchester, Institute of Cancer Sciences, Manchester, United Kingdom, 4 University of Manchester, Faculty of Life Sciences, Manchester, United Kingdom Background: Chronic inflammation is a hallmark of most, if not all cancers and is a major risk factor in the development of many cancer types. Immune cells create a microenvironment that promotes the survival and sustained proliferation of cancer cells as well as encouraging malignant transformation, angiogenesis and resistance to therapy. Consequently, it is vital to develop a pre-clinical model in which we can assess tumour-related inflammation and correlate this with disease progression and therapeutic response. To address this need we are developing the two-stage carcinogenesis model, for use in pre-clinical PET and MR-imaging. In this model, heterogeneous tumours with complex microenvironments develop in situ, in immune-competent animals. Metastatic progression to invasive squamous cell carcinoma (SCC) occurs in 10−15% of tumours, permitting the assessment of malignant transformation

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in the context of an intact immune system. Because the model generates multiple tumours, the effects of different topically applied treatments can be evaluated on the same animal, enabling efficient pharmacodynamic biomarker validation. Treatment of these tumours with an established pharmacological inhibitor (XMD8-92) of the fundamental cell-signalling protein ERK5, decreases intra-tumoural inflammation (particularly macrophages) eliciting a substantive decrease in tumour burden. Materials and Methods: Anti-ERK5 therapy (XMD8-92) was employed as a tool to modify the inflammatory tumour microenvironment. We then evaluated the effect of XMD8-92 on intra-tumoural inflammation using the PET tracers [11C]PK11195 and [18F]FDG. We also employed DW-MRI (ADC) to evaluate changes in cellularity in order to determine if this technique can detect changes in tumour immune cell infiltrate. Results: Inhibition of intra-tumoural inflammation, via anti-ERK5 therapy, resulted in a reduction in tumour-associated uptake of [11C]PK11195 and [18F]FDG. Diffusion-weighted MRI revealed that control tumours show increases in ADC over time, consistent with a change in cellularity that may reflect inflammation. XMD8-92-treated tumours show stabilisation of ADC relative to control. Conclusions: The TSPO-based PET tracer [11C]PK11195 can track specific changes in intra-tumoural macrophage populations. Anti-ERK5 therapy may also alter tumour glucose consumption. DW-MRI can give rise to biomarkers indicative of inflammation. No conflict of interest. 497 The role of lysyl oxidase activity in ccRCC cell adhesion and mobility in vitro Q. Wang1 , A. Jeylani1 , A.K. Fallah1 , L. Lee-Jones1 , S. Kumar1 , M. Slevin1 . 1 Manchester Metropolitan University, School of Healthcare Sciences, Manchester, United Kingdom Background: Lysyl oxidase (LOX), an extracellular matrix remodelling enzyme, has a role in normal embryonic development and connective tissue function. However, LOX promotes tumour progression and metastases although it may also have tumour-inhibitory effects, which depend on the location and the transformation status of the tumour, and the cell type involved. Our previous studies found higher expressions of LOX in clear cell renal cell carcinoma (ccRCC) compared to adjacent normal renal tissues. Clear cell renal cell carcinoma arise from epithelial cells of the proximal convoluted tubules of the nephron and account for approximately 80% of all renal malignancies. Unfortunately, the link between LOX activity and ccRCC cell adhesion and metastasis remains unclear. Material and Methods: ccRCC cell line, Caki-2 cells were cultured in McCoy’s 5A complete growth medium supplemented with 10% FBS in a humidified 5% CO2 atmosphere at 37 ºC. Various concentrations (50 to 200 mM) of beta-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, were used to inhibit LOX activity in ccRCC cells for 24 to 48 hours in vitro. Untreated cells cultured under the same condition were used as control. The activities of LOX were tested using a fluorometric method. BAPN treated and untreated cells adhesion to fibronectin were analyzed. The expression of genes involved in tumour metastatic signalling pathways were assessed by quantitative RTPCR. Western blotting analyses were performed to evaluate the expressions of matrix metalloproteinases, MMP2 and MMP9, following LOX inhibition in Caki-2 cells. Results and Discussion: Our results showed that BAPN inhibited LOX activity in ccRCC cells in a dose-dependent manner in vitro. LOX inhibition was associated with a significant increase in ccRCC cell adhesion to fibronectin in vitro. The inhibition of LOX enzyme elevated the expression of metastatic regulatory genes, MTSS1 and NME in ccRCC cells. In contrast, expressions of MMP2 and MMP9 proteins in ccRCC were reduced following LOX inhibition. These results indicate that LOX may promote ccRCC cell progression and metastasis by modulating metastatic gene expressions. Thus, targeting LOX by inhibiting its activity may be a new strategy in preventing ccRCC metastasis. Conclusion: LOX activity affects ccRCC cell adhesion to extracellular matrix, and is associated with cancer cell metastasis. This study suggests LOX may be a potential therapeutic target in ccRCC patients. No conflict of interest. 498 GRP94 protein and prosurvival autophagy, the Achilles heel on brain metastasis progression A. Sierra1 , N. Santana-Codina2 , R. Sanz-Pamplona3 , L. Muix´ı4 . 1 IDIBAPS, Molecular and Translational Oncology, Barcelona, Spain, 2 Bellvitge Biomedical Research Institute-IDIBELL, Molecular Oncology, L’ Hospitalet de Llobregat, Spain, 3 Bellvitge Biomedical Research Institute-ICO, Unit of Biomarkers and Susceptibility, L’Hospitalet de Llobregat, Spain, 4 Bellvitge Biomedical Research Institute-IDIBELL, Molecular Oncology, L’Hospitalet de Llobregat, Spain Introduction: GRP94 is a 94-kDa glycoprotein abundant in the endoplasmic reticulum (ER) and is induced by glucose starvation. Overexpression of GRP94

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has been associated with cellular transformation in a variety of cancer cell lines and human cancer biopsies. We used a system approach involving computational modeling to identify the central role of GRP94 to ER stress responses in brain metastasis (BrM) (Sanz, et al. 2011). These observations motivated the study of pathways endoplasmic reticulum stress resistance (ERSR). Material and Methods: BR-eGFP-CMV/Luc-V5CA (BRV5) cells were generated after 5 in vitro/in vivo passages from 435-Br1 cells containing the retroviral vector preGFP-CMV-PLuc, under the control of the 5 LTR, and the photinus luciferase (PLuc) gene, under the control of the cytomegalovirus (CMV) promoter (Mart´ınez et al., 2013). Stable GRP94 protein knockdown. 5 constructs containing different short hairpin RNA (shRNA) against GRP94 and a non-target shRNA control vector were used to obtain shGRP94/shControlBrV5 cells. Clonal selection was performed and Western blot and Immunofluorescence were used to validate the knockdown of GRP94. Experimental breast tumors and BrM in athymic Nude-Foxn1nu female mice were induced with clones containing one of the sequences selected. Results and Discussion: BRV5 cells have a metabolic ERSR orchestrated by GRP94 that confers survival in hypoglycemic conditions. siGRP94-BRV5 cells showed a modified UPR response that regulates transcription (ATF6) and translation (TRAF2) of genes to restore functions and re-establish homeostasis in stress conditions. The organ-specific pro-survival phenotype of brain metastatic cells was corroborated by comparative expression of genes involved in cell death mechanism. We further investigated the consequences of GRP94 levels in vitro and in experimental brain metastasis. Those mice injected with CTR-pool cells developed brain masses faster than mice injected with shGRP94 clones, as was registered by the cellular burden in two different clones, clone 2 (p = 0.0772) and clone 8 (p = 0.0132), whereas all the control mice died by the disease in a couple of months. Statistical analysis using a linear model algorithm to compare GRP94 knockdown tumors showed that, either clone 2, p < 0.0001 or clone 8, p = 0.0002, had difficulties to grow up with regard to control tumors. Additional studies indicate that induction of antiapoptotic proteins and pro-survival autophagy might be the direct mechanisms implicated in GRP94-dependent brain metastasis progression. Conclusions: In the light of these results we can conclude that GRP94 has a cause-effect role in breast cancer development and in the achievement of brain metastatic progression. Besides its role as an ER chaperone, GRP94 induces ERSR coupled to pro-survival autophagy that can relieve metabolic stress in low glucose conditions. No conflict of interest. 499 DNAJB1 renders cancer stem cell-like and metastatic traits to colorectal cancer cells dependent on b-catenin Y. Guo1 , T. Zhang1 , Y. Liu2 , C. Zhao3 . 1 The Affiliated Hospital of Southwest Jiaotong University- The Second Chengdu Hospital Affiliated to Chongqing Medical University, Medical Research Center, Chengdu, China, 2 The Affiliated Hospital of Southwest Jiaotong University- The Second Chengdu Hospital Affiliated to Chongqing Medical University, Department of Gastrointestinal Surgery, Chengdu, China, 3 The Affiliated Hospital of Southwest Jiaotong University- The Second Chengdu Hospital Affiliated to Chongqing Medical University, Department of Digestive Diseases, Chengdu, China Background: Cancer stem cells are considered to be the engine for metastasis and recurrence. They hence are expected to be the ideal targets for radical cure of cancers. The aim of this study is to understand the effect of DNAJB1, a heat shock protein 40 family member, on colorectal cancer stem cells and metastasis of colorectal cancers (CRC). Methods: Immunohistochemistry and microarray-based comparative genomic hybridization were employed to observe the relationship between DNAJB1, b-catenin and metastasis of 250 CRC patients. After overexpression or RNAi of DNAJB1 in CRC cell lines SW480 or SW620 individually, which have the same genetic background but different metastatic ability, in vivo and in vitro metastasis were observed; Spheroid and clone formation, colorectal cancer stem cell markers CD133, CD44v6 and Lgr5 and their regulation signal b-catenin were detected. Further, b-catenin was knocked down to know its necessity to DNAJB1. Results: DNAJB1 was found to be genetically amplified and highly expressed in colorectal cancer tissues (127/250), which was significantly related to the early recurrence of patients. Also, DNAJB1 was expressed more highly in metastatic CRC cells SW620 with much more stem-like cells (44.1%) than SW480 (0.4%) (Sukhdeo et al.,PLoS One 2013, 8, e53015). When DNAJB1 was knocked down, the protein levels of CD133, CD44v6, Lgr5 and b-catenin were suppressed in SW620, and its ability of spheroid, clone formation and migration was responsively inhibited. In contrast, overexpression of DNAJB1 confer the cancer stem cell-like traits and metastatic potential on SW480 cells. Furthermore, silence of b-catenin withdrawn those features of SW480 induced by DNAJB1. That was consistent with the positive correlation of DNAJB1 expression with the nuclear levels of b-catenin in cancer tissues of CRC patients which means an active state of b-catenin signal. Finally, repression of DNAJB1 could significantly interrupted the metastasis of SW620 in mice.

Conclusions: Those finding suggested that dependent on b-catenin signal, DNAJB1 could affect the cancer stem cell-like traits and metastasis of colorectal cancer, and present as a candidate target against cancer stem cells of colorectal cancers. No conflict of interest. 500 Benzyl butyl phthalate (BBP) induces the expression of CD44+/CD24− leading to enhanced colony formation in MCF-7 mammosphere cell T.H. Hsieh1 , T. Eing-Mei1 . 1 Department of Obstetrics and Gynecology, Kaohsiung Medical University, Kaohsiung city, Taiwan Background: The breast cancer phenotype of CD44+/CD24− have been demonstrate to have tumor-initiating capability with stem cell-like. Nevertheless, the biological function by which benzyl butyl phthalate (BBP) mediates colony formation in MCF-7 mammosphere cell remains unclear. Material and Methods: BBP, a plasticizer, which was weakly estrogenic and promote tumorigenesis in human breast cancer. Here, we isolated mammosphere cell by sphere culture, it maintained the cell long term with stem cell marker CD44+/CD24− and with tumor-initiating capability. Results: We confirmed the expression of CD44+/CD24− by flow cytometry and stem cell marker Musashi-1, CK7 and CK19 by immunofluorescence. In addition, we found that the E2 and BBP mediated the increasing of CD44 and repression of CD24 proportion by flow cytometry in mammosphere cell. Subsequently, E2 and BBP also increased the ability of colony formation in MCF-7 mammosphere cell. Conclusion: We demonstrate a possible new biological function by which BBP influence colony formations via CD44+/CD24− expression. This study provides the molecular impact of the plasticizer, BBP and suggests possible strategies for preventing and treating human breast cancer. No conflict of interest. 503 Comparison of antitumour effects of tributyltin chloride and triphenyltin chloride in different human breast cancer cell lines, MCF-7 and MDA-MB-231 L. Hunakova1 , L. Toporova2 , D. Macejova2 , J. Brtko2 . 1 Cancer Research Institute, BMC, Slovak Academy of Sciences, Bratislava, Slovak Republic, 2 Institute of Experimental Endocrinology, BMC, Slovak Academy of Sciences, Bratislava, Slovak Republic Introduction: Triorganotin compounds induce hormonal alterations, i.e., endocrine-disrupting effects in mammals, including humans. Tributyltin chloride (TBT-Cl) and triphenyltin chloride (TPT-Cl) are known to function as nuclear retinoid X receptor (RXR) agonists. Their cytotoxic effects in ER(+) luminal human breast cancer cell line MCF-7 and ER(−) basal-like human breast cancer cell line MDA-MB-231 were examined. Material and Method: Flow cytometry was used to detect and quantify apoptosis, semiquantitative real-time PCR for nuclear retinoid and retinoid-X receptor subtypes expression. Kinetic activation of caspase-3/7 was monitored morphologically using live cell imaging and quantified using the IncuCyte™ FLR object counting algorithm (Essen BioScience, UK) and migration assay was based on the relative wound density measurements by scratch wound application of the IncuCyte ZOOM™ Kinetic Imaging System. Results and Discussion: We observed significantly higher toxicity of TBTCl in comparison with TPT-Cl in both cell lines. Comparable, apoptosisinducing concentrations were 200 and 800 nM, respectively, as shown by PARP cleavage and FDA staining. Both compounds activated executive caspases in the concentration-dependent manner in MDA-MB-231 cells, but the onset of TPT-Cl-induced caspase-3/7 activation was delayed in comparison with TBTCl. Both compounds slowed down the migration of these highly invasive cells, which was accompanied by RARbeta upregulation. Other RAR and RXR expressions were differentially modulated by studied organotins in both cell lines. Conclusion: Our data show the differences of the TBT-Cl and TPT-Cl action in both toxicity and selected RAR and RXRsubtype mRNA expression patterns in human breast cancer MCF-7 and MDA-MB-231 cell lines. We described up-regulation of RARbeta in association with reduced migration of MDAMB-231 cells. Our findings may contribute to the new potential therapeutic utility of RARbeta modulation to better manage triple-negative breast cancer progression. Acknowledgement: This work was supported by APVV-0160-11, VEGA 2/0171/14, VEGA 2/0080/15, and VEGA 02/0092/16 grants. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 505 Ceramide is a key factor that regulates the crosstalk between TGF-b and sonic hedgehog signaling at the basal cilia to control cell migration and tumor metastasis S. Gencer1,2,3 , B. Ogretmen4 . 1 Uskudar University-, Molecular Biology and Genetics, Istanbul, Turkey, 2 Abdullah Gul University, Molecular Biology and Genetics, Kayseri, Turkey, 3 Medical University of South Carolina, Biochemistry and Molecular Biology, Charleston, USA, 4 Medical University South Carolina, Biochemistry and Molecular Biology, Charleston, USA Bioactive sphingolipid ceramide is a second messenger in cell membrane in response to inflammation and stress. Recent studies indicate that ceramide species with different fatty-acid chain lengths play diverse biological functions in various cellular processes, highlighting the importance of ceramide synthases (CerS) in these processes. Migration and cell mobility, a part of these processes, also are effected by ceramide metabolism. However, the molecular mechanism of CerS/and ceramide involved is unknown. Here, we investigated the effect of CerS/and ceramide on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4/ceramide is involved to cell migration and tumor metastasis. Here, we also have generated CerS4−/− mice for in vivo studies. Interestingly, we observed that genetically loss of CerS4 resulted in severe irreversible alopecia, which was associated with hyper-proliferation and migration of keratinocytes. Mechanistically, we show here that genetic loss or shRNA-mediated knockdown of CerS4 enhances cell migration by which ligand-independent signaling of TGF-beta receptors I and II in various cell types, including keratinocytes, mouse embryonic fibroblasts and cancer cells. Moreover, we found that ceramide directly interact with Smad7 and this interaction was decreased by shRNA-mediated knockdown of CerS4. Thus, ceramide-Smad7 binding modulates plasma membrane association of TGF-bR1 at primary basal cilia, and inhibits its signaling through SonicHedgehog (Shh) for migration. Furthermore, Ceramide accumulation at the primary basal cilia was decreased by knockdown of CerS4, and this was associated with direct interaction of TGF-bR1 and SMO receptors in cilia. In fact, inhibition of TGF-bR /Shh signaling or cilia formation using molecular or pharmacologic inhibitors almost completely prevented cell migration in response to CerS4 knockdown. These data revealed that CerS4/ceramide signaling plays key roles in the regulation of cell migration and metastasis via controlling the TGF-bR and Shh axis at primary basal cilia. No conflict of interest. 506 Development of an organotypic system for the study of cancer dormancy M. Montagner1 , E. Sahai1 . 1 The Crick Institute LRI, Tumour Cell Biology, London, United Kingdom Introduction: Metastasis is a complex multistep process that is the major source of cancer-related death. In many of the major cancer types, such as breast and prostate, the process of metastatic outgrowth occurs rather slowly. Actively growing metastases can arise many years after the original primary tumour was removed. To explain this phenomenon it has been proposed that disseminated cancer cells can enter a state of ‘dormancy’ in organs such as the lungs and bone. In this state they persist, but do not proliferate. To date, the investigation on the mechanisms that regulate the dormant state has been significantly hampered by the difficulty in studying very small numbers of cells deep within an animal. The aim of this project is developing a lung organotypic system that recapitulates some of the in vivo features of the dormant niche. Material and Methods: We have created an in vitro model for lung tissue by co-culturing alveolar type I-like (AT1), type II-like (AT2), and lung fibroblasts on an air-permeable support. To optimise an in vitro model that recapitulates dormancy we compared the growth of D2.OR and D2.A1 breast cancer cell lines when cultivated in the presence of the lung organotypic system. Results and Discussion: We are able to recreate the in vivo behaviour of the D2.OR and D2.A1 cells using our co-culture system. 12 days after plating, D2.A1 develop into big colonies of cells while D2.OR remain as single cells. This is in marked contrast to the steady growth of both D2.OR and D2.A1 cells in conventional cell culture conditions. The next steps will be the study of the role of the different stromal cells in the control of the dormant status of the D2.OR cells and the dissection of the molecular pathways involved in the process. Finally, we have used this system to evaluate the efficacy of existing therapies to target dormant cells and identify new strategies that we will present. Conclusion: By developing an in vitro model of cancer dormancy, we are able to studying the problem with unprecedented resolution and greater throughput than in vivo analysis. This has enabled the identification of effective strategies for killing dormant cells. No conflict of interest.

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507 MicroRNA induced autophagy confers chemoresistance in glioblastomas U. Baumgartner1,2 , N. Wirth1 , S. Haemmig3 , A. Zulliger1 , M.P. Tschan1 , E. Vassella1 . 1 Institute of Pathology, University of Bern, Switzerland, 2 University of Bern, Graduate School for Cellular and Biomedical Sciences, Switzerland, 3 Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Glioblastoma multiforme (GBM) is a prognostically most discouraging neoplasia in human. Temozolomide (TMZ) is the only therapeutic agent for the treatment of GBM, but not all patients benefit from therapy and resistance develops rapidly. We provided a novel mechanism, by which oncogenic miR125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2, two negative regulators of the NFúB pathway (Haemmig et al., 2014). GBM cells overexpressing miR125b showed increased NFúB activity, which was associated with TMZ resistance of GBM cells. Although those cells have a survival benefit, apoptosis was unaffected. This is in line with published data suggesting that TMZ induces autophagy rather than apoptosis in glioma cells (Kanzawa et al., 2004). Interestingly, TNFAIP3 prevents K63-linked ubiquitination of BECN1 and thereby inhibits autophagy (Shi et al., 2010). Thus, we hypothesize that miR125b confers TMZ resistance via TNFAIP3 inhibition and induction of autophagy. Indeed, ectopic miR125b expression increased autophagic flux as assessed by LC3B Western blotting. This result was confirmed using a mcherryGFP-LC3B tandem construct and LC3B dot formation assay. In addition, combined treatment of cells with TMZ and the autophagy inhibitor bafilomycinA1 significantly reduced cell viability of miR125b-transfected cells compared to TMZ-treated cells. This suggests that enhanced autophagy by miR125b is a resistance mechanism to TMZ. Most importantly, ectopic expression of miR125b-refractory-TNFAIP3 completely abrogated miR125binduced autophagy indicating that TNFAIP3 has a major function in autophagy regulation. We are currently investigating if this is due to enhanced NFúB activity or reduced ubiquitination of BECN1. Finally, we plan to assess whether autophagy correlates with miR125b expression in GBM patients. In sum, our results provide a novel mechanism of TMZ-resistance, which could open a new therapeutic application by inhibiting autophagy in combination with TMZ. No conflict of interest. 510 Pluripotent genes role in normal melanocyte lineage commitment and malignant transformation D. Sheinboim1 , I. Maza2 , I. Dror3 , J. Hanna2 , C. Levy1 . 1 Department of Human Genetics & Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 2 Department of Molecular Genetics and The Helen and Martin Kimmel Institute for Stem Cell Research, Weizmann Institute of Science, Rehovot, Israel, 3 Computational molecular biology, Technion, Institute of Technology, Haifa, Israel Lineage conversions (e.g. transdifferentiation) could be obtained by the expression of defined lineage transcription factors (TFs) while the induction of embryonic stem cells (ESCs) differentiation requires an additional set of custom-designed cocktail of extrinsic factors. Here, we validated a transgenic system that enabled us to evaluate the ability of ESCs and primary cells to undergo differentiation and transdifferentiation into melanocytes in response to MITF, the melanocyte lineage master regulator. We demonstrate that somatic cells are more prone to switch lineage compared to ESCs. We show that Oct4, the pluripotent master regulator, counteracts prodifferentiation of MITF in several ways thus impeding ESCs differentiation into melanocytes. Oct4 intervenes with MITF transcriptional activity, physically interacts with MITF and blocks MITF binding to its consensus site. In a large scale, we demonstrate that Oct4 occupies the promoter region of part of MITF target genes and shares the same contact domains with MITF hence blocking the access for MITF to target its genes. Moreover, Oct4 also occupies the promoter region of other lineage TFs target genes and shares topological domains with these TFs as well. In vivo examination for Oct4 effect showed that upon Oct4 induction, somatic cells tend to dedifferentiate as exhibited by lineage specific gene downregulation. Our findings exemplify how Oct4 and MITF activity clash to maintain cell identity. This emphasize Oct4 role as ESCs gatekeeper. Next, we aim to examine these observations in the context of melanoma. Dedifferentiation towards ESCs like cells is one of the main characters of tumor progression. Interestingly, pluripotent genes are upregulated during melanoma progression while MITF levels are reduced. Based on preliminary results we hypothesized that Hypoxia-inducible factor (HIF) downregulates MITF expression and interrupts its activity via upregulation of Oct4. We want to establish a link between hypoxia and pluripotent genes role in melanoma progression by exploring HIF and Oct4 impact on MITF activity. This could provide a basis for new therapeutic strategies for melanoma. No conflict of interest.

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511 Metabolic targeting and antagonism of the EGFRvIII/PDK1 axis in temozolomide resistant glioblastoma models K.K. Velpula1,2 , M.R. Guda1 , K. Sahu3 , J. Tuszynski3 , S. Asuthkar1 , S.E. Martin4 , J.D. Lathia5 , A.J. Tsung1,2,6 . 1 Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL, USA, 2 Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA, 3 Department of Oncology, University of Alberta, Edmonton, Canada, 4 Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, IL, USA, 5 Department of Cellular and Molecular medicine, Cleveland Clinic, Cleveland, OH, USA, 6 Illinois Neurological Institute, Peoria, IL, USA Glioblastomas are characterized by amplification of epidermal growth factor receptor (EGFR). Half of EGFR-amplified tumors also express a constitutively active ligand independent EGFR variant III (EGFRvIII). This phenotype represents a tumor specific target correlating with a high growth potential. While current treatments emphasize surgery followed by radiation and chemotherapy with Temozolomide (TMZ), acquired chemoresistance is a universal feature of recurrent GBMs. Mimicking the GBM resistant state, we generated an in vitro TMZ resistant model by continuous exposure of U373 cells constitutively expressing EGFRvIII (U373vIII) to 150mM TMZ for 6 months (U373vIIIR). Here, we documented that dichloroacetate (DCA), a metabolic inhibitor of pyruvate dehydrogenase kinase 1 (PDK1), reverses the Warburg effect by increasing oxygen consumption and reduction of lactate production in the aforementioned cell lines. Correlating with this, we observed that DCA decreased GBM survival with reversion to “normal” metabolism. Microarray analysis on U373vIII or U373vIIIR cells lines and their subsequent treatment with DCA revealed PDK1 as its sole target. DCA induced mitochondrial membrane potential change and apoptosis as evidenced by JC-1 staining and electron microscopic studies. Computational homology modeling studies demonstrated that DCA binds to EGFR and EGFRvIII with strong binding affinity apart from its binding to PDK1. Expression of EGFRvIII was more correlative to PDK1 compared to EGFR in GBM surgical specimen’s further supports the homology data. In a mouse xenograft model, DCA was effective in regression of tumors induced by U373vIII and U373vIIIR cells. In summary, the current study provides the first in vitro proof of concept that DCA reverts the Warburg effect in the setting of TMZ resistance leading to GBM cytotoxicity. No conflict of interest. 512 Pro-survival p53 target genes have evolved clusters of interacting polymorphic response elements that can affect cancer risk P. Zhang1 , G. Stracquadanio1 , X. Wang2 , M. Pybus3 , J. Zeron-Medina3 , S. Nornes1 , S. Moore1 , Y. Bi1 , M. Wallace1 , E. Bond1 , B. Davies4 , N. Sacilotto1 , S. De Val1 , B. Schuster-Boeckler1 , D. Bell2 , G. Bond1 . 1 Ludwig Institute for Cancer Research, University of Oxford, Nuffield Department of Clinical Medicine, 2 Environmental Genomics Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences-National Institutes of Health, 3 Vall d’Hebron University Hospital, Oncology Department, Passeig de la Vall D’Hebron 119, 4 Transgenic Technology Research Group, The Wellcome Trust Centre for Human Genetics, University of Oxford The p53 tumour suppressor is best known for its inhibition of cell survival through regulating transcription of anti-survival genes and ~50% of cancers exhibit mutated p53, which promotes tumorigenesis. However, p53 also has pro-survival target genes and recent data from mice suggest that cancers retaining wild type p53 benefit from the activation of these genes. In humans, we find that pro-survival p53 target genes are more likely than anti-survival targets to have evolved clusters of polymorphic response elements that interact and affect cancer susceptibility. Using targeted genome editing, we evaluated a cluster of four genetically linked polymorphic p53 response elements and show that p53-dependent up-regulation of a pro-survival gene can promote cancer cell survival through c-kit-mediated signaling. Our results provide human genetic evidence supporting a tumor-promoting role of pro-survival activities of p53. This urges re-thinking of p53’s role in tumorigenesis and could support the development of more effective therapy combinations. No conflict of interest. 513 A central role for the syntenin exosomal pathway on the SRC metastatic function in colorectal cancer C. Lecointre1 , N.S. Imjeti2,3 , F. Lembo2 , V. Simon1 , P. Zimmermann2,3 , S. Roche1 . 1 Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS UMR5237, University of Montpellier, France, 2 Centre de ´ ´ Recherche en Cancerologie de Marseille (CRCM), Aix-Marseille Universite, Marseille, 3 Department of Human Genetics, K. U. Leuven, Leuven, Belgium The oncogene and non-receptor tyrosine kinase SRC as it is a master regulator of cell growth and adhesion and hyperactive SRC is a potent driver of human colorectal metastasis. How SRC promotes this malignant process remains unclear. Exosomes are small vesicles secreted by cells, with an

important role in intercellular communication and numerous studies indicate that cancer cells exploit the exosomal pathway to promote metastasis. Yet, little is known about the molecular mechanisms that regulate the biogenesis of cancer exosomes. Recently, we have characterized a molecular machine involving heparanase, syndecan heparan sulfate proteoglycans, syntenin-ALIX as important for the biogenesis of a subpopulation of exosomes (syntenin exosomes) and potentially controlling the in trans activity of heparan-sulfate dependent signaling complexes. By phosphoproteomic analyses of SRCdependent colon carcinogenesis, we also identified Syntenin and ALIX as prominent SRC substrates in this cancer. Here we addressed the role of the Syntenin pathway on SRC oncogenic activity in CRC. We first observed a large increase of Syntenin protein level in metastatic samples of patients with CRC. Second, we found that Syntenin knock-down in CRC cells dramatically affected anchorage-independent cell growth and invasion. The increased transforming properties observed following SRC expression in these tumor cells was also totally dependent upon Syntenin expression. Importantly, the functional defects observed following Syntenin depletion was largely restored when incubating these cells with a conditioned medium (CM) from the corresponding parental CRC cells, suggesting a role for Syntenin exosomes in SRC transforming activity. Finally, we have started addressing the underlying mechanism of these Syntenin pro-tumoral functions and identified important SRC phosphorylation sites in the Syntenin sequence. Their role in SRC oncogenic activity is under current investigation. Overall, our results would support an important role of Syntenin on SRC metastatic function in CRC. No conflict of interest.


Experimental/Molecular Therapeutics, Pharmacogenesis I 514 Thermally responsive ELP-dnMAML protein and its effect on U251 cells in vitro T. Opacak-Bernardi1,2 , J.S. Ryu2 , D. Raucher2 . 1 Faculty of Medicine, Department of Chemistry- Biochemistry and Clinical Chemistry, Osijek, Croatia, 2 University of Mississippi Medical Center, Department of Biochemistry, Jackson, USA Introduction: The Mastermind-like (MAML) family of proteins has gotten its name because of the similarities with the Drosophila Mastermind protein in both structure and function. MAML proteins are transcriptional co-activators in Notch signaling and several other pathways. MAML is essential for the assembly of transcription activation complex of Notch target genes. MAML proteins are structurally simple and can be the base for binding more complex proteins and getting them in close contact necessary for proper function. dnMAML1 mutant is a truncation of MAML1 protein consisting of 62 amino acids (13−74) from the N-terminal basic domain of MAML1. Since the N-terminus is the most conserved part of all MAML proteins, it can inhibit all four receptors and MAML proteins. Depending on the part of MAML involved, dnMAML is presumed to be able to mimic MAML in Notch-independent functions as well. That is true for p53, where binding is accomplished between the N-terminal part of MAML and the DNA binding domain of p53. MAML family of co-activators make an excellent candidate for targeting since they modulate a wide number of signaling pathways. Materials and Methods: The N-terminal fragment of MAML1 protein, further named dnMAML, was cloned into a vector carrying elastin like polypeptide (ELP) and SynB1 cell penetrating peptide. SynB1-ELP-dnMAML inhibition potential was tested in U251 glioblastoma derived cell line. Cells were treated with increasing concentrations of SynB1-ELP-dnMAML for 1 h at different temperatures in two 72 h cycles. The same was done with SynB1-ELP to show that the vehicle protein itself is not cytotoxic. Precise mechanism of inhibition was explored by testing levels of apoptosis induction and cell cycle distribution. Results and Discussion: SynB1-ELP-dnMAML shows cytoplasmic accumulation in the cells. Protein uptake in U251 cells doubles when heat is applied. Treatment with dnMAML inhibits cell growth significantly (up to 60%). The effect can partially be explained by increased apoptosis (up to 20% in heated samples). There is no significant effect on cell cycle or expression levels of Notch canonical targets Hes and Hey. dnMAML also negatively affects expression levels of key regulatory proteins MAPK, pAKT and p53 (mutated in the tested cell line). Conclusion: The effect of SynB1-ELP-dnMAML on cell growth and signaling is evident. It affects cytoplasmic targets due to the lack of nuclear penetration. The ability of this peptide to be modified and tailored more specifically further increases its potential for development. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 515 Cinacalcet inhibits neuroblastoma tumor growth and upregulates cancer-testis antigens 2 C. De Torres1 , C.J. Rodr´ıguez-Hernandez ´ , S. Mateo-Lozano2 , M. Garcia2 , C. Casala` 2 , N. Castrejon ´ 2 , E. Rodr´ıguez2 , M. Sunol3 , C. Lavarino1 , ´ Pediatric Oncology- Developmental J. Mora1 . 1 Hospital Sant Joan de Deu, ´ Tumor Biology Laboratory, Barcelona, Spain, 2 Hospital Sant Joan de Deu, Developmental Tumor Biology Laboratory, Barcelona, Spain, 3 Hospital Sant ´ Pathology, Barcelona, Spain Joan de Deu,

Background: The calcium-sensing receptor (CaSR) is G-protein coupled receptor that exerts cell-type specific functions in numerous tissues and some cancers. We have previously reported that this receptor exhibits tumor suppressor properties in neuroblastoma, a developmental tumor of the peripheral nervous system. We have now assessed cinacalcet, an allosteric activator of the CaSR approved for clinical use, as targeted therapy for this developmental tumor. Materials and Methods: The in vitro effects of cinacalcet were examined in nine neuroblastoma cell lines with different CaSR, MYCN and TP53 status. In vivo analysis included also patient-derived xenografts (PDX). Results: In vitro, acute exposure to cinacalcet induced endoplasmic reticulum stress coupled to apoptosis via ATF4-CHOP-TRB3 in CaSR-positive, MYCNamplified cells. Both phenotypes were partially abrogated by phospholipase C inhibitor U73122. Prolonged in vitro treatment also promoted dose- and timedependent apoptosis in CaSR-positive, MYCN-amplified cells and, irrespective of MYCN status, differentiation in surviving cells. Cinacalcet significantly inhibited tumor growth in MYCN-amplified xenografts and reduced that of MYCN-non amplified PDX. Morphology assessment showed fibrosis in MYCNamplified xenografts exposed to the drug. Microarrays analyses revealed up-regulation of cancer-testis antigens (CTAs) in cinacalcet-treated MYCNamplified tumors. These were predominantly CTAs encoded by genes mapping on chromosome X, which are the most immunogenic. Other modulated genes upon prolonged exposure to cinacalcet were involved in differentiation, cell cycle exit, microenvironment remodeling and calcium signaling pathways. CTAs were up-regulated in PDX and in vitro models as well. Moreover, progressive increase of CaSR expression upon cinacalcet treatment was seen both in vitro and in vivo. Conclusions: In summary, cinacalcet reduces neuroblastoma tumor growth and up-regulates CTAs. This effect represents a therapeutic opportunity and provides surrogate circulating markers of neuroblastoma response to this treatment. Conflict of interest: Ownership: CdT holds a patent, WO 2013144397 (A1), US-2015-0342907-A1, and a patent submission (PCT/ES2015/070561) on cinacalcet. 516 Dual roles of MMP2 activity on brain tumor progression and invasion C.S. Chiang1 , C.F. Yu2 . 1 National Tsing Hua University, Department of Biomedical Engineering and Environmental Sciences, Hsinchu, Taiwan, 2 Chang-Gung Memorial Hospital, Department of Radiation Oncology, Taoyuan, Taiwan Background: Gliomas are the most common type of primary brain tumor. They are highly invasive and patients with gliomas have a poor prognosis. We have previously shown that a murine astrocytoma cell-line, ALTS1C1, displays a highly invasive pattern similar to clinical anaplastic astrocytoma. This cell line express relative high levels of matrix metalloproteinase-2 (MMP-2) mRNA. This study aims to study the role of MMP2 on tumor progression and invasion. Material and Methods: Orthotopical murine ALTS1C1 brain tumor model was used throughout the study and immunohistochemical (IHC) staining was used to examine the expression of MMP-2 and its association with tumor progression and invasion. The expression level of MMP-2 in ALTS1C1 cells was inhibited using lentiviruses carrying specific shRNA. Results: IHC results show that the intensity pattern of MMP-2 protein expression in ALTS1C1 tumor was an uneven distribution across tumor sections with higher intensity on invading tumor fronts than tumor center core. MMP-2-knockdown (MMP-2kd) cells showed low invasive ability, however no differences in cell migration and proliferation rates were observed compared with parental cells in vitro. The suppression of MMP-2 expression prolonged the median survival days of tumor-bearing mice associated with tumors having smooth tumor margins, decreased Ki67 proliferating index, and downregulation of GLUT-1 antigen. Although the suppression of MMP-2 expression did not alter the vessel density of tumors in comparison to parental ALTS1C1 tumors, vessels in MMP-2kd tumors were less functional as evidenced by the low ratio of pericyte coverage and reduction in Hoechst33342 dye perfusion. Conclusions: This study illustrated that tumor-derived MMP-2 has at least two roles in tumor malignancy − to enhance tumor invasiveness by degrading extracellular matrix and to enhance tumor growth by promoting vessel maturation and function. Acknowledgment: This work is supported by NHRI-EX105-10514BI grant. No conflict of interest.

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517 Enhancement of doxorubicin cytotoxicity by histone deacetylase inhibition in human sarcoma cells M.G. Tupone1 , D. Trisciuoglio1 , M. Desideri1 , M. Di Martile1 , S. Buglioni1 , R. Loria1 , V. Ferraresi1 , N. Baldini2 , R. Biagini1 , D. Del Bufalo1 . 1 Regina Elena National Cancer Institute, Sarcoma Translational Group, Roma, Italy, 2 Istituto Ortopedico Rizzoli IOR, Orthopaedic Pathophysiology and Regenerative Medicine Unit, Bologna, Italy Introduction: Soft tissue and bone sarcomas are rare malignancies of mesenchymal origin that account for about 1% of all adult solid tumors. Surgical resection is the first treatment option but is heavily hampered by delayed diagnosis. Due to few therapeutic treatments available, novel and efficacious therapy is urgently needed. Histone deacetylase inhibitors (HDACi) are emerging as a prominent class of therapeutic agents for several cancers; however, little is known about HDACi activity in sarcomas. By using human sarcoma models, we studied the therapeutic activity of ITF2357, a novel histone deacetylase inhibitor, as an anti-cancer agent alone or in combination with doxorubicin, on both wild-type and mutated p53-bearing sarcoma cell lines. Material and Methods: A wide panel of sarcoma cell lines, including both established and patient-derived sarcoma from different histotypes, were used. Western Blot analysis was performed to evaluate the effect of ITF2357 on histone and non-histone protein acetylation and expression of potential downstream targets. Cell viability was tested by MTT and CellTiterGlo assays. Apoptosis induction and cell cycle perturbation were assessed by flow cytometry and Western Blot analysis. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells and Western Blot analysis. Pharmacological interaction between ITF2357 and doxorubicin was assessed by conservative isobologram analysis. In vivo efficacy of drug combination was evaluated in nude mice bearing sarcoma xenografts. Results: In sarcoma cell lines, HDAC inhibition induced H3 histone and alpha-tubulin acetylation, cell proliferation inhibition and activation of apoptotic program in a dose-dependent manner. ITF2357 cytotoxicity was independent on p53 status, despite its ability to inhibit the expression of mutated p53 protein. Moreover, ITF2357 induced a canonical-autophagic process, which protects sarcoma cells from apoptotic cell death and enhances cell survival. The combination of doxorubicin and ITF2357 was found to be highly synergistic in terms of cell proliferation inhibition and was related to the engagement of the apoptotic mitochondrial pathway. Noteworthy, treatment with both drugs also showed synergistic inhibition of cell viability in a doxorubicin-resistant osteosarcoma line, as compared with either agent alone. The effect of the drug combination was corroborated in sarcoma stem cells obtained from sarcoma patients. In vivo experiments in mouse xenograft models confirmed that co-treatment increased both inhibition of tumor growth and apoptosis, as compared with either agent alone. Conclusions: Overall, HDAC inhibition in sarcoma may provide an effective therapeutic modality capable of overcoming the doxorubicin resistance, and thus may lead to more durable responses in the clinic. No conflict of interest. 518 Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells M. Di Martile1 , M. Desideri1 , T. De Luca1 , S. Buglioni1 , D. Del Bufalo1 , D. Trisciuoglio1 . 1 Regina Elena National Cancer Institute, ResearchAdvanced Diagnostics and Technological Innovation Department, Roma, Italy Introduction: Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene[4-(4 -chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Material and Methods: The biological effect of CPTH6 was analysed in a panel of NSCLC cell lines (H1299, A549, Calu-1, Calu-3, H1650, H460, HCC827, A427, H1975) and in patient-derived Lung Cancer Stem Cell (LCSC) (LCSC136, LCSC36, LCSC18, LCSC143, LCSC229, LCSC196, LCSC223) models. Cell viability was tested by MTT and CellTiter-Glo. Apoptosis induction, cell cycle perturbation and modulation of cancer stem cells markers were assessed by flow cytometry. Pharmacological interaction between CPTH6 and Pemetrexed or Cisplatin were assessed by conservative isobologram analysis. Nude and NOD/SCID mice were used for in vivo experiments. Results: The HATi CPTH6 reduced in vitro cell viability of both NSCLC and LCSC lines. Interestingly, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide

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clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 was able to inhibit the growth of LCSCderived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation was also confirmed in vivo. Conclusions: Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC. No conflict of interest. 519 Delivery of microRNA-26a by CD38-conjugated nanoparticles into chronic lymphocytic leukemia cells of the Em-TCL1 mouse model L. D’Abundo1 , E. Callegari1 , A. Bresin1 , C. Bassi1 , P. Guerriero1 , G. Russo2 , S. Sabbioni3 , F. Malavasi4 , M. Negrini1 . 1 University of Ferrara, Morphology-Surgery and Experimental Medicine, Ferrara, Italy, 2 Istituto Dermopatico dell’Immacolata, Laboratorio di Oncologia Molecolare-, Roma, Italy, 3 University of Ferrara, Life Sciences and Biotechnology, Ferrara, Italy, 4 University of Torino, Life and Health Sciences, Torino, Italy Introduction: Despite its indolent nature, chronic lymphocytic leukemia (CLL) is an incurable disease. The dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of CLL, thus suggesting their use as potential anti-CLL molecules. To improve delivery of miRNA molecules into CLL cells, we developed a novel CD38-conjugated nanoparticles. Here, we used the Em-TCL1 transgenic mouse model, which reproduces leukemia with a similar course and distinct immunophenotype as human B-CLL, to test the potential therapeutic effect of miR-26a. Materials and Methods: Delivery bio-distribution and efficiency of miRNA delivery by nanoparticles was investigated in different tissues by ddPCR. The anti-leukemic activity of CD38-NP-miR-26a was evaluated in transplanted TCL-1 FVB mice after treatment with single strand miR-26a mimic (100 mg/injection i.p., three times a week for 3 weeks). To analyze the progression of leukemia, the human TCL-1 gene was quantified from genomic DNA of murine blood cells by ddPCR and furthermore the expansion of leukemic B cells (B220+/CD5dim) was monitored through flow cytometry analysis. In addition, the apoptotic effects of miR-26a on murine leukemic splenocytes were assessed by Annexin V. Results and Discussion: The use of CD38-NP was highly effective in delivering miRNA molecules to the spleen of diseased mice. Indeed, the level of mature miRNA increased 70-fold in leukemic splenocytes after CD38-NP-miR treatment compared to treatment with PEI-miR formulation and a 3-fold increase compared to treatment with NP-miR formulation (nonCD38-conjugated). In vivo, treatments with miR-26a increase the apoptosis of leukemic spleen cells, which could be related to the modulation of multiple targets. Finally, a 3-weeks treatment with CD38-NP-miR-26a could elicit a significant delay in leukemic cell expansion. Conclusions: These results provide a novel in vivo delivery approach of miRNAs, which improves efficiency and specificity of delivery to CD38+ leukemic cells. CD38-NP could effectively deliver functional miR-26a, resulting in target down-regulation and anti-leukemic activity and suggests that the use of microRNAs could represent a novel potential therapeutic approach for the treatment of CLL. No conflict of interest. 520 Development of novel tirapazamine-loaded liposomes for hypoxia targeted therapy V.L. Silva1 , W. Al-Jamal1 . 1 University of East Anglia, School of Pharmacy, Norwich, United Kingdom Background: Hypoxia is considered a hallmark of cancer and a common characteristic of locally advanced solid cancers. Tumour hypoxia plays a key role in promoting angiogenesis, metastasis, and drug resistance. It is also linked to poor patient survival. Hypoxia-activated prodrugs have opened the door for specific treatment of the solid and metastatic tumours with lower side effects. Tirapazamine (TPZ) is the most advanced hypoxia-activated prodrug. TPZ has shown great specificity and potency in inhibiting tumour growth at moderate to severe hypoxic conditions. It is currently in phase III clinical trials to treat non-small cell lung cancer and cervical cancer. It’s efficacy in vivo has been limited due to its inadequate diffusion in the tumour mass and fast metabolism. This project aims to enhance the therapeutic effectiveness of TPZ by developing a novel liposome-based delivery system with enhanced penetration in tumour tissues, following the systemic administration. Material and Method: To improve the encapsulation of TPZ in liposomes, TPZcopper complexes (Cu(TPZ)2) were prepared and characterised using different analytical techniques (HPLC, IR, UV/Vis, spectrofluorometry and MALDI-TOF). Next, a remote loading method was developed to encapsulate stably Cu(TPZ)2 complexes in different liposomal formulations. Cu(TPZ)2-loaded liposomes were characterised using dynamic light scattering, spectrofluorometry and HPLC. The cytotoxicity of TPZ and Cu(TPZ)2 complexes, and Cu(TPZ)2-

loaded liposomes was assessed in vitro, using three-dimensional tumour spheroids, as models for tumour hypoxia. The cytotoxicity was evaluated by monitoring spheroid growth over time, and cell viability using Alamar Blue® assay. Results and Discussion: Cu(TPZ)2 complexes exhibited a red-shift in their optical properties. In addition, the Cu(TPZ)2 complexes were more hydrophobic in nature, which led to an efficient drug loading in the liposomal formulations. The drug loading was dependent on the lipid composition, drug: lipid and the hydrating buffers used. 3D tumour spheroid models were an interesting model to assess the toxicity of Cu(TPZ)2-loaded liposomes under hypoxic conditions. Our results showed that the cytotoxicity of the Cu(TPZ)2loaded liposomes was dependent on the cell line, drug concentration, and the incubation time. Conclusion: In this work we showed that the complexation of TPZ with copper is an attractive approach to promote TPZ encapsulation in a novel liposomebased system. This novel nanomedicine could overcome TPZ shortcomings in vitro and in vivo, and offers a promising approach to target advanced cancer in patients. No conflict of interest. 522 Cediranib affects tumor dissemination and prolongs the survival of mice bearing patient-derived ovarian cancer xenografts (EOC-PDX) A. Decio1 , M. Cesca1 , F. Bizzaro1 , M.R. Bani1 , D. Belotti1 , R. Giavazzi1 . 1 IRCCS − Istituto di Ricerche Farmacologiche “Mario Negri”, Oncology, Milano, Italy Background: Vascular Endothelial Growth Factors (VEGFA/VEGFC) are overexpressed and secreted in large amount in epithelial ovarian cancer (EOC). Cediranib is a VEGF receptor tyrosine kinase inhibitor that affects tumor angiogenesis and shows benefit in women with relapsed platinum sensitive ovarian cancer (ICON6 trial). The aim of this study was to investigate the efficacy of cediranib alone or in combination with cisplatin (DDP) on tumor growth and dissemination in the peritoneal cavity on patient-derived human ovarian carcinoma xenografts (EOC-PDX) with different degree of sensitivity to chemotherapy. Material and Methods: EOC-PDX (n = 5) were transplanted orthotopically in the peritoneal cavity of nude mice and treated with cediranib monotherapy (3 mg/kg, p.o.) or in combination with the standard-of-care chemotherapy for ovarian cancer (we used cisplatin, DDP, 4 mg/kg, i.v.). The effect on ascites formation, tumor dissemination and metastases (interim analysis) and overall survival (end point) was investigated. Results: The response of EOC-PDX to cediranib was heterogeneous, with a life span increment superior to 100% in 3 out of 5 xenografts (HOC22, ILS 250%; HOC8, ILS 122%; Y-HOC8, ILS 143%; HOC76, ILS 72%, HOC119, 8%). The EOC-PDX expressed VEGF receptors (VEGFR2/VEGFR3) and released VEGFA and VEGFC in their ascites; however the level of these biomarkers could not predict the response to cediranib. Cediranib reduced ascites production from all the EOC-PDX, but its effect on tumor dissemination into the organs of the peritoneal cavity (e.g. ovaries, pancreas, liver, diaphragm) was limited. Only combined with DDP, ascites and metastases were both reduced. The reduction of dissemination to peritoneal organs varied between EOC-PDX and was associated to the increase of overall survival. Cediranib added to DDP improved the response to chemotherapy without increasing toxicity. Conclusions: The response of EOC-PDX to cediranib likely reflects the heterogeneous response observed in patients. Cediranib potentiated chemotherapy, significantly inhibiting tumor progression and dissemination to metastatic organs, even in tumors poorly responsive to cisplatin. EOC-PDX models with different metastatic phenotype and responsiveness to cediranib may help in identifying determinants of response to cediranib. AD is a fellow of the Umberto Veronesi Foundation, Milan, Italy. No conflict of interest. 523 The effects and molecular mechanisms of crown ethers on drug efflux pumps I. Guberovic1 , M. Marjanovic1 , K. Ester1 , A.M. Mikecin1 , I. Martin-Kleiner1 , T. Sumanovac-Ramljak2 , K. Mlinaric-Majerski2 , M. Kralj1 . 1 Rudjer Boskovic Institute, Department of Molecular Medicine- Laboratory for Experimental Therapy, Zagreb, Croatia, 2 Rudjer Boskovic Institute, Department of Organic Chemistry and Biochemistry- Laboratory of Synthetic Organic Chemistry, Zagreb, Croatia Background: Cancer stem cells (CSCs) are regarded as a leading cause of cancer relapse due to their ability to reconstitute its heterogeneity even after tumor eradication. It has been suggested that CSC resistance to chemo- and radiotherapy could be related to enhanced survival pathways, decreased levels of reactive oxygen species and increased expression of drug efflux pumps. Nevertheless, related molecular mechanisms remain elusive. Recent research has pinpointed salinomycin, a naturally occurring potassium ionophore, as a

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 promising compound for targeting CSCs. Moreover, several studies indicate that one of the mechanisms of action of salinomycin towards CSCs is the inhibition of P-gp drug efflux pump activity. Pertaining to the mentioned studies and results previously published by our group regarding anticancer activity of proprietary crown ethers that act as K+ ionophores, we hypothesized that these compounds could show selectivity towards CSC. Therefore, we set about to elucidate their mechanism of action, with the focus on the effect towards the function and activity of drug efflux pumps. Materials and Methods: The impact of compounds on the expression of P-gp was evaluated by Western Blot and qRT-PCR. Moreover, MTT, cell cycle and annexin V assays were used to determine the effect of compounds on cancer cells with respect to P-gp expression, while the functional activity of P-gp was evaluated by Rhodamine efflux assay. Mechanisms of action of tested compounds were evaluated by UIC2 shift assay, Western Blot and immunofluorescence. Results and Discussion: Cytotoxicity evaluated by MTT showed increased resistance of P-gp overexpressing cells to P-gp substrates (e.g. doxorubicine, paclitaxel), as expected. Results obtained by Rhodamine efflux assay showed strong P-gp inhibition by proprietary compounds, while combined treatment with paclitaxel indicated cell sensitization towards this chemotherapeutic. Additionally, implications of crown ether molecular mechanisms of action were obtained by UIC2 shift assay and by analysis of the impact of these compounds on P-gp expression and upstream regulators of this drug efflux pump. Conclusions: Results indicate stronger P-gp inhibition obtained by proprietary crown ether treatment than by the commercially available P-gp inhibitor, verapamil. Additionally, plausible molecular mechanisms of action of crown ether compounds will be presented. No conflict of interest. 524 Effects of telomerase blockage on the advanced triple negative breast cancer cells culture by activated lymphocytes A. Tavartkiladze1 , R. Khutsishvili2 , A. Gogiberidze2 . 1 Institute for Personalized Medicine, Medical Oncology, Tbilisi, Georgia, 2 Institute for Personalized Medicine, Molecular Biology, Tbilisi, Georgia Background: Telomerase is commonly expressed in human cancer cells. Increased telomerase expression produces vulnerability of cancer cells, distinguishing them from normal cells in the body, although normal cells do also have some active telomerase. Recent studies also suggest that telomerase is implicated in tumor progression in unexpected ways. Material and Methods: The aim of our research is the utilization of biological therapy as an anti-telomerase cancer therapy. We observed cells cultures of 20 patients with advanced triple negative breast cancer (TNBC). In each culture, we observed the affect of genetically close relative’s lymphocyte mass, activated in special conditions. The control group was the non-affected cells culture of the same 20 patients. Incubation/cultivation went on for 90 days. Telomerase gene expression was studied by RT-PCR. Results: As the result of the research, we achieved the 45.3% blockage of telomerase expression in cells, compared to control group. Conclusions: Hence from the above, we can plan a large-scale pre-clinical study in in vivo conditions using experimental animals, that in fact will bring new perspectives in anti-telomerase target research. No conflict of interest. 525 Salinomycin affects Golgi apparatus function in cancer stem-like cells A.M. Mikecin1 , M. Marjanovic1 , I. Guberovic1 , M. Kralj1 . 1 Rudjer Boskovic Institute, Division of Molecular Medicine, Zagreb, Croatia Background: Cancer stem cells (CSC) are an immortal tumor-initiating population that can self-renew and has pluripotent capacity. Despite their small number, CSCs are believed to be critical for tumor initiation, progression and metastasis, as well as for the treatment failure and disease relapse. CSCs have been confirmed to exist in various types of tumors which include leukemia as well as various solid cancers. There are several obstacles involved with CSC research among which are their low number within tumor cell population and their relative instability in culture. Recently, a breast CSC model was established by experimental induction of epithelial to mesenchymal transition (EMT) in immortalized human mammary epithelial cells (HMLE). Using this model salinomycin was identified to be selectively toxic towards epithelial CSCs. Salinomycin is a K+/H+ exchanger that can affect cation transport across different membranes present in the cell. However the exact mechanism of the observed selectivity remains largely unknown. We have noticed that salinomycin affects posttranslational modifications of membrane proteins in the fore mentioned CSC model. This lead us to the hypothesis that salinomycin induces Golgi apparatus stress that affects secretory pathway which may represent a key vulnerability of EMT cells. Of note, monensin, another ionophore with chemical structure similar to that of salinomycin, is a well described inhibitor of Golgi function.

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Materials and Methods: We used HMLE cells with knocked-down E-cadherin (HMLEshEcad) that went through EMT and display characteristics of stem cells. To test our hypothesis we performed western blotting, confocal microscopy and flow cytometry after staining with fluorescently labeled Golgirelated antibodies. Results: The treatment of stem-like HMLEshEcad cells with submicromolar concentrations of salinomycin causes dysfunctional maturation of N-cadherin. In addition, we observed similar changes in processing of other plasma membrane proteins that could be accounted for aberrant Golgi apparatus function. The same effect was not observed in control cells. Immunostaining confirmed changes of Golgi apparatus morphology and function. Conclusion: Our results indeed point that salinomycin differentially affects mesenchymal and epithelial cells due to their different dependence on the Golgi apparatus function. We believe that agents that promote endoplasmic reticulum and Golgi apparatus malfunction should be further explored for their potential to selectively eradicate cancer cells that have undergone an EMT. No conflict of interest. 526 1-Methylhistidine and high dose methotrexate influence on triple negative breast cancer in experiment A. Tavartkiladze1 , R. Khutsishvili2 , A. Gogiberidze2 . 1 Institute for Personalized Medicine, Medical Oncology, Tbilisi, Georgia, 2 Institute for Personalized Medicine, Molecular Biology, Tbilisi, Georgia Background: As the result of our observations, we stated that in blood plasma, the level of 1-methylhistidine is extremely decreased in oncologic cases in humans as well as in animals, especially in Triple Negative Breast Cancer (TNBC). Material and Methods: For observation, we used 50 rats with breast cancer (Carcinogen was N-Methyl-Nitrosourea) which were daily given the infusion of Methotrexate (MTX) 1-Methylhistidine combination. The control group consisted of 25 healthy rats without any intervention. 25rats with TNBC which were infused only Methotrexate and 25 rats with TNBC which were infused only 1-Methylhistidine. Dosage was as following: Methotrexate 100 mg/m2, on every 14th day and 1-methylhistidine 50 mg/kg, on every 4th day. The treatment went on for 120 days. Results: 1. Tumor regression in the investigated group (MTX + 1-methylhistidine) came to 87.2%; 2. In the free control group the disease progressed and 4 rats died within 120 days; 3. In MTX monotherapy group, the tumor regression came to 29.7% (1 rat died within 120days); 4. In 1-methylhistidine monotherapy group, the tumor regression came to 12.3% (2 rats died within 120 days). Conclusions: The achieved results are undoubtedly interesting on the example of low-molecular bio-regulators: 1-methylhistidine, as a new target, anti-tumor agent in remodeling and diagnostic approach to the treatment of Triple Negative Breast Cancer. No conflict of interest. 528 The role of reactive oxygen species in photodynamic therapy combined with acetylsalicylic acid in colon and esophagus cancer cells M. Laranjo1 , N. Almeida2 , A. Serra3 , A.M. Abrantes1 , M. Pineiro4 , A.C. Gon¸calves5 , J. Casalta-Lopes1 , A.B. Sarmento-Ribeiro6 , M.F. Botelho1 . 1 Faculty of Medicine of University of Coimbra, Institute of Biophysics/Biomathematics- CIMAGO- CNC.IBILI, Coimbra, Portugal, 2 Faculty of Medicine of University Coimbra, Institute of Biophysics/Biomathematics, Coimbra, Portugal, 3 Faculty of Sciences and Technology of University of Coimbra, Department of Chemical Engineering, Coimbra, Portugal, 4 Faculty of Sciences and Technology of University of Coimbra, Department of Chemistry, Coimbra, Portugal, 5 Faculty of Medicine of University of Coimbra, Applied Molecular BiologyUnit- FMUC, Coimbra, Portugal, 6 Faculty of Medicine of University of Coimbra, Applied Molecular Biology Unit, Coimbra, Portugal Introduction: One of the key features for the overall efficacy of photodynamic therapy is the generation of reactive oxygen species (ROS), which are the main effectors in terms of cell death. Non-steroidal anti-inflammatory drugs, like acetylsalicylic acid are referred often in studies as promoters of ROS generation although others paradoxically refer its properties in terms of cytoprotection by antioxidative mechanisms. Having this in mind, the objective of this study is to understand if acetylsalicylic acid interacts with photodynamic therapy enhancing the ROS generation in WiDr and OE19 cell lines leading to cell death. Methods and Materials: WiDr cell line was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotics and sodium piruvate, while OE19 cell line was propagated in culture with RPMI also with 10% FBS, 1% antibiotics and sodium piruvate. For the studies of ROS inhibition 80,000 cells/mL were seeded in 48 well plated, with the addition of a photosensitizer, previously synthetized by us, in concentration range from 5nM to 500nM. After 24 h of incubation, 5mM Sodium Azide (Superoxide

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anion inhibition) or 40mM D-Mannitol (Hydroxyl radical inhibition) were added to the plates. Past 2 h the plates were irradiated with a flow 7.5 mW/cm2 to achieve 10 J, followed by the addition of aspirin at the concentration of 2.5mM. The metabolic activity was measured by MTT Assay 24 h later. For the flow cytometry studies, the cells were stained with DCF probe for evaluating the peroxide content, DHE probe for evaluating the superoxide anion formation and JC-1 probe for evaluating the mitochondrial membrane potential. Results: Following the inhibition of superoxide anion and hydroxyl radical the efficacy of photodynamic therapy is limited. The intracellular peroxide content seems to decrease with higher photosensitizer concentrations particularly in WiDr cell line, decreasing almost 4-fold in the higher concentration. In terms of superoxide anion, the higher photosensitizer concentration leads to an increase of its intracellular concentration. Finally, is observed a decrease in mitochondrial membrane potential in both cell lines, with a 2-fold decrease for the WiDr cell line and a 3-fold decrease for OE19 cell line, which is a marker of cell death by apoptosis. Conclusion: Reactive oxygen species are of paramount importance in photodynamic therapy, being singlet oxygen, typical of type II photodynamic reaction, considered the most prevalent one. However with this work we reinforce the importance of type II ROS in anticancer activity. Furthermore, acetylsalicylic acid may have a role in modulating the ROS generation after photodynamic therapy. Acknowledgment: The authors would like to thank the Foundation for Science and Technology (FCT) (Strategic Project CNC.IBILI: UID/NEU/04539/2013), COMPETE-FEDER for financial support. No conflict of interest. 529 miRNA pattern modifications in bladder cancer R.M. Cojocneanu Petric1 , C. Braicu1 , L. Budisan1 , C. Ivan2 , S. Chira1 , B. Petrut1 , L. Raduly1 , A. Jurj1 , G.A. Calin2 , I. Berindan-Neagoe1 . 1 Iuliu Hatieganu University of Medicine and Pharmacy, Research Center for Functional Genomics- Biomedicine and Translational Medicine, Cluj Napoca, Romania, 2 The University of Texas MD Anderson Cancer Center, Center for RNA Interference and Non-Coding RNAs, Houston, USA Introduction: miRNAs are short, stable RNA molecules, shown to have biomarker potential in cancer. Bladder cancer (BC) is one of the most common malignancies of the urinary tract, ranking 6th in men and 9th in both sexes according to Globocan 2012. Due to its nonspecific clinical symptoms, a precise diagnosis for most BC cases is achieved in its late stages, and mostly following a surgical procedure, thus the development of specific biomarkers for the diagnosis, prognosis and response to therapy of bladder cancer is of utmost importance. We investigated the miRNAs profile of BC tissue in order to identify an altered molecular profile specific for BC which could have biomarker potential. Material and Methods: We performed a one-color microarray study on 24 matched tumor versus normal tissue samples collected from patients with urinary bladder cancer prior to chemotherapy. Data were analyzed using the GeneSpring GX® software from Agilent® , considering a fold change cut-off of 1.5 and a corrected p-value d-tocopherol (TP) g- and a-TP. d-TP phosphate-5 -gemcitabine (NUC050) was synthesized to improve upon the pharmacology and activity of gemcitabine (Gem). Materials and Methods: Growth inhibitory (GI) effects of Gem and NUC050 were evaluated in 96-well plates by exposing cells to drug for 72 hr. To determine whether NUC050 could bypass nucleoside transport downregulation, control wells did not include dipyridamole (DP) and treated wells included a final DP concentration of 1 mM. To determine whether NUC050 delivered a nucleoside MP intracellularly, testing was performed in CCRF-CEM cells either deoxycytidine kinase (dCK) (+) or (-). Mouse PK were performed by Eurofins/Panlabs. Mice were administered 2 mg/kg IV of NUC050, blood drawn on 3 mice at 6 different time points, and plasma analyzed by LC/MS/MS. MTD was determined for different doses and schedules. A pilot efficacy study was performed in nude mice implanted with the LoVo cell line (colon cancer). Results and Discussion: NUC050 was tested in three cell lines against Gem with and without DP. MDA-MB231 (breast), NCI-H460 (NSCL) and HCT-116 (colon). GI50 (in mM) were as follows for each cell line, respectively: Gem: 3.08, 0.02, 0.03; Gem+DP: 56.77, 0.82, 2.39; NUC050: 17.16, 2.14, 3.07; NUC050+DP: 23.30, 1.47, 6.74. DP increased the GI50 of gemcitabine from 18 to 80-fold, while that of NUC050 was increased at most 2.2-fold. Gem GI50 went from 0.002 mM in dCK(+) cells to 124.5 mM in dCK (-) cells, an increase of 62,250-fold. NUC050 GI50 went from 0.59 mM to 19.2 mM, an increase of only 32.5-fold, compatible with the delivery of Gem MP. In mice, the halflife of NUC050 is 3.9 hours compared to 0.28 hours reported for gemcitabine (increase of 13.9-fold). MTD in mice was 15 mg/kg (5.4 mg/kg Gem equimolar) when administered q3d × 5, and 50 mg/kg (18.1 mg/kg Gem equimolar) when administered qwk × 3. A xenograft model with LoVo using the latter dose and schedule (n = 2) showed tumor weight reduction of 50.6% compared to saline controls (n = 5) 10 days after the last dose. Conclusions: NUC050 penetrates the cell by a nucleoside transport independent mechanism and delivers intracellularly a nucleoside MP and dTP. The half-life of NUC050 in mice is 13.9-fold longer than that of Gem, while the pharmacodynamics of NUC050 are compatible with the increased duration of drug exposure. NUC050 has demonstrated activity in an animal model of colon cancer. Conflict of interest: Ownership: I am the sole partner in Epigenetics Pharma. Corporate-sponsored Research: Research is funded in part by AmorChem Venture Fund (Montreal, Canada). Other Substantive Relationships: Research is funded in part by the State of Washington Life Sciences Discovery Fund (Seattle, WA, USA). 550 In vitro and in vivo testing of a novel DNA methyl transferase inhibitor (DNMTI) with activity against hematogenous and solid tumors whether p53 null or wild type (WT) R. Daifuku1 . 1 Epigenetics Pharma, Research and development, Mercer Island, USA Background: Aberrant DNA methylation is an epigenetic mechanism that can inactivate the expression of genes that suppress tumorigenesis: tumor suppressor genes, genes that suppress apoptosis, metastasis and angiogenesis, DNA repair genes and genes that express tumor-associated antigens. NUC013 (2 ,2 -difluoro-5-azadeoxycytidine) is a DNMTI which has been found to be safer and more active than decitabine both in vitro and in vivo.

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Materials and Methods: DNMTI was measured colorimetrically in a 96-well microplate kit. In vitro testing of NUC013 for activity was performed in the NCI60 panel at 10 mM in 59 cell lines of different tissues of origin: breast, colon, CNS, renal, lung, melanoma, ovarian, prostate, and hematogenous. Tumor xenograft models were performed at Southern Research Institute in nude mice implanted with HL-60 (human leukemia, p53 null) or LoVo (colon cancer, p53 WT) cell lines. Drug or saline were administered IV for three consecutive days a week for three weeks (n = 10/group). Results and Discussion: In HCT116 (colon cancer), NUC013 has been found to be approximately 10-fold more potent than 5-azacytidine at DNMT inhibition. In the NCI60 cell line panel, NUC013 was more active than decitabine in most cell lines: decitabine had growth inhibition 50% (GI50) 90% of melanomas in all stages of the disease and by other difficult-to-treat cancers such as TripleNegative Breast Cancer. Material and Methods: Bispecific antibody (bsAb),MCSPxDR5 was constructed by fusing the binding domains of a high-affinity anti-MCSP antibody (MAb 9.2.27) to those of the agonistic DR5 antibody tigatuzumab and complemented with a fully functional human IgG1 Fc domain. Antitumor activity of MCSPxDR5 was evaluated using a panel of cancer cell lines, primary patient-derived melanoma cells and in co-cultures of cancer cells and FcRpos myeloid effector cells. Results: MCSPxDR5 selectively induced MCSP-directed apoptosis in a panel of MCSPpos/DR5pos cancer cell lines and primary patient-derived melanoma cells. Moreover, the antitumor activity of MCSPxDR5 was potently enhanced after cross-linking of its IgG1 domain by FcRpos myeloid immune effector cells. Thus, MCSPxDR5 exerts strong and selective DR5-dependent cytotoxic activity against MCSPpos melanoma cells. Cross-linking MCSPxDR5 by FcR’s increases its cytotoxic potential further without compromising its selectivity. Conclusion: Mode-of-action of bsAb MCSPxDR5 comprises 3 cooperating processes: delivering a DR5 agonist to malignant cells, potent activation of DR5-mediated cell death and recruitment of FcgRpos immune cells to mount an immune response. Therefore, MCSPxDR5 may be used to enhance the efficacy of current DR5-targeted cancer therapy. No conflict of interest.

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552 Ex vivo culture of circulating tumour cell derived explants to facilitate rapid therapy testing in small cell lung cancer

554 Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport

A. Lallo1 , U. Warpman Berglund2 , K. Frese1 , D. Potter1 , T. Helleday2 , C. Dive1 . 1 CRUK Manchester Institute, Clinical and Experimental Pharmacology Group, Manchester, United Kingdom, 2 Science for Life Laboratory- Translational Medicine and Chemical Biology- Karolinska Insitute, Medical Biochemistry and Biophysics, Stockholm, Sweden

M. Michaelis1 , F. Rothweiler2 , M. Wurglics3 , N. Aniceto4 , M. Wiese5 , M. Wass1 , T. Ghafourian6 , M. Schubert-Zsilavecz3 , J. Cinatl Jr2 . 1 University of Kent, School of Biosciences, Canterbury, United Kingdom, 2 Goethe¨ Institut fur Universitat, ¨ Medizinische Virologie, Frankfurt am Main, Germany, 3 ¨ Institut fur Goethe-Universitat, ¨ Pharmazeutische Chemie, Frankfurt am Main, Germany, 4 Universities of Kent and Greenwich in Medway, Medway School of Pharmacy, Chatham, United Kingdom, 5 University of Bonn, Pharmaceutical Institute, Bonn, Germany, 6 University of Sussex, School of Life Sciences, Brighton, Germany Introduction: Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARa, PPARg, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARa, and PPARg represent potential anti-cancer drug targets we here investigated the effects of pirinixic acid derivatives on cancer cells. Material and Method: The effects of 39 pirinixic acid derivatives were determined on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability. Subsequently, the effects of selected compounds were investigated on drug-resistant neuroblastoma cells. In silico docking studies were performed to examine the drug interactions with ABCB1. Results and Discussion: Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anticancer effects and the effects on 5-LO, mPGES1, PPARa, or PPARg. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATPbinding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2mM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Conclusion: Pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport. No conflict of interest.

Background: Small cell lung cancer (SCLC) is a highly aggressive disease with dismal prognosis. Whilst 80% of patients respond to first line therapy with platinum/etoposide +/− radiotherapy, 95% of responders relapse within 3−18 months. The molecular mechanism(s) unpinning disease relapse and acquired drug resistance are poorly understood, due in part to the paucity of SCLC biopsies available for mechanistic studies. To overcome this limitation, we generated circulating tumour cell explant models (CDX) in immunocompromised mice that faithfully represent patients’ disease and mirror response to chemotherapy. However, in vivo pharmacology studies in CDX take several months, therefore, I developed an ex vivo CDX culture system to more rapidly test novel therapies and select candidates for further analysis in vivo. Material and Methods: SCLC cells were isolated from disaggregated CDX tumours derived from treatment na¨ıve donor patients and grown ex vivo. CDX cultures were tested for sensitivity to cisplatin/etoposide to confirm responses of donor patients and matched CDX models. Immunohistochemical analysis and drug screening were repeated at multiple time points to assess changes associated with duration of ex vivo culture. To examine changes imposed by ex vivo culture, CDX-derived cells were cultured for up to 4 weeks and samples for RNAseq were collected weekly. At 4 weeks, cells were reimplanted into immunocompromised mice and RNAseq performed to compare gene expression in the original CDX with the cells re-implanted after ex vivo culture. Additional drug screening with novel targeted agents were performed and compared with the corresponding CDX in vivo study. Results: Cells were obtained from 3 CDX models representing the range of sensitivities to chemotherapy. Optimization of culture conditions revealed that HITES media supplemented with FBS and ROCK inhibitor Y-27632 supports CDX ex vivo growth. Expression of SCLC markers and response to therapies were maintained over time and RNAseq analysis demonstrated 80% similarity between CDX tumours and derived CDX cultures. A pilot experiment assessing efficacy of the experimental MTH1 inhibitor TH1579 demonstrated that the in vitro activity of TH1579 in CDX3 cultures predicted its preclinical efficacy in CDX3 in vivo. Similar fidelity in responses in CDX models in vivo and ex vivo were observed for DNA damage repair inhibitors, cell cycle checkpoint abrogators, BH3 mimetics and PI3K inhibitors. Conclusion: This CDX ex vivo platform now presents the opportunity to test multiple novel therapies in reliable and consistent, patient relevant preclinical models, facilitating the identification of novel therapeutic strategies for clinical testing in a disease where improved patient outcomes are urgently required. No conflict of interest. 553 Flubendazole as potential anti-neuroblastoma therapy option 2 , Y. Voges2 , ¨ M. Michaelis1 , B. Agha2 , R. Florian2 , N. Loschmann F. Westermann3 , M. Wass1 , J. Cinatl Jr2 . 1 University of Kent, School of ¨ Institut fur Biosciences, Canterbury, United Kingdom, 2 Goethe-Universitat, ¨ Medizinische Virologie, Frankfurt am Main, Germany, 3 German Cancer Research Center, Division Tumor Genetics, Heidelberg, Germany

Background: Flubendazole was shown to exert anti-leukaemia and antimyeloma activity through inhibition of microtubule function. Material and Method: Flubendazole was tested for anti-cancer in cancer cell lines and in the chick chorioallantoic membrane assay. Protein levels were determined by Western blot and flow cytometry. RNAi-mediated depletion was used to inhibit gene expression. Results and Discussion: Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. Conclusion: Flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells. No conflict of interest.

555 The Resistant Cancer Cell Line (RCCL) collection M. Michaelis1 , M. Wass1 , J. Cinatl Jr2 . 1 University of Kent, School of ¨ Institut fur Biosciences, Canterbury, United Kingdom, 2 Goethe-Universitat, ¨ Medizinische Virologie, Frankfurt am Main, Germany Introduction: The heterogeneity and individuality of cancer diseases is tremendously high. Recent genomic investigations revealed a tremendous genetic complexity in the cells from solid cancer diseases. Cancer cell (sub)populations may differ substantially between primary tumours and metastases as well as within primary tumours. This heterogeneity is a consequence of cancer clonal evolution processes. Among other models, comprehensive cancer cell line collections will be required to address this wide complexity. Resistance acquisition to anti-cancer therapies represents a major obstacle to the development of effective anti-cancer therapies. Major cancer cell drug resistance mechanisms have been discovered in drugadapted cancer cell lines including the ABC transporters ABCB1 (also known as P-glycoprotein or MDR1) and ABCC1 (also known as MRP1) and clinically relevant resistance mechanisms to so-called “targeted therapeutics” (e.g. EGFR tyrosine kinase inhibitors, oncogenic BRAF inhibitors, anti-androgens). Material and Method: Initially chemosensitive cancer cell lines are adapted to growth in the presence of clinical concentrations of anti-cancer drugs. Results and Discussion: The Resistant Cancer Cell Line (RCCL) collection consists of >1000 cell lines from 15 different cancer entities with acquired resistance to a broad range of cytotoxic and targeted anti-cancer drugs (www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html). Conclusion: The RCCL collection is a readily available tool for the studying of drug-induced cancer cell resistance mechanisms, the investigation of anti-cancer agents, and the examination of drug-induced clonal evolution processes. No conflict of interest. 556 Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status 2 , M. Sharifi3 , T. Ghafourian4 , ¨ M. Michaelis1 , F. Rothweiler2 , N. Loschmann J. Cinatl Jr2 . 1 University of Kent, School of Biosciences, Canterbury, United ¨ Institut fur Kingdom, 2 Goethe-Universitat, ¨ Medizinische Virologie, Frankfurt am Main, Germany, 3 Universities of Kent and Greenwich, Medway School of 4 Pharmacy, Chatham, United Kingdom, University of Sussex, School of Life Sciences, Brighton, United Kingdom Introduction: The PKCb inhibitor enzastaurin is under investigation as anti-cancer drug. Here, we tested its efficacy against neuroblastoma and rhabdomyosarcoma cells.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Material and Method: The efficacy of enzastaurin was tested in a panel of parental neuroblastoma and rhabdomyosarcoma cell lines, their vincristineresistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2transduced, and p53-depleted cells. The interaction of enzastaurin and ABCB1 was investigated by computational docking experiments. Results and Discussion: Enzastaurin IC50s ranged from 3.3 to 9.5mM in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125mM interfered with ABCB1-mediated drug transport. PKCa and PKCb may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations 1.25mM. The investigated cell lines did not express PKCb. PKCa depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Docking experiments detected a direct interaction of enzastaurin and ABCB1. Conclusion: Our data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. These findings are important for the further development of therapies combining enzastaurin with ABC transporter substrates. No conflict of interest. 557 Systems level analysis of T-DM1 resistance in in vitro and in vivo models of HER-2 overexpressing breast cancer O. Sahin1 , O. Saatci1 , S. Durmus1 , E. Eyupoglu1 , S. Wiemann2 . 1 Bilkent University, Department of Molecular Biology and Genetics, Ankara, Turkey, 2 German Cancer Research Center DKFZ, Division of Molecular Genome Analysis, Heidelberg, Germany Introduction: Most of the human epidermal growth factor receptor 2 (HER2)positive breast cancer patients respond well to trastuzumab, a monoclonal antibody against HER2, but only 10% has complete remission, and the rest develops resistance. T-DM1, an antibody–drug conjugate (ADC) of trastuzumab and an anti-mitotic agent, DM1 is the second line therapy being used in trastuzumab refractory patients. However, improvement in survival is limited to 5−6 months after which patients experience disease recurrence. Therefore, it is crucial to identify novel targets overcoming T-DM1 resistance. Here, we aimed to integrate genomics and proteomics approaches to build T-DM1 resistance network and to overcome resistance by targeting the identified candidates. Material and Methods: We have developed in vitro models of T-DM1 resistance by continuously treating two HER2-positive breast cancer cell lines, BT474 and SK-BR-3 with escalating doses of T-DM1 over 8 months. We have characterized the resistance by viability assays and performed RNA sequencing. We obtained the enriched pathways among genes differentially expressed between wild-type (WT) and resistant clones by Metacore Pathway Analysis, and generated a network of resistance in CytoScape. Finally, we have performed an siRNA screen to identify essential mediators of resistance whose knockdown increases T-DM1 response and did RPPAs to examine the effects of perturbations in the resistance network. Results: In line with the mechanism of action of T-DM1, cell cycle progression was found to be the top enriched process among genes differentially expressed between WT and resistant cells. In silico analysis with the differentially expressed genes revealed several gene clusters that regulate or are regulated by similar protein networks in TCGA dataset. Through extensive literature mining and analysis of protein-protein interactions, a network of resistance was generated comprising these gene clusters. A small scale siRNA screen targeting proteins in the resistance network not only identified genes in cell cycle progression as mediators of resistance in both models, but also identified a few novel genes as sensitizers upon being knocked down which will further be mechanistically analyzed. Conclusions: Our study revealed several proteins in cell cycle progression as important candidates mediating T-DM1 resistance in HER2-positive breast cancer. In vivo studies with pharmacological inhibition of the identified candidates are on-going. Ultimately, this study will provide valuable information for the treatment of T-DM1 refractory patients for which there is currently no available therapy. Acknowledgment: This study is supported by TUBITAK-BMBF with project number: 214Z130 No conflict of interest. 558 Development of tumour spheroid models for cancer research and drug discovery R. Polanski1 , M. Vazquez-Chantada1 , S. Peel1 , P. Hopcroft1 , F. Ghari1 , C. Roberts1 , K. Roberts1 . 1 Astrazeneca, Discovery Sciences, Cambridge, United Kingdom Background: Tissue culture in two dimensions (2D) is highly artificial but, due to simplicity and cost efficiency compared, has been broadly used in

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research. Yet, evidence suggests that 2D systems do not always sufficiently represent in vivo biology. Cultivation of tumour spheroids remains one of the simplest 3D techniques that bridges the gap between 2D and in vivo; multiple studies report their better predictibility of in vivo results than results obtained in 2D. High-throughput approaches were also attempted though, due to specific experimental design, they were biased towards analysing quiescent cells. Tumour spheroid assays could be used to comprehensively study diverse aspects of cell biology and pharmacology, and drive drug discovery programs. However, clear guidelines on spheroid production and assay development are lacking, and fundamental isses remain unclear, including: optimal number of cells seeded to form a spheroid, experiment duration, need and rationale for changing media over the course of the experiment. Therefore, we have performed extensive optimisation of a number of tumour spheroid models and have defined a series of guidelines for their use in cancer research and drug discovery. Materials and Methods: Spheroids were grown in ultra-low-adhesion 384-well plates and bright-field images were analysed with a proprietary image analysis tool. Different seeding densities were compared in long-term cultures. Western blotting and immunohistochemical biomarker analysis of proliferation, hypoxia and epithelial-to-mesenchymal transition were performed at multiple time points. Changes in media composition were measured by mass spectrometry. Results: We show that the number of cells seeded per spheroid defines the dynamic range of the spheroid growth assay. In most cell line models studied, spheroid growth reached a plateau and size largely independent on the number of cells seeded. Whilst most studies use large densities of >5000 cells/well, we found that seeding substantially fewer cells (500 mm to cm), it is suitable to use agents that are active in the nearinfrared (NIR) region (650–900 nm) of the radiation spectrum to minimize the light extinction by intrinsic chromophores in native tissue. The NIR region also corresponds to the therapeutic window. Gold nanorods (GNR) with suitable aspect ratios (length divided by width) can absorb and scatter strongly in the NIR region. From many investigations, the NPs are, however, usually not followed up and traced during the in vivo studies. The challenge is to investigate if the NPs are specifically released at the targeted sites and specifically kill the targeted cells. This study aims to develop and monitor simultaneously an imaging and drug delivery carrier system for anti-cancer drugs in vivo. This is feasible by incorporating the drugs as well as attaching a biding ligand such as a substituted tetrazine that would enable the system to bind to the enzyme or protein, a fluorophore such as BODIPY, fluorescein, rhodamine, coumarin or cyanine that would enable to localize the drug in the cells by emitting fluorescence, on the surface of GNR. Synthesis and optimization of GNR (seed and seedless methods), characterization (UV, TEM, Zeta size and potential) was done and cytotoxicity assays were performed using Hela cells and MTT assay. The synthesized GNR were optimized at a size of 50 nm and appeared in the NIR region (800 nm) while showing good stability from the zeta analysis. The preliminary cytotoxicity results are quite promising and need to

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be repeated. Overall this results give right and hope to further our project on the development of a potential versatile theranostic nanocarrier for cancer. No conflict of interest. 580 Mechanistic differentiation of PI3K/mTOR, Aurora kinase and EZH2 inhibitor classes by comparative cancer cell line profiling J.C.M. Uitdehaag1 , J.A.D.M. De Roos1 , M.B.W. Prinsen1 , J.R.F. De Vetter1 , J. Dylus1 , A.M. Van Doornmalen1 , S.J.C. Van Gerwen1 , J. De Man1 , R.C. Buijsman1 , G.J.R. Zaman1 . 1 Netherlands Translational Research Center B.V., Discovery, Oss, Netherlands Background: Profiling of drug candidates in cell line panels is an important tool to compare the selectivity and targeting of new anti-cancer agents. In addition, comparative profiling may be used to repurpose established therapeutics by identifying new mechanisms of action or cross-selectivities. Material and Methods: A collection of more than 120 anti-cancer agents, targeting all important oncogenic signaling pathways, including classic cytotoxic agents as well as many targeted kinase inhibitors and epigenetic modulators, was profiled on a panel of 44 or 66 parallel cell line proliferation assays (Oncolines™) [1,2]. The profiles of the compounds were compared by Pearson correlations of their inhibitor responses. Inhibitor sets were clustered using hierarchical trees. Response profiles were correlated to the genetic background of the cell lines by Analysis of Variance (Anova). Results: Reproducibility of the cell panel data was validated by monitoring cell growth rate and the variation in IC50s of replicate profiles over a period of three years. The Pearson correlation between replicates ranged between 0.60 and 0.99 for 16 different inhibitors. For selected reference compounds, correlations of between 0.9 and 0.5 are seen with the profiling data set of the National Cancer Institute (NCI60). Separate clusters are seen of, a.o., taxanes, platins, topo-isomerase inhibitors, and EGFR, ABL, MEK and BRAF inhibitors. The profile of the BTK inhibitor ibrutinib correlates with EGFR inhibitors. In biochemical experiments we show that this due to its cross-reactivity with EGFR. The panel profiling was used to classify inhibitors of three important drug targets. Aurora kinase inhibitors (six) fall into two separate clusters, which are related to their biochemical selectivity. Aurora A-selective inhibitors are relatively more active in cell lines with mutations in cell cycle checkpointrelated genes such as TP53 and RB1; whereas pan-Aurora inhibitors are more active in cell lines with mutations in growth factor signaling pathways, such as NRAS. Profiling of eleven PI3 kinase and mTOR inhibitors revealed four distinct clusters. PI3Kalpha and PI3Kdelta isoform selective inhibitors each target genetically distinct subgroups of cell lines. Rapamycin-analogs, such as everolimus, specifically target PTEN-mutant cell lines. Finally, profiling of five EZH2 inhibitors indicates that there are essentially two EZH2 inhibitor subclasses. These have a cellular profile that is distinct from HDAC, DOT1L or BET inhibitors. Conclusions: Comparative cancer cell line profiling is a robust and powerful tool to rapidly explore the pharmacogenomics of drug action in cancer cells and to identify new or previously unnoted activities of compounds. Reference(s) [1] Uitdehaag et al. (2014) PLOS ONE 9(3) e92146. [2] Uitdehaag et al. (2015) PLOS ONE 10(6) e0132230. Conflict of interest: Board of Directors: RCB and GJRZ are directors and shareholders of NTRC B.V. Corporate-sponsored Research: all other authors are employees of NTRC B.V. 581 Evaluation of pre-analytical procedures for the detection of BRAF V600 mutations in melanoma patients: comparison between Sanger sequencing and Competitive allele-specific TaqMan PCR (Cast-PCR) R. Barbano1 , B. Pasculli1 , M. Coco1 , A. Fontana2 , M. Copetti2 , M. Rendina1 , V.M. Valori3 , P. Graziano3 , E. Maiello3 , V.M. Fazio1 , P. Parrella1 . 1 IRCCS Casa Sollievo Della Sofferenza, Laboratorio Di Oncologia, San Giovanni Rotondo FG, Italy, 2 IRCCS Casa Sollievo Della Sofferenza, Unita’ di Biostatistica, San Giovanni Rotondo FG, Italy, 3 IRCCS Casa Sollievo Della Sofferenza, Onco-Ematologico, San Giovanni Rotondo FG, Italy Introduction: BRAF mutation analysis is now mandatory to identify patients affected by metastatic melanoma who may benefit from Tyrosine Kinase Inhibitors. We report a methodological approach with the potentiality to fulfil the requirements which may impact on the efficiency of the BRAF mutation analysis in the routine clinical setting. Materials and Methods: 66 FFPE specimens from the 54 patients were analysed. DNA was extracted by phenol chloroform and purified by GENECLEAN® II Kit. Samples were analysed by Cast-PCR using the BRAF_476_mu and BRAF_473_mu probes for the detection of V600E and V600K mutations, respectively. Sanger sequencing was performed using primer pairs designed to amplify a fragment including BRAF codon 600. Results and Discussion: k-statistic showed a significant agreement between Sanger sequencing and Cast-PCR (k = 0.85, CI: 0.710–0.990; p < 0.001). All

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the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. Cloning and sequencing of the clones was used to confirm mutations. The limit of detection of the two techniques were evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Mutations could be detected until a 1:100 mutated/not mutated ratio for CAST-PCR but only at 1:25 ratio for Sanger sequencing. Among the technical issues related to BRAF mutation detection in melanoma, melanin content is overlooked. Indeed, melanin coisolates with DNA and it is a strong inhibitor of DNA polymerase. Thus we compared the performance of Cast-PCR and Sanger analyses before and after GC-purification. Melanin content strongly affected the number of cases successfully analysed by Sanger sequencing, and interpretable electropherograms were obtained only in 4% of not purified cases. Likely due to the use of less amount of starting DNA, Cast-PCR was less affected, however, DCt values were higher in not purified samples (Mean±SD 2.55±1.61) than in purified ones (Mean±SD 3.60±2.19) (p value = 0.14), and in 1 case a BRAFV600E mutation was only detected after purification. With respect to intratumour heterogeneity Cast-PCR showed concordant results in all samples for which different regions on the same section were available. Whereas, in one case Sanger sequencing was unable to detect the mutation in 1 out 4 regions. Conclusions: We demonstrated that the use of highly sensitive techniques such as Cast-PCR is able to improve the ability to correctly genotype melanoma patients. Our results strongly suggest that diagnostic laboratories should evaluate whether their pre-analytical procedures are able to reduce the impact of melanin content on subsequent analytical methods. No conflict of interest. 582 Molecular determinants of sensitivity and resistance to FGFR inhibition in FGFR2-amplified gastric cancer I. Babina1 , R. Cutts1 , J. Ning2 , E. McKnight2 , A. Pearson1 , A. Swain2 , N. Turner1 . 1 Institute of Cancer Research, Molecular Oncology, London, United Kingdom, 2 Institute of Cancer Research, Cancer Biology, London, United Kingdom Background: Despite improvements in diagnostics and chemotherapy regiments in gastric cancer, there is still an urgent need for novel biomarkers and second-line treatment interventions. High-level FGFR2 amplification is found in ~5% gastric cancers, and responses in FGFR2-amplified gastric cancer have been observed in a phase II study of FGFR inhibitor. In this study we used patient-derived xenografts (PDX) to understand the mechanisms of sensitivity and resistance in FGFR2-amplified gastric cancer. Methods: PDX models were generated from the baseline biopsies of two Caucasian patients with junctional FGFR2-amplified tumors who had durable clinical responses to AZD4547. To investigate adaptive molecular changes to AZD4547 treatment, PDX tumors and PDX-derived spheroid cultures were profiled using various immunodetection approaches. Resistant PDX models were generated via continuous treatment with AZD4547. Results: Both PDX recapitulated the histology of the original cancers, and whole exome sequencing demonstrated 85−90% similarity in mutations between the patient biopsies and the PDX tumors. Comparable to the patients, both models were highly sensitive to AZD4547, with regression of ~60% seen after 10 days of treatment and subsequent stability on chronic dosing. FGFR inhibition resulted in prolonged MAPK signaling suppression in primary spheroids, with initial acute PI3K-mTOR pathway suppression. However chronic exposure to the drug resulted in an increase in phospho-S6 and phospho-4EBP1. Similarly, phospho-S6 expression was elevated in PDX tumours after continuous exposure to AZD4547. Adapted stable PDX tumours additionally demonstrated increased phosphorylation of alternative RTKs, increased ERBB3 and insulin receptor phosphorylation, and elevated levels of MCL1 compared to vehicle controls. This data implicated restoration of mTOR signaling as a key adaptive mechanism in the persistent PDX tumours. Chronically treated residual PDX tumours acquired resistance to AZD4547 after a median of 180 days. Resistant PDX demonstrated an increase in FGFR2 copy number and reproducible loss of 4p and 9p. Whole exome sequencing of individual resistant tumours suggested independent acquisition of resistant genotypes in xeno-patients, demonstrating convergent evolution from stable residual disease into acquisition of resistance. Conclusion: We show that re-activation of mTOR signaling limits sensitivity of FGFR2-amplified tumors to AZD4547. Subsequent acquired resistance originates stochastically from the stable adapted disease, emphasizing the importance of therapeutic strategies to deepen response and reduce persistence of tumour cells available to acquire resistance. No conflict of interest.

583 Effects of doxorubicin-loaded dextran coated magnetic nanoparticles: on gene and protein expression profile of p53, survivin and bcl-2 in doxorubicin sensitive/resistant MCF-7 cell lines S. Yalcın1 , O. Onguru2 , U. Gunduz3 . 1 Faculty of Engineering and ArchitectureAhi Evran University, Department of Food Engineering, Kırsehir, Turkey, 2 Gulhane Military Medical Academy, Deparment of Pathology, Ankara, Turkey, 3 Faculty of Art and Sciences- Middle East Technical University, Department of Biology, Ankara, Turkey Background: In the present study, Dextran coated magnetic nanoparticles (Dox-Dex-MNPs) were prepared to obtain an effective targeted delivery system for Doxorubicin on breast cancer cell line. Material and Methods: Dox-Dex-MNPs were synthesized and characterized by TEM, SEM, FTIR, VSM and TGA analyses. Drug loading efficiencies and release characteristics were investigated. On the other hand, drug-sensitive and drug-resistant MCF-7 cell lines were grown in RPMI 1640 medium supplemented with 10% FBS at 37oC and 5% CO2. Total RNA content was isolated by TRI Reagent according to the manufacturer’s instructions. cDNA was synthesized from 1 ug of total RNA and random hexamer primers. The expression levels of p53, survivin and bcl-2 genes related to cell survival and apoptosis were shown using qRT-PCR. In order to confirm the results obtained by qRT-PCR analyses, the protein levels of Survivin, p53 and bcl-2 were demonstrated by immunocytochemistry(ICC). Results: According to SEM, TEM, VSM and TGA results, the synthesized Dox-Dex-MNPs possess desired shape and size range (10−15 nm) as they can be internalized into the cell and also have superparamagnetic properties. FTIR analysis confirmed that Doxorubicin was successfully loaded on dextran coated MNPs. On the other hand, Dox-Dex-MNPs have pH-responsive release characteristics. Nanoparticles were efficiently taken up by both sensitive and Doxorubicin resistant MCF-7 cell lines (MCF/Dox) and this increases the efficacy of the drug and maintains overcoming the resistance of Doxorubicin in MCF-7/Dox cells. According to the gene expression results, survivin expression was 3 fold upregulated in MCF-7/Dox cells. However, it was 3 fold and 6 fold downregulated due to the application of free Doxorubicin and Dox-DexMNPs respectively. The anti-apoptotic Bcl-2 gene was approximately 10 fold downregulated in response to Dox-Dex-MNPs with respect to the application of free Doxorubicin on MCF-7/Dox cell lines. The immunoreactivity of p53 levels of MCF-7/Dox cells increased up to 95% with 3+ intensity after treatment with Dox-loaded Dex-MNPs. Conclusion: The results of this study demonstrated that Dox-Dex-MNPs can be a potential targeted therapeutic agent to overcome drug resistance. No conflict of interest. 584 miR-625-3p regulates oxaliplatin resistance by directly targeting MAP2K6/MKK6 in human colorectal adenocarcinoma I. Lyskjær1 , M. Heilskov Rasmussen1 , R. Rakownikow Jersie-Christensen2 , L. Schmidt Tarpgaard3 , M. Muhlig Nielsen1 , J. Skou Pedersen1 , T. Falck Ørntoft1 , C. Lindbjerg Andersen1 . 1 Aarhus University Hospital- Center for Molecular Clinical Cancer Research, Department of Molecular Medicine MOMA, Aarhus N, Denmark, 2 University of Copenhagen, The Novo Nordisk Foundation Center for Protein Research- Proteomics Program, Copenhagen, Denmark, 3 University of Southern Denmark, Clinicical Institut, Odense, Denmark Introduction: Oxaliplatin resistance in colorectal cancer (CRC) is a major medical problem, and predictive markers are urgently needed. Recently, we reported miR-625-3p as a candidate biomarker. We showed in two independent cohorts of patients with metastatic CRC that patients with a high miR-625-3p transcript level had a more than 6 fold higher risk of not responding to oxaliplatin than other patients. Now, we provide evidence that miR-625-3p is directly and functionally involved in resistance to oxaliplatin. Material and Method: In order to investigate the role of miR-625-3p in modulating oxaliplatin sensitivity in CRC cells in vitro we constructed a transposon-based doxycycline inducible vector. This was used for generation of three different stable and inducible CRC cell line models. The models were constructed in HCT116 (mitch-match repair deficient (MMR) and p53wt), SW620 and HCC2998 (both MMR proficient and p53mut). These models, as well as additional CRC cell lines, were used to dissect the mechanism of action for miR-625-3p in oxaliplatin resistance by use of reporter systems, rescue experiments, functional assays, siRNA knockdown, chemical inhibitor experiments, microarray profiling, and advanced proteomics. Results and Discussion: Our results indicate that miR-625-3p induces resistance by abrogating the stress and DNA damage-response signalingaxis MAP2K6-p38, which then in turn leads to increased cell viability by decreasing apoptosis. We show that miR-625-3p directly binds and downregulates MAP2K6 and that this primes blockage of the central kinase p38. Inhibiting the pathway both at and downstream of MAP2K6 closely mimicked the effect of the miRNA. Rescue experiments showed that the sensitivity towards oxaliplatin could be restored by expression of a miR-625-3p insensitive MAP2K6 variant.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Conclusion: Our study shows that miR-625-3p induces oxaliplatin resistance by abrogating MAP2K6-p38 regulated apoptosis and cell cycle control networks, and corroborates the predictive power of miR-625-3p. This has significant clinical potential as the expression level of miR-625-3p, possibly combined with the expression level of MAP2K6, has the potential to serve as a predictive biomarker. Since ~20% of metastatic CRC patients have high miR-625-3p expression, the number of patients potentially benefiting from the use of miR-625-3p/MAP2K6 is substantial. No conflict of interest. 585 Gli1/DNA interaction is a druggable target for Hedgehog-dependent tumors P. Infante1 , M. Mori1 , R. Alfonsi2 , C. Ingallina1 , B. Botta3 , L. Di Marcotullio2 . 1 Istituto Italiano di Tecnologia, CNLS@Sapienza, Rome, Italy, 2 Sapienza University, Molecular Medicine, Rome, Italy, 3 Sapienza University, Chimica e Tecnologie del Farmaco, Rome, Italy Background: Hedgehog (Hh) pathway is essential for tissue development and stemness, and when deregulated leads to tumorigenesis. Although many Hedgehog-driven human cancers involve upstream pathway activation (i.e. either loss-of-function of the receptor Ptch1 or gain-of-function mutations of the transmembrane transducer Smo), Smo-independent hyperactivation of the downstream Gli transcription factor is responsible for the development of several tumors and resistance to therapy. This raises the need to identify novel Gli1 inhibitors, a challenging issue mostly due to the lack of information on the structural requirements of Gli1/DNA interaction. Material and Methods: Molecular characterization of Gli1/DNA binding was performed by a mix of computational and experimental structure-based in vitro studies. Molecular dynamics simulations were carried out to identify the residues in Gli1 zinc-finger involved in DNA binding. The data obtained were then used to set up the docking-based virtual screening of a natural products library available in house, with the aim to discover pharmacological agents able to interfere with Gli1/DNA interaction. The molecules identified as potential Gli1 inhibitors were investigated for their functional activity through a Glidependent luciferase reporter screening assay. The most active was tested for its effectiveness to counteract Hh-dependent tumor growth by medulloblastoma and basal cell carcinoma allograft model from Ptch+/− mice and ortothopic medulloblastoma xenograft. Results: We identified a small molecule, Glabrescione B (GlaB), an isoflavone naturally found in the seeds of Derris glabrescens (Leguminosae), able to impair Gli1/DNA binding as revealed by Chip and EMSA assays. In agreement with these molecular results, GlaB revealed great antitumor efficacy. Indeed, we observed that GlaB strongly inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. Conclusions: Our study highlighted the relevance of structural details of Gli1/DNA interaction as a promising tool to discover small molecules able to inhibit Hh pathway by directly targeting Gli1. Here we identified GlaB as a potent and specific Gli1 inhibitor able to interfere with Gli1/DNA binding, resulting in the inhibition of Hh-dependent tumor cells and cancer stem cells growth, thus becoming a profitable pre-clinical candidate. No conflict of interest. 586 The unique binding mode of NTRC 0066-0, a novel inhibitor of the spindle assembly checkpoint kinase TTK (Mps1), leads to long target residence time and potent anti-tumor activity G. Zaman1 , J. Uitdehaag1 , J. De Man2 , N. Willemsen-Seegers1 , J.G. Sterrenburg2 , J. De Wit1 , J. De Roos1 , M. Prinsen1 , R. Buijsman2 . 1 Netherlands Translational Research Center B.V., Biology, Oss, Netherlands, 2 Netherlands Translational Research Center B.V., Chemistry, Oss, Netherlands Introduction: An abnormal number of chromosomes, or ‘aneuploidy’, is a common feature of solid human tumors and a predictor of poor prognosis in breast, lung, brain and colorectal cancer. Aneuploidy is caused by malfunctioning of the Spindle Assembly Checkpoint (SAC), a surveillance mechanism that ensures the fidelity of chromosome segregation. The protein kinase TTK (commonly referred to as Mps1) is a component of the SAC. Inhibition of TTK gene expression by RNA interference and inhibition of TTK kinase activity by small molecule kinase inhibitors causes chromosome missegregation and cancer cell death. Material and Method: A novel class of compounds was identified that potently inhibits TTK enzyme activity and cancer cell line proliferation [1]. Its binding mode and that of reference inhibitors was characterized by protein crystallography. Binding kinetics and target residence time were determined by surface plasmon resonance. Anti-proliferative activity was measured on a broad panel of cancer cell lines [2,3]. Results and Discussion: The clinical candidate, NTRC 0066-0, inhibits TTK with subnanomolar potency (IC50) in a kinase enzyme assay and is more than 200 times selective over 276 kinases examined, including mitotic and cell

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cycle dependent kinases (CDKs). X-ray structures of the TTK kinase domain in complex with NTRC 0066-0 and analogs indicate that this class of compounds induces a large conformational shift in the glycine-rich loop, invoking an inactive kinase conformation. In surface plasmon resonance experiments, NTRC 0066-0 exhibited slow dissociation kinetics, resulting in a long target residence time. Parallel surface plasmon resonance experiments confirmed the exquisite selectivity of NTRC 0066-0 for TTK over Aurora and Polo-like kinases. NTRC 0066-0 inhibited the proliferation of a wide variety of human cancer cell lines in the same potency range as marketed cytotoxic agents. The crystal structure, binding kinetics and cellular potency of NTRC 0066-0 were compared to that of other TTK inhibitors such as Mps1-IN-2, AZD-3146, MpsBAY2b, Bay 1161909 as well as analogs from the NTRC 0066-0 series. This suggests that the unique binding mode of NTRC 0066-0 results in long target residence time which contributes to its strong anti-tumor activity. In subsequent mouse xenograft models of human cancer cell lines, NTRC 0066-0 inhibited tumor growth as a single agent after oral administration at 20 mg per kg. Conclusions: NTRC 0066-0 is a novel TTK inhibitor with outstanding in vitro properties and potent anti-tumor activity in mouse xenograft models. Our data suggest that long target residence time corresponds with potent cellular activity for TTK inhibitors. Reference(s) [1] Maia et al. (2015) Annals of Oncology 26, 2180–2192. [2] Uitdehaag et al. (2014) PLOS ONE 9(3) e92146. [3] Uitdehaag et al. (2015) PLOS ONE 10(5) e0125021. Conflict of interest: Ownership: I am a shareholder of Netherlands Translational Research Center B.V. (NTRC). Board of Directors: I am a managing director of Netherlands Translational Research Center B.V. (NTRC). Other Substantive Relationships: I am a co-founder of Netherlands Translational Research Center B.V. (NTRC). 587 Thermally triggered theranostics for pancreatic cancer M. Malekigorji1 , P. Kong Thoo Lin2 , M. Lees3 , M. Gueorguieva4 , A. Curtis5 , C. Hoskins5 . 1 Keele University, Institute of Science and Technology, Keele, United Kingdom, 2 Robert Gordon University, School of Pharmacy and Life Sciences, Aberdeen, United Kingdom, 3 University of Warwick, Physics Department, Warwick, United Kingdom, 4 University of Dundee, Institute for Medical Sciences and Technology, Dundee, United Kingdom, 5 Keele University, School of Pharmacy, Keele, United Kingdom Background: Pancreatic cancer is the 4th most aggressive cancer in the western world with less than 34% of patients surviving past 5 years. Lack of specific symptoms results in delayed diagnosis. Theranostics are new platforms, which offer simultaneous diagnosis and therapy resulting in a decrease in treatment time. Here treatments are conjugated onto diagnostics by stimuli responsive binding allowing for controlled drug release resulting in a rapid and localised clinical effect. Hybrid nanoparticles are composed of an iron oxide core surrounded by a rigid gold shell. These particles undergo manipulation due to inherent magnetism of the core whilst laser irradiation of their gold shell results in localised heating due to surface plasmon resonance. Hence, they can be utlilised as diagnostics using MRI and laser irradiation can be used as a trigger for drug release. Methods: Proof of concept studies have been carried out using a novel bisnaphthalamido (BNIP) based drug series. BNIPs are a series of novel compounds, which have exhibited exciting potential as chemotherapy agents. HNPs were fabricated and characterised using PCS, TEM, MRI, SQUID and zeta potential measurement. Drug conjugation and release was quantified using reverse phase HPLC. Cellular response and cytotoxicity assays were carried out using trypan blue exclusion, MTT assay and atomic force microscopy. Results and Discussion: In our studies, we designed hybrid nanoparticles (50 nm) capable of drug loading onto their surface (3:1:0.25, Drug:Fe:Au). By exploiting the gold surface-to-drug interaction of a range of novel Bisnaphtalamido based agents a system with heat triggered drug release was produced. In vitro studies of these formulations showed the novel formulations possess a 10-fold lower IC50 value when compared with the free drug after only 24 h. These cytotoxicity studies combined with cellular uptake studies showed the formulations to be significantly more effective compared with gemcitabine. In vivo trials have commenced to further elucidate their viability for use as theranostics. Conclusion: These data highlight the potential of HNPs as dual imaging agents and contrast agents for pancreatic cancer therapy. No conflict of interest.

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Experimental/Molecular Therapeutics, Pharmacogenesis II 589 Stratification of breast cancer patients using Tip60 staining paired with evaluation of a designed Tip60 specific histone acetyltransferase inhibitor for targeted treatment A. Shalaby1 , H. Emma2 , E. Bourke1 , C. Gao3 , S. Martin4 , K. Michael5 , L. Eriksson6 , H. Thomas4 , J. Brown5 . 1 Lambe Institute for Translational Research, Discipline of Pathology- School of Medicine, Galway, Ireland, 2 National University of Ireland Galway, School of Mathematics- Statistics and Applied Mathematics, Galway, Ireland, 3 University of Gothenburg-, ¨ Department of Chemistry and Molecular Biology, Goteborg, Sweden, 4 Karolinska Institute, Division of Translational Medicine and Chemical Biology- Department of Medicine-, Stockholm, Sweden, 5 Lambe Institute for Translational Research, Discipline of Surgery- School of Medicine, Galway, Ireland, 6 University of Gothenburg, Department of Chemistry and Molecular ¨ Biology, Goteborg, Sweden Introduction: Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism. Dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently few HAT inhibitors have been described or robustly characterised. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional coactivator. Additionally, Tip60 haploinsufficiency was reported in breast cancer. This suggests a Tip60 targeted treatment could significantly impact breast cancer treatment. Materials and Methods: Using in silico modeling we rationally designed and synthesized a HAT inhibitor specifically targeting Tip60 (TH1834). We evaluated the specificity and efficacy of TH1834 in vitro using MCF7 (Luminal A breast cancer line) and MCF10A (non-tumourigenic breast cell line). The molecular effects of TH1834 were evaluated using: an in vitro HAT assay, apoptosis/cytotoxicity assays and imaging of the DNA damage response. Additionally, a breast cancer tissue microarray (>400 samples) was stained for Tip60 and scored. Statistical analysis and modeling was performed, evaluating the Tip60 staining pattern (cytoplasmic, nuclear or both) and clinicopathological details (subtype, stage, survival, etc). Results and Discussion: Modeling of Tip60 identified opposing charges at each end of the active binding pocket, with the positively charged end attributed to specific side chains. We used structure-based design to develop an inhibitor (TH1834) to specifically fit this pocket. We demonstrated TH1834 significantly inhibits Tip60 activity in an in vitro HAT assay. Treating cells with TH1834 results increased unrepaired DNA damage (following ionizing radiation treatment) and apoptosis in breast cancer, but not control cells. Furthermore, TH1834 did not affect the activity of related HAT MOF, indicated by H4K16Ac, demonstrating specificity (Gao et al., 2014). Ongoing work is investigating the synergistic effects of combining TH1834 and current chemotherapeutics. Using tissue microarrays, we identified breast cancer subtypes with dysregulated Tip60 levels and are analysing the prognostic and diagnostic utility of Tip60 staining. Conclusion: Identifying breast cancer subtypes with atypical Tip60 levels pinpoints a subtype specific therapeutic target for our inhibitor. TH1834 treatment specifically led to cancer but not normal cell death. The validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60. Our small molecule inhibitor, paired with our stratification of breast cancer patients (by Tip60 expression) provides an exciting potential new and improved treatment for breast cancer. No conflict of interest. 590 Trastuzumab-based photoimmunotherapy integrated with viral HER2 transduction inhibits HER2-negative gastric cancer S. Kagawa1 , M. Ishida1 , T. Fujiwara1 . 1 Okayama University Graduate School of of Medicine- Dentistry and Pharmaceutical Sciences, Department of Gastroenterological Surgery, Okayama, Japan Background: Peritoneal dissemination is the most frequent metastasis in gastric cancer and is associated with poor prognosis. The lack of particular target antigens in gastric cancer other than human epidermal growth factor receptor 2 (HER2) has hampered the development of treatments for peritoneal dissemination of gastric cancer. We hypothesized that HER2-extracellular domain (HER2-ECD) gene transduction combined with trastuzumab-based photoimmunotherapy (PIT) might provide excellent and selective anti-tumor effects for peritoneal dissemination of gastric cancer.

Material and Methods: Anti-HER2 PIT integrated with adenoviral HER2-ECD gene transfer was applied in mice bearing peritoneal dissemination of HER2negative gastric cancer (MKN1 and MKN45) in vitro and in vivo. Results: In vitro, adenovirus/HER2-ECD (Ad/HER2-ECD) efficiently transduced HER2-ECD into HER2-negative gastric cancer cells. TrastuzumabIR700 (Tra-IR700)-mediated PIT induced selective cell death of HER2-ECDtransduced tumor cells. Ad/HER2-ECD also induces homogenous expression of HER2 in heterogeneous gastric cancer cells, resulting in uniform sensitivity of the cells to Tra-IR700-mediated PIT. In vivo, intraperitoneal administration of Ad/HER2-ECD and Tra-IR700 with PIT inhibited peritoneal metastasis and prolonged the survival of mice bearing MKN45 peritoneal dissemination. Furthermore, minimal side effects allowed the integrated therapy to be used repeatedly, providing better control of peritoneal dissemination. Conclusions: The novel therapy of molecular-targeted PIT integrated with gene transfer technology is a promising approach for the treatment of peritoneal dissemination in gastric cancer. No conflict of interest. 591 Lichen compound protolichesterinic acid inhibits DNA synthesis and DNA repair in cultured cancer cells H. Ogmundsdottir1 , H. Hauksdottir2 , J. Thorsteinsdottir1 . 1 University of Iceland, Faculty of Medicine, Reykjavik, Iceland, 2 University of Iceland, Faculty of Pharmaceutical Sciences, Reykjavik, Iceland Background: Protolicheterinic acid (PA), an aliphatic a-methylene g-lactone isolated from the lichen Iceland moss (Cetraria islandica) has been shown previously to inhibit the proliferation of a variety human cancer cells. An inhibitory effect on 5- and 12-lipoxygenase was also established, but recently we demonstrated that the anti-proliferative effect is not mediated by lipoxygenase inhibition. PA also affects fatty acid synthase in association with overexpression of HER2 in breast cancer cells. Effects on ERK2/2 and AKT signalling were secondary and not likely to be involved in mediating the antiproliferative effects. Earlier studies had indicated an inhibitory effect of PA on HIV reverse transcriptase and DNA polymerase. Material and Methods: In this study we explored the effects of PA on DNA replication and repair in the human pancreatic cancer cell line AsPC-1. DNA synthesis was assessed by flow cytometric analysis of BrDU incorporation. Double-strand DNA breaks were induced by ionizing irradiation and detected by immunofluorescence staining for gH2AX foci, examined by confocal microscopy and quantified using ImageJ. Furthermore, ultrastructural changes were evaluated by electron microscopy (EM). Results: Flow-cytometric analysis of BrDU incorporation indicated significant inhibition by PA of DNA synthesis in S-phase. Following exposure to PA a significantly greater number of gH2AX foci remained unrepaired after 24 hours, compared with controls. EM examination revealed mitochondrial changes similar to those described for agents that affect mitochondrial DNA polymerase. Conclusions: Taken together, the results indicate that PA may inhibit proliferation and DNA repair through DNA polymerase inhibition but this remains to be tested directly. No conflict of interest. 592 A 3D image-based phenotypic screen of bi-specific antibodies targeting stem cells in a panel of patient derived colon carcinoma organoids B. Herpers1 , R. Roovers2 , B. Ebbink2 , M. Van de Wetering3 , K. Yan1 , L. Salinaro1 , H. Clevers3 , W. De Lau3 , R. Vries3 , M. Throsby2 , L. Price1 . 1 OcellO BV, OcellO BV, Leiden, Netherlands, 2 Merus BV, Merus BV, Utrecht, Netherlands, 3 Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, Netherlands Background: Organoid cultures grown in 3D increase the likelihood for accurately predicting drug responses in patients, discriminating different drug responses and flagging toxicity. We used a high content screening platform and a panel of 40 patient derived colorectal cancer (CRC) organoids to characterize the responses to a large panel of (545) bispecific antibodies. These antibodies comprised a HER3 or EGFR targeting arm combined with a LGR4, LGR5, ZNRF3 or RNF43 targeting arm to target CRC stem cells. Results: The broad mutational spectrum of the organoids was reflected in a broad heterogeneity of organoid phenotypes. Some CRC organoids formed well-differentiated spheroids with a single lumen that resembled the phenotype of normal wild type organoids, whereas others had multiple lumens or were poorly differentiated without a luminal cavity. A rich set of morphological features was extracted from 3D image data, including organoid size and shape, planar cell polarity, lumen formation as well as cell number and nucleus shape. Some features, such as those that described lumen formation, were more sensitive in detecting drug treatment than features associated with cell proliferation, improving the sensitivity of the assay to detect active molecules. A set of 10 features was selected to create a drug response profile. The bispecific antibody screen was performed in two stages: a primary screen

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 in three different tumoroid models (18,908 wells) and a validation screen in 25 different tumoroid models (23,040 wells). These screens simultaneously measured morphological alterations associated with growth, differentiation and survival (e.g. apoptosis) and identified a panel of bispecific antibodies that potently inhibited the growth of a significant majority of colorectal cancer tumoroid models tested. Conclusions: These results demonstrate that high content screening of CRC organoids is an effective strategy to identify novel inhibitors of CRC tumor outgrowth and enable identification of bispecific antibodies that target colorectal cancer stem cells with different mutational backgrounds. Conflict of interest: Ownership: Leo Price is a founder and co-owner of OcellO BV, a company that performs fee-for-service contract research for the drug discovery industry. 593 Targeting HER3 by interfering with its Sec61-mediated cotranslational insertion into the endoplasmic reticulum A. Ruiz-Saenz1 , M. Sandhu1 , Y. Carrasco2 , R. Maglathlin2 , J. Taunton2 , M. Moasser1 . 1 University of California San Francisco, Department of Medicine, San Francisco, USA, 2 University of California San Francisco, Department of Cellular and Molecular Pharmacology, San Francisco, USA Introduction: There is increasing evidence implicating HER3 in several types of cancer, as a resilient copartner for HER2 in HER2-amplified cancers, and as a driver of resistance in many other types of cancer. However, the development of targeted therapies to inactivate HER3 function has been a challenging endeavor. Its tumor-driving functions are only sometimes driven by ligandactivation, and targeting its extracellular domain with antibody molecules has only limited effects. Its kinase domain functions in allostery, not catalysis, and we have previously shown that the classical ATP-analog class of tyrosine kinase inhibitors fail to inactivate it. Here we describe a novel approach to target HER3 using a small molecule, CT8, that blocks its Sec61-mediated cotranslational insertion into the endoplasmic reticulum. Materials and Methods: In this study, we have shown that treatment with the cotransin CT8 eliminates HER3 expression in different HER2-amplified cell lines. Using site-directed mutagenesis, a doxycycline-inducible luciferase system, and a CT8-insensitive Sec61-expressing cell line, we have found that CT8 prevents HER3 translocation into the endoplasmic reticulum in a signal sequence and Sec61 dependent manner. We have also tested the effect of the combination CT8-lapatinib in HER2-HER3 signaling and apoptosis in HER2overexpressing cancer cells. Results and Discussion: Here we describe a novel approach to target HER3 that eliminates its expression. This involves interfering with mechanisms critical for its cotranslational translocation into the endoplasmic reticulum (ER), resulting in the elimination of HER3 protein expression and loss of HER3 function. The small-molecule cotransin CT8 binds the Sec61 translocon channel in the ER membrane and prevents the signal peptide of the nascent HER3 protein from initiating its cotranslational translocation into the lumen of the ER, resulting in the degradation of HER3. Interestingly, CT8 treatment prevents HER3 translocation while the other HER proteins are unaffected. In addition, CT8 treatment suppresses the compensatory induction of HER3 that accompanies lapatinib treatment of HER2-amplified cancer cells, making for a durable suppression of HER2-HER3 signaling with the lapatinib-CT8 combination. Consistent with the effective inactivation of HER2HER3 signaling, CT8 treatment synergistically enhances the apoptotic effects of lapatinib in HER2-overexpressing cancer cells. The target selectivities of cotransins are highly dependent on their structure and the signal sequence of targeted proteins and can be narrowed through structure-function studies. Conclusion: Our results highlight Sec61-dependent processing as a novel strategy to eliminate HER3 expression and function with considerable potential for the treatment of HER2-amplified and other types of cancers. No conflict of interest. 594 Insulin/insulin-like growth factor-1 receptors mediate acquired resistance to anti-EGFR therapy in human cholangiocarcinoma cells by regulating an epithelial to mesenchymal transition/cancer stem cell axis 1 , M. Mergey1 , C. Desbois-Mouthon1 , ´ J. Vaquero1 , C. Lobe1 , A. Claperon ´ F. Praz1 , L. Fouassier1 . 1 INSERM- UMR_S 938, Sorbonne UniversitesUPMC Univ Paris 06- Centre de Recherche Saint-Antoine F-75012, Paris, France Background: Cholangiocarcinoma (CCA) is a tumor that arises from biliary epithelial cells. CCA has a very poor prognosis due to its late clinical presentation and the lack of effective non-surgical therapies. Epidermal Growth Factor Receptor (EGFR), a tyrosine kinase receptor, is overexpressed in CCA tumors and plays a major role in CCA progression. Thus, EGFR has been envisaged as a molecular target for CCA therapy. However, clinical trials using the tyrosine kinase inhibitor (TKI) erlotinib, did not provide a therapeutic benefit in patients with CCA, suggesting the emergence of resistance mechanisms. In

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the present study, we intend to unravel the underlying cellular and molecular mechanisms involved in acquired resistance to erlotinib in CCA. Material and Methods: Cell pools resistant to erlotinib were obtained by treating four human CCA cell lines (HuCCT1, EGI-1, SK-ChA-1 and MzChA-1) with increasing concentrations of erlotinib (0−20 mM) for at least 6 months. Cell viability was determined by MTT assay after exposure to erlotinib for 72 h. Signaling pathways were analyzed by phosphoprotein arrays, immunoprecipitation and WB. Two dual TKI, BMS-536924 and linsitinib, were used to specifically inhibit the insulin/insulin-like growth factor-1 receptors (IR/IGF1R). Expression of epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) markers was investigated by RT-qPCR, WB and immunofluorescence. Adhesion and migratory ability of cells were evaluated by xCELLigence and videomycroscopy, respectively. Cell clonogenicity was evaluated by colony formation assay. Results and Discussion: The four erlotinib resistant CCA cells showed reduced sensitivity to erlotinib toxicity compared to their parental counterparts. All EGFR family members were inhibited, whereas IR and IGF1R were activated and their ligand, IGF2, was overexpressed in resistant cells compared to parental cells. Phenotypically resistant cells displayed a scattered phenotype, which was accompanied by a disruption of the adherens junctions as attested by the decreased expression of E-cadherin and/or b-catenin at the plasma membrane. In resistant cells, the expression of EMT-inducing transcription factors (EMT-TFs), as well as mesenchymal markers, was induced, along with an increase of adhesion and migratory properties of the cells. Additionally, the expression of CSC markers was also upregulated in erlotinib-resistant cells. Consistenly, resistant cells showed increase clonogenicity than parental cells in presence of erlotinib. Treatment with BMS536924 and linsitinib restored the sensitivity to erlotinib, reduced the expression of EMT-TFs, and reduced the clonogenicity of resistant cells. Conclusions: The activation of the IGF signaling axis could contribute to the resistance to erlotinib through the regulation of an EMT program and stemness in CCA cells. No conflict of interest. 595 Trefoil Factor 3 (TFF3) is HER2-regulated and substitutes for HER2 signalling in acquired trastuzumab resistance in HER2+/ER+ breast cancer Q.Y. Chong1 , V.K. Pandey1 , A. Banerjee1 , Y.J. Chen1 , M.L. You1 , P.E. Lobie1 . 1 National University of Singapore, Cancer Science Institute of Singapore, Singapore, Singapore Background: The bidirectional crosstalk between HER2 and estrogen receptor (ER) contributes to resistance towards HER2-targeted therapy in breast cancers. In this project, the potential role of TFF3, as an estrogen responsive oncogene, in mediating trastuzumab resistance in HER2+/ER+ breast cancer is investigated. Materials and Methods: The modulation of TFF3 by HER2 activity was studied using luciferase reporter assay, real-time PCR and western blot in BT474, MDA-MB-361, and SKBR3 cells. BT474 cells with forced or depleted expression or inhibition of TFF3, and cells with acquired resistance to trastuzumab were used in trastuzumab sensitivity and resistance studies respectively. Cell functional and cancer stem cell assays performed include monolayer cell viability, 3D matrigel growth, soft agar colony formation, mammosphere formation and Aldefluor assay. Results: TFF3 promoter activity, mRNA and protein levels were observed to be downregulated by activation of HER2, and conversely, upregulated by trastuzumab, at least partially in an ERa-independent manner. Forced expression of TFF3 activated the HER family of receptor tyrosine kinases (HER1 to HER4). As trastuzumab-induced upregulation of TFF3 acts on the HER receptors signalling, the same pathway that is targeted by trastuzumab, only a minor effect of TFF3 in modulating trastuzumab sensitivity was observed. Nevertheless, TFF3 was significantly upregulated in trastuzumab resistant cells as compared to control cells. Knockdown or inhibition of TFF3 in the trastuzumab resistant cells significantly decreased cell viability, 3D matrigel and anchorage-independent growth, without re-sensitizing the cells to trastuzumab. In contrast, combined trastuzumab and TFF3 inhibition, exhibited a synergistic effect in reducing the aldehyde dehydrogenase (ALDH) positive population in trastuzumab resistant cells. The absence of an additional inhibitory effect of trastuzumab on cell viability and growth can be attributed to decreased HER receptors signaling in the trastuzumab resistant cells. Hence, a change in oncogene addiction from dependence on HER2 to TFF3 is proposed, in which upregulated TFF3 act through alternative pathways to contribute to acquired trastuzumab resistance. Conclusion: TFF3, independent of ERa, is a potential biomarker and therapeutic target in acquired trastuzumab resistance in HER2+/ER+ breast cancer. Conflict of interest: Other Substantive Relationships: P.E.L. has previously consulted for Perseis Therapeutics Limited. P.E.L. is named on PCT application numbers WO 2006/69253 and WO 2008/042435 and US provisional application number 61/059558 and derivatives thereof.

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596 Docosahexaenoic acid induces cell death through downregulation of hedgehog signaling via the activation of SIRT6 in human non-small cell lung carcinomas harboring EGFR mutations S. Jeong1,2 , K. Jing1,2,3 , S. Shin1,2 , Y.J. Jeon1 , J.Y. Heo1,2 , G.R. Kweon1,2 , S.K. Park1 , J.I. Park1 , K. Lim1,2,4 . 1 Chungnam National University, Biochemistry, Daejeon, Korea, 2 Chungnam National University, Infection Signaling Network Research Center, Daejeon, Korea, 3 Affiliated Hospital of Guangdong Medical College, Stem Cell Research and Cellular Therapy Center, Zhanjiang524001, China, 4 Chungnam National University, Cancer Research Institute, Daejeon, Korea Background: Docosahexaenoic acid (DHA) induces apoptotic cell death through several mechanisms in cancer cells. Here we report that DHA-induced apoptotic cell death is related to the inhibition of Hedgehog (Hh) signaling via the activation of SIRT6 in human PC9 and H1975 non-small cell lung cancer (NSCLC) cells. Material and Method: PC9 and H1975 cells were treated with w3-PUFAs, and cell viability and apoptotic parameters were examined. To investigate the effect of w3-PUFAs endogenously, PC9 stable cell lines of fat-1 (w3-desaturase) gene, which express w3-desaturase and thus contain a higher amount of endogenous w3-PUFAs, were estabished by stably transfection and selection. The cell growth of the fat-1 stable PC9 (f-PC9) was compared with control vector expressing cells (c-PC9) both in vitro and in vivo. Results: DHA induced the increase in the level of Bax, in the cleavage of PARP as well as the number of TUNEL-positive cells and activation of caspase-3, indicating cell death. DHA significantly increased the SIRT6 levels. Knockdown of SIRT6 by siRNAs inhibited apoptosis induced by DHA, while SIRT6 overexpression increased apoptotic cell death, indicating that DHAinduced SIRT6 activation promotes apoptosis. In addition, SIRT6 activation by DHA treatment was associated with downregulation of Hh signaling and knockdown of SIRT6 resulted in augmentation of Hh signaling, suggesting elevation of SIRT6 levels in DHA-treated NSCLC cells leads to downregulation of the Hh signaling. Smo activator SAG increased the protein levels of Hh signaling molecules by DHA, and SAG plus DHA treatment decreased cleaved PARP levels, implying that DHA-inhibited Hh signaling is related to apoptotic cell death. To unveil the effects of endogenous DHA on apoptosis via SIRT6mediated Hh signaling, we used PC9 stable cell lines of fat-1 gene. The SIRT6 levels were significantly increased and Hh signaling molecules were diminished in fat-1 stable PC9 (f-PC9) cells, compared with the cells transfected with the control vector (c-PC9). Moreover, SIRT6 expression was elevated; on the other hand, Gli and smo levels were inhibited in tumor tissues from f-PC9 cellsinjected mice, demonstrating that DHA also regulates SIRT6 and Hh signaling in vivo. Conclusion: These results suggest that DHA may induce apoptotic cell death through the suppression of SIRT6-modulated Hh signaling in human NSCLC cells. These findings implicate that w3-PUFAs may represent a therapeutic potential for the chemoprevention and treatment of human NSCLC. Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008). No conflict of interest. 597 Docosahexaenoic acid induces ROS-mediated apoptosis through the Nrf2–Klf9–Txnrd2 pathway in HeLa cells K. Jing1,2,3 , S. Shin1,3 , S. Jeong1,3 , S. Kim1,3 , Y.J. Jeon1 , J.Y. Heo1,3 , G.R. Kweon1,3 , S.K. Park1 , J.I. Park1 , K. Lim1,3,4 . 1 Chungnam National University, Biochemistry School of Medicine, Daejeon, Korea, 2 Affiliated Hospital of Guangdong Medical College, Stem Cell Research and Cellular Therapy Center, Zhanjiang524001, China, 3 Chungnam National University, Infection Signaling Network Research Center, Daejeon, Korea, 4 Chungnam National University, Cancer Research Institute, Daejeon, Korea Background: We have previously demonstrated that the w3-polyunsaturated fatty acid (w3-PUFA), docosahexaenoic acid (DHA) triggers apoptosis by increasing reactive oxygen species (ROS) accumulation in cervical cancer HeLa cells; however, the underlying mechanism remains poorly understood. Here, we report that the ROS-mediated apoptosis caused by DHA is associated with Nrf2 signaling activation as judged by increases in Nrf2 protein levels, nuclear translocation and tansactivation of its target genes, heme oxygenase-1 and Kruppel-like factor 9 (Klf9). Material and Method: The HeLa cervical cancer cells were treated with DHA, and DHA-induced apoptosis was analyzed using the Annexin V staining, TUNEL assay, caspase activity assay, and Western blot. Dihydroethidium and MitoSOX were used for reactive oxygen species (ROS) measurement. Results: The activation of Nrf2 signal seems to result from decreased Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1), because DHA remarkably attenuated Keap1 expression levels. Moreover, silencing Nrf2 by small interfering RNAs inhibited the cytotoxic effect of DHA, indicating that Nrf2

activation plays a positive role in the process of DHA-induced apoptosis. We also found that DHA time- and dose-dependently suppressed the expression of thioredoxin reductase 2 (Txnrd2) which is known to be negatively regulated by Klf9 and acts to eliminate cellular ROS. Conclusion: These results suggest that Nrf2–Klf9–Txnrd2 pathway contributes to DHA-induced ROS accumulation and apoptosis in HeLa cells. Thus, utilization of w3-PUFA may represent a promising therapeutic approach for chemoprevention and treatment of human cervical cancer. Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008). No conflict of interest. 598 Rational design of combination therapies and blockage of acquired targeted drug resistance H. Reuveni1 , L. Kupershmidt2 , S. Carmi2 , N. Moskovitz2 , M. Rozen2 , M. Shlapoberski2 , E. Golomb3 , M. Lotem4 , S.M. Stemmer5 , I. Haviv2 . 1 TyrNovo Ltd., CEO, Herzliya, Israel, 2 Bar Ilan University, Medical school, Safed, Israel, 3 Shaare Zedek Medical Center, Institute of Pathology, Jerusalem, Israel, 4 Hadassah Medical Center, Oncology, Jerusalem, Israel, 5 Rabin Medical Center, Institute of Oncology, Petach Tiqwa, Israel Background: Recent successes of targeted therapies on advanced stage cancer patients highlight the value of mutation-based biomarkers for drug response. However, the impact of these drugs is often temporary and the tumor progresses due to acquired resistance. A common mechanism of drug evasion involves feedback mechanisms that increase expression or phosphorylationdriven activation of an alternative oncogenic pathway. The objective of this work was to set up a streamlined methodology (kinome profiling, shRNA negative RNAi screens, evolution tracking etc.) for rational drug combination designs. Material and Methods: The research includes pre-clinical assessment of novel drugs focused on human tumors refractory to biomarker predicted targeting and seeking combination therapies that would block the spontaneous drug evasion. We explored the efficacy of combination approach on different malignancies derived xenografts in mice including colon, pancreatic, head&neck cancers, melanoma and others. Each tumor was subject to target somatic mutation screening, which resulted in a targeted drug recommendation, and mice were treated accordingly. As a blocker of the evasion mechanism and epithelial to mesenchymal transition, we characterized the utility of a novel molecule NT219, which uniquely eliminates Insulin Receptor Substrates 1/2 (IRS1/2) and shuts down both IRS1/2-to-AKT and STAT3 feedback pathways. Results: Personalized treatments using targeted drugs for central oncogenic pathways, such as the EGFR inhibitor Erlotinib or the EGFR antibody Cetuximab, the mTOR inhibitor Everolimus, the mutated BRAF inhibitor Vemurafenib and the MEK inhibitor Trametinib induced temporary regression of the patient-derived tumors followed by acquired resistance and tumor progression. Combined therapy with NT219, the dual blocker of STAT3 & IRS1/2 feedback mechanisms, prevented acquired resistance to these drugs, leading to sustained efficacy, and even led to regression of the recurrent tumor mass. Conclusions: The unique mode of action of NT219, a dual blocker of the STAT3 and IRS-to-AKT feedback mechanisms induced by many anticancer drugs, as well as its significant effects in overcoming drug-resistance, may open a new venue of combined targeted therapy in a wide range of malignancies. Conflict of interest: Ownership: TyrNovo is a startup company that develops NT219 to the clinic. Dr. Reuveni & Dr. Kupershmidt are employees of the company. Advisory Board: Prof. Haviv and Prof. Stemmer are consultants of TyrNovo. 600 Esculentin-2PLa, a frog skin antimicrobial peptide, causes necrotic cell death in breast cancer cell lines S. Sancar-Bas1 , S. Bolkent1 . 1 Istanbul University, Department of Biology, Istanbul, Turkey Background: Antimicrobial peptides are molecules which play important roles in the natural immune system from insects to mammals. These peptides show significant differences in terms of amino acid sequence and secondary structure. The net charges of these peptides vary between +2 to +9 in neutral pH and their cationic characters based on the basic amino acids such as arginine and lysine. On the other hand, these peptides are short peptides composed of 5−40 amino acids and 30% or more of these amino acids have hydrophobic character. Beside bacteria killing and immunomodulatory effects, these peptides have gained attention in recent years due to their anticancer activities. In this study we aimed to research the anticancer activity of Esculentin-2PLa on breast cancer cell lines, MCF-7 and MDA-MB 231. Esculentin-2PLa is a 37-mer peptide that is firstly defined in the skin secretion

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 of Rana palustris and the net charge and hydrophobicity is +5 and 48%, respectively. Material and Methods: Estrogen receptor (ER) positive cell line, MCF-7 and ER(−) cell line, MDA-MB 231 were used in this study. Antitumor activity was determined with cell proliferation assay and cell cytotoxicity assay after treatment with 0–200 mg/ml Esculentin-2PLa for 24 hours. The cell death mechanism was investigated by measuring activity of caspase-3 and counting stained the apoptotic and necrotic cells with acridine orange-ethidium bromide. The cell morphology after the peptide treatment was observed by scanning electron microscopy. In addition, possible electrostatic interactions between cell membranes and the peptide were researched by solid-phase heparan sulfate and chondroitin sulfate binding assay and by measuring the affinity of peptide to cancer cell membranes. Results: Esculentin-2PLa reduced cell viability in a dose dependent manner in the two cell lines and MDA-MB 231 cell line was more sensitive. Beside this, the peptide caused the release of lactate dehydrogenase (LDH) from the cells. In addition, necrotic cell death was detected in both cell lines by observing membrane pores with scanning electrone microscope and this finding was consistent with the increase in the number of cell nuclei stained with etidium bromide. Moreover, Esculentin-2PLa attached to heparan sulfate and chondroitin sulfate proteoglycans which are abundant on cancer cell membranes in a dose dependent manner. Furthermore, this peptide has a high affinity to cancer cell membranes via electrostatic interaction. Conclusions: Overall, these results suggest that Esculentin-2PLa induced cell death through membranolytic effects and its anticancer activity may arise from its cationic and hydrophobic characters which facilitate the interaction with cancer cell membranes. Our findings indicate that Esculentin-2PLa has the potential for development as anticancer agent. No conflict of interest. 601 Bioactivities of novel boehmeriasin derivatives in liver cancer cells E. Akhan Guzelcan1 , M. Baumann2 , I. Baxendale2 , R. Cetin Atalay1 . 1 Middle East Technical University, Bioinformatics Department, Ankara, Turkey, 2 University of Durham, Department of Chemistry, Durham, United Kingdom Introduction: Hepatocellular carcinoma (HCC) is the second most deadly cancer type and the fifth most common cancer in the world which occurs in patients with chronic liver disease/infection or cirrhosis. The incidence of HCC has increased over the past decades due to obesity but still an effective therapy has not been developed. Sorafenib, which is the only FDA approved agent, can improve the patient survival just for a few months, therefore liver transplantation is the most efficient way of treatment up to date. Hence, more efficient therapeutic agents must be developed. Boehmeriasin A, an alkaloid isolated from extracts of Boehmeria siamensis Craib has been shown to possess promising anticancer activities in various cancer types such as lung, breast, kidney, colon, prostate and leukemia. However, the bioactivities of boehmeriasin A and several of its novel synthetic derivatives remain unclear in liver cancer. In this study, we demonstrated that boehmeriasin A and other boehmeriasin derivatives boehmeriasin-OH, -ketone and -K-salt have cytotoxic bioactivities in HCC cells. Material and Methods: The initial cytotoxicity of boehmeriasin A and -OH, -ketone -K-salt were assessed with Sulforhodamine B (SRB) assay on epithelial cancer cells: Huh7, HCT116 and MCF7. Then the compounds were tested in a panel of HCC cell lines: Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU475. The cytotoxicity of the boehmeriasin A and its hydroxyl-derivative were further confirmed by Real-time cell electronic sensing assay (RT-CES) over 96 hours. The cell cycle analysis of treated HCC cells was assessed with flow cytometry assays. Finally, the cell signaling mechanisms leading to cells death with boehmeriasin derivatives were analyzed by immunofluorescence and western blot assays. Results: In this study, we showed highly potent cytotoxic activities of boehmeriasin A and boehmeriasin-OH, and -ketone on HCC cells. The IC50 values of both boehmeriasin A and its -OH derivative were in the nanomolar range in all HCC cells tested. The real time cell growth analysis also supported this significant anti-proliferative effect of the compounds on HCC cells in a time dependent manner. The compounds were also shown to induce apoptosis as demonstrated by cytochrome-c release and induction of other apoptotic proteins. Apoptotic arrest was further confirmed by cell cycle analysis. In parallel ROS and SIRT pathways, which was previously described in boehmeriasin action were analyzed in detail. Conclusion: Novel boehmeriasin derivatives were identified as powerful therapeutics due to their strong cytotoxicity and apoptotic effect on HCC cells. These compounds can be considered as potential therapeutic agents in primary liver cancer treatment. No conflict of interest.

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602 Improving nelarabine efficacy in refractory/relapsed T-cell acute lymphoblastic leukemia (T-ALL) by targeting aberrant PI3K/mTOR signaling F. Chiarini1 , A. Cappellini2 , A. Lonetti3 , A. Bertaina4 , F. Locatelli4 , F. Melchionda5 , A. Pession5 , A.M. Martelli3 . 1 National Research Council, Institute of Molecular Genetics, Bologna, Italy, 2 University of Cassino, Department of Human Social and Health Sciences, Cassino, Italy, 3 University of Bologna, Department of Biomedical and Neuromotor Sciences, Bologna, Italy, 4 IRCCS Bambino Gesu` Children’s Hospital, Department of Pediatric Hematology and Oncology, Rome, Italy, 5 S. Orsola-Malpighi HospitalUniversity of Bologna, Pediatric Oncology and Hematology Unit “Lalla Seragnoli”, Bologna, Italy Background: The introduction of novel chemotherapy protocols has improved the outcome of T-ALL patients, but refractory and/or relapsing disease remains a major concern. In this context, a major contribution was provided by the introduction of the nelarabine, approved for salvage treatment of refractory/relapsed T-ALL patients. Aims: Nelarabine could induce a dose-dependent neurotoxicity. To improve its efficacy, it is essential to study its molecular targets, testing selective inhibitors of such targets, to be administered in combination with nelarabine, allowing for a lower dosage of the drug. Methods: Human T-ALL cell lines and primary T-ALL refractory/relapsed lymphoblasts from patients were incubated with increasing concentrations of nelarabine alone or combinated with PI3K/mTOR inhibitors for cell viability assays. Apoptosis and phenotyping analyses were performed by flow cytometry. Protein expression was evaluated by Western Blot. ENT1/2 gene expression was measured by quantitative real time PCR in T-ALL settings. Results: Cell viability assays indicated the presence of T-ALL cell lines sensitive to nelarabine (IC50 15 mM). Nelarabine sensitive cells showed a significant increase of apoptotic cells after 48 h treatment with 2−5 mM nelarabine, as demonstrated by caspases and PARP cleavage. In contrast, resistant T-ALL cells were not perturbated. Levels of expression of ENT1/2 nucleoside transporters could be related to in vitro nelarabine sensitivity of T-ALL cells, and could be also dependent on interactions between leukemic cells and tumor microenvironment. No significant differences in ENT1/2 mRNA levels between samples sensitive or resistant to nelarabine were seen. Modulation of ENT1/2 gene expression was not related to nelarabine treatment, even if in some cases the co-culture with human stromal HS-5 cells supported cell survival. Upregulated PI3K/mTOR signaling is a common feature of T-ALL, where it portends a poorer prognosis by influencing leukemic cell proliferation/survival/drug-resistance. Sensitive T-ALL cells treated with nelarabine showed a strong decrease in the phosphorylation of Ser473 p-Akt and Ser235/236 p-S6 ribosomal protein. In contrast, resistant cells, showed a hyperactivation of PI3K/mTOR signaling pathway. The combination of nelarabine with the pan PI3K inhibitors ZSTK474 or BKM120 was synergistic in reducing proliferation and in inducing strong apoptosis in all the resistant cell lines and in relapsed T-ALL patient samples with upregulated PI3K/mTOR. Conclusions: Nelarabine combined with PI3K inhibitors efficiently reduced cell viability and induced apoptosis in T-ALL settings, allowing for a lower dosage of nelarabine and therefore, synergizing with conventional therapies in relapsed/refractory T-ALL patients with upregulation of PI3K signaling. No conflict of interest. 603 S-phase arrest independent activation of the DNA-damage response under hypoxic conditions M. Likhatcheva1 , R. Gieling1 , C. Demonacos1 , K. Williams1 . 1 The University of Manchester, Pharmacy, Manchester, United Kingdom Introduction: Hypoxia is a common feature of all solid tumours that arises due to a high proliferation rate and aberrant angiogenesis. Hypoxia is associated with poor prognosis, a more aggressive tumour phenotype as well as radio and chemoresistance. Recently, it has been proposed that tumour hypoxia leads to the activation of the DNA damage response (DDR) during tumour growth and development. Evidence suggests that the activation of DDR in hypoxia occurs as a direct consequence of S-phase cell cycle arrest. This arrest leads to the activation of ATM via ATR. Furthermore, changes in chromatin structure have been associated with the activation of ATM. It has been shown that ATM activation is dependent on the chromatin context, under normoxia as well as under hypoxia. Specifically this is related to the induction of H3K9me3. In response to the DNA damage in normoxia, Tip60 is the link between the DNA damage induced-changes in the chromatin structure and the activation of ATM. Whether the same mechanism operates under hypoxic conditions in not known and is under evaluation in this study. Materials and Methods: The activation of ATR and ATM was assessed in U87 and FTC133 cell lines by Western blot under hypoxic (0.1%) and anoxic (0%) conditions. Cell cycle distribution was analysed at different time points by FACS. The induction of H3K9me3 marker under hypoxic and anoxic conditions was established by Western blot and immunofluorescence. The activity of

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Tip60 under experimental conditions was verified by western blot using p53k120(Ac) as the downstream marker in U87 cell line. Tip60 knock down was performed using siRNA. Results: The expression of pATM is higher in anoxic conditions than in hypoxia after 18 h of exposure. Cell cycle analysis suggest that the activation of ATM under these experimental conditions occurs in time points prior to S-phase arrest in hypoxia and is independent of S-phase arrest in anoxia. Cell cycle analysis in FTC133 cell line showed that S-phase arrest in hypoxia occurs only in time point 24 h. Anoxia induced G1/G0 cell cycle arrest with no evidence of S-phase arrest under these conditions. H3K9me3 is induced in hypoxia and anoxia. Tip60 is active under hypoxic and anoxic conditions in U87 cell line. Knockdown of Tip60 leads to a down regulation of pATM in hypoxia and anoxia. Conclusions: The preliminary results suggest a Tip60 and chromatin dependent mechanism for the activation of ATM prior to S-phase arrest under low oxygen conditions in FTC133 cell line. Further validation of this hypothesis will be the focus of future research. No conflict of interest. 604 Mutant p53 modulates the signal of hepatocyte growth factor (HGF) to endow cancer cells with drug resistance Y. Stein1 , A. Jacob1 , N. Goldfinger1 , R. Straussman1 , V. Rotter1 . 1 The Weizmann Institute of Science, Molecular Cell Biology, Rehovot, Israel Background: Approximately 50% of human cancers harbor p53 mutations, which often endow cancer cells with novel oncogenic functions, such as promoting resistance to various drugs. It was recently demonstrated that secreted molecules from the microenvironment can promote drug resistance as well. However, whether cancer cell determinants such as mutant p53 can modulate tumor–stroma interactions remains poorly understood. In the current study, we were interested to investigate whether mutant p53 can act in a cooperative manner with secreted molecules from the microenvironment to promote resistance to pathway-targeted therapy. Materials and Methods: We utilized the human lung adenocarcinoma cell-line PC9, which harbors an EGFR hyperactivating mutation and an endogenous “hotspot” mutation in p53 DNA binding domain, R248Q. To study mutant p53dependent effects, we created PC9 sub-lines which stably express turbo-RFP (tRFP) and either an shRNA targeting mutant p53 (PC9 shp53) or shRNA targeting irrelevant sequence as control (PC9 Mut-p53). We then subjected both PC9 sub-lines to either gefitinib, an EGFR inhibitor, or DMSO as control, together with a library of 300 recombinant secreted molecules. We then followed the growth of each of the sub-lines in the different conditions by monitoring changes in their tRFP signal for another 6 days. Results and Discussion: We did not observe significant mutant p53dependent resistance to gefitinib without cytokines. However, we identified several cytokines that endowed the Mut-p53 sub-line with higher survival compared to shp53 sub-line when treated by gefitinib. Interestingly, the cytokine which exhibited the most significant mutant p53-dependent rescue from gefitinib was hepatocyte growth factor (HGF). We further observed that mutant p53 indeed modulated the signaling elicited by HGF, as reactivation of the downstream effector phosphorylated-ERK after combined gefitinib and HGF treatment was diminished in shp53 sub-line as compared to Mut-p53 sub-line. Moreover, we observed that mutant p53 facilitated HGF mediated rescue in additional cell-line-drug combinations, suggesting a general effect for mutant p53 on HGF-dependent drug resistance. Stromal-secreted HGF was previously shown to confer various cancer cells with resistance to different drugs, including gefitinib. However, we show here that this rescue seems to be enhanced by mutant p53, which is consistent with another report of mutant p53 driving invasion by enhancing c-MET dependent signaling. In all, our findings may imply that mutant p53 can modulate stromal-derived signals to endow tumor cells with resistance to targeted therapy. Conclusion: We describe here a novel mechanism for mutant p53-mediated drug resistance, which is not solely dependent on cell-autonomous factors, but rather on a synergistic effect between mutant p53 and HGF. No conflict of interest. 605 Identification of CDK4/6-response biomarkers using estrogen receptor-positive breast cancer patient-derived xenografts (PDX) M. Palafox1 , M.T. Herrera2 , M. Bellet1 , J. Arribas3 , C. Saura1 , E. Di Tomaso4 , ´ 1 , J. Baselga5 , V. Serra1 . 1 Vall d’Hebron Institute N.C. Turner2 , J. Cortes of Oncology, Experimental Therapies Laboratory, Barcelona, Spain, 2 The Institute of Cancer Research, Breast Unit, London, United Kingdom, 3 Vall d’Hebron Institute of Oncology, Growth Factors Laboratory, Barcelona, Spain, 4 Novartis Pharmaceutical Corporation, Novartis Institutes for BioMedical Research, Cambridge, USA, 5 Memorial Sloan-Kettering Cancer Center, Human Oncology & Pathogenesis Program, New York, USA Background: Endocrine resistance is a clinical challenge for the treatment of estrogen receptor positive (ER+) breast cancer (BC). CDK4/6 blockade in

combination with endocrine therapy has shown clinical activity in metastatic ER+ BC. However, there is a need for biomarkers that can predict the response to this treatment and improve patient stratification. We aimed to address this issue using xenograft models established from samples of ER+ BC patients. Materials and Methods: Six ER+ PDXs were treated with continuous doses of a CDK4/6 inhibitor (LEE011, 75 mg/kg, 6IW) and a PI3K-alpha inhibitor (BYL719, 35 mg/kg, 6IW) as single agents and in combination, and intrinsic sensitivity to these agents was evaluated. The models were then genomically characterized using a capture-based sequencing panel and by digital PCR. Results: One PDX model was intrinsically sensitive to single-agent CDK4/6 inhibition and experienced tumor regression, but all individual tumors eventually escaped therapy after 50 days of treatment. This particular model harbored at baseline an ESR1-mutation and concomitant losses of CDKN2A/B. At relapse, we identified the acquisition of an RB1 frameshift mutation. Whole Exome Sequencing (WES) analysis is being performed for additional sensitive and acquired-resistance tumors of this model. Interestingly, upfront combined treatment with a PI3K-alpha inhibitor delayed the onset of tumor progression. Triple combination with endocrine therapy increased the antitumor response. Two out of the remaining five CDK4/6-resistant PDXs harbored at baseline either a frameshift mutation in RB1 (plus loss of heterozygosity) or had low pRb protein expression. Two other resistant models harbored CCND1 and MYC amplifications. The remaining one harbored a TSC1 loss. In all the CDK4/6resistant PDX, however, the combination of CDK4/6 and PI3K-alpha inhibition resulted in tumor regression. Conclusions: From our results, we conclude that loss of G1-cell cycle checkpoint control, such as mutation/loss of RB1 and CCND1-amplification, is associated with lack of response to CDK4/6 blockade in ER+ BC PDX. The addition of a PI3K-alpha inhibitor results in improvement of disease control in all experimental models tested. No conflict of interest. 606 A novel role for Top2a as a mediator of resistance to ribosomal DNA transcription inhibition D. Cameron1 , E. Sanij2 , M. Bywater3 , N. Hein1 , J. Lim4 , G. McArthur2 , G. Poortinga2 , R. Hannan1 . 1 John Curtin School of Medical Research, Cancer Biology & Therapeutics, Canberra, Australia, 2 Peter MacCallum Cancer Centre, Oncogenic Signalling & Growth Control, Melbourne, Australia, 3 Cambridge University, Biochemistry, Cambridge, United Kingdom, 4 Senhwa Biosciences, Project Management, San Diego, USA Background: RNA Polymerase I (Pol I) transcription sits at the nexus of many cellular checkpoints and we have previously shown that inhibition of Pol I transcription by CX-5461 (a first-in-class inhibitor of Pol I transcription; Senhwa Biosciences) selectively kills malignant cells over normal cells. This work led to a Phase I clinical trial in patients with refractory haematological malignancies. Further investigation into acquired resistance to CX-5461 will be invaluable for future trials of CX-5461 and other Pol I inhibitors. Material and Methods: We used the Em-Myc mouse model as a discovery tool to investigate resistance as the CX-5461 response is well defined, with tumours relapsing with true drug-resistant disease. Independent Em-Myc tumours that had relapsed on CX-5461 were sequenced following whole exome capture and compared to drug-na¨ıve Em-Myc tumours. Analysis of these data identified Top2a as the most frequently mutated gene (seven mutations in three tumours) in CX-5461 resistant tumours. The role of Top2a in mediating resistance to Pol I inhibition was investigated by shRNA knockdown, CRISPR gene editing and a range of biochemical assays including the novel combination of an assay for detecting proteins covalently bound to DNA (TARDIS) and rDNA-FISH. Results: Top2a relieves DNA torsional stress by enabling the passage of one DNA strand through a temporary double-strand break in another. It has been shown that Top2a is required to induce de novo Pol I transcription, but not elongation nor re-initiation from a primed rDNA repeat. The Top2a mutants detected in the CX-5461 resistant tumours were heterozygous and led to reduced Top2a expression and activity. These tumours also remained resistant to CX-5461 when rechallenged in vivo. Resistance to CX-5461 was recapitulated by knocking down Top2a expression by shRNA. Reduced Top2a expression specifically caused resistance to inhibition of Pol I initiation, but not Pol I elongation, suggesting the resistance was mediated via the rDNA promoter. We detected a transient increase in Pol I transcription with low dose, short CX-5461 treatment suggesting recruitment of Pol I to the rDNA in response to Pol I inhibition, which is not detected in the Top2a mutant cells. Furthermore, the apoptotic checkpoint response is muted in the Top2a mutant cells in vivo. Conclusion: Our data show that Top2a mutations lead to reduced Top2a expression and mediate resistance to CX-5461. We hypothesise that the tumour cell initally responds to Pol I inhibition via a Top2a-dependent recruitment of Pol I to the rDNA, which then triggers an apoptotic response. In the absence of excess Top2a, as in the Top2a mutant cells, the apoptotic checkpoint response is reduced, thereby conferring resistance to CX-5461.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Thus, Top2a expression may provide a valuable biomarker of response to Pol I inhibitors in the clinic. Conflict of interest: Other Substantive Relationships: JL is an employee of Senhwa Biosciences. 607 Induction of apoptosis in cancer cells by p-coumarate ester derivatives O. Firuzi1 , F. Borges2 , J.C. Menezes3 , N. Edraki1 , S. Kamat3 , M. Khoshneviszadeh1 , H.H. Mirzaei1 , R. Miri1 , J.A. Cavaleiro3 , L. Saso4 . 1 Shiraz University of Medical Sciences, Medicinal and Natural Products Chemistry Research Center, Shiraz, Iran, 2 University of Porto, CIQ/Department of Chemistry and Biochemistry, Porto, Portugal, 3 University of Aveiro, Department of Chemistry & QOPNA, Aveiro, Portugal, 4 Sapienza Univerisity of Rome, Department of Physiology and Pharmacology “Vittorio Erspamer”, Rome, Italy Background: Cancer is a major cause of death worldwide and novel anticancer agents with improved efficacy are much needed. Hydroxycinnamic acids are naturally-occurring compounds with important biological activities including cytotoxicity against cancer cells. Previous studies have shown that esterification increases the lipophilicity and in consequence improves the biological effects of these compounds in different disease models. Material and Methods: We synthesized a total of 19 various HCAs alkyl ester derivatives of p-coumaric, ferulic, sinapic and caffeic acids and tested their cytotoxicity against 4 different human cancer cells as well as one noncancer cell line. Effects of the active derivatives were analyzed on cell cycle with propidium iodide-based analysis by flow cytometry and on apoptosis with evaluation of caspase-3 activation by western blot. Results and Discussion: Most of the ester derivatives, but not the 4 parent compounds, were selectively active against MOLT-4 (human lymphoblastic leukemia) cells; The most effective ones were C14 (1c) and C16 (1d) alkyl esters of p-coumaric acid and C14 alkyl ester of caffeic acid (4c) with IC50 values of 0.123, 0.301 and 1.0 mM, respectively. Interestingly, most of the esters also exhibited anticancer effects against multidrug resistant MES-SADX5 uterine sarcoma cells, some of them being several times more potent than standard anticancer agents, doxorubicin and cisplatin. p-Coumarate esters were either ineffective or much less potent against NIH/3T3 non-cancer fibroblast cells. Compounds 1c, 1d and 4c were able to induce a significant increase in sub-G1 phase of the cell cycle in MOLT-4 cells, which indicate the induction of apoptosis in these cells. Furthermore, induction of apoptosis by the same compounds was also confirmed by the observation of caspase-3 enzyme activation in immunoblots. Conclusion: The present findings demonstrate that long ester derivatives of p-coumaric represent promising scaffolds for discovery of novel anticancer agents capable of selective induction of apoptosis in cancer cells. No conflict of interest. 608 Urokinase kringle-derived peptide UP7 suppresses tumor angiogenesis and breast cancer metastasis Y.A. Joe1 , H.K. Kim1 , P. Naidansuren1 , S.W. Lee1 . 1 The Catholic University of Korea, Department of Medical Lifescience and Cancer Research Institute, Seoul, South Korea Background: The kringle domain of urokinase has been shown to inhibit angiogenesis in vitro and in vivo and suppress brain tumor growth in vivo. In order to explore functional core sequence of the kringle domain and its possible application, we examined the several kringle-derived peptides for antiangiogenic and anti-cancer activities. Materials and Methods: The kringle domain-derived seven peptides were designed, synthesized, and tested for anti-angiogenic activities. The most potent anti-angiogenic peptide was selected and further investigated for antiangiogenic and anti-cancer properties at cellular level or in experimental animal models using endothelial cells (ECs), NCI-H460 lung cancer cells, and LM-MDA-MB-231 breast cancer cells. Proliferation (MTS assay), migration, invasion (Boyden chamber assay), cell adhesion, and signaling were assessed at cellular and molecular levels. Results: Among the tested peptides, UP-7 potently inhibited the proliferation and migration of ECs and also suppressed in vivo angiogenesis in the mouse Matrigel plug assay. At molecular level, UP-7 inhibited VEGF- or bFGFinduced phosphorylation of FAK and ERK1/2 and formation of stress fibers and focal adhesions in ECs. In a subcutaneous xenograft mouse model of NCIH460 cells, daily intraperitoneal administration of UP-7 (50 mg/kg) for 16 days resulted in suppression of in vivo tumor growth with angiogenesis inhibition. Interestingly, UP7 inhibited the proliferation of LM-MDA-MB-231 cells albeit at higher concentrations, whereas it showed no inhibition of NCI-H460, U87 cells, and HEK293 cells. Moreover, it potently inhibited migration and invasion of LM-MDA-MB-231 cells. It showed no suppression of MMP-9, but suppressed the attachment and spreading of these cells onto immobilized fibronectin. It also inhibited serum-induced FAK activation of LM2-MDA-MB-231 cells.

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Accordingly, UP-7 potently inhibited the lung metastasis of LM2-MDA-MB-231 in an experimental metastasis model. Conclusion: Taken together, these results suggest that UP-7 peptide can be effectively used for treatment of cancer growth and metastasis. Acknowledgement: National Research Foundation of Korea grants NRF2012R1A1A2007175, NRF-2012R1A5A2047939. No conflict of interest. 609 Inhibition of neuroblastoma growth by the thiadiazolidinone compound TDZD-8 D. Aguilar-Morante1 , R. Pardal1 . 1 Instituto de Biomedicina de Sevilla IBiS- Hospital Universitario Virgen del Roc´ıo/CSIC/Universidad de Sevilla, ´ Departamento de Fisiolog´ıa Medica y Biof´ısica, Seville, Spain Background: Neuroblastoma (NB) is one of the most lethal pediatric tumors. Currently, there is no effective cure for the most aggressive cases, which are unfortunately the most abundant (45% of total). In neuroblastoma and other solid tumors, there is a subpopulation of undifferentiated cells (NB-Undiff cells), with high tumorigenic potential and stemness features. Probably, these cells are responsible for the maintenance and recurrence of these tumors, as well as for tumor resistance to various therapies. Data from our laboratory point to glycogen synthase kinase 3b enzyme (GSK-3b) as a novel candidate for combined therapy for neuroblastomas. Thiadiazolidinones (TDZD) are small heterocyclic compounds first described as non-ATP competitive inhibitors of GSK-3b. In this study, we analyzed the effects of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) on human neuroblastoma cell growth in vitro and in vivo. This work aims to identify new approaches to neuroblastoma therapies, targeting NB undifferentiated or total cells. Material and Methods: We first performed cell viability analysis using MTT assay in different human NB cell lines. Detection of apoptosis was done with Annexin-V and MitoTracker and luciferase experiments were made to analyze transcriptional regulation of NF-úB. Immunofluorescence, qPCR and immunoblotting of genes involved in DNA damage response were also performed. In vivo studies were done in SCID mice using 5 mg/kg of TDZD-8. Results and Discussion: Our data show that TDZD-8 decreases proliferation and induces apoptosis on human NB cells in vitro, and delays tumor growth in vivo. We also demonstrate that TDZD-8 treatment in human NB cells reduces NF-úB activity. In this regard, other groups have shown that survival of different tumor cells depends on GSK-3b activity trough a NF-úB-dependent pathway. Therefore, gene expression analysis show that these effects are associated with high level expression of p27 and p21 genes and decreased expression of genes involved in DNA damage response. In this line, our data show that TDZD-8 treatment promotes an increase in DNA damage in NB cells, visualized by active gH2AX detection. Finally, treatment of NB-Undiff cells with TDZD-8 resulted in an inhibition of proliferation and self-renewal of these cells, highlighting the importance of these cells as specific targets for the drug. Conclusions: Our data demonstrate that GSK-3b inhibitor, TDZD-8, blocks neuroblastoma growth by means of activating checkpoints and intoxicating DNA repair pathways, showing a high potential for treatment improvement in pediatric neuroblastoma. Acknowledgement: This research has been supported by ‘Asociacion ´ Espanola ˜ contra el Cancer’ ´ (AECC), Spanish Ministry of Science (SAF), and European Union (ERC). No conflict of interest. 610 The MRN complex: A potential target for MYCN amplified neuroblastoma ` Roncero2 , C. Heil2 , P. Infante1 , B. Ricci2 , M. Petroni1 , F. Sardina2 , M. Sahun E. Petricci3 , E. Locatelli4 , M. Comes-Franchini4 , G. Giannini2 . 1 Istituto Italiano di Tecnologia, CLNS@Sapienza, Rome, Italy, 2 ’La Sapienza’ University of Rome, Dip Medicina Molecolare, Rome, Italy, 3 Universita` degli Studi di Siena, Department of Biochemistry- Chemistry and Pharmacy, Siena, Italy, 4 Alma Mater Studiorum − University of Bologna, Department of Industrial Chemistry “Toso Montanari”, Bologna, Italy Introduction: Finding an effective treatment for MYCN amplified (MNA) high-risk neuroblastoma patients represents an open challenge in pediatric oncology. The high rate of replication stress in Myc-addicted cancer cells makes them more sensitive to inhibition of ATR and CHK1, the main kinases activated in response to replication stress-associated DNA Damage Response (DDR). A prominent role of the MRE11/RAD50/NBS1 (MRN) complex in replication stress response is also emerging. We have recently shown that MYCN upregulates the three components of the MRE11/RAD50/NBS1 (MRN) complex during granule cell progenitors expansion in postnatal cerebellar development to restrain the deleterious effects of MYCN-dependent replication stress. Here we investigated how inhibition of the MRN complex impacts on MNA neuroblastoma in vitro and in vivo. Material and Methods: We genetically inactivated the MRN complex in NBs cells and we evaluated the effects in terms of proliferation and colony formation.

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Subsequently we treated NBs cell lines with the pharmacological inhibitor of MRE11 exo-nuclease activity, mirin. We monitored its biological effects on proliferation (MTS assay, FACS), DNA damage (Neutral comet assay) and survival (Trypan blue exclusion Test, Tunel assay, PARP and Caspase 3 cleavage) and its effects on the DDR pathway (WB). Moreover we tested mirin in the treatment of human neuroblastoma (LAN5) xenografts, in vivo. Tumors were measured and then analyzed for DDR and apoptotic markers by WB and IHC. Results: Analysis of multiple datasets in the R2 database, indicated that MRE11 and RAD50 are significantly more expressed in MNA compared to MYCN single copy primary neuroblastomas. MRE11 or NBS1 RNAi-mediated knock down impaired proliferation and colony formation in a MYCN-dependent way. Pharmacological inhibition of the MRN complex via mirin, selectively induced cell death in MNA compared to MNSC cells or non-neuroblastoma cancer models. While mirin caused accumulation of 53BP1 foci, a marker of DNA damage associated with replication stress, it also inhibited the ATR/CHK1-dependent checkpoint/s, preventing any arrest in the S and G2 phases of the cell cycle. In contrast, mirin induced early occurrence of DNA double strand breaks and a typical DDR characterized by ATM, H2AX and p53 phosphorylation culminating in the accumulation of pro-apoptotic p53 target genes. Gain and loss of function experiments confirmed that mirin-induced cell death in MNA cells is p53 dependent. Injection of mirin encapsulated in nanoparticles significantly inhibited tumor growth in a neuroblastoma xenograft model. Drug-treated tumors were characterized by an high rate of apoptosis and DDR activation. Conclusion: Overall, we believe that our experiments provide a solid proof of principle that targeting the MRN complex can have significant effects in the treatment of MNA NBs. No conflict of interest. 611 The challenges of an autologous cell therapy product in clinical trials R. Smith1 , M. Bryan1 , K. Campbell2 , L. Cooney1 , F. Gilbert3 , T. Hamill1 , A. Scott4 , K.J. Williams1 . 1 TC BioPharm, Clinical, Motherwell, United Kingdom, 2 TC BioPharm, Quality Control, Motherwell, United Kingdom, 3 TC BioPharm, Production, Motherwell, United Kingdom, 4 TC BioPharm, Operations, Motherwell, United Kingdom Introduction: The cell therapy product is culture-expanded autologous gd T lymphocytes which presents many challenges for clinical trials. These include collecting starting material from each patient, shipping to the production facility, manufacturing the product specifically for each patient, then releasing and administering final product at the clinical trial sites. These challenges limit sites that can be included in a clinical trial and the patients who can be enrolled. Material and Method: Studies have shown that the proliferative capacity of gd T cells varies between individuals. We have developed a pre-screen proliferation assay to identify the patients who proliferate to the capacity required for autologous product manufacture. Shelf-lives of the proliferation assay blood sample and final product are limited, therefore shipment time between the clinic and manufacturing facility geographically restricts sites that can be involved. The Company has been conducting studies to extend shelf-life of the sample and product. Results and Discussion: To manufacture this autologous product, starting material is collected from each patient using an apheresis procedure to extract their own cells. Apheresis can be a demanding procedure, thus to ensure patients do not undergo the process unnecessarily, a pre-screening proliferation assay is performed to confirm proliferative capacity of gd T cells for each patient. This involves obtaining a 10 ml blood sample from a patient, with a limited shelf-life, currently qualified to 6 hours. This therefore geographically limits which clinical sites can be included in a clinical trial. The nature of the product also causes logistical issues. Manufacture of the product is a continuous 14-day process, thus the manufacturing facility must maintain close communication with both final product shipment courier and clinicians at the trial sites, to ensure that a patient is available for treatment on the specified day. If a patient misses their treatment visit, that dose cannot be retained, and a new dose will be manufactured. The final product also has a limited shelflife of 36 hours, and is therefore shipped under quarantine to clinics pending final QP certification, allowing for some geographically challenging sites to be included in the trial. In 6 months we have been able to improve the shelf life of the final product from 24 hours to 36 hours. Conclusion: The main challenge to success of any clinical trial with this product, is the shelf-life of the pre-screening blood sample and the final product. By conducting more shelf-life studies, it is considered that the shelf-life of the materials can be extended. This will expand the potential geographical area for clinical trial sites, and allow more patients to be enrolled and ultimately treated with autologous cell therapy. Conflict of interest: Ownership: Angela Scott, Karen J Williams and Rachel Smith

612 Idelalisib sensitivity and mechanisms of disease progression in relapsed TCF3-PBX1 acute lymphoblastic leukemia S. Eldfors1 , H. Kuusanmaki ¨ 1 , M. Kontro2 , M.M. Majumder1 , A. Parsons1 , 3 ¨ , H. Edgren1 , T. Pemovska1 , O. Kallioniemi1 , K. Wennerberg1 , N. Gokbuget T. Burmeister4 , K. Porkka2 , C. Heckman1 . 1 University of Helsinki − FIMM, Institute for Molecular Medicine Finland, Helsinki, Finland, 2 University of Helsinki and Helsinki University Central Hospital Cancer Center, Department of Hematology, Helsinki, Finland, 3 Goethe Unversity, Department of Medicine ¨ II, Frankfurt, Germany, 4 Charite´ Universtatsmedizin Berlin, Department of Hematology- Oncology and Tumorimmunology, Berlin, Germany Background: TCF3-PBX1 is a recurrent gene fusion in B-cell-precursor lymphoblastic leukemia (BCP-ALL) resulting from translocation t(1;19). The TCF3-PBX1 protein is a transcription factor that directly up-regulates pre-B-cell receptor genes, causing constitutive activation of pre-B-cell receptor signaling. TCF3-PBX1 BCP-ALL patients typically respond to chemotherapy; however, many relapse and develop resistant disease with few effective treatment options. In this study, we aimed to identify novel targeted drugs for treating TCF3-PBX1 BCP-ALL by profiling leukemic cells from a 25-year-old patient with relapsed disease. In addition, we sought to identify molecular mechanisms of disease pathogenesis and progression. Material and Methods: Bone marrow (BM) aspirates and a skin biopsy were collected from the patient at diagnosis and relapse. The sensitivity of BM cells was assessed against a library of 302 anti-neoplastic drugs. To identify molecular mechanisms underlying the pathogenesis and progression of the disease, exome and RNA-seq was performed. Drugs effective at inhibiting the viability of the patient cells were further investigated using TCF3-PBX1+ BCP-ALL and control cell lines. Results and Discussion: Drug sensitivity testing showed that index patient’s leukemic cells were sensitive to several classes of targeted drugs, including PI3K, mTOR, BET and HDAC inhibitors. Among the most effective was PI3K delta (p110d) inhibitor idelalisib approved for CLL and follicular lymphoma. Testing of samples from the patient, positive control CLL and healthy controls, showed that the BCP-ALL and CLL cells were similarly sensitive to idelalisib. To determine whether idelalisib sensitivity is common to all BCP-ALLs, we tested the sensitivity of TCF3-PBX1 positive (n = 3) and negative (n = 3) BCPALL cell lines. Two TCF3-PBX1+ cells lines were sensitive while negative cell lines were resistant. Idelalisib sensitivity of TCF3-PBX1+ BCP-ALL cells was further supported by evidence showing TCF3-PBX1 directly regulates expression of PIK3CD, the gene encoding p110d. RNA sequencing of the relapse samples showed high CXCR4 expression, which was not observed in a cohort of diagnostic phase TCF3-PBX1 BCP-ALLs (N = 15). CXCR4 mediates interactions with CXL12 expressing BM stromal cells and induces contact mediated drug resistance. Idelalisib inhibits CXCR4 signaling, providing a rationale for using this drug to counter drug resistance. The patient’s leukemia acquired mutations to TP53 at relapse. In addition, the patient’s leukemic cells had an MTOR mutation, which was associated with high sensitivity to mTOR inhibitors, which has not been observed before in TCF3-PBX1 BCP-ALL. Conclusions: Our results suggest idelalisib is a promising treatment for patients with TCF3-PBX1 BCP-ALL, while other drugs could be useful depending on the genetic context of individual patients. No conflict of interest. 613 Digoxin is a modifier increasing platinum drug anticancer activity E. Dudko1 , V. Chernov1 , T. Bogush1 , Y. Dyakova1 , V. Kirsanov2 , Z. Shprakh3 , A. Kamensky4 , B. Polotsky5 , S. Tjulandin6 , E. Shestakova1 . 1 N.N. Blokhin Russian Cancer Research Center, Laboratory of medical chemistry, Moscow, Russian Federation, 2 N.N. Blokhin Russian Cancer Research Center, Department of surgery, Moscow, Russian Federation, 3 N.N. Blokhin Russian Cancer Research Center, Deputy director, Moscow, Russian Federation, 4 Lomonosov Moscow State University, Department of human and animal physiology, Moscow, Russian Federation, 5 N.N. Blokhin Russian Cancer Research Center, Department of thoracic surgery, Moscow, Russian Federation, 6 N.N. Blokhin Russian Cancer Research Center, Department of clinical pharmacology and chemotherapy, Moscow, Russian Federation Introduction: Aerobic glycolysis (Warburg effect) is a fundamental hallmark of cancer cells. Glycolysis inhibition by cardiac glycosides increases anticancer drug toxicity towards tumor cells in vitro. The purpose of the study was to reveal such an effect in vivo, studying the influence of cardiac glycoside digoxin (Dig) on anticancer effect of platinum-drug cisplatin (Cis), which activity is associated with energy-dependent reparation of DNA damages. Materials and Methods: Breast cancer Ehrlich ascitis tumor was chosen as a model because of known high level of aerobic glycolysis in the cells. The investigation was conducted on female CBA/Lac mice. Dig 1 mg/kg was injected one hour before Cis administration. Both drugs were injected intraperitoneally (ip). The results were analyzed using Mann–Whitney and Student criteria (significant difference at p < 0.05). Results and Discussion: 1. Linear dosage-effect dependence was shown in 2.5 and 5.0 mg/kg of Cis, and the latter one is a maximal effective dose

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 because of Cis toxicity at dosage escalation. 2. Dig enhanced antitumor activity of Cis: survival time increase of tumor-bearing mice treated with Dig+Cis 2.5 mg/kg was 79%, as compared to 42% under Cis 2.5 mg/kg action only. 3. Cis 2.5 mg/kg+Dig efficacy was higher in comparison with Cis 5.0 mg/kg only: survival time increase of tumor-bearing mice treated with Dig+Cis 2.5 mg/kg was 79% as compared to 51% under Cis 5.0 mg/kg action only. 3. The differences in survival time of tumor-bearing mice between the groups «Cis 2.5 mg/kg» and «Cis 2.5 mg/kg+Dig», as well as between the groups «Cis 2.5 mg/kg+Dig» and «Cis 5.0 mg/kg», were highly significant (p < 0.0001) independently of which control was taken for comparison: tumor-bearing mice without treatment or with Dig injection. Conclusion: On the ascitic Ehrlich tumor-bearing mice, it was shown that ip injection of digoxin one hour before ip injection of cisplatin increases survival time of the tumor-bearing mice compared to cisplatin monotherapy. The amelioration of cisplatin efficacy under digoxin action exceeded the treatment effect of two-fold higher dose, and was more exhibited than the highest anticancer effect revealed in this experimental model. So, digoxin is an effective modifier increasing platinum drug anticancer activity. According to design and results of the study, we believe that clinical trial of cisplatin combination with digoxin in intraperitoneal chemotherapy of ascitic ovarian cancer is important in terms of revealing amelioration of treatment efficacy. Acknowledgment: Supported by grants: RFBR (15-04-06991-a, 16−34-01049mol-a) and President of Russian Federation (MK-7709.2016.7). No conflict of interest. 615 Induction of EMT is WNT-independent in cisplatin resistant urothelial carcinoma cell lines M.A. Skowron1 , G. Niegisch1 , G. Fritz2 , J.G.H. Van Roermund3 , A. Romano4 , P. Albers1 , W.A. Schulz1 , M.J. Hoffmann1 . 1 Heinrich-Heine-University, Urology, Duesseldorf, Germany, 2 Heinrich-Heine-University, Toxicology, Duesseldorf, Germany, 3 Maastricht University Medical Centre, Urology, Maastricht, Netherlands, 4 Maastricht University Medical Centre, Obstetrics and Gynaecology, Maastricht, Netherlands Background: Failure of cisplatin therapy in urothelial carcinoma (UC) patients originates from chemoresistance. Since the underlying mechanisms have not been clearly defined yet, we have generated cisplatin resistant UC cell lines (UCCs) by long-term treatment and characterized phenotypical and molecular changes associated with the development of cisplatin resistance. We observed morphological changes resembling epithelial–mesenchymal transition (EMT) indicating that UCCs might circumvent cisplatin induced apoptosis by phenotypic plasticity. As EMT is considered to be associated with stemness, chemoresistance, and metastatic potential, we determined the proliferative and clonogenic potential of cisplatin resistant UCCs and investigated the activity of stemness-associated signalling pathways. As combination chemotherapy is used to overcome resistance, cross-resistance towards doxorubicin and gemcitabine was also studied. Material and Method: Cisplatin-resistant UCCs were selected by long-term treatment (LTT) with escalating doses over several months. Sensitivity towards several cytotoxic drugs and population doubling time were assessed by MTT assay. Clonogenic potential and cell cycle distribution were evaluated by Giemsa staining and FACS analysis, respectively. Phenotypical changes were followed on the molecular level by qRT-PCR, immunofluorescence, and reporter assay for EMT markers and WNT-signalling components. Results: LTT-UCCs RT-112 and J82 grew more slowly compared to parental cells, but survived cisplatin treatment better over shorter or longer periods. Strong cross-resistance to gemcitabine, but not to doxorubicin was observed in RT-112-LTT. Treatment with increased doses of cisplatin disturbed the cell cycle of LTT cells, but they did not undergo apoptosis and recovered fully after 10 days. At the molecular level increased expression of Vimentin, Twist, and Zeb1 and decreased E-Cadherin expression corresponded with their EMTlike morphology. In addition, WNT pathway target genes (CTNNB1, AXIN2, CCND1, MYC, PITX2) were induced. However, TOPflash reporter assays revealed no significant activation of canonical WNT-signalling. Accordingly, the WNT-inhibitor niclosamide did not revert cisplatin resistance. Conclusions: Cisplatin-resistant UCCs appear to evade cell death by a phenotypic plasticity resembling EMT that may extend to other therapeutic compounds. Although overexpression of some WNT target genes was observed, phenotypic plasticity appeared not to be substantially mediated by canonical WNT signalling. No conflict of interest.

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616 Elucidation of molecular action mechanisms of cytotoxic palladium derivatives on various cancer cells O. Kacar1 , I. Hatipoglu1 , V. Yilmaz2 , N. Arda3 , E. Ulukaya4 , C.A. Acilan1 . TUBITAK, Marmara Research Center, Genetic Engineering and Biotechnology Institute, Turkey, 2 Uludag University, Faculty of Arts and Sciences, Departments of Chemistry, Turkey, 3 Istanbul University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, Turkey, 4 Uludag University, Medical School, Department of Medical Biochemistry, Turkey 1

Background: The discovery of cisplatin, which is prescribed to almost 50% of cancer patients, has accelerated the design of many novel metal complexes. In this study, two novel palladium (Pd) complexes, [PdCl(terpy)](sac).2H2O (complex 1) and [Pd(sac)(terpy)](sac).4H2O (complex 2) were investigated for their anti-cancer properties. Material and Method: Cell viability was determined using the WST-1 proliferation assay. Apoptosis was assessed based on condensed/fragmented nuclei, gross morphological changes via microscopic analysis, and DNA laddering visualized on agarose gel electrophoresis. DNA double strand breaks were measured. HeLa, MDA-MB-231, and HCT-116 cells were arrested at G1, at the G1/S boundary or in mitosis. Synchronized cells were treated with the Pd compounds for 24, 48, and 72 h. Synchrony was evaluated based on morphology and flow cytometry, and cell viability was measured. Results: The tested compounds induced cytotoxicity in cancer cell lines that originated from various organs, suggesting a broad spectrum of activity. Cells treated with the Pd(II) complex exhibited typical characteristics of apoptosis based on increase in caspase 3/7 activity, presence of fragmented nuclei, DNA laddering, cellular shrinkage and blebbing. DSBs were formed in response to Pd(II) treatment both in vitro and in vivo. Furthermore, our initial data indicated that whilst arresting cells through serum starvation at G1/S had no effect on Pd(II) induced cytotoxicity, cells arrested in M phase appeared to be more resistant to drug treatment. Our results showed that cells were efficiently arrested at the expected cell cycle phase. Treatment of cells with the Pd(II) complexes at specific stage of the cell cycle resulted in similar death rates. The main form of cell death was found to be apoptosis in all treatments tested. Conclusions: The new Pd(II) complex appears to represent a new potent anticancer agent that acts through DNA damage and can induce apoptosis. Arresting cells at the cell cycle phases was accomplished successfully; consequently, these cells were treated with Pd(II) complexes which yielded no significant variation in cell death for HCT-116 and MDA-MB-231; however, HeLa was more sensitive in S/G1. Apoptosis was found to be the leading cause of cell death in all treatments. Indeed, further studies in animal models are required for proof-of-concept before undertaking clinical studies. No conflict of interest. 618 Synthesis, biological characterization and evaluation of molecular mechanisms of novel copper complexes as anticancer agents C. Acilan1 , B. Cevatemre2 , Z. Adıguzel1 , D. Karakas2 , E. Ulukaya3 , N. Arda4 , N. Ribeiro5 , I. Correiae5 , J.C. Pessoa5 . 1 TUBITAK, Marmara Research Center, Genetic Engineering and Biotechnology Institute, Turkey, 2 Uludag University, Faculty of Arts and Sciences, Department of Biology, Turkey, 3 Uludag University, Medical School, Department of Medical Biochemistry, Turkey, 4 Istanbul University, Moelcular Biology and Genetics Department, ´ Istanbul, Turkey, 5 Centro de Qu´ımica Estrutural, Instituto Superior Tecnico, Universidade de Lisboa Background: Three copper(II) complexes, Cu(Sal-Gly)(phen), Cu(SalGly)(pheamine), Cu(Sal-Gly)(phepoxy) are synthesized as potential anticancer agents, and characterized for their interaction with DNA, cytotoxicity, and mechanism of action. Material and Method: The binding ability of the complexes to Calf Thymus DNA was evaluated by competition fluorescence studies with thiazole orange, a known intercatator, UV-Vis spectrophotometric titrations and circular dichroism spectroscopy. DNA damage was determined by cleavage of supercoiled DNA in vitro and induction of 8-oxo-guanidine and gH2AX staining in cells. Apoptosis was assessed by DNA condensation/fragmentation, assessment of mitochondrial membrane potential, Annexin V staining and caspase 3/7 activity. Total ROS measurements were made by DCFDA analysis. Gene expression was evaluated via RT-qPCR analysis. Results and Discussion: Binding constants were evaluated as 1.7×106 , 2.5×106 and 3.2×105 M−1 , for Cu(Sal-Gly)(phen), Cu(Sal-Gly)(pheamine) and Cu(Sal-Gly)(phepoxy), respectively. All compounds induce DNA damage both in vitro and in cell culture. Apoptosis is the main form of cell death based on morphological analyses. Caspase 3/7 activity is elevated in response all Cucomplexes, supporting apoptotic cell death. There is an increase in reactive oxygen species (ROS), suggesting that ROS may mediate DNA damage and antitumor activity. Although the compounds were cytotoxic to all tested cancer cell lines, only Cu(Sal-Gly)(pheamine) displayed significantly lower toxicity towards non-cancer cells, demonstrating selectivity for cancer cells. Therefore, Cu(Sal-Gly)(pheamine) was further tested for molecular changes in response

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to drug treatment. Our results indicate that the expression of genes involved in apoptosis were significantly elevated, supporting our previous findings. Presence of p53 was dispensable for apoptosis in response to Cu-complexes. Conclusion: As copper is an essential element, these complexes may be considered promising anticancer drugs with activity in cancer cells even with deficient p53 status. No conflict of interest. 619 Biological and biophysical characteristics of a new polymer platform for drug delivery systems M. Jiratov ´ a´ 1 , A. Galisov ´ a´ 1 , M. Paˇr´ızek2 , A. Posp´ısˇ ilova´ 3 , M. Rabyk3 , D. Jirak ´ 1, M. Hrubý3 . 1 Institute for Clinical and Experimental Medicine, Department of Diagnostic and Interventional Radiology, Prague, Czech Republic, 2 Institute of Physiology of the Academy of Sciences of the Czech Republic, Department of Biomaterials and Tissue Engineering, Prague, Czech Republic, 3 Institute of Macromolecular Chemistry of the Academy of Sciences of the Czech Republic, Supramolecular Polymer Systems Department, Prague, Czech Republic Introduction: Nano-sized drug delivery systems targeted to tumors by EPR (enhanced permeability and retention) effect are currently developing among many scientific groups. Our new polymer platform for such purpose is unique due to use of a glycogen. In this study, we characterized and tested in vitro/in vivo our novel drug delivery systems intended as a theranostic agent. Material and Methods: In vitro tests were performed on a hepatocellular carcinoma cell line (HepG2). Cytotoxicity was tested by MTT assay, cytoplasmic localization (with/without glucose addition into the incubation media) was visualized by confocal microscopy. Colocalization studies with lysosomes were performed using confocal microscopy. Preliminary in vivo test was done on healthy immunodeficient CD1-Nude mice. These tests were focused on biodistribution and biodegradation rate of GG-Gd DOTA-Dy615. After these tests s. c. tumors were induced by injection of a 5(±1)x106 hepatocellular carcinoma cell line (HuH7) into the subcutaneous area above the right hind leg of RNU rats. Three weeks after tumor induction GGGd DOTA-Dy615 was applied into the tail vein of the animals. Contrast enhancement and accumulation of GG-Gd DOTA-Dy615 into the tumor were tested in vivo using either MR or fluorescence imaging and compared with commercially available contrast agent. Results and Discussion: Cytotoxicity test did not showed any significant difference between control cells and treated cells. However cells viability continuously rises with lower GG-Gd DOTA-Dy615 concentrations. These nonsignificant differences in the cells viability could be caused by Gd3+. Confocal microscopy studies confirmed that GG-Gd DOTA-Dy615 is endocyted inside the cells. No strong colocalization with lysosomes was observed. Glucose addition into the incubation media did not affect cytoplasmic localization of GG-Gd DOTA-Dy615. All these results indicate that physiological degradation pathway as for native glycogen is used also for GG-Gd DOTA-Dy615. Preliminary in vivo test on the healthy animals showed relatively fast biodistribution in a whole organism and accumulation of fluorescent probe Dyomics 615 (Dy615) in urine within three hours. MR and fluorescence imaging in vivo on the animals with s. c. tumors revealed that GG-Gd DOTA-Dy615 was accumulated in the tumor tissue and the contrast enhancement was long lasting and higher compared to commercially available contrast agent. Conclusion: GG-Gd DOTA-Dy615 possesses beneficial characteristics for its use as a drug delivery system, namely nontoxic character, biodegradability and multimodal imaging properties. Acknowledgement: This project was supported through the Grant Agency of the Ministry of Health, Czech Republic (grant # 15-25781a), MH CR-DRO (Institute for Clinical and Experimental Medicine IKEM, IN00023001) and and Charles University Grant Agency, project No. 282216. No conflict of interest. 620 Investigating a novel binuclear palladacycle complex for anti-cancer activity in rhabdomyosarcoma cells J. Bleloch1 , R. Ballim1 , S. Aliwaini1 , S. Mapolie2 , S. Prince1 . 1 University of Cape Town, Human Biology, Cape Town, South Africa, 2 University of Stellenbosch, Department of Chemistry and Polymer Science, Cape Town, South Africa Rhabdomyosarcoma is the most common soft tissue sarcoma in children and adolescents and in comparison to other cancers causes a substantial loss of years of life. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Recently, a novel binuclear palladacycle, AJ-5, was shown to exert potent cytotoxicity in melanoma and breast cancer cells and to have negligible adverse effects in in vitro and in vivo studies. This study therefore investigated the potential of AJ-5 as an anti-cancer agent in embryonal and alveolar rhabdomyosarcoma. To this end, cytotoxicity assays were performed and an IC50 of 0.2mM and 0.14mM was calculated respectively. Clonogenic assays show that AJ-5 greatly compromises the ability of rhabdomyosarcoma cells to

survive and proliferate. To determine the underlying molecular mechanisms by which AJ-5 exerts its cytotoxicity, western blot analyses were performed with antibodies to key proteins involved in the DNA damage response. The results show that AJ-5 induced levels of gH2AX, a marker of double-strand DNA breaks, phosphorylated p38 and p21 in both cell types, which correlated with cell cycle arrests. Furthermore, Annexin V−FITC/propidium iodide staining, western blotting and Caspase Glo assays demonstrated that AJ-5 induces intrinsic and extrinsic apoptosis. AJ-5 treatment also led to autophagy as confirmed by the formation of autophagosomes, increased levels of LC3-II and the presence of LC3 puncta in cells transfected with a GFP-LC3 expression construct. Together these findings suggest that AJ-5 may be an effective chemotherapeutic for treating rhabdomyosarcomas. No conflict of interest. 621 Resistance to sunitinib in clear cell renal cell carcinoma results from drug sequestration in lysosomes and inhibition of autophagic flux ´ 1 , P. Colosetti3 , A. Belaid2 , S. Giuliano1,2 , Y. Cormerais1 , M. Dufies2 , R. Grepin J. Parola4 , P. Auberger3 , B. Mograbi2 , G. Pages ` 2 . 1 Centre Scientifique de Monaco, Biomedical Department, Monaco, Monaco, 2 University of Nice Sophia Antipolis- IRCAN, UMR CNRS 7284- INSERM, Nice, France, 3 ´ ´ ´ University of Nice Sophia Antipolis- Center Mediterran een de Medecine ´ Moleculaire, INSERM, Nice, France, 4 Centre Antoine Lacassagne, Nice, France Background: Metastatic renal cell carcinomas (mRCC) are highly vascularized tumours that are a paradigm for the treatment with anti-angiogenesis drugs. The available drugs increase the time to progression but are not curative and patients inevitably relapse. Here we focused our attention on the first line treatment of mRCC, sunitinib (an ATP mimetic), which inhibits VEGFR 1−3, CSF1R, PDGFR and c-Kit. Sunitinib is also a hydrophobic weak base (pKa 8.95), which presents lysosomotropic characteristics. We aimed to decipher the link between lysosomal sequestration, autophagy and the mechanisms of resistance to sunitinib in mRCC. Materials and Methods: The anarchic vascularization of tumours results in the core of mRCC tumours receiving suboptimal concentrations of the drug. To mimic this in vivo situation, we exposed two mRCC cell lines (786-O and RCC10) to concentrations of sunitinib below the level that inhibits cell proliferation by 50% (IC50). Additionally, sunitinib resistant cells were established to evaluate the molecular mechanisms of this resistance. Lysosomal sequestration and regulation of autophagy by sunitinib were analysed by different markers (lysotracker, Lamp1, intralysosomal pH or LC3B/p62 (SQSTM1)) by immunoblotting and immunofluorescence. Intracellular autophagosomes/autophagolysosomes were observed by electron microscopy. The expression of efflux transporters such as ABCB1 was quantified by immunoblotting. The efficacy of treatments combining sunitinib with specific drugs directed against new relevant targets highlighted by our study was evaluated by the quantification of cell death. Results: At sub-optimal concentrations, sunitinib accumulated in lysosomes, inhibited the activity of lysosomal proteases and led to incomplete autophagic flux. Deprivation of amino acids initiated autophagy and enhanced sunitinib resistance through the amplification of autolysosome formation. Sunitinib also stimulated the expression of ABCB1, which participates in the accumulation of the drug in autolysosomes and favours its cellular efflux. Inhibition of this transporter by elacridar and the permeabilization of lysosome membranes with Leu-Leu-O-methyl induced the apoptosis of mRCC cells resistants for sunitinib. Proteasome inhibitors also induced death of resistant cells suggesting that the ubiquitin-proteasome system compensates for the inhibition of autophagy to maintain cellular homeostasis. Conclusion: Lysosomal sequestration of sunitinib can directly influence its activity by preventing access of the drug to the tyrosine kinase receptors present in the cytoplasm, thus participating in the loss of efficacy of this drug. Based on our results we propose new therapeutic approaches that combine sunitinib with molecules that prevent lysosomal accumulation, efflux transporter or inhibition of the proteasome. No conflict of interest. 622 Thymoquinone confers higher cytotoxicity against triple negative breast cancer compared to estrogen receptor positive breast cancer through modulation of TNF receptor superfamily genes and P65 activity M. Cakir1 , C. Sakalar1 , S. Sezen1 , H. Aksu1 , B. Kurt1 , H. Canatan1 . 1 Erciyes University Faculty of Medicine, Basic Medical Sciences, Kayseri, Turkey Background: Breast cancer is the most common malignancy in females after midlife. MCF-7 is an estrogen receptor positive, luminal, paclitaxel sensitive breast cancer cell line and MDA-MB-231 is a triple negative, paclitaxel resistant breat cancer cell line. Thymoquinone is the active component of Nigella sativa. Thymoquinone has been shown to induce cytotoxicity and apoptosis in various breat cancer models. Tumor Necrosis Factor (TNF) binds to TNF receptor

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 superfamily (TNFRSF) and induces apoptosis or NF-úB or c-Jun signaling depending on the context. Materials and Methods: MCF-7 and MDA-MB-231 cell lines were grown using standart cell culture methodology. Cytotoxicity and apoptosis induced by different doses of TQ (3.125–100 mM) were evaulated by trypan blue and giemsa staining, respectively. Effect of TQ on invasion and migration of cancer cells was studied utlizing poylcarbonate membrane inserts. RNA was isolated from MCF-7 and MDA-MB-231 cells treated with low and high dose of TQ (12.5 and 50 mM) and cDNA was synthesized. Gene expression analysis was performed using a real-time PCR panel including 84 genes involved in TNF signaling and basic bioinformatics analysis was performed using online software DAVID. Protein levels were quantified by western blot. Results: TQ induces cytotoxicity in MCF-7 and MDA-MB-231 (MDA) cells with IC50 values of 16 and 5 mM, respectively. TQ (25 mM) induces apoptosis in 79(±7.8)% of MDA-MB-231cells and TQ (20 mM) induces apoptosis in 14(±4.9)% of MCF-7 cells at 24 h. TQ (12.5 mM) inhibited the migration of MDA-MB-231 cells by 52(±2)%, however inhibited the migration of MCF-7 cells only by 4(±1)%. When genes that were simultaneously affected by TQ in MCF-7 and MDA cells were analyzed, TQ has been shown to modulate the expression of TNFR1 and FAS signaling genes in both cell lines (P = 6.2×10−12 for TNFR1 and P = 7.8×10−10 for FAS). TQ (50 mM) modulates the expression 14 genes that belong to TNF receptor superfamily (TNFRSF) in MDA, however, TQ (50 mM) modulates only 6 genes of TNFRSF in MCF-7. When genes that were modulated in MDA by TQ but not modulated in MCF-7 were analyzed, TNFRSF binding genes were found to be modulated with high significance (P = 3.6×10−14 ). Since TNF receptor superfamily genes also activate NF-úB signaling, levels of P65 and its phosphorylation was evaulated in MCF-7 and MDA. P65 phosphorylation was not detected in MCF-7, however TQ reduced both full length and phosphorylated P65 in MDA. Conclusion: TQ shows higher cytotoxicity against MDA compared to MCF-7. TQ modulates TNF receptor superfamily genes in MDA differentially and in a stronger manner. Therefore, TNF receptor superfamily genes and their downstream signaling network might have a role in the impact of TQ against MDA. No conflict of interest. 623 Characterisation of a novel and potent STAT3 inhibitor, VS-43 H. Valentine1 , V. Satam2 , P. Patil2 , R. Sjoholm2 , M. Lee2 , M. Lee2 , J.A. Hartley1 , K. Kiakos3 . 1 UCL Cancer Institute, CR-UK Drug-DNA Interactions Research Group, London, United Kingdom, 2 Department of Chemistry, Georgia State University, Atlanta, USA, 3 Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria Introduction: Signal Transducer and Activator of Transcription 3 (STAT3) is constitutively activated in many cancer cell lines, leading to survival, proliferation, angiogenesis and metastasis and is therefore a promising anticancer target. Curcumin is a well-known natural STAT3 inhibitor however it lacks the potency and bioavailability to succeed in the clinic; therefore we have developed a novel STAT3 inhibitor, VS-43. Here we characterise VS-43, determining the in vitro efficacy both alone and in combination with cisplatin. Materials and Methods: Experiments were performed in both DU145 prostate cancer and A549 non-small cell lung cancer cell lines. Immunoblotting was utilised to characterise VS-43 in terms of potency, specificity, STAT3 target regulation and mode of cell death induction. The TransAM® STAT Family ELISA was used to assess selectivity of VS-43 for STAT3, and the effect of VS-43 on STAT3 DNA binding was assessed by EMSA. SRB assay was used to determine cell growth inhibition by VS-43 alone and in combination with cisplatin, and quantification of synergy was performed by combination index analysis. DNA interstrand crosslinking in cells was determined using a modification of the alkaline comet assay and cellular g-H2AX levels were assessed by confocal microscopy to determine the effect of VS-43 on cisplatininduced DNA damage. Results and Discussion: VS-43 is a potent inhibitor of STAT3 phosphorylation and activation, capable of blocking STAT3 DNA binding at concentrations as low as 0.8mM. VS-43 selectively inhibits STAT3 activation over STAT1, STAT5a and STAT5b and down-regulates STAT3 downstream target genes such as Survivin and Bcl-2. VS-43 inhibits growth of cancer cell lines DU145 and A549 with a GI50 of 1.4mM and 2.5mM respectively following an 18 hour incubation and treatment of cancer cell lines with VS-43 induces cell death via apoptosis as shown by cleaved-PARP induction. We determined by combination index analysis that VS-43 acts synergistically in combination with cisplatin, significantly reducing cisplatin GI50 values and enhancing levels of apoptosis. In comparison, STAT3 inhibitors Stattic and Curcumin displayed a lower degree of synergy with cisplatin. VS-43 also effectively inhibits pSTAT3 levels after an acute 1 hour exposure to 5mM or 10mM in DU145 and A549 cells respectively, and can also chemosensitise these cells to cisplatin using this shorter treatment schedule. VS-43 inhibits the unhooking of cisplatin-induced DNA interstrand crosslinks and significantly enhances the DNA damage response as assessed by cellular g-H2AX levels as has previously been shown with stattic and curcumin.

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Conclusion: VS-43 is a potent and selective novel STAT3 inhibitor, exhibiting chemosensitising properties. No conflict of interest. 624 A platinum-blue complex exerts its cytotoxic activity via DNA damage and induces apoptosis in cancer cells Z. Adiguzel1 , S. Ozalp-Yaman2 , G. Celik3 , S. Salem2 , T. Bagci-Onder4 , Y.C. Cetin1 , N. Arda5 , C. Acilan1 . 1 TUBITAK, Marmara Research Center, Genetic Engineering and Biotechnology Institute, Turkey, 2 Atilim University, Faculty of Engineering, Department of Chemical Engineering and Applied Chemistry, Turkey, 3 Sentegen Biotech, Bilkent Cyberpark, Cankaya/Ankara, ¸ Turkey, 4 Koc University, Medical School, Sariyer/Istanbul, Turkey, 5 Istanbul University, Molecular Biology and Genetics Department, Vezneciler/Istanbul, Turkey Background: Since the discovery of cisplatin, metal based agents have gained significant interest as potential anticancer drugs. Several drugs have been rationally synthesized to overcome the hurdles of cisplatin, majorly its toxicity and resistance. Search for less toxic, but still potent anti-cancer platinum compounds revealed a class of platinum complexes with pyrimidines called platinum–pyrimidine complexes, also known as “platinum blue” (Pt-blue) complexes. Here, we describe the characteristics of a Pt-blue complex [Pt4(2atp)8(H2O)(OH)] (2-atp: 2-aminothiophenol) as a prodrug for its DNA-binding properties and its use in cancer therapy. Material and Method: Spectroscopic measurements were made to evaluate the electronic absorption spectra, thermal behavior, viscosity, fluorometric titration and agarose gel migration of the Pt-blue treated DNA. Reactive oxygen species (ROS) were determined via DCFDA analysis. WST-1 assay was used to evaluate cell viability. DNA condensation/fragmentation analysis, live cell imaging microscopy and TUNEL analyses were used to assess apoptosis. Gene expression was evaluated through RT-qPCR. Results and Discussion: Our results suggested that the compound was able to partially intercalate DNA and appeared to induce both single and double stranded nicks on DNA in vitro, but no double stranded breaks in cells. The ability of the compound to induce DNA damage was dependent on ROS in vitro. There was elevated formation of ROS and SOD expression in response to drug treatment in cell culture. The complex was cytotoxic to a variety of cancer cells in comparison to non-cancer controls. The mean of cell death was determined to be through apoptotic pathways as assessed via biochemical, morphological and molecular observations, as well as increase in the levels of pro-apoptotic genes such as Bag3, Bak, Bik, Bmf and Hrk. Conclusion: The Pt-blue complex under study appears to be a promising molecule for anticancer therapy exerting its effect through DNA damage and grants premise for further studies. No conflict of interest. 625 Statin-induced metabolic modulation in cancer: a biomarker for targeting monocarboxylate transporters M. Mehibel1 , F. Ortiz-Martinez1 , A. Boyers1 , B. Telfer1 , K. Williams1 , I. Stratford1 . 1 University of Manchester, Manchester Pharmacy School, Manchester, United Kingdom Background: Statins are currently recommended for the treatment of hyperlipidaemia as well as prevention of cardiovascular events. Studies have suggested that statin-induced myopathy could partly be attributed to the ability of statins to interfere with metabolic pathways essential in maintaining muscle homeostasis. Interestingly, statins also possess powerful pleiotropic effects in a wide range of pathological disorders including cancer. Metabolism in tumours is largely supported by a family of transporters known as monocarboxylate transporters (MCTs), notably MCT1 and MCT4. The aim of this project is to investigate the effect of statins on metabolic modulation in head and neck cancers with a vision to predict a personalised anticancer therapy. Poor survival rates and lack of clinical biomarkers highlight an urgent need to develop more effective therapies for this deadly disease. Method: In this study, the effect of simvastatin on protein expression of MCTs and metabolic phenotypes was investigated in a panel of human head and neck cancer cells. This was followed by an evaluation of the effect of simvastatin therapy on tumour growth in human xenografts. Ex-vivo, impact on expression of MCTs was investigated by immunohistochemistry and spacial concentrations of metabolites were determined by induced metabolic bioluminescence imaging. Finally, the effect of combination therapy of simvastatin and MCT inhibition on tumour growth was assessed in vivo. Results: Simvastatin treatment resulted in an upregulation of MCT1 and MCT4 expression and changes in the metabolic phenotypes of the cell lines tested. In vivo, significant tumour growth delay was only observed when simvastatin was given prior to tumour injection and this was associated with an upregulation of MCT1 expression and a significant reduction in tumour lactate content (30.3±6.4 mmol/g for simvastatin-treated tumours versus 11.5±0.6 mmol/g for vehicle-treated tumours). This is suggestive of an association between statin therapy and a low glycolytic phenotype,

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possibly due to the addiction of simvastatin-treated tumours to lactate as a compensatory survival mechanism. Interestingly, combining simvastatin with MCT1 inhibition resulted in a significant inhibition of tumour growth as compared to simvastatin alone, probably through inhibition of lactate uptake into the cells. Conclusion: In recent years, there has been a shift towards personalised, patient-tailored treatment in cancer therapy and predictive biomarkers are potentially the most useful for clinical decision-making. Here, we propose that prediagnostic statin use warrants further clinical investigation as a predictive biomarker for targeting metabolic pathways in tumours through inhibition of MCT function. No conflict of interest. 626 MicroRNA-31 regulates chemosensitivity in malignant pleural mesothelioma via altered intracellular drug localisation H. Moody1 , M. Lind2 , S. Maher3 . 1 Hull York Medical School, School of Biological- Biomedical and Environmental Sciences, Hull- East Yorkshire, United Kingdom, 2 Hull and East Yorkshire NHS Trust, Centre for Oncology and Haematology- Castle Hill Hospital, Hull- East Yorkshire, United Kingdom, 3 University of Hull, School of Biological- Biomedical and Environmental Sciences, Hull- East Yorkshire, United Kingdom Introduction: Malignant pleural mesothelioma (MPM) is associated with extremely poor prognosis and many patients are unresponsive to treatment; developing resistance to chemotherapeutics. MicroRNA (miRNA/miR) are small, non-coding RNA that function to regulate gene expression and have been demonstrated to alter cellular sensitivity to cytotoxic agents in various cancers. MiR-31 is encoded on chromosome 9p21.3, which is reportedly the most frequently deleted genomic location in MPM tumours. Here, we examined if dysregulation of miR-31 alters MPM chemosensitivity. Material and Methods: The miR-31 deficient NCI-H2452 and miR-31 positive P31 epithelioid cell lines were used as in vitro models of MPM. Stable miR-31 overexpression or suppression was achieved via liposomal-based transfection of plasmid-based vectors and antibiotic selection. Clonogenic assay was employed as a measure of cellular sensitivity to cisplatin and carboplatin; colonies were enumerated in an unbiased manner using a GelCount device. Inductively coupled plasma mass spectrometry (ICP-MS) was utilised to quantify cellular cisplatin flux. Subcellular fractionation via sucrose gradient centrifugation was used to isolate intracellular organelles. Immunofluorescent microscopy was used to visualise intracellular localisation and protein density. Gene expression was assessed by qPCR and protein expression by Western Blot. Results: Surprisingly, reintroduction of miR-31 into cisplatin- and carboplatintreated NCI-H2452 cells significantly increased chemoresistance compared to vector controls; conversely, suppression of miR-31 in P31 cells increased cellular sensitivity to cisplatin. Additionally, miR-31 reintroduction mediated a delay in the cytotoxic activity of chemotherapy. Interestingly, a higher relative intracellular concentration of platinum was observed in miR-31 transfected cells, potentially as a result of increased expression of the plasma membranebound cisplatin influx transporter CTR1. However, a concurrently significantly decreased intranuclear concentration of platinum was determined in miR-31 expressing cells, suggesting altered nuclear transport or sequestration within the cytosolic compartment. Subsequently, we identify that a miR-31-mediated increase in the lysosomal-associated drug transporter ABCB9, in conjunction with reduced expression of the associated bipotential transcription factor OCT-1, may promote the extranuclear sequestration of chemotherapeutic agents in lysosomes in MPM cells expressing miR-31. Conclusions: Here, miR-31 expression was found to significantly enhance chemoresistance in MPM cells in vitro. While deletions in the genomic location encoding miR-31, 9p21.3, may be associated with an overall poor prognosis, the loss of miR-31 may not actually contribute to the chemoresistance observed in MPM patients. No conflict of interest. 627 Oxidized products of a-linolenic acid negatively affect cell survival and motility of breast cancer cells J. Gutierrez-Pajares1 , C. Oger2 , J.M. Galano2 , T. Durand2 , S. Chevalier1 , P. Frank1 . 1 Universite Francois Rabelais, INSERM1069, Tours, France, 2 ´ Universite´ de Montpellier, Institut des Biomolecules Max Mousseron IBMM UMR 5247 − CNRS − UM − ENSCM, Montpellier, France Background: Cancer is a major cause of death in the world, and more than six million new cases are reported every year. Despite recent advances in our understanding of the biological processes leading to the development of cancer, there is still a need for new and effective agents to treat this disease. Nature is an attractive source of new therapeutic compounds, as a tremendous chemical diversity is found in millions of species of plants, animals, and microorganisms. Plant-derived compounds have played an important role in the development of several clinically useful anti-cancer agents. Phytoprostanes (PhytoP) and phytofurans (PhytoF) are non-enzymatically

oxidized products derived from of a-linolenic acid (C18:3 n-3, ALA) that are present in seeds and vegetable oils. They have been shown to possess antiinflammatory and apoptosis-promoting activities in macrophages and leukemia cells, respectively. In this work, seven PhytoPs and one PhytoF were tested on breast cancer cell lines to determine their effect on cell viability and motility. Materials and Methods: PhytoP and PhytoF were synthesized according to previously published methods. These compounds were evaluated for cytotoxic, chemosensitization and anti-migratory activities using the MCF-7 and MDA-MB-231 breast cancer cell lines. Cell survival, proliferation and chemosensitization analysis were conducted using Crystal violet staining assay. Wound-healing and the Boyden chamber assays were performed to study 2D and 3D migration, respectively. Additionally, cell adhesion to serum extracellular matrix protein was also measured. Results and Discussion: Among the compounds tested, only three PhytoPs had a significant effect on cell viability compared to the control group: Ent-9L1-PhytoP decreased cell viability in both cell lines, while 16-F1t-PhytoP and 9-L1-PhytoP decreased viability in MCF-7 and MDA-MB-231 cells, respectively. When combined with a sub-cytotoxic dose of Doxorubicin, these three PhytoPs significantly enhanced the cytotoxic effect on MCF-7 cells while the chemotherapeutic drug alone had no effect on the cells. In cell motility assays, Ent-9-(RS)-12-epi-ST-delta10-13-PhytoF was able to significantly inhibit cell migration of MDA-MB-231 cells in both the wound-healing and Boyden chamber assays. In addition, Ent-9-(RS)-12-epi-ST-delta10-13-PhytoF also enhanced cell adhesion of MDA-MB-231 cells. Considering the structural similarities between PhytoP/PhytoF and isoprostanes, it is possible that these compounds may be modulating prostaglandin signaling pathways and may therefore interfere with cell survival and motility. Conclusion: This study shows for the first time that the plant-derived compounds phytoprostanes and phytofurans could be further exploited alone or in combination with chemotherapy to improve the arsenal of therapies available against breast cancer. No conflict of interest. 628 Identification of a pyrrolo-pyrimidin derivative to overcome the resistance to apoptosis in non-small cell lung cancer cells P. Gilson1 , F. Mahuteau2 , C. Beauvineau2 , J.L. Coll1 , A. Hurbin1 , B. Busser1 . 1 UGA/INSERM U1209/CNRS UMR5309, Institut Albert Bonniot, Grenoble, ´ France, 2 Institut Curie UMR9187/U1196, CMIB Chimie- Modelisation et Imagerie pour la Biologie, Orsay, France Background: Non-small cell lung cancer (NSCLC) is associated with a poor prognosis and a high resistance to apoptosis that makes it a strong therapeutic challenge. In this context, we designed a cell-based assay to identify new molecules restoring apoptosis in NSCLC cells resistant to serum starvation. We screened a chemical library composed of 7520 molecules and ultimately identified 15 compounds with a common pyrrolo-pyrimidin pharmacophore, supposed to inhibit tyrosine kinase activity. We investigated their anti-tumor effects revealing a new strategy for the treatment of patients with resistant NSCLC. In particular, we focused on a pyrrolo-pyrimidin member, named molecule IAB-11. Material and Methods: Using a wide panel composed of cancer cell lines (colon, breast, . . . ) including NSCLC cell lines (containing wild type and mutant EGFR cell lines), we determined the cytotoxicity of molecule IAB-11, its proapoptotic activity and its impact on cell cycle, by MTT assays and flow cytometry analyses. We also investigated the targets of molecule IAB-11 by a kinome array, immunofluorescence assays, tubulin polymerization tests and western blotting. The toxicity and antitumor effects (both on lung tumor growth and metastasis invasion) of molecule IAB-11 were evaluated in vivo. Results: At low doses, molecule IAB-11 restored serum deprivation-induced apoptosis, whereas no noticeable effect was observed in complete medium, suggesting a specific action on NSCLC resistant cells. Moreover, the molecule IAB-11 promoted mitotic cell cycle arrest by impairing mitotic spindles organization and consequently the proper orientation of the chromosomes in metaphase. The spindle pole composition was normal whereas kinetochore microtubules were misoriented and the astral microtubules were highly developed. It also modified microtubule dynamics with a small depolymerizing effect. Our preliminary results also suggested a perturbation of a signalling pathway involved in the resistance to apoptosis and previously described by our laboratory. A kinome array experiment established that kinases (including mitotic kinases) were not the direct targets of molecule IAB-11. Interestingly, in vivo experiments showed that the molecule IAB-11 had antitumor and antimetastatic effects without any noticeable toxicity on chicken embryos. Conclusions: We identified a novel molecule that exerts cytostatic and proapoptotic effects on a specific lung cancer survival pathway. Our preclinical studies also established its ability to reduce both tumor growth and invasiveness that makes it a promising strategy for the treatment of resistant NSCLC. We are currently looking deeper into the characterization of molecule IAB-11’s targets and mechanism of action. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 629 Targeting the PI3K/AKT/mTOR pathway with a dual mTORC1/2 inhibitor in bladder cancer. Rationale for its testing in clinical trials 1,2 1,2 A. Hernandez-Prat ´ , A. Martinez1,2 , O. Arp´ı1,2 , S. Menendez ´ , N. Iarchoukc3 , F. Rojo1,4 , J. Albanell1,2,5 , R. Brake3 , A. Rovira1,2 , J. Bellmunt1,6,7 . 1 Cancer Research Program, IMIM Hospital del Mar Research Institute, Barcelona, Spain, 2 Medical Oncology Department, Hospital del Mar, Barcelona, Spain, 3 Milllennium Pharmaceuticals Inc., Millennium, Cambridge, MA, USA, 4 Hospital Universitario Fundacion Jimenez DiazMadrid- Spain, Molecular pathology department, Madrid, Spain, 5 Medical School, Universitat Pompeu Fabra, Barcelona, Spain, 6 Dana-Farber Cancer Institute, Dana-Farber Cancer Institute, Boston, MA, USA, 7 Bladder Cancer Center- Dana-Farber Cancer Institute- Brigham and Women’s Cancer Center, Bladder Cancer Center, Boston, MA, USA

Background: PI3K/AKT/mTOR pathway is a promising target for cancer treatment being commonly deregulated in human bladder tumors. Pathway activation results in the promotion of tumor cell growth, survival, and resistance to chemotherapy. The aim of this study is to characterize the effects of TAK228, a dual mTOR inhibitor targeting the PI3K/AKT/mTOR pathway in bladder cancer. Materials and Methods: TAK-228 is a novel ATP-competitive inhibitor of the serine/threonine kinase mTOR. TAK-228 inhibits the activity of both complexes mTORC1 and mTORC2. We evaluated the effects of the drug as single agent or in combination with an upstream targeted therapy MLN1117 (PI3Ka inhibitor) or with chemotherapy (Paclitaxel). The effects of the agent alone or in combination were analysed in a panel of six selected bladder cancer cell lines and in tumor xenografts. These models were selected based on specific genomic alterations considered to be relevant for therapeutic intervention (PIK3CA and TSC1 mutations). Effects of TAK-228 and of the combinations on cell viability, on PI3K/AKT/mTOR signal pathway, cell-cycle, apoptosis, and autophagy were tested in vitro and in vivo. Previous reported biomarkers of sensitivity or resistance to TAK-228 were analysed. Results and Discussion: TAK-228 strongly inhibited cell proliferation through PI3K/AKT/mTOR pathway inhibition in bladder cancer cell lines with diverse genetic backgrounds. TAK-228 inhibited tumor growth and angiogenesis in the in vivo xenographs. Both TAK-228 + MLN1117 or TAK-228 + Paclitaxel combinations, produced synergistic antiproliferative effects in cell lines and improved the effect of each drug alone in vitro and in vivo. A decline on 4E-BP1 and on the ratio of p-4E-BP1/4E-BP1 was correlated with cellular responses to TAK-228. Conclusions: Our preclinical studies in bladder cancer models show that TAK228 is a promising investigational agent that warrants further investigation as novel anti-cancer agent alone or in combination with chemotherapy in bladder clinical trials. No conflict of interest. 630 Resistance to photodynamic therapy in glioblastoma cancer cells S. Shahmoradi Ghahe1 , B. Majchrzak1 , K. Kopania1 , A. Ciuba1 , B. Tudek1,2 . 1 Institute of Genetics and Biotechnology, Faculty of Biology- University of Warsaw, Warsaw, Poland, 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Glioblastoma is the most frequent and deadliest brain tumor of astrocytic origin. It is resistant to all current therapies and is associated with a high rate of recurrence. Glioblastoma was shown to respond to treatment by the method of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). Since there are still some cells which escape the treatment, our study was designed to determine the markers of resistance, characterize them and sensitize glioblastoma PDT resistant cells to PDT. Glioblastoma (U-87) cell line resistant to PDT (U-87R) was isolated from parental, sensitive line (U-87P) by applying several cycles of ALA-PDT, and further growing surviving cells. Doubling time of U-87R cells was considerably longer in than that of U-87P. Cell cycle analysis showed longer G1 phase in U-87R cells. Moreover, accumulation in G2 phase following PDT was observed 24 hours earlier in resistant cells than in sensitive. Western blotting analysis showed that in untreated cells histone H2AX was phosphorylated at Ser139 only in U-87R line, and quickly disappeared following PDT. H2AX phosphorylation arose a couple of hours after PDT in parental cells. This suggests early activation of DNA damage response and increase in double strand breaks repair capacity in resistant cells. Resistant glioblastoma cells were sensitized to PDT by applying inhibitor of one of the main kinases of DNA damage response, ATM. Moreover, over-expression of RNA, and protein and higher activity of some other DNA repair enzymes were observed in glioblastoma PDT resistant cells. In addition to studying the role of DNA repair in resistance to PDT in glioblastoma cancer cells, the level of oxidative stress, accumulation of protoporphyrin IX as the photosensitizer in ALA-PDT and whole protein profile of cells were studied. Our data showed that many factors are responsible for conferring resistance to PDT in glioblastoma cells, and repair of double strand breaks is one of important characteristics of these cells.

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Acknowledgements: This research is supported by National Science Center of Poland, grant number: UMO-2014/15/B/NZ5/01444. No conflict of interest. 631 Combination of photodynamic therapy with doxorubicin in osteosarcoma: Cell death and the role of oxidative stress B. Serambeque1 , G. Brites1 , M. Laranjo2 , G. Chohfi de Miguel3 , A. Serra4 , M. Pineiro5 , A.M. Abrantes2 , J. Casalta-Lopes1 , A. Rocha-Gonsalves5 , A.C. Gon¸calves6 , A.B. Sarmento-Ribeiro7 , D. Priolli3 , M.F. Botelho2 . 1 Faculty of Medicine of University of Coimbra, Institute of Biophysics/Biomathematics, Coimbra, Portugal, 2 Faculty of Medicine of University of Coimbra, Institute of Biophysics/Biomathematics- CIMAGO- CNC.IBILI, Coimbra, Portugal, 3 ˜ Francisco, Bragan¸ca Paulista, Brazil, 4 Faculty of Universidade de Sao Sciences and Technology of University of Coimbra, Department of Chemical Engineering, Coimbra, Portugal, 5 Faculty of Sciences and Technology of University of Coimbra, Department of Chemistry, Coimbra, Portugal, 6 Faculty of Medicine of University of Coimbra, Applied Molecular BiologyUnit, Coimbra, Portugal, 7 Faculty of Medicine of University of Coimbra, Applied Molecular Biology Unit, Coimbra, Portugal Introduction: Osteosarcoma (OS) is the most common malignant tumor that arises from primitive mesenchymal bone-forming cells. Doxorubicin (DOX) remains among the most widely used antineoplasic agents. Photodynamic therapy (PDT) is a non-mutagenic anticancer therapy. Several studies reported that the association of chemotherapy with PDT may contribute to the outcome, to overcome drug resistance and to provide an approach to unresectable tumors. Therefore, the main goal of this work was to evaluate cell death and the oxidative stress in OS cells treated with the combination of doxorubicin and photodynamic therapy (DOX-PDT). Methods: The MNNG-HOS human OS cell line was cultured as recommended and used in all studies. We used 2millions cells for each condition, namely, non-treated cells (control), cells submitted to DOX, cells treated with PDT and cells subjected to DOX-PDT. The evaluation was performed 72 hours after DOX administration. In order to evaluate the types of cell death (annexin-V and propidium iodide labeling), the mitochondrial membrane potential (JC-1 probe), cell cycle (propidium iodide) and oxidative stress (DCFDA and dihydroethidium probes) we proceeded to flow cytometry technique. Results and Discussion: DOX-PDT induced a sharper loss of mitochondrial membrane potential and the predominant type of cell death induced was later apoptosis/necrosis. In terms of cell cycle, there was a retention in G2/M phase in cells subjected to DOX and in cells subjected to DOX-PDT, which is also an indication of cell death by apoptosis. The cells subjected to the combined treatment also showed a higher intracellular production of peroxides and of superoxide anion and there was also an increase in antioxidant defenses. Therefore, the DOX-PDT seems to have a synergistic cytotoxic effect on OS cells. Conclusion: These results show that the predominant cell death was later apoptosis/necrosis and the production of reactive oxygen species was in part rectified by the production of antioxidant defenses in cell cultures subjected to combined treatment. This work also reveals the potential of this combination as a future therapeutic approach in osteosarcoma. Acknowledgment: Funding: FCT, Portugal (UID/NEU/04539/2013), COMPETEFEDER and Liga Portuguesa Contra o Cancro/ANA. No conflict of interest. 632 MicroRNA SNPs as novel markers of methotrexate toxicity in pediatric acute lymphoblastic leukemia L. Iparraguirre1 , M. Umerez1 , A. Gutierrez-Camino1 , I. Mart´ın-Guerrero1 , N. Garc´ıa de Andoin2 , A. Sastre3 , A. Navajas4 , I. Astigarraga4,5 , A. Garc´ıa-Orad1,4 . 1 University of the Basque Country, GeneticsPhysical Anthropology and Animal Physiology, Bilbao, Spain, 2 Hospital Donostia, Pediatrics, San Sebastian, Spain, 3 University Hospital La Paz, Oncohaematology, Madrid, Spain, 4 BioCruces Health Research Institute, Pediatrics, Barakaldo, Spain, 5 University Hospital Cruces, Pediatrics, Bilbao, Spain Introduction: Methotrexate (MTX) is a drug used at high doses in pediatric acute lymphoblastic leukemia (ALL) therapy, which is known to improve survival. Nevertheless, it often causes toxicity, requiring a dose reduction or treatment cessation, so it is necessary to predict which patients will develop MTX toxicity. Most of MTX pharmacogenetic studies including GWAS have focused mainly in coding genes involved in drug absorption, distribution, metabolism and excretion (ADME genes) with promising results. It is known that several of those genes are regulated by microRNAs (miRNAs). Variants in miRNAs have already been associated with drug toxicity. In fact, in a preliminary study in 46 miRNA variants, we found significant associations with MTX levels. In this context, we try to determine the role of miRNA variants in MTX toxicity in a larger amount of SNPs and samples.

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Material and Methods: Blood samples of 167 B-cell ALL patients treated with LAL/SHOP protocol were analyzed. We selected all the SNPs described in pre-miRNAs with a MAF >1% (213 SNPs in 206 miRNAs). Genotyping was performed with VeraCode GoldenGate platform. Results and Discussion: We found a SNP in miR-595 significantly associated with high MTX plasma levels. Interestingly, in silico analysis determined that this miRNA could regulate three of the transporters involved in MTX uptake: SLC19A1, SLCO1A2 and SLC46A1. Moreover, other 6 miRNAs with significant results in this study target two of these transporters; miR-1206, miR-4432 and miR-5007 bind to SLCO1A2 and miR-5189, miR-604 and miR-4268 to SLC46A1. Conclusion: SNPs in miRNAs that regulate genes of the MTX pharmacokinetics pathway could be novel markers for high MTX levels in patients with pediatric B-ALL. These results open a very promising area in pharmacogenetics if we consider that miRNA expression could be exogenously controlled. Acknowledgment: This project was supported by RETICS (RD/12/0036/0060 and RD/12/0036/0036) and Basque Government (IT661-13). No conflict of interest. 633 Identification and development of novel Smoothened antagonists for the treatment of Hh-dependent tumors R. Alfonsi1 , M. Mori2 , P. Infante2 , F. Ghirga2 , B. Botta3 , L. Di Marcotullio1 . 1 Universita` La Sapienza, Molecular Medicine, Roma, Italy, 2 Istituto Italiano di Tecnologia, CLNS@Sapienza, Roma, Italy, 3 Universita` La Sapienza, Chimica e Tecnologia del Farmaco, Roma, Italy Background: The Hedgehog (Hh) pathway has recently emerged as a “druggable” therapeutic target in cancer, since its crucial role in tissues development, proliferation and in the maintenance of cancer stem cells (CSCs) in tumors. The Smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/ErivedgeTM) for the treatment of basal cell carcinoma. However, novel Smo mutations that confer constitutive activity and drugs resistance have been reported during Vismodegib treatment. For this reason, the development of additional Smo inhibitors represents a major challenge for cancer therapy. Material and Methods: In order to identify new Hh inihibitors, we performed a molecular docking simulation experiment. An in house library of natural, synthetic or semi-syntetic compounds was screened in silico towards the crystallographic structure of the Smo receptor in complex with the natural antagonist cyclopamine. Only compounds virtually capable of fitting the antagonist binding site within the heptahelical bundle of Smo were tested for the ability of inhibit the signaling by a luciferase functional assay. The most active compound was validated by proliferation assays in vitro in different Hedgehog-dependent tumor cells as well as in cerebellar granule cells progenitors (GCPs) and medulloblastoma stem-like cells. Results: Hh functional-based assay identified the 2 ,4 ,5 ,3,4-pentamethoxychalcone, namely Chalcone, as the most effective Hh inhibitor within the test set. Chalcone displayed a high percentage of displacement of BodipyCyclopamine, in both Smo-WT and the drug-resistant mouse orthologous SmoD477G mutant, in a dose dependent manner. Moreover, it was able to inhibit the growth of both cerebellar granule cells progenitors and Hhdependent tumor cells, as well as the proliferation of primary medulloblastoma cells derived from Ptch1+/− mice tumors, and the clonogenic self-renewal ability of medulloblastoma stem cells. Conclusions: In this work we discover a new specific Smo inhibitors that impairs the Hh-dependent tumor and cancer stem cells growth, and stands as a valuable starting point to develop potential therapeutic agents for Vismodegib- or Sonidegib-resistant tumors. No conflict of interest. 634 Design and evaluation of novel theranostic fluorogenic dual probe-prodrug in cancer S. Mathur1 , D. Mincher1 , A. Turnbull1 , C. Stevens1 , A. Poole1 . 1 Edinburgh Napier University- Edinburgh- UK, Molecular Drug Research Laboratory, Edinburgh, United Kingdom Background: In spite of major advances in the diagnosis and treatment of cancer, there remains a paucity of biomarkers for early detection. Legumain is a potential cancer biomarker and a molecular target for imaging and drug targeting. Legumain is a lysosomal protease with a remarkably restricted specificity, as it cleaves only substrate sequences having an asparagine (Asn) at the P1 site. We have previously shown that a legumain substrate probe (SM9), Pro-Ala-Asn~PEG-AQ, is quenched until activated by proteolytic action of legumain. Design of first generation molecular probe SM9 has been extended to a second generation theranostic dual probe-prodrug SM20 (RhoPro-Ala-Asn~Lys-PEG-AQ) that serves as both a diagnostic and therapeutic (theranostic) tool for cancer. Material and Methods: Solution and solid phase peptide methods have been used to create a novel rhodamine-labelled tetra peptide second

generation theranostic dual probe-prodrug (SM20) of legumain, which was purified by chromatographic methods and characterised by high resolution mass spectrometry, together with all synthetic intermediates. Fluorescence spectroscopic methods were used to determine the efficiency of FRET in the prodrug (SM20). In vitro fluorimetric assay was developed with human recombinant legumain at 370C in MES assay buffer (pH 5.0). The lipophilic character (Log D) of the SM20 was assessed by distribution coefficient measurements. Confocal microscopy studies were performed to investigate the cellular uptake and sub-cellular localization of both SM9 and SM20 and their legumain-mediated cleavage fragments in PC3 prostate cancer cells. Results: The fluorogenic second generation prodrug (SM20) is an efficient FRET substrate and affords good restoration of fluorescence when incubated at 10mM with rh-legumain (40 ng) in legumain assay buffer (pH-5.0; lex 544 nm, lem 585 nm). Cleavage at the designed Asn~Lys (SM20) cleavage ‘hotspot’ was demonstrated at concentrations of legumain in the 5−40 ng range in in vitro metabolism experiments using recombinant legumain. The second generation theranostic prodrug (SM20) is structurally similar to the SM9 probe, with a different aminoanthraquinone quencher, which is lysosomotropic. Measurement of the distribution coefficients have shown that SM20 is more lipophilic than the Lys-PEG-AQ cleavage product from which it is derived. Confocal studies for 0−1 hr at 63× magnification have shown that both SM9 (1M) and SM20 (1M) were localized in the lysosomes of PC3 prostate cancer cells. Conclusions: The theranostic dual probe-prodrug (SM20) is an efficient substrate for sensitive and early detection of legumain and a smart therapeutic agent for cancer. Work is ongoing to optimize the dual probe-prodrug and to extend the studies in vivo. No conflict of interest. 635 Unveiling the mechanisms of exemestane-acquired resistance: The role of autophagy and PI3K pathway C. Amaral1 , T. Augusto1,2 , E. Tavares-da-Silva3,4 , F.M.F. Roleira3,4 , G. Correia-da-Siva1 , N. Teixeira1 . 1 UCIBIO.REQUIMTE- Faculty of PharmacyUniversity of Porto, Laboratory of Biochemistry- Department of Biological Sciences, Porto, Portugal, 2 Faculty of Sciences- University of Porto, FCUP, Porto, Portugal, 3 Faculty of Pharmacy- University of Coimbra, Pharmaceutical Chemistry Group, Coimbra, Portugal, 4 CNC.IBILI- University of Coimbra, Pharmaceutical Chemistry Group, Coimbra, Portugal Introduction: Aromatase inhibitors (AIs) are one of the therapeutic approaches used for estrogen-dependent (ER+) breast cancer, being Exemestane (Exe) the third-generation steroidal AI used in clinic. Despite its therapeutic success, Exe-acquired resistance is a major drawback, causing tumor relapse. In order to find new targets to improve breast cancer treatment our group has been studying the Exe effects in sensitive and resistant breast cancer cells. We showed that Exe induces apoptosis and autophagy in breast cancer cells, being autophagy a pro-survival process that may be involved in resistance [1,2]. Besides, PI3K/Akt is considered the major pathway involved in endocrine resistance. Therefore, in this work, it was investigated the link between autophagy and PI3K pathway in Exe-acquired resistance. Material and Methods: Using an AI-resistant breast cancer cell line (LTEDaro) treated with Exe, it was explored the biological effects of 3-methyladenine (3MA) and Ly294002 (LY), an autophagic and PI3K inhibitors, respectively. The effects on cell viability were assessed by MTT/LDH assays and the occurrence of apoptosis by the activation of caspases-9 and -7. Autophagy was evaluated by the formation of acid vesicular organelles (AVOs) by flow cytometry and through the LC3 expression by Western-blot. The involvement of PI3K and MAPK pathways were analyzed by Western-blot through the expression of phosphorylated Akt, p44/42, p38 and PI3K. Results: Contrary to Exe, the autophagic inhibitor 3-MA caused a reduction in viability of Exe-treated LTEDaro cells and an increase in caspase-9/-7 activities. As expected, a decrease in AVOs formation and inhibition of LC3 turnover were observed. Additionally, it was detected an increase in p38 phosphorylation and a decrease in PI3K activation. Thus, LY, which inhibits PI3K activation and Akt phosphorylation, was used to explore the involvement of PI3K pathway. The LY decreased viability of Exe-treated LTEDaro cells and increased caspase-9/-7 activities. So, results suggest that LY enables Exeresistant cells to have a similar behavior as cells treated with 3-MA. Conclusion: By targeting autophagy and PI3K pathway, it may be possible to sensitize acquired-resistant breast cancer cells to Exe-treatment, by inducing apoptosis through mitochondrial pathway. This work contributes to understand the link between autophagy and PI3K pathway in AIs-resistance, providing new insights on the mechanisms involved in Exe-acquired resistance. Moreover, it highlights new targets that can improve breast cancer treatment. Acknowledgements: FCT: Amaral C. grant (SFRH/BPD/98304/2013) and (UID/MULTI/04378/2013−POCI/01/0145/FERDER/007728); Prof. S. Chen (Beckman Research Institute, USA) for LTEDaro cells. Reference(s) [1] Amaral C. et al. (2012), PLoS ONE, 7(8):e42398. [2] Amaral C. et al. (2013), J Steroid Biochem Mol Biol, 135:51−9. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 636 Performance evaluation of a new integrated blood collection/sample preparation system for the stabilization and extraction of circulating cell-free DNA (ccfDNA) ¨ 1 . 1 Qiagen GmbH, PreAnalytiX Product A. Ullius1 , T. Voss1 , D. Grolz Development, Hilden, Germany Background: The application of circulating cell-free DNA (ccfDNA) as biomarker is an emerging field in cancer research, e.g. to monitor and detect tumors. The sensitivity of rare mutant allele fraction analysis and quantification of circulating tumor DNA (ctDNA) is, however, compromised by the release of genomic DNA from lymphocytes due to mechanical lysis or apoptosis during collection, transport and storage of the blood sample. Moreover, the extraction of the low concentrated and highly fragmented ccfDNA is technically challenging. PreAnalytiX has developed the PAXgene® Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic BD Vacutainer® tube with unique, non-crosslinking chemistry preserving extracellular levels of ccfDNA and preventing the release of intracellular DNA from cells into the plasma, and the QIAsymphony® PAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. The fully automated QIAsymphony PAXgene Blood ccfDNA workflow offers two protocols to choose between purification of predominately small ccfDNA fragments or co-isolation of large ccfDNA fragments to cover the needs for diverse research applications such as organ disease and cancer. Here we present the stabilization and extraction efficiency of the new system compared to blood samples collected in EDTA tubes. Methods: Whole blood from up to 20 donors in three replicates was collected into PAXgene Blood ccfDNA tubes or spray-dried K2EDTA tubes and stored for up to 7 days at 25ºC. For transport studies, blood tubes were rotated for 5 hours, followed by horizontal storage for up to 7 days. ccfDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit or the QIAsymphony PAXgene Blood ccfDNA Kit and the obtained ccfDNA was analyzed by quantitative real-time PCR, using assays with validated performance. Two automated extraction protocols were compared for their efficiency to purify in addition to small ccfDNA also longer ccfDNA fragments. Results: ccfDNA yield from PAXgene Blood ccfDNA tubes was comparable to blood drawn into EDTA tubes, when plasma was processed directly after blood draw. In contrast to blood from EDTA tubes, ccfDNA yield did not increase significantly during simulated transport and storage. A direct comparison between the two protocol lines (STA and LAF) demonstrated efficient coisolation of large ccfDNA fragments in addition to unbiased isolation of small ccfDNA fragments for the LAF protocol. Conclusions: The PAXgene Blood ccfDNA Tube stabilizes blood cells for transport and short time storage. The fully automated QIAsymphony PAXgene Blood ccfDNA workflow offers two protocols for purification of predominately small ccfDNA fragments or co-isolation of large ccfDNA fragments to provide optimal sample conditions for various research applications. For Research Use Only. Not for use in diagnostic procedures. Conflict of interest: Other Substantive Relationships: QIAGEN employee.

638 Speeding up the S5 XL sequencing system: Sequencing in an hour enables sample to answer in a 8 hr workday R. Qi1 , C. Parikh1 , A. Luchetti2 , D. Mandelman3 , H. Latif3 , A. Harris3 , S. Ghosh1 . 1 Thermo Fisher Scientific, Ion Torrent, South San Francisco, CA, USA, 2 Thermo Fisher Scientific, Ion Torrent, Carlsbad, CA, USA, 2 Thermo Fisher Scientific, Ion Torrent, San Diego, CA, USA The Ion S5 XL™ Sequencer currently sequences 200 bp in 2.5 hrs with about 1 hr analysis time. Here we focused on optimization of the sequencing and data analysis workflow in an effort to reduce total turn around time (TAT). Since it largely depends on and the number of reagent flows (one flow produces ~0.5 base) and the speed of the flows, we improved the flow speed and utilized the minimum number of flows optimized for the length of the amplicons. We used an existing streamlined library preparation workflow based on AmpliSeq, clonal amplification (templating) with Isothermal Amplification, and the new rapid Ion S5 XL™ Sequencer procedure. The new rapid approach was utilized with two different types of applications that have the potential to benefit from rapid sequencing turn-around times. (1) A rapid infectious disease identification and antibiotic susceptibility panel based on the AmpliSeq highly-multiplexed PCR approach. (2) A whole genome amplification based approach for detection of aneuploidies for pre-implantation Genetic Screening of fresh to allow a same or next day decision for implantation of in vitro fertilized embryos. With Ion S5 XL™ Sequencer and modifications to the on-instrument flow times and total number of flows, we can reduce the time for sequencing and accurate data analysis for applications requiring rapid TAT. For AmpliSeq and ReproSeq assays, sequencing and Torrent Suite analysis could be completed in as short as 55 minutes (200 flows). Paired with fast library and template preparation, the total TAT was at or below a standard workday. We demonstrated that accuracy of detection was not compromised with the reduced TAT. No conflict of interest.

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639 Cold atmoshperic plasma as an approach to retinoblastoma R. Silva-Teixeira1 , M. Laranjo2 , G. Brites1 , A.M. Abrantes2 , T. Rodrigues3 , A.C. Gon¸calves4 , S. Raquel3 , A.B. Sarmento-Ribeiro4 , P. Matafome5 , F. Caramelo1 , M.F. Botelho2 . 1 Faculty of Medicine of University of Coimbra, Institute of Biophysics/Biomathematics, Coimbra, Portugal, 2 Faculty of Medicine of University of Coimbra, Institute of Biophysics/BiomathematicsCIMAGO- CNC.IBILI, Coimbra, Portugal, 3 Faculty of Medicine of University of Coimbra, Laboratory of Physiology- CNC.IBILI, Coimbra, Portugal, 4 Faculty of Medicine of University of Coimbra, Applied Molecular Biology Unit, Coimbra, ´ Portugal, 5 Instituto Politecnico de Coimbra- Coimbra Health School ESTeSC, Department of Complementary Sciences, Coimbra, Portugal Background: Retinoblastoma is the most common primary intraocular tumor in children. Histologically, necrosis found in relatively avascular areas demonstrate the dependence of retinoblastoma on its blood supply. Unfortunatelly, available therapies lack selectivity and are associated to serious side effects such as loss of vision and secondary neoplasias. In order to circumvent these effects our group is studying plasma, an ionized gas, as an option. Therefore, the aim of this work was to evaluate the effect and selectivity of cold atmospheric plasma (CAP) in human retinoblastoma. Materials and Methods: An electronic device capable of generating high output voltage (HV; ~4kV) was designed to produce an electrical discharge between the HV electrode and multiwell plates. Human retinoblastoma Y79 cells were seeded in multiwell plates. CAP was generated in open air, 2 mm above the surface of the cell cultures medium, during several short periods of time, ranging from 15 s to 180 s. In order to evaluate the cytotoxicity of the plasma flair, Alamar Blue and MTT assays were performed. In addition, cell viability was assessed by SRB assay. Moreover, annexin V/ propidium iodide (PI) apoptosis detection kit and PI/RNAse staining solution for Flow Citometry were used to study type of cell death and cell cycle, respectively. Concerning selectivity, human fibroblasts HFF1 were treated similarly and Alamar Blue was performed. Furthermore, to evaluate the potential anti-angiogenic effects of CAP, an aortic ring assay was carried out in excised mouse thoracic aorta segments. All the tests were performed 24 hours after CAP exposure. Results and Discussion: The metabolic activity of retinoblastoma cells significantly decreased accordingly to the CAP exposure time. After 60 s of CAP exposure, the metabolic activity was (37.5±19.7)% (p < 0.001) and (57.2±21.2)% (p = 0.047) as evaluated by Alamar Blue and MTT assay, respectively. Furthermore, the time to reduce this activity in 50% (IT50) was 68.1 s and 79.2 s, accordingly to the previous techniques. Moreover, after 60 s of CAP exposure a viability of (56.3±19.6)% (p = 0.048) was obtained SRB assay, which reveals cell death as the main cause of obtained results. Preliminary results show both the contribution of apoptosis and necrosis to cell death and a shift of cells populations in each phase of the cell cycle. However, regarding the fibroblasts, after 60 s of CAP exposure the metabolic activity was still (74.5±13.1)% (p = 0.058) with an IT50 of 109.6 s. Furthermore, our preliminary results on aortic ring assay revealed that CAP might have antiangiogenic effects. Conclusions: These results suggest that CAP is cytotoxic and might be selective to tumor cells (p < 0.001). Furthermore it might have a deleterious effect on tumor neoangiogenesis. Acknowledgment: Funding: FCT, Portugal (UID/NEU/04539/2013), COMPETEFEDER. No conflict of interest. 640 Selective inhibition of HDAC1 and HDAC2 counteracts medulloblastoma cell growth in mouse models through Gli acetylation S. Coni1 , A.B. Mancuso1 , L. Di Magno2 , G. Sdruscia2 , D. Rotili3 , A. Mai3 , G. Canettieri1 . 1 Sapienza University, Molecular Medicine, Rome, Italy, 2 Italian Institute of Technology, Center for Life Nanoscience@Sapienza, Rome, Italy, 3 Sapienza University, Department of Drug Chemistry and Technologies, Rome, Italy Introduction: Medulloblastoma (MB) is one of the most aggressive pediatric brain tumor and, despite a combination of surgery and chemotherapy, the prognosis is still poor for a significant fraction of patients. Hedgehog pathway is a crucial regulator of cerebellar development and its aberrant activation is a leading cause of MB. Pharmacological inhibition of Hedgehog pathway using Smo inhibitors has been proven effective in clinical trials, although resistance eventually occurs. Thus, targeting downstream Hh effectors appears to be a preferable option. In this view, we previously identified Gli acetylation as a critical druggable inhibitory modification for Hedgehog signaling, selectively regulated by HDAC1 and HDAC2. Aim of this work was to test the efficacy of HDAC1/2 inhibitors in MB growth in vitro and in preclinical mouse models. Material and Methods: Allografts, RNAi, proliferation curves, were performed as described in D’Amico et al. 2015. RNA studies, BrDu proliferation assays, immunohistochemistry and western blots were performed as described in Canettieri et al. 2010. Results: We first tested whether inhibition of HDAC1 and 2 is sufficient to suppress Hh signaling. Knockdown of HDAC1 or HDAC2 caused a strong

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repression of Hedgehog target gene expression and proliferation rate in MB tumor cells (Med1-Mb and primary cultures of mouse MB). We then tested the selective HDAC1/2 inhibitor Mocetinostat at appropriate concentrations in MB cells. Treatment with Mocetinostat led to a significant decrease of proliferation rate that was accompanied to an increase of Gli acetylation. Importantly, the proliferation rate of MB cells stably expressing Gli was inhibited by Mocetinostat, while cell stably expressing Gli K/R (consistutively deacetylated), were not affected by the drug, indicating that Gli acetylation is the critical event for the effect of mocetinostat. Finally Mocetinostat treatments in allografted nude mice caused a 60% reduction of tumor growth after 3 weeks of administration, accompanied to increased Gli acetylation and repression of Hedgehog target genes. Conclusion: Together our results indicate that selective inhibition of HDAC1 and HDAC2 causes a robust reduction of Medulloblastoma growth in vitro and vivo through Gli acetylation, implying that this approach could be used for the treatment of SHH MB in patients. No conflict of interest. 641 Hyperbaric oxygen therapy combined with photodynamic therapy as a new therapeutic approach against retinoblastoma A. Neves1 , A. Abrantes2 , A. Pires2 , R. Teixo2 , M.F. Botelho2 . 1 Faculty of Science and Technology- Biophysics and Biomathematics InstituteIBILI-Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 2 Biophysics and Biomathematics Institute- IBILI-Faculty of MedicineCIMAGO- CNC.IBILI, University of Coimbra, Coimbra, Portugal Introduction: Retinoblastoma (RB) is the most common primary ocular malignancy tumor in childhood with an incidence of nearly 11 cases per million. Photodynamic therapy (PDT) is presented as an emerging therapeutic approach for the treatment of cancer. PDT involves photochemical activation of the photosensitizing agent, that generates highly toxic singlet oxygen and other reactive oxygen species (ROS), which can cause intracellular death. Hyperbaric oxygen therapy (HBOT) consists in oxygen delivery to a patient, at pressures greater than atmospheric pressure. HBOT also induces the formation of ROS or other free radicals which can damage tumors by inducing excessive oxidative stress. We aim to evaluate the combination of HBOT and PDT as a new alternative therapy to treat RB and to ascertain the best therapeutic approach produced by different conditions of both therapies combination. Materials and Methods: Our experimental design included human RB Y79 cells that were cultured and submitted to PDT alone, PDT+HBOT and HBOT+PDT. Cells were seeded in 96-multiwell plates, at a density of 0.5×106 cells/mL. Then cells were incubated with several concentrations of PS (ACS88F1): 5, 50, 100 and 500 nM. Irradiation was performed 24 h after cells were treated with PS. Furthermore, HBOT was performed during 30 or 60 min, before (HBOT+PDT) and after (PDT+HBOT) light activation (10 J) of PS. To evaluate metabolic activity and total protein content, alamar blue and SRB assays, respectively, were performed 24 h after treatments. Results and Discussion: After combined therapies, metabolic activity and protein content of Y79 cells decreased with increasing PS concentration and HBOT exposure time in comparison with PDT alone. With alamar blue assay, we verified that combined therapy showed a better outcome for RB treatment, specifically PDT+HBOT60min. To confirm these preliminary results, we used SRB assay, which revealed a lower amount of protein content with combined therapies in comparison with PDT alone. Conclusion: In conclusion, we found that PDT leads to a decrease in cell proliferation. The obtained results revealed that combination of PDT and HBOT is promising, namely PDT+HBOT60min approach. Protein content evaluation confirmed the previous results. These studies may contribute to the development of a promising therapy for RB. No conflict of interest. 642 Targeting cell metabolism to improve prostate cancer therapeutics M. Bedaj1 , K. Rao1 , C. Robson1 , S. McCracken1 . 1 Newcastle University, Northern Institute for Cancer Research, Newcastle upon Tyne, United Kingdom Background: Metabolic reprogramming has recently been proposed as an emerging hallmark of cancer. In the unstable tumour microenvironment, cancer cells drive associated fibroblasts to undergo such metabolic reprogramming (“Reverse Warburg effect”) and produce lactate “fuel” for sustained cancer cell proliferation. Monocarboxylate transporters MCT1 and MCT4, permitting such cross-talk, are implicated as functional and prognostic markers in prostate cancer (PCa). As advanced PCa is refractory to treatment, we aimed to explore the potential link between lactate metabolism and drug resistance in PCa. Materials and Methods: Drug-sensitive LNCaP and derivative Enzalutamide (MDV) resistant cell lines (LNCaP-EnzR) were employed as PCa models, with primary fibroblasts representing tumour stroma. Western blotting and lactate assay were employed to assess MCT expression and activity, respectively. MCT1 inhibition was achieved using siRNA technology and separately with the use of a specific MCT1 inhibitor. Immunohistochemical staining of prostate cancer tissue microarrays was used to evaluate both MCT1 and MCT4

expression in cancerous and benign clinical tissue samples, including matched paired patient tissue samples (before and after relapse on conventional antiandrogen treatment). Results: MCT1 and MCT4 were overexpressed in the anti-androgen drugresistant line and fibroblasts, respectively. Fibroblast treatment with cancer cellconditioned media promoted a shift towards enhanced lactate export. Under hypoxic conditions (1% O2), the Enzalutamide drug-resistant line employed MCT1 and upregulated MCT4 transporters for efficient lactate elimination, whereas MCT1 knockdown or inhibition resulted in the intracellular lactate build-up and subsequent decline in cell proliferative potential. In addition, MCT1 and MCT4 were overexpressed in PCa tissue samples and correlated with clinic-pathological indicators, including tumour grade, local invasion and metastasis. Finally, expression of MCT-1 appeared to be higher than that of MCT-4 in hormone na¨ıve prostate cancer, and there was no indication that this profile of relative expression was different in those patients treated with hormone ablation. However, analysis of immunohistochemical staining demonstrated that MCT-1 expression increased, while MCT-4 expression decreased in patients with later stage “castrate-resistant” disease. Conclusions: Alterations in MCT transporter levels allow LNCaP resistant lines to efficiently adapt their metabolism and promote “Reverse Warburg” phenotype in fibroblasts. MCT1 transporter in particular is essential for metabolic balance and cell viability, and presents as an attractive therapeutic target for overcoming or delaying drug resistance. No conflict of interest. 644 Photodynamic therapy in EGFR targeted nanoparticles for lung cancer M. Laranjo1 , R. Teixo2 , A.M. Abrantes1 , A.C. Gon¸calves3 , A.B. Sarmento Ribeiro3 , M. Pineiro4 , A. Serra5 , S. Oliveira6 , M.F. Botelho1 . 1 Faculty of Medicine of University of Coimbra, Institute of Biophysics/BiomathematicsCIMAGO- CNC.IBILI, Coimbra, Portugal, 2 Faculty of Medicine of University of Coimbra, Institute of Biophysics/Biomathematics, Coimbra, Portugal, 3 Faculty of Medicine of University of Coimbra, Applied Molecular Biology Unit, Coimbra, Portugal, 4 Faculty of Sciences and Technology of University of Coimbra, Department of Chemistry, Coimbra, Portugal, 5 Faculty of Sciences and Technology of University of Coimbra, Department of Chemical Engineering, Coimbra, Portugal, 6 Utrecht University, 5Division of Molecular Oncology and Cell Biology, Utrecht, Netherlands Background: Lung cancer (LC) is one of the major causes of cancer worldwide. Photodynamic therapy (PDT) depends on the photosensitizer (PS), a drug whose effect is triggered only after irradiation with visible light. This therapeutic was previously used in the management of LC showing conservation of lung function. Liposomes, due to their encapsulation ability, are efficient drug carriers. We previously developed liposomes appropriate to use in the pulmonary network. Moreover, nanobodies are the smallest functional antigen binding fragments which bind with great specificity and high affinity to their antigens. Therefore, we intend to develop and evaluate a novel transport system to carry photosensitizers (PS) to treat lung cancer (LC) based on endothelial growth factor receptor (EGFR) targeted nanobodies coupled to lipossomes, which were developed on purpose to apply in pulmonary network. Material and Methods: A lipidic mixture composed by maleimidepolyethyleneglycol 2000 disterylphosphatidylethanolamine, disteroylphosphatidylcholine, phosphatidylglicerol, N-acetyl muramic acid and cholesterol was evaporated to form a lipid film. The PS 5,15-bis(2-bromo-3-hydroxyphenyl)chlorin synthetized by our group was added in hydration prior extrusion through 100 nm pore size membranes. The nanobody Ega1 was conjugated to the liposomes overnight and efficiency was evaluated in SDS-PAGE. The formulation obtained was administrated in the H1299 and A549 human lung cancer cell lines and, after 2 hours, washing and irradiation was performed. MTT assay was performed to access metabolic activity. Flow cytometry with annexin V/propidium iodide double labeling analyzed cell viability. Cell lines EGFR expression was confirmed by western blot. Cell nanoparticle interactions were evaluated through fluorescence microscopy and flow cytometry. Results and Discussion: The conjugation of the nanobody with the lipossomes was succefully performed with a conjugation yield of 40%. For this, a ratio of 1.6 nmol nanobody per 1 mmol total lipid was used. It was found that metabolic activity was limited by photodynamic treatment with the formulation prepared. Furthermore the decrease of viability of H1299 and of A549 cells was up to 60% being observed both apoptosis and necrosis. EGFR expression was detected in both lung cancer cell lines and interaction of these cells with the nanoparticules was observed. Conclusions: This initial study showed the feasibility of a new formulation which interacted with EGFR expressing cells leading to cell death after irradiation. These results encourage the continuation of this work. Acknowledgment: Funding: The authors would like to thank Foundation for Science and Technology (FCT PTDC/BIMONC/0979/2012 and Strategic Project CNC.IBILI: UID/NEU/04539/2013), COMPETE-FEDER and Liga Portuguesa Contra o Cancro. No conflict of interest.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 645 Investigation of the anti-cancer potential of natural products and their synthetic analogues B. Adegbesan1 , C. Demonacos1 . 1 University of Manchester, Manchester Pharmacy School, Manchester, United Kingdom Introduction: NRH quinone-oxidoreductase 2 is one of the two members of the quinone-oxidoreductase gene family; it is an antioxidative enzyme involved in the two-electron reduction of quinones directly to hydroquinones. The biological function of NQO2 has not yet been elucidated. NQO2 is overexpressed in some types of cancer therefore inhibiting its enzymatic activity could potentially be used for the treatment of such types of cancer. The mammalian sirtuins are a class of proteins possessing either monoribosyltransferase or deacylase activity and have been reported to be involved in cellular stress resistance. Many natural agents and synthetic analogues targeting both NQO2 and sirtuins have been used as experimental drugs in cancer prevention and treatment. Resveratrol (3, 4 5-trihydroxy-transstilbene), a member of polyphenols has been shown to inhibit the enzymatic activity of NQO2 and activate sirtuins. Sirtinol, a cell-permeable inhibitor of sirtuins has been reported to inhibit the growth of cancer cells. This study is designed to explore pathways that could be used to design novel cancer therapeutics based on the biological effects mediated by NQO2 and Sirt-1 using resveratrol, sirtinol and other synthetic analogues in breast cancer cells. Materials and Methods: The interrelation between Sirt-1 and NQO2 was investigated by probing for p53 and E2F1 protein levels in MDA-MB-231, MDAMB-468, MCF7 and T47D breast cancer cell lines using western blot analysis; the effect of these activators and inhibitors on cell cycle progression and ROS generation were determined by FACs; the potential NQO2 and/or Sirt-1 mediated regulation of p53 and E2F1 transcriptional activities was investigated using Cyclin-E luciferase reporter assay. Results and Discussion: The results from this study indicate that NQO2 and Sirt-1 potentially regulate p53 and E2F1 protein levels in a cell type dependent manner. Also, treatment with resveratrol, sirtinol and other synthetic compounds in these cells affect p53 and E2F1 levels differently suggesting that they may serve as important tools to dissect the synergistic and antagonistic effects of Sirt-1 and NQO2 on these cell cycle and apoptosis main regulators. Cell cycle analyses reveal that resveratrol, sirtinol and other synthetic analogues cause arrest of cell cycle at either the G1 or S phase. ROS analyses indicate that these compounds affect the level of intracellular ROS differentially. Cyclin-E Luciferase assays reveal that the transcriptional activities of E2F1 and p53 are also differentially regulated by the different treatments in the cell lines. Conclusion: Resveratrol, sirtinol and other synthetic analogues cause arrest of cell cycle at either the G1 or S phase in breast cancer cells an effect that make them suitable to be explored as therapeutic tools in breast cancer. No conflict of interest. 646 AAV-shRNA vectors as an alternative therapy for human basal-like breast cancer C. Pinto1,2 , A.S. Ribeiro3 , G. Silva1 , M. Oliveira3 , A.S. Coroadinha1,2 , C. Peixoto1,2 , A. Barbas1 , C. Brito1,2 , J. Paredes3 , P.M. Alves1,2 . 1 IBET´ Instituto de Biologia Experimental e Tecnologica, Animal Cell Technology ´ ´ Unit, Oeiras, Portugal, 2 Instituto de Tecnologia Qu´ımica e Biologica Antonio Xavier- Universidade Nova de Lisboa, Animal Cell Technology Unit, Oeiras, Portugal, 3 IPATIMUP, Epithelial Interactions in Cancer, Porto, Portugal Background: Basal-like breast cancer (BBC) is mainly comprised by triple-negative breast cancers (TNBC) which display a highly aggressive and metastatic phenotype. These tumors rapidly acquire resistance to chemotherapy, are refractory to endocrine therapy and HER2 inhibitors, and have no targeted therapeutic options. As a consequence, they present a poor clinical outcome. Therefore, there is a need to find alternative therapies against this type of tumors. In this study, we aimed to develop a novel gene therapybased treatment for BBC based on viral vector-mediated delivery of shRNA targeting BBC cell survival genes, such as MCL1, PSMA2 and PSMB4. Materials and Methods: pAAV2-shRNA plasmids were generated by cloning shRNA sequences targeting previously identified BBC dependency genes, or validated scramble control shRNA sequences, together with GFP reporter gene between AAV2 inverted terminal repeats in an AAV plasmid backbone. AAV2-shRNA vectors were produced by 2-plasmid co-transfection of HEK293T cells with pAAV2-shRNA plasmids and the pDG plasmid, and purified by affinity chromatography. Target gene knockdown efficiency and functional effects of the different AAV2-shRNA vectors in BBC cells were evaluated by qRT-PCR, Presto Blue analysis and flow cytometry. The anti-tumorigenic effect of AAV2PSMA2sh vector was evaluated on mouse orthotopic BBC cell xenografts. Results: AAV2-shRNA vectors against several BBC genes and control scramble vectors (negative controls) were produced with high titers and high purity. Transduction of two BBC cell lines (MDA-MB-468 and HCC1954) with AAV2-PSMA2sh decreased the expression of its target gene by 80%, as compared to control cells transduced with AAV2 vector encoding a scramble shRNA (negative control). In MDA-MB-468 cells, knockdown of PSMA2 gene

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was associated with significant decrease in cell viability and 2 fold increase in the percentage of apoptotic cells. In vivo studies to assess the anti-tumorigenic effect of AAV2-PSMA2sh in mouse orthotopic BBC cell xenografts showed that intratumoral injections with this vector caused a significant decrease in tumor growth, when compared to negative scramble shRNA control vector or PBS injected mice. No major macroscopic alterations were observed in the liver of mice treated with AAV-shRNA vectors, as compared to PBS control. Assays are currently in progress to further characterize the tumors, e.g. proliferation and apoptotic status. Conclusions: These results indicate that AAV2-PSMA2sh vector decreases BBC tumorigenesis and suggest that PSMA2 gene may represent a potential therapeutic target for BBC therapy. No conflict of interest. 647 In-vitro and in vivo preclinical evaluation of a 5T4 specific antibody–drug conjugate for treating ovarian cancer Y.L. Wan1 , P. Sapra2 , D. Gilham3 , H.C. Kitchener1 , P.L. Stern1 . 1 University of Manchester, Gynaecological Oncology Research Group- Institute of Cancer Sciences, Manchester, United Kingdom, 2 Pfizer Worldwide Research & Development, Bioconjugates Discovery & Development- Oncology Research Unit, Pearl River, NY, USA, 3 University of Manchester, Clinical and Experimental Immunotherapy Group- Institute of Cancer Sciences, Manchester, United Kingdom Background: The oncofoetal glycoprotein, 5T4, is strongly expressed by many different cancers but has little expression in normal adult tissues. Its expression is associated with advanced disease and poorer clinical outcome in a number of cancer types, including ovarian cancer. 5T4 is mechanistically associated with the directional movement of cells through the regulation of epithelial mesenchymal transition, facilitation of chemotaxis and modulation of Wnt signaling. The tumour selective expression of 5T4 has prompted the development of several immunotherapies; including a 5T4 antibody– drug conjugate (A1mcMMAF). The latter is a 5T4 humanized monoclonal antibody (A1) linked by sulfydryl-based conjugation to deliver a microtubuledisrupting agent, monomethyl auristan F (MMAF) via a maleimidocaproyl linker. A1mcMMAF has shown potent in vivo activity in a variety of tumour models, with induction of long term regression after the last dose. Here we test activity versus ovarian cancer cells. Methods: In vitro IC50 values of 5T4-ADC in SKOV3 cells expressing 5T4 and 5T4 knock out cells (SKOV3-5T4KO) were determined using MTS assays. Apoptosis and viability were assessed by Apotox-Glo assay. Anti-tumor activity of A1mcMMAF with or without carboplatin chemotherapy against SKOV3luciferase xenografts in NSG mice models was assessed using bioluminescent imaging. Results: A1mcMMAF selectively killed SKOV3 cells with an IC50 20 fold lower than that seen in SKOV3-5T4KO cells. Within the selective range, A1mcMMAF caused apoptosis in SKOV3 but not SKOV3-5T4KO cells. A1mcMMAF (5 mg/kg IP) treatment of SKOV3 xenografts reduced tumour load, delayed growth and significantly increased survival compared to untreated animals. A combination of A1mcMMAF with optimized dosing of carboplatin showed no toxicity and a greater reduction in tumour load and improved survival compared to A1mcMMAF monotherapy. A1mcMMAF plus carboplatin (25 mg/kg IP) showed a median survival increase of 80% compared to carboplatin alone. Conclusions: A1mcMMAF is selective for cells expressing 5T4 and shows potent anti-tumor activity in SKOV3 xenograft models. These data support the use of A1mcMMAF in ovarian cancer treatment potentially in combination with carboplatin. Conflict of interest: Other Substantive Relationships: A1McMMAF was developed by Pfizer Inc and kindly donated for use in this project which was funded by the Wellcome Trust. Puja Sapra is an employee of Pfizer Inc. and owns company’s stock options and/or units. Peter Stern has received honoraria from Pfizer Inc. 648 Membrane Hsp70 as a biomarker for aggressive prostate cancer and therapeutic target G.A. Foulds1 , N. Dunning-Foreman1 , S. Stangl2 , M. Gehrmann2 , J. Vadakekolathu1 , D. Boocock1 , R. Rees1 , G. Multhoff2 , A.G. Pockley1 . 1 Nottingham Trent University, John van Geest Cancer Research Centre, ¨ Munchen, Nottingham, United Kingdom, 2 Technische Universitat ¨ Department of Radiation Oncology, Munich, Germany Introduction: Professor Gabriele Multhoff first identified the selective expression of a membrane form of Hsp70 (memHsp70) on the plasma membrane of tumour cells (but not normal tissue) using a unique monoclonal antibody (mAb, cmHsp70.1, multimmune GmbH). The expression of memHsp70 has since been shown to associate with aggressive disease (e.g. metastasis) and an unfavourable prognosis and poorer survival in patients with cancer. An ongoing screening program of solid tumours at the Technische Universitat ¨ Munchen ¨ is revealing that more than 50% of all tumours express memHsp70.

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As a therapeutic target, memHsp70 acts as recognition structure for activated natural killer (NK) cells, with NK cellular cytotoxicity being mediated via the memHsp70-specific uptake of NK cell-derived granzyme B (grB). Administration of ex vivo activated NK cells, the cmHsp70.1 mAb or a recombinant form of human grB reduces tumour growth in animals bearing memHsp70 positive tumours. The therapeutic potential of ex vivo activated NK cells is currently being evaluated in a Phase II clinical trial in patients with non small lung cell carcinoma. This study examined the relationship(s) between memHsp70 expression and ‘aggressive’ prostate cancer. Materials and Methods: memHsp70 expression by human prostate cancer cell lines representing differing metastatic potential (PC3, DU145, LnCap) was determined using flow cytometry. Given the accumulating evidence that cancer cells acquire the invasive and migratory properties that are necessary for the invasion-metastasis cascade by reactivating a latent embryonic programme known as epithelial to mesenchymal transition (EMT), the relationship between memHsp70 and EMT was also examined using a recently-developed spontaneous model of EMT. This uses a primary tumour-derived human prostate cancer cell line (OPCT-1), from which have been derived four phenotypically distinct clones, the drug resistance, clonogenicity, sphereforming capacity, ALDH1 expression (stem cell marker), invasion and migration capacity, and in vivo tumourigenicity of which have been characterised. Results and Discussion: PC3, DU145 and LnCap cells expressed memHsp70, with expression being higher in those cells that represent an aggressive phenotype. The membrane Hsp70 status of the OPCT-1 clones also correlated with their ‘aggressiveness’ and in vivo tumorigenicity. Furthermore, cells expressing memHsp70 can be killed using recombinant grB. These findings confirm that memHsp70 is a marker of aggressive disease and suggest that ‘aggressive’ prostate tumours will be sensitive to memHsp70targeted therapeutics such as activated NK cells, the cmHsp70.1 mAb (via the induction of ADCC) or recombinant human GrB. Conclusion: Membrane Hsp70 offers an exciting target on which to base new therapeutic strategies for aggressive prostate cancer. Conflict of interest: Other Substantive Relationships: AGP has a consultancy with multimmune GmbH. 649 Vascular endothelial growth factor (VEGF) isoform mediated tumour response to combination treatment with a blocking antibody to VEGFR-2 and radiotherapy D. Mukherjee1 , S.J. Lunt2 , M. Fisher2 , W. English2 , C. Kanthou2 , G. Tozer2 . 1 The University of Manchester Targeted Therapy Group, Institute of Cancer Sciences, Manchester, United Kingdom, 2 Tumour Microcirculation Group, Department of Oncology and Metabolism, Sheffield, United Kingdom Introduction: Vascular Endothelial Growth Factor-A (VEGF) is a key component of vascular development and exists as multiple splice variants, VEGF120/121, VEGF164/165 and VEGF188/189 in mice/humans respectively. Differential VEGF isoform expression in murine fibrosarcomas leads to differences in vascular maturity [1]. Here, we determined the influence of isoform expression on tumour response to a VEGF pathway inhibitor and radiotherapy. Materials and Methods: Murine fibrosarcomas expressing only VEGF120 (fs120), VEGF164 (fs164) or VEGF188 (fs188) were implanted subcutaneously into SCID mice. We examined tumour response to DC101, a blocking antibody against VEGFR-2 (30 mg/kg every 3 days), either alone or in combination with fractionated radiotherapy (8×2.5 Gy over 4 days). Immunohistochemical staining for CD31 was carried out on a separate cohort of excised tumours. Results and Discussion: Fs188 tumours were more responsive than fs120 to radiotherapy (12 versus 9 day growth delay at 800 mm3 ), which corresponded to them being marginally less hypoxic. Fs188 tumours were also significantly more responsive than fs120 tumours to DC101 treatment, especially when combined with radiotherapy (17 versus 8 day growth delay at 700 mm3 ). Growth response of fs164 tumours was similar to that of fs120. Despite a poor growth response in fs120 tumours, blood vessel counts (CD31 staining) were significantly reduced in these tumours 24 h after the second dose of DC101 (from 53±4 to 22±3 vessels per high-power field in viable tumour). A similar reduction in fs120 tumours was found at early times after irradiation, with no additive effect for the combined treatment. Conclusions: Differential tumour cell expression of VEGF isoforms strongly influences tumour growth response to DC101±radiotherapy, suggesting that fs120/fs164 tumours are less dependent on VEGFR-2 than fs188 tumours for growth. Investigation of vascular versus growth effects is on-going. VEGF isoform expression has potential for selection of soft tissue sarcoma patients for treatment with VEGFR-2 inhibitors in combination with radiotherapy. Acknowledgement: This work is supported by Cancer Research UK grant A18538. We thank Eli Lilly for supply of DC101. Reference(s) [1] Tozer G.M. et al. Cancer Res., 68: 2301–11 (2008). No conflict of interest.

650 Photodynamic therapy in osteosarcoma. Is there involvement of the apoptosis pathway mediated by p53, or the proliferative pathway mediated by beta-catenin? What is the action mechanism of PDT? G. De Miguel1 , B. Camporeze2 , A.Y.K. Grizotto2 , A.M. Abrantes3 , ˜ Francisco University, M.F. Botelho3 , J.A. Pereira4 , D.G. Priolli1 . 1 Sao ˜ Francisco Stricto sensu in Health Science, Bragan¸ca Paulista, Brazil, 2 Sao University, Medicine Course. Scientific Initiation Programme, Bragan¸ca 3 ˜ Paulista, Brazil, Coimbra University, Biophysics, Coimbra, Portugal, 4 Sao Francisco University, Medicine Course, Bragan¸ca Paulista, Brazil Osteosarcoma is a malignant tumor with low response to chemotherapy, which aims to tumor cell necrosis. Its prognosis is determined by the local recurrence. For successful treatment, new therapies are needed, especially for tumors located in areas that tumor extirpation is impossible. Osteosarcoma in skullcap and vertebrae is fatal because complete excision is impossible and chemotherapy alone is not enough to cure. Thus, it seems interesting new forms of adjuvant treatments for this type of cancer, such as Photodynamic Therapy (PDT). PDT aim to sensitize tumor cells to chemotherapy and it has been presented as a promising agent in cancer treatment, although there is insufficient literature regarding PDT application in osteosarcoma. Possible forms of PDT antitumor action, would be its effect on p53 expression, involving in apoptosis pathway or in proliferative pathway mediated by beta-catenin. Objective: To evaluate PDT effect on p53 and beta-catenin expression, in an animal model of skullcap and spine osteosarcoma. Material and Methods: Mice (n = 20) were used, randomized in four groups. The animals were inoculated with osteosarcoma human cells/MNNG-HOS previously processed in cell culture. Tumor volume was monitored by daily measurement. Animals received the photosensitizer (porphyrin BBr2HPP) when tumor volumes reached 150 to 200 mm3 . After 72 h of the photosensitizer administration, the PDT group underwent tumor irradiation with 10J in a single cycle. Confirmation of the presence of tumor was made by histopathological, x-Ray and MIBI-Scintilografy. After euthanasia, tumor resection, conventional histopathological and immunohistochemical study (p53 and beta-catenin) was done. The analysis was carried out by adopting a significance level of 5%. Results: The orthotopic xenografts in skullcap showed 100% implant success rate, while in the spinal group was about 65% of animals. There was tumor reduction in PDT-treated tumors (p = 0.00). Radiographic examination showed the predominance of lithic character of the OS, with areas in solution of continuity of the skullcap and cortical destruction. Skullcap treated group with PDT showed decrease in MIBI uptake. Pathological examination showed tumor cell clusters of large size with acidophilus and scant cytoplasm, cells with round nuclei of condensed chromatin and clear nucleoli. It was found greater area of tumor necrosis and lower deposition of osteoid matrix in the group treated with PDT than in untreated. beta-catenin and p53 expression showed no significant difference between the treated and untreated groups. Conclusion: PDT is an osteosarcoma antitumor new agent. PDT action does not occur by apoptotic pathway mediated by p53 or by inhibition of proliferation mediated by beta-catenin. More studies are needed in order to clarify PDT antitumor mechanism. No conflict of interest. 651 Antiangiogenic potential of flavonoids in an animal model of human colon adenocarcinoma D. Priolli1 , G.D.C. Orfali2 , I.R.O. Assun¸cao ˜ 2 , I.M. Marchesi2 , N.P. Martinez1 , D.D.C. Silva1 , J.A. Pereira3 , P.D.O. Carvalho1 , A.Y.K. Grizotto2 . 1 Universidade ˜ Francisco, Stricto sensu in Health Science, Bragan¸ca Paulista, Sao ˜ Francisco, Medicine Course. Scientific Initiation Brazil, 2 Universidade Sao ˜ Francisco, Programme, Bragan¸ca Paulista, Brazil, 3 Universidade Sao Medicine Course, Bragan¸ca Paulista, Brazil The antiangiogenic therapy is a promising target for new anti-cancer therapies, assuming that tumor growth is an angiogenesis-dependent variable. Angiogenesis is a complex process dependent of many factors. The vasoinhibins (VASH) are a family of peptides that suppress the nitric oxide-dependent vasodilation and angiogenesis. Flavonoids are polyphenolic compounds widely distributed in the plant kingdom and, besides their important nutritional role, they demonstrate an extensive pharmacological activity; however, few studies address its antiangiogenic potential on tumor models. The aim of this study is to evaluate the prophylactic and therapeutic antiangiogenic potential of flavonoids − quercetin-3-glucoside (Q3G), rutin and hydrolyzed rutin (HR) − in colon adenocarcinoma/HT-29 in animal model by VASH immunoexpression and microvascular quantification. Material and Method: 35 Balb-c nude mices, with six to eight weeks of age, were randomly divided into seven distinct groups. The animals were implated with colon adenocarcinoma/HT-29 processed in cell culture. Tumor volume was monitored by direct measurement. After sacrificing tumors were resected. The surgical specimen was fixed in 10% formalin and fixed in paraffin block for subsequent histological and immunohistochemistry analysis. The statistical analysis adopted a significance level of 5% (p < 0.05). Results and Discussion: HR prophylactic administration was able to inhibit tumor growth during the first week after implantation (p < 0.01), leading to


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a reduction of the final tumor volume (p = 0.03) and an overexpression of VASH (p = 0.03), but without affecting the vascular amount (p > 0.05). When administered therapeutically, all the tested compounds were able to inhibit tumor growth (p < 0.01), assuming that the Q3G group had decreased final tumor volume (p = 0.04) as well increased VASH expression (p = 0.03) and decreased vascular proliferation (p < 0.05). It was found an inversely proportional relationship between the tumor growth of human colon adenocarcinoma/HT-29 and VASH expression (p < 0.05). Conclusion: Flavonoids HR and Q3G demonstrated antiangiogenic potential in colon adenocarcinoma/HT-29 when administered prophylactic and therapeutically, respectively. Q3G showed direct inhibition of the neovascular proliferation. No conflict of interest.

of tumor growth in all groups tested in the control group (p = 0.00). Immunohistochemical analysis revealed a reduction in the expression of p53 (p = 0.00) in all groups compared to control group. There was no difference in the expression of ING2 (p = 0.97). The drugs tested exhibited tumor growth reduction and involvement in antiproliferative pathways mediated by the p53 protein, corroborating literature data showing activation of apoptotic mitochondrial pathway of flavonoid. Conclusions: Flavonoids Q3G and HR demonstrated antitumor activity. Reduction of tumor growth was observed in all groups, moreover it was directly proportional to the reduction in mutated p53 expression in the therapeutic Q3G group (p = 0.03), indicating a potential pro-apoptotic action and cell cycle arrest (G1 / S phase) through p53 signaling pathways. No conflict of interest.

653 Anti-cancer properties of secondary metabolites derived from marine bacteria L. Lee-Jones1 , Q. Wang1 , S. Bohan1 , O. Martin1 , P.E. Linton1 . 1 Manchester Metropolitan University, Healthcare Science, Manchester, United Kingdom

655 New biocompound for the treatment of glioma: Is rutin hydrolyzed by hesperinase an alternative?

Background: Natural combinatorial chemistry has been occurring in plants and microorganisms throughout evolutionary history and consequently is far more sophisticated than that achievable in the laboratory by current combinatorial chemical procedures. This makes natural products an invaluable source of novel bioactive molecules which could lead to the development of novel pharmaceutical compounds to treat cancer. Material and Methods: Samples were collected from Heysham, West Lancashire. Marine bacteria were isolated to pure culture from marine tidal surfaces including marine invertebrates, molluscs and marine plants using actinomycete-seawater culture media. Resultant pure cultures were grown in liquid media and cell-free crude extracts where prepared by culture centrifugation and filter-sterilisation. Bacterial species were identified by 16S rDNA PCR-sequencing methods. Crude extracts were then screened for bioactivity in the human haematological cell lines (Jurkat, U937 and K562) to determine their effects on cell proliferation, cell cycle and induction of apoptosis. Results and Discussion: From an initial pilot study of 12 extracts derived from several strains of actinomycetes, 5/12 demonstrated anti-proliferative properties for both 24 and 48 hour timepoints. A further extract demonstrated anti-tumour activity at the 48 hour time point only. Conclusion: The pilot study successfully investigated 12 extracts derived from marine bacteria and had a high success rate (42% for both 24 and 48 hour time points, 50% for 24 hour and/or 48 hour timepoints). One of the extracts demonstrated anti-proliferative properties that were more potent than the hydroxyurea (positive) control. Evaluation of the bioactive properties of the remaining panel of extracts (n = 82) is now underway. Marine bacteria represent a largely untapped resource with enormous potential as a source of novel bioactive compounds. No conflict of interest. 654 Flavonoids Q3G and hydrolyzed rutin demonstrated antitumor activity in colon adenocarcinoma − in vivo study D. Priolli1 , D.D.C. Silva1 , A.C. Duarte2 , G.D.C. Orfali2 , N.P. Martinez1 , ˜ Francisco, Stricto P.D.O. Carvalho1 , A.Y.K. Grizotto2 . 1 Universidade Sao ˜ sensu in Health Science, Bragan¸ca Paulista, Brazil, 2 Universidade Sao Francisco, Medicine Course. Initiation Scientific Programme, Bragan¸ca Paulista, Brazil Background: Flavonoids are polyphenolic compounds widely distributed in the plant kingdom, whose importance lies in its beneficial physiological effects, including antioxidant and anti-proliferative effects. Carcinogenesis is a complex process that involves metabolic and cell proliferative changes by alteration in organic circuits, including oncogenes and tumor suppressor genes such as P53 and ING2. Method: For the experiment, 25 athymic mice were used and randomly divided into 5 groups: control, therapeutic Q3G and HR, prophylactic Q3G and HR. All animals were implanted with colon adenocarcinome (HT-29) tumor cell line under subcutaneous tissue. Control animals had their tumor growth assessed and used as a standard curve for comparative analysis. Prophylactic groups were gavados with their respective drugs for 7 days prior to tumor implantation, while animals from therapeutic groups were implanted and gavados only when their tumors reached volumes equal to or greater than 100 mm3 . After an evaluation of tumor growth the animals were euthanized, their tumors resected and stored for histological analysis and immunohistochemistry. The mechanism of action of the compounds was evaluated by immunohistochemical analysis of paraffin embedded tumor pieces, investigating the expression of proteins that participate in cell regulation process and progression of carcinogenesis, p53 and ING2. The analysis of the results was carried out by adopting a significance level of 5% (p 0.05). Results and Discussion: Histological analysis showed poorly differentiated adenocarcinoma with the presence of signet ring cell. There was a reduction

C. Pacheco1 , P.M. Moro1 , R. Colenci1 , A.C. Duarte1 , M.C.F. Messias2 , ˜ Francisco G. Mamprim3 , C.T. Parisi2 , D.G. Priolli2 , T.W. Santos2 . 1 Sao University, Medicine Course. Initiation Scientific Programme, Bragan¸ca ˜ Francisco University, Stricto sensu in Health Science, Paulista, Brazil, 2 Sao ˜ Francisco University, Medicine Course, Bragan¸ca Paulista, Brazil, 3 Sao Bragan¸ca Paulista, Brazil Flavonoids have shown biological effects, including antitumor activity by cell cycle arrest, DNA repair and apoptosis induction. Quercetin-3-Oglucoside (Q3G) has been the aim of several studies; however, it is scarce in nature. Q3G can be obtained with enzymatic de-glycosylation of quercetin3-rutinoside (Rutin), resulting in a mixture addressed as Hydrolized Rutin (HR), with significant increase in bioavailability and antitumor potential. This study sets out to evaluate HR’s cytotoxic activity in vitro, and its antiproliferative, pro-oxidative and genotoxic potential in animal model of glioma/U-251. Method: Tumor cell lines panel were evaluated for HR’s antiproliferative activity using Sulforhodamine B assay. HR’s action mechanisms were also investigated in cells apoptosis and cell cycle by flow cytometry. 15 nude mice randomly were divided in 3 groups: control (n = 7), HR treatment (n = 4) and HR prophylaxis (n = 4). All animals were grafted with human Glioma/U-251. The control group didn’t receive any treatment. The HR prophylaxis group received the biocoumpond for 5 days prior to tumor graft, while the HR treatment group first received the graft, initiating coumpound administration only when tumors reached volumes 100 mm3 . All animals had tumor growth daily monitored. The control group was used as a standard for comparative analysis. The animals were euthanized and the tumors ressected. The histopathological analysis used to confirm diagnosis, morphological characteristics, number of mitosis and the presence of necrosis. Lipid peroxidation was done by TBARS method and genotoxicity tests by COMET assay. The results were carried out adopting a significance level of 5%. Results: Among the tumor cell lines tested, HR obtained the best antiproliferative effect on Glioma/U251, able to reach significant results of total growth inhibition (TGI) with 3.6 mg/mL of drug concentration. No results were found in apoptosis flow cytometry. However, HR showed action of cell cycle arrest in the G1 phase, demonstrating a cytostatic effect. There was tumor growth inhibition on treated (p = 0.03) and prophylaxis group (p = 0.01). There was a decrease in mitoses (p < 0.04) in the treated groups. There was an increase in lipid peroxidation of both prophylactic and therapeutic HR groups. There was no significant variation in oxidative DNA damage level. Conclusion: HR exhibited moderate antiproliferative action in glioma. Cytoxicity was proven by TBARS assay; although, no genotoxic effect was shown. The evaluation of possible action mechanisms of HR in glioblastoma demonstrated cytostatic effect, but no apoptosis induction. Therefore, HR presents antiproliferative effect in high grade glioma/U-251. Results suggest that mitosis reduction and celular morphology changes are due to mechanisms related to drug action through a process of apparent oxidative damage on the cell membrane. No conflict of interest. 656 Hydrolyzed rutin in an animal model of human glioma P. Prado1 , C. Pacheco1 , C.T.P. De Oliveira2 , P.D.O. Carvalho2 , G. Mamprim3 , ˜ Francisco University, Medicine Course. D. Priolli2 , A.Y.K. Grizotto1 . 1 Sao ˜ Francisco Initiation Scientific Programme, Bragan¸ca Paulista, Brazil, 2 Sao ˜ University, Stricto sensu in Health Science, Bragan¸ca Paulista, Brazil, 3 Sao Francisco University, Medicine Course, Bragan¸ca Paulista, Brazil Among the various types of cancers, glioma is configured as a neoplasm of high power of invasion, proliferation and recurrence, being among the most lethal human cancers. The average survival time is usually less than one year after diagnosis. Thus, the need to search for new drug to combat this tumor improves survival of patients. Flavonoids are phenolic compounds of natural origin that are present in most foods of the human diet and have antitumor activity.

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Objective: To assess the cytotoxic potential of hydrolyzed rutin (RH) in an animal model (nude mice) implanted with tumor cell line U-251/human glioma and its possible mechanism of action by immunostaining p53 related to tumor apoptosis. Methods: We used 15 Balb c nu/nu mice six to eight weeks old, from Charles River Laboratory. The animals were divided into three groups: prevention, control and treatment. The three groups were subjected to the xenograft with U-251 strain cells, while the treatment group received the biocompound (flavonoid alcoholic solution at a dose of 0.032 ml/g) after implantation, the prophylaxis group before the implant and the control does not received treatment. The animals were followed daily and tumor volumes determined. After sacrificing the animals, the histopathology of the tumor was performed by pathological diagnosis by hematoxylin and eosin staining and immunohistochemical analysis, seeking expression of p53 protein. Results: It was observed decrease in tumor size of the treatment groups (p = 0.03) and prophylaxis (p = 0.01) compared to the animals in the control group. There was decrease in mitosis number in the prophylaxis group over the control group (p = 0.04). There was inverse correlation between tumor growth and the number of mitosis (p = 0.03, with rs = −0.513). There was no difference in the immunoreactivity of p53 protein in the treated groups. Conclusions: Was proven antiproliferative potential of the hydrolyzed rutin in animals implanted with human glioma/U-251. The antitumor mechanism of action of the flavonoid was not mediated by p53 apoptotic pathway. No conflict of interest. 657 Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK positive neuroblastoma cells, Drosophila and mice J. Siaw1 . 1 Institute of biomedicine, Gothenburg University, Medical biochemistry and cell biology, Gothenburg, Sweden Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutation is reported in about 5−7% of neuroblastoma cases but the ALK positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALKpositive lung cancer has had less dramatic responses in neuroblastoma, precipitating investigation of second generation ALK inhibitors. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activities by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments. No conflict of interest. 658 Clearing mutant p53 with natural compounds: implication for anticancer therapeutical approaches G. D’Orazi1 , A. Garufi2 , M. Cirone3 . 1 University “G. d’Annunzio”, MedicalOral and Biotechnologic Sciences, Chieti Scalo Chieti, Italy, 2 Regina Elena National Cancer Institue, Department of Research- Advanced Diagnosticsand Technological Innovation, Rome, Italy, 3 Institute Pasteur-Fondazione Cenci Bolognetti- Sapienza University, Department of Experimental Medicine, Rome, Italy TP53 oncosuppressor is frequently mutated in cancer. Several p53 mutant proteins escape proteolytic degradation and are highly expressed in an aberrant conformation often acquiring pro-oncogenic activities that promote tumor progression and resistance to therapy. It has been vastly proposed that reactivation of wild-type (wt) function(s) from mutant (mut) p53 may have therapeutic significance. Pharmacological targeting of mutp53 is a rapidly developing field at the forefront of translational cancer research and many small molecules that can rescue mutp53 proteins, by either restoring wild-type protein folding or inducing mutp53 degradation, have been tested. Although the efficient antitumor activity of these molecules has been confirmed, their utility in clinical practice is partially limited by toxicity. Thus, a new field of research aims at studying the antitumor effects of natural compounds, as they rarely exhibit toxic side-effects. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a significant pungent ingredient in a variety of red peppers of the genus Capsicum has been shown to exert biological activities (anticarcinogenic, antimutagenic and chemopreventive) in many cancer cell lines through different mechanisms and recently it has been shown to induce autophagy in cancer cells. The aim of this study is to investigate whether capsaicin is able to target mutp53 proteins in cancer cells and inhibit their oncogenic function in the attempt to identify novel natural compound to be used in clinical practice of p53-based therapies. Brest cancer and glioblastoma cells lines, bearing

several different mutp53 proteins will be used. Biochemical experiments will be performed to evaluate the effect of capsaicin on mutp53 protein expression levels; molecular experiments will be performed to evaluate the restoration of wtp53/DNA binding and transcription of target genes. The role of autophagy will be evaluated by using pharmacological and genetic autophagy inhibitors. The role of p53 on the capsaicin-induced biological effect (i.e., cell death) will be evaluated by pharmacologic inhibition of wtp53 transcriptional activity. Combination treatment experiments will be also performed with capsaicin and chemoptherapy. Preliminary experiments showed that capsaicin downregulates mutp53 protein levels, restoring wtp53 transcriptional activity. Pharmacological inhibition of autophagy showed that autophagy is the main mechanism of capsaicin-induced mutp53 degradation. Our results could open the way to exploit the activity of natural compounds to develop novel mutp53-targeting pro-drugs with less toxic activity, to be used in clinical practice. Therefore, the use of natural compounds such as Caspaicin alone or combination with chemotherapy could be a valuable strategy for p53based cancer therapy to be translate in clinical practice for the benefit of patients. No conflict of interest. 661 Combination of BGJ398 with either a pan-PI3K inhibitor or a specific PIK3CA inhibitor shows synergy in FGFR2 mutant endometrial cancer cell lines L. Packer1 , X. Geng1 , V. Bonazzi1 , C. Mahon1 , R. Ju1 , S. Stephenson1 , P. Pollock1 . 1 Queensland University of Technology, School of Biomedical Sciences, Institute of Health and Innovation, Translational Research Institute, Brisbane, Australia Introduction: Over 12% of endometrioid endometrial cancers (ECs) harbour mutations in Fibroblast Growth Factor Receptor 2 (FGFR2). The majority of ECs also harbour genetic aberrations in the PI3K/AKT pathway resulting in constitutive activation. In other tissue types, PTEN loss has been shown to provide resistance to specific PIK3CA inhibition. We have previously reported that FGFR inhibition with PD173074 induced cell death despite loss of PTEN. We hypothesize that dual inhibition of FGFR2 and the PI3K pathway will lead to increased cell death and more effective tumour growth inhibition. Materials and Methods: Two FGFR2 mutant EC cell lines were treated with BGJ398 (pan FGFR inhibitor) alone or in combination with either a pan-PI3K inhibitor (GDC0941) or a specific PI3KA inhibitor (BYL719). Synergy was assessed using the fixed-ratio method proposed by Chou and Talalay. Cell death was determined by Annexin V staining and colony formation assays using low clinically-relevant drug concentrations. Downstream signalling pathways were analysed by Western blot analysis and BGJ398 in combination with either GDC0941 or BYL719 was assessed in vivo. Results and Discussion: Synergy was observed between BGJ398 and either the pan-PI3K inhibitor (GDC0941), or the specific PI3KA inhibitor (BYL719). Regression of tumour xenografts was observed in mice treated with BGJ398 and GDC0941 as well as BGJ398 and BYL719, despite PTEN inactivation. Conclusions: Dual targeting of the FGFR and PI3K pathways is more effective than targeting either of them alone. PTEN loss does not provide resistance to PIK3CA inhibition in our EC models suggesting context dependent differences in PI3K signalling. Conflict of interest: Pamela Pollock has acted as a paid consultant for Novartis. 662 The anti-leukemic drug nilotinib inhibits metastatic properties of colorectal cancer cells by targeting the receptor tyrosine kinase DDR1 P. Tosti1 , C. Leroy2 , M. Jeitany1 , V. Simon1 , D. Bonenfant2 , L. Thouennon3 , S. Elmessaoudi3 , C. Mollevi4 , A. Thierry3 , P. Martineau3 , B. Robert3 , A. Sirvent1 , S. Roche1 . 1 Centre de Recherche de Biologie Cellulaire de Montpellier (CRBM) CNRS UMR5237-Universite´ de Montpellier, Montpellier, France, 2 Novartis Institutes for Biomedical Research, Basel, Switzerland, 3 ´ Institut de Recherche en Cancerologie de Montpellier (IRCM) INSERM U896-Universite´ Montpellier-ICM, Montpellier, France, 4 Department of Biostatistics, Institut du Cancer de Montpellier (ICM)-Val d’Aurelle, Montpellier, France Compelling evidence demonstrate that clinical-approved drugs designed to target a well-defined oncogene may be of broader clinical interest through off-target-dependent mechanisms. Accordingly, we observed that the clinically available tyrosine kinase (TK) inhibitor nilotinib, originally developed to target imatinib-resistant BCR-ABL oncoprotein in chronic myeloid leukemia, also inhibits the invasive properties of colorectal cancer cells (CRC), including those harboring a RAS oncogenic signaling. Remarkably, nilotinib also strongly reduces liver metastasis formation following injection of HCT116 and HT29 CRC cells in the spleen of nude mice. As ABL is not deregulated in CRC, we speculated the involvement of an alternative target. A recent chemicalproteomic analysis identified DDR1 as the highest affinity target of nilotinib (Rix et al., Blood, 2007) suggesting that this protein could mediate nilotinib activity

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 in CRC cells. DDR1 is a poorly-characterized receptor TK, whose ligands are collagens, ones of the major components of the extracellular matrix. Consistent with this hypothesis, our data demonstrate that DDR1 is abundantly expressed in CRC and promotes invasive and metastatic properties of CRC cells. Importantly, this pro-invasive function of DDR1 absolutely requires receptor TK activity. We next demonstrated that nilotinib responses observed in CRC are largely mediated through inhibition of DDR1 TK activity: remarkably, expression of the nilotinib-resistant DDR1/T807I receptor, in which the gatekeeper threonine in the nilotinib-binding pocket was mutated into the bulkier isoleucine, counteracts all the inhibitory effects of this drug in CRC cells. We also found a dramatic anti-metastatic activity of nilotinib in mice that have already developed DDR1-dependent metastatic nodules, thus revealing an unsuspected role of DDR1 during late metastasis. The clinical relevance of these observations was supported by transcriptomic data demonstrating that the level of DDR1 expression is associated with a shorter relapse free survival in patients with CRC and by biochemical analyses showing that DDR1 catalytic activity is highly increased in metastatic nodules compared to the primary tumor and the healthy tissue of the same patient. Additionally, phosphoproteomic analyses revealed the RAS independent nature of DDR1 signaling and pointed BCR as an essential DDR1 substrate for its pro-invasive activity. Altogether, these data suggest that the targeting of DDR1 by nilotinib may be of therapeutic value in metastatic CRC, including those harboring a RAS oncogenic signaling. No conflict of interest. 663 Antiproliferative and anti-inflammatory activities of a dichloromethane-methanol extract from Pterocarpus mildbraedii leaves C. Otuechere1 , G. Santosh2 , O. Farombi3 . 1 Redeemer’s University, Department of Chemical Sciences- Biochemistry Programme, Ede TownOsun State, Nigeria, 2 Indian Institute of Integrative Medicine, Cell Signaling and Pharmacology, Jammu, India, 3 University of Ibadan, Department of Biochemistry, Ibadan- Oyo State, Nigeria Background: Inflammation is related to several chronic diseases, including cancer whose incidence is progressing rapidly in sub-Saharan Africa. Pterocarpus mildbraedii is a special leafy vegetable in Nigeria. Our aim was to evaluate the anticancer and anti-inflammatory properties of the dichloromethane-methanol extract from Pterocarpus mildbraedii (PME) in vitro and in vivo experimental models respectively. Materials and Methods: The antiproliferative activity was evaluated on 5 human cancer cell lines namely, promyelocytic leukemia (HL-60), monocytic leukemia (THP-1), prostrate adenocarcinoma (PC-3), colon adecarcinoma (COLO-205) and lung carcinoma (A549) by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Furthermore, we evaluated the anti-inflammatory activity of the extract against pesticide-induced alteration of inflammatory proteins in albino rats. Results: PME had the most anti-growth activity on the cancer cell line HL-60 cells with an IC50 value of 8.5 mg/ml. The extract also showed much less cytotoxicity against the other cell lines. The increased expressions of COX-2 and iNOS, accompanied by NFúB up-regulation, and correlating with elevated levels of myeloperoxidase and nitric oxide in the liver of pesticide-treated animals were ameliorated by PME at a dose of 200 mg/kg. Total phenolic and flavonoid contents were calculated using the Folin-Ciocalteu and the aluminum chloride colorimetric methods and found to be 0.215±0.005 and 0.082±0.003 mg GAE/g respectively. PME showed a maximum 1,1-diphenyl2-picryl hydrazyl (DPPH) radical scavenging activity of 55%. Conclusions: The results of these assays indicated that Pterocarpus mildbraedii can be a candidate for prevention and treatment of many diseases related to inflammation and malignancy. No conflict of interest.


Molecular and Genetic Epidemiology I 666 Cancer of unknown primary is associated with diabetes ¨ Sweden X. Li1 . 1 Lund University, Clinical Research Center, Malmo, Background: Both type 1 diabetes (T1D) and type 2 diabetes (T2D) increase in incidence worldwide. T2D is associated with many cancers. However, no data are available on cancer of unknown primary (CUP), a relatively common, fatal cancer for which tobacco smoking is the only known risk factor. At diagnosis of CUP metastases are found in various organs which has implications for prognosis. We carried out a nationwide study on the association of CUP with T1D and T2D. Patients and Methods: 32,600 T1D patients and 178,000 T2D patients were identified from the national Hospital Discharge Register, Outpatient Register and Primary Health Care Register and these were linked to the Swedish

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Cancer Registry. Standardized incidence ratios (SIRs) were calculated for CUP from 1997 through 2010 using anyone without diabetes as a reference. Results: The SIR of CUP in 421 diabetic patients was 1.71, highest for CUP with liver (2.17) and respiratory system metastases (1.95). The SIR was 2.91 for T1D but with small number of patients, 1.38 for T2D with insulin treatment and 1.78 for the main group of T2D. CUP with liver and respiratory system metastases were increase for each diabetic type but for T2D also CUP with gastrointestinal and bone metastases were increased. The highest SIRs for CUP were recorded in the three types of diabetes during the first year of follow-up, most likely due to diagnostic bias during medical work-up. Conclusions: The results provide the first demonstration that CUP is one of the cancers associated with diabetes, with definite evidence on T2D. CUP has a poor prognosis, which may be even worse when diabetes is the underlying co-morbidity. A mechanistic question for future work is to resolve whether diabetes promotes primaries that escape detection or their metastatic spread. No conflict of interest. 669 Effects of WW domain-containing oxidoreductase (WWOX) gene polymorphisms in the Kozak sequence on the risk and progression of oral cancer C. Ying-Erh1,2 , S.F. Yang2 , C.W. Lin3 . 1 Chung Shan Medical University, School of Medicine-, Taichung City, Taiwan, 2 Chung Shan Medical University Hospital, Department of Medical Research, Taichung, Taiwan, 3 Chung Shan Medical University, Institute of Oral Sciences, Taichung, Taiwan Introduction: In Taiwan, oral cancer is the fourth leading cancer in males and is associated with exposure to environmental carcinogens. WW domaincontaining oxidoreductase (WWOX), a tumor suppressor gene, is associated with the development of various cancers. We hypothesized that genetic variants of WWOX influence the susceptibility to oral cancer. Material and Method: We genotyped 5 single nucleotide polymorphisms of WWOX from 761 male patients with oral cancer and 1199 male cancer-free controls. The association between WWOX expression and WWOX genetic polymorphism was analysed in the Encyclopedia of DNA Elements (ENCODE) data from the NCBI gene database and confirmed by quantitative real-time PCR. Results: We observed that the WWOX rs11545028 polymorphism carriers involving the TT or CT + TT genotype were associated with oral cancer susceptibility. Furthermore, patients with advanced-stage oral cancer were associated with a higher prevalence of WWOX rs11545028 polymorphisms with the variant genotype TT than did patients with the wild-type gene. An additional integrated in silico analysis confirmed that rs11545028 affects WWOX expression, which significantly correlates with tumor expression and subsequently with tumor development and aggressiveness. Conclusion: In conclusion, genetic variants of WWOX contribute to the occurrence of oral cancer, and the findings regarding these biomarkers provided a prediction model for risk assessment. No conflict of interest. 670 The effect of the CYP1A1*2A variant on colorectal cancer susceptibility in a British population E. Ozta¸s1 , G. Ozhan1 , A.K. Daly2 . 1 Istanbul University, Faculty of PharmacyDepartment of Pharmaceutical Toxicology, Istanbul, Turkey, 2 Newcastle University, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom Background: Colorectal cancer (CRC) is keeping its majority on public health for decades due to increasing incidence and high mortality despite of the overall improvements in diagnostics and treatment. Indeed, it is well known CRC has a complex aetiology and been still not well defined. Genetic polymorphisms on the biotransformation or DNA repair genes alone and as a contributor to dietary and environmental factors probably affect carcinogenic process. CYP1A1 gene is associated with tobacco-related cancers, including CRC, and extensively studied due to their key role on to metabolic activation of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene. CYP1A1*2A (6235 T/C, rs4646903, MspI) variant thought to be associated with increased risk of CRC, however, results of previous studies are conflicted [1,2]. In the present study, we examined a possible association between the CYP1A1*2A allele and CRC and the effect of cigarette smoking on this risk in a British population. Material and Methods: A prospective case–control study with British participants from Newcastle and North Tyneside Hospitals. 200 cases diagnosed with histologically confirmed adenocarcinoma of colon or rectum and 254 age-sex matched controls were included [3]. Genotyping of CYP1A1*2A was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotyping analyse was performed based on a dominant model with the wild type was chosen as reference group. Data comparisons were done by using Fisher’s exact test. Results: Genotyping analysis based on dominant model showed that individuals with C allele had about 1.5-fold risk of developing CRC

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(OR = 1.566), however the association did not reach statistical significance (p = 0.1197). We also evaluated a possible genotype-smoking association and found that C allele carrying individuals with smoking within the last 5 years had a significant risk of CRC (OR = 2.284; p = 0.0427). Conclusion: It has been revealed that CYP1A1 allele frequencies show large differences across different populations. Evaluation of an association between CYP1A1*2A and cancer susceptibility is more difficult and needs large number of participants in Caucasians. The data from this new study should be useful for understanding CRC aetiology and in identifying individual risk of developing CRC. Reference(s) [1] Inoue H, et al (2000). Cancer research, 60(14), 3749–3752. [2] Sivaraman L, et al (1994). Cancer research, 54(14), 3692–3695. [3] Welfare MR, et al (1997). Carcinogenesis, 18(7), 1351–1354. No conflict of interest. 671 Carcinogenic and non-carcinogenic risk assessment for children exposed to DDTs residues in pasteurized cow milk from Iran market N. Rastkari1 , M. Zare Jeddi2 , R. Ahmadkhaniha3 , M. Yunesian4 . 1 Tehran University of Medical Sciences, Center for Air Pollution Research CAPRInstitute for Environmental Research IER, Tehran, Iran, 2 Tehran University of Medical Sciences, Institute for Environmental Research, Tehran, Iran, 3 Tehran University of Medical Sciences, Department of Human EcologySchool of Public Health, Tehran, Iran, 4 Tehran University of Medical Sciences, Department of Environmental Health − School of Public Health, Tehran, Iran Background: Milk-producing animals accumulate pesticides from contaminated feed and by inhaling contaminated air. Organochlorine pesticides due to their lipophilic properties are initially stored in fat-rich tissues and subsequently translocated and excreted in milk. Therefore, the consumption of dairy products together with other contaminated food may expose consumers to unexpected levels of organochlorine pesticides. Organochlorine pesticides being endocrine disrupting chemicals are believed to produce a wide variety of adverse health outcomes such as cancers. Dichlodiphenyl-trichloroethane (DDT), a previously widely used OCP, and some of its major metabolites, is officially classified according to Integrated Risk Information System (IRIS) of United States Environmental Protection Agency (USEPA) as probable human carcinogens (Group B2) and responsible for causing damage to organs through repeated and prolonged exposure. Material and Methods: The objectives of the present study were to evaluate children’s exposure to DDTs (DDT and its metabolites) via dietary milk consumption in Iran, and to assess the respective potential risks to children’s health in terms of carcinogenic and non-carcinogenic effects. Residue levels were determined by GCMS analysis in 60 cow milk samples of Full fat (3%) pasteurized commercial types. The estimated daily intake (EDI) was calculated for different ages (1, 3, 5 and 7 years old children) based on the respective milk consumptions and finally Hazard quotient (HQ) for non-carcinogenic effects and hazard rate for carcinogenic effect were estimated. Results and Discussion: In all of the samples DDT metabolites were detected in the range of 0.0015 to 0. 2796 mg/L with the mean of 0.047±0.03, 0.15±0.08, 0.085±0.05, 0.05±0.02 mg/L for o, p -DDE, p,p -DDE, p,p -DDT, p,p -DDD, respectively. The calculated EDIs (0.002–0.03 mg/kg-day) for all age categories and all compounds were lower than the RfDs. Consequently, the hazard quotients calculated in these groups for DDTs were far less than 1. For estimation of the cancer risk values, the cancer benchmark concentrations (CBC) were derived using oral slope factors and their hazard ratios (HR) in all age categories. Hazard ratio obtained for o, p -DDE, p,p -DDE, p,p -DDT, p,p -DDD in Full fat (3%) pasteurized milk could not pose potential carcinogenic risk to young children (1, 3, 5 and 7 years old) since all of the HRs were far less than 1. Conclusion: This study has shown that cow milk is not a major source of human exposure to DDTs and from this perspective is safe for consumption. No conflict of interest. 672 Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms exacerbate bladder cancer risk associated with alcohol drinking: Gene−environment interaction H. Masaoka1,2 , H. Ito3,4 , N. Soga5 , A. Yokomizo2 , M. Eto2 , K. Matsuo1,4 . 1 Aichi Cancer Center Research Institute, Division of Molecular Medicine, Nagoya, Japan, 2 Kyushu University Graduate School of Medical Sciences, Department of Urology, Fukuoka, Japan, 3 Aichi Cancer Center Research Institute, Division of Epidemiology and Prevention, Nagoya, Japan, 4 Nagoya University Graduate School of Medicine, Department of Epidemiology, Nagoya, Japan, 5 Aichi Cancer Center Hospital, Department of Urology, Nagoya, Japan Background: Although a range of chemical exposures (cigarette smoking and occupational exposure) are recognized risk factors for the development of

bladder cancer (BCa), many epidemiological studies have demonstrated that alcohol drinking is not associated with BCa risk. Aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) polymorphisms impact the accumulation of acetaldehyde, resulting in an increased risk of various cancers. To date, however, no studies evaluating the association between BCa risk and alcohol drinking have considered these polymorphisms. Here, we conducted a matched case–control study to investigate whether ALDH2 and ADH1B polymorphisms influence BCa risk associated with alcohol drinking. Material and Methods: Cases were 74 BCa patients and controls were 740 first-visit outpatients without cancer at Aichi Cancer Center Hospital between January 2001 and December 2005. Odds ratio (OR), 95% confidence interval (CI) and gene–environment interaction were assessed by conditional logistic regression analysis with adjustment for potential confounders. Results: ALDH2 Glu/Lys was associated with a significantly increased risk of BCa compared with Glu/Glu (OR 2.03, 95% CI 1.14–3.62). In contrast, ALDH2 Glu/Lys showed no increase in risk among the stratum of never drinkers compared with Glu/Glu, indicating a gene–environment interaction. ADH1B His/Arg had an OR of 1.98 (1.20–3.24) compared with His/His. ADH1B Arg+ showed a similar OR and 95% CI. Individuals with ALDH2 Glu/Lys and ADH1B Arg+ had the highest risk of BCa compared with ALDH2 Glu/Glu and ADH1B His/His (OR 4.00, 95% CI 1.81–8.87)]. Conclusions: Those with the ALDH2 Glu/Lys and ADH1B Arg+ genotype are at increased risk of BCa. Gene–environment interaction between ALDH2 Glu/Lys and alcohol drinking might suggest acetaldehyde contributes to the carcinogenesis of BCa. No conflict of interest. 673 Association between night shift work and methylation status of circadian rhythm genes among Polish nurses and midwives − preliminary results A. Bukowska1 , E. Wieczorek2 , B. Peplonska1 , M. Przybek2 , E. Jablonska2 , E. Reszka2 . 1 Nofer Institute of Occupational Medicine, Department of Environmental Epidemiology, Lodz, Poland, 2 Nofer Institute of Occupational Medicine, Department of Toxicology and Carcinogenesis, Lodz, Poland Background: It has been suggested that rotating night shift work may contribute to epigenetic changes in the circadian rhythm genes. The results of previous studies in this topic are sparse and inconsistent. The aim of the study was to assess the potential relationship between current rotating night shift work and changes in levels of 5-methylcytosine in the promoter regions of selected circadian rhythm genes. Material and Method: The cross-sectional study included as many as 710 nurses and midwives (348 working on rotating night shifts and 362 working only during the day) aged 40−60 years. During in person interviews, information about occupational history and potential confounders was collected. DNA was isolated from peripheral blood samples and promoter methylation status of core circadian rhythm genes (PER1, PER2, PER3, BMAL1, CLOCK, CRY1, CRY2, NPAS2) was determined using quantitative methylation-specific real-time PCR assay (qMSP) method. Univariate and multivariate regression models were used to assess the association between current work at night and epigenetic changes, with adjustment for age and folate intake. Results: Current rotating night work was significantly inversely associated with hypermethylation of promoter region PER2, with OR = 0.7 (95% CI: 0.5−1.0). Moreover, increased odds ratio for high promoter methylation level of gene CRY2 (OR = 1.4 (95% CI: 1.0−2.1) was observed. There were no statistical significant association between night shift work and epigenetic changes in other circadian rhythm genes. Conclusions: Our study suggests that rotating night shift work may modify methylation level of PER2 and CRY2 genes. Further analyses are warranted to explore this under investigated topic. No conflict of interest.


Molecular and Genetic Epidemiology II 674 IKZF1 gene deletions are strongly associated with Ikaros dominantnegative isoforms expression in acute lymphoblastic leukemia V. Vshyukova1 , A. Meleshko1 . 1 Belarussian Center for Paediatric Oncology and Haematology, Laboratory of genetic biotechnologies, Minsk, Belarus Background: In most cases IKZF1 deletions lead to the expression of dominant-negative Ikaros splice variants (Ik-DN isoforms), and significantly correlate with an increased relapse rate and poor outcome in acute lymphoblastic leukemia (ALL) patients. Since detection of IKZF1 deletions by PCR and sequencing associated with a number of technical difficulties due to oligoclonality of tumor cells, RQ-PCR quantification of Ik-DN isoforms is on potential benefits for IKZF1 status assessment.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Material and Methods: Totally 220 bone marrow (BM) samples from primary ALL patients were included in the study. Control group included of 32 BM samples from healthy donors. RQ-PCR quantification of Ikaros isoforms expression was performed in 9 separate reactions for each sample. We worked up RQ-PCR estimation of functional (Ik1, 2, 3) and Ik-DN (Ik4, 5, 6, 8, 9 and 10) isoforms expression relative to control gene − Abelson’s tyrosine-kinase. Ik-Ratio between a functional isoforms (Ik1+Ik2+Ik3) and the sum of all isoforms expression levels was calculated. IKZF1 deletions of exons 1−6 (Ex1−6) and exons 3−6 (Ex3−6) were assessed by PCR amplification followed by sequencing. Results: The thresholds for Ik-DN overexpression were set empirically on the level 0.2 for Ik6, 0.1 for Ik9 and Ik10, and 0.8 for Ik-Ratio. Totally, IkDN overexpression has been identified in 96 ALL samples. Determined by the threshold, 29.2% of ALL were characterized by overexpression of Ik6 only, and 8.3% overexpressed Ik9. Combined overexpression of Ik6 and 9 was estimated in 50.0% of ALL samples, Ik6 + 9 + 10 − in 8.3%, and Ik6 + 10 − in 4.2%. The presence of clonal IKZF1 deletion was followed by Ik6−10 overexpression in 100% of cases, whereas subclonal deletions came to Ik-DN in 40% of cases only. To be noticed is that 10.9% of ALL with IKZF1 wild type also registered overexpression Ik6 or 9. Compared to Ik-DN isoforms quantification, the IkRatio have demonstrated major specificity for the presence of IKZF1 deletions, including subclonal deletions: only 4.2% (n = 4) of cases with IKZF1 wild type were determined as false positive results. Conclusions: Our results demonstrate feasibility of RQ-PCR quantification of Ik-DN, in particular the calculation of Ik-Ratio, as an additional method for IKZF1 gene testing in ALL patients. No conflict of interest. 675 Development of a prediction model and estimation of cumulative risk for upper aerodigestive tract cancer based on aldehyde dehydrogenase 2 (ALDH2) genotype and alcohol consumption in a Japanese population K. Matsuo1 , Y. Koyanagi2 , H. Ito3 . 1 Aichi Cancer Center Research Institute, Division of Molecular Medicine, Nagoya, Japan, 2 Nagoya University Graduate School of Medicine, Department of Epidemiology, Nagoya, Japan, 3 Aichi Cancer Center Research Institute, Division of Epidemiology and Prevention, Nagoya, Japan Background: Alcohol consumption and aldehyde dehydrogenase 2 (ALDH2) polymorphism are associated with Upper aerodigest tract (UAT) cancer risk, and a significant gene–environment interaction between the two has been confirmed in a Japanese population. To aid the development of a personalized prevention strategy, we developed a risk prediction model and estimated absolute risks stratified by a combination of ALDH2 genotype and alcohol consumption. Materials and Methods: We conducted two age- and sex-matched case– control studies at Aichi Cancer Center, one (630 cases and 1,260 controls) for model derivation and the second (654 cases and 654 controls) for external validation. Based on data from the derivation study, a prediction model was developed by fitting a conditional logistic regression model using the following predictors: age, sex, smoking, drinking, and ALDH2 genotype. A fitting of models were done for UAT cancer, esophageal cancer and head and neck cancer separately. Then, fitted models were applied in the validation study. Discriminative ability was assessed by the value of the area under the receiver operating characteristic (ROC) curve (AUC), which is also known as the concordance (c) statistic. Cumulative risks were estimateded by combining odds ratios estimated from the risk model with the age-specific incidence rate and population size as described by Peto et al (Peto et al. BMJ 2000;321:323– 329). Results: The risk model, including a combination of ALDH2 genotype and alcohol consumption, provided high discriminatory accuracy and good calibration in both the derivation and validation studies for UAT cancer: C statistics were 0.82 (95% confidence intervals 0.80–0.84) and 0.83 (0.81– 0.85), respectively, and the calibration plots of both studies stayed close to the ideal calibration line. Discrimination accuracy was better in esophageal cancer (0.94 for derivation and 0.91 for validation) and slightly worse in head and neck cancer (0.72 for derivation and 0.73 for validation). For heavy drinkers with a heterozygous genotype, cumulative risk at age 80 for UAT cancer was above 20%. In contrast, risk in the other groups was less than 5%. Conclusion: We developed simple risk prediction models of UAT cancer among Japanese population and they were acceptable accuracy. Estimation of cumulative risk revealed that a specific population, heavy drinkers with ALDH2 heterozygous, would be highly benefitted from personalized modification of alcohol consumption. Although application of genetic information is still on the way in cancer prevention, our results would be one of the nice applications. No conflict of interest.

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676 Genome-wide AFB1-induced mutational signature in cells, mice and human tumors − implications for molecular epidemiology W. Yu1 , M. Huang1 , W.W. Teoh2 , M. Ardin3 , S. Villar3 , A. Jusakul4 , R. Othman2 , K. Sabapathy2 , J. Zavadil3 , S. Rozen1 . 1 Duke-NUS Medical School, Centre for Computational Biology and Programme in Cancer and Stem Cell Biology, Singapore, Singapore, 2 National Cancer Centre Singapore, Division of Cellular & Molecular Research- Humphrey Oei Institute of Cancer, Singapore, Singapore, 3 International Agency for Research on Cancer, Molecular Mechanisms and Biomarkers Group, Lyon, France, 4 Duke-NUS Medical School, Programme in Cancer and Stem Cell Biology, Singapore, Singapore Introduction: Aflatoxin B1 (AFB1), found in foodstuffs throughout the world, is an IARC Group 1 carcinogen that causes hepatocellular carcinoma (HCC). The mutagenic effects of AFB1 on TP53 and reporter genes have been studied experimentally and in HCCs. Here we present first-of-its-kind data on the extended, genome-wide AFB1 mutagenesis using a set of complementary human and mouse in vivo and in vitro experimental systems. Materials and Methods: We determined genome-wide mutation patterns induced by AFB1 in two clonally expanded human liver cell-lines HepaRG and HepG2, in a mouse model of HCC driven by AFB1 treatment, and in own and public data from primary HCCs associated with aflatoxin exposure. Results and Discussion: The cell-line mutational patterns were remarkably stable across replicates, but differed somewhat between cell lines. Mutational patterns in the mouse tumours were more variable across replicates, possibly reflecting variability in the physiological clearance of the toxin in mice or random events during tumourigenesis. However, the overall pattern was consistently dominated by G>T mutations with a preference for TGC>TTC and substantial mutation enrichment on the non-transcribed strand. We next integrated these results with publicly available human HCC data and newly generated genomic HCC data from a known geographical region of aflatoxin exposure. Like the experimental systems, the human HCCs showed high rates of G>T mutations and strong transcriptional strand bias, providing evidence that the HCCs were direct consequences of AFB1 exposure. However, they differed from the experimental systems in that the most prominent mutations were GGC>GTC. This difference may be due to exposure to other aflatoxins, to other mutagens, or to differences in biochemical processing of AFB1. Human HCCs may harbour mutational signature of tobacco smoking that is similarly dominated by G>T mutations although it is typically associated with an enrichment in GG>TT dinucleotide substitutions. Indeed, we observed low GG>TT to G>T ratios (TT to G>T ratio (>0.01) suggested additional smokingassociated DNA damage on the target guanines. Conclusions: The experimental cell-based clonal expansion systems and mouse models used in our study of genome-wide mutagenic impact of AFB1 present innovative tools allowing to determine mutational signatures of candidate mutagenic carcinogens. We propose that the described experimental, multi-system approach can be used more broadly in support of molecular epidemiology studies aimed at cancer prevention. Acknowledgment: Funding obtained from IARC; ITMO CANCER − INSERM Plan Cancer 2015; NIH/NIEHS 1R03ES025023-01A1; Singapore A*STAR and MOH via Duke-NUS and NMRC/CIRG/1422/2015. No conflict of interest. 677 Implementation of liquid biopsies for detection of activating EGFR mutations in Bulgarian patients with NSCLC S. Bichev1 , A. Savov1 . 1 National Genetics Laboratory, University Hospital Of Obstetrics And Gynecology, Sofia, Bulgaria Background: Activating somatic mutations in tyrosine kinase domain of EGF receptor are well established predictive biomarker for patient’s response to targeted therapies. For this reason NSCLC patients have to be tested for these mutations. The preferred material for EGFR testing is FFPET samples or cytology samples. However in many cases the initial material is insufficient and often re-biopsy is not possible due to patient’s status or patient’s will. In such cases an alternative method for EGFR genotyping should be used. In recent years it becomes clear that tumor cells release cell free tumor DNA (CtDNA) in bloodstream. Molecular characterization of ctDNA may provide a strategy for EGFR genotyping. Material and Method: For a period of 3 months 15 patients have been screened for EGFR activating mutations. From all patients, ctDNA was isolated from plasma samples, immediately after collecting the blood. Cell free tumor DNA was extracted with QiaAmp Circulating Nucleic Acid kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. To achieve better results from DNA extraction we used QiaVac 24 plus system (Qiagen, Hilden, Germany). EGFR genotyping was performed with therascreen EGFR Plasma RGQ PCR Kit. Results and Discussion: DNA extraction was successful in all 15 cases. The quality and concentration of extracted DNA fulfilled the requirements of

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EGFR Plasma PCR kit and therefore all samples were screened for activating mutations in EGFR gene. We found only one mutation (L858R) from all 15 analyzed samples (6.6%). The expected frequency of EGFR mutations in Bulgarian population is 9.5%, the lower frequency in this cohort could be explained by the small group of analyzed patients and by the sensitivity of the method. Conclusion: According to different studies the sensitivity of this method is about 65%, so the preferred material for EGFR testing should be tumor tissue where possible. However liquid biopsies are great options, in rare cases, where the initial biopsy is exhausted. This approach allows more patients with NSCLC to benefit from targeted therapies. Conflict of interest: Corporate-sponsored Research: The research was sponsored by Astra Zeneca. 678 Diagnostic and prognostic value of long non-coding RNA expression in urothelial carcinoma J. Droop1 , T. Szarvas2 , W.A. Schulz1 , C. Niedworok2 , G. Niegisch1 , K. Scheckenbach3 , M.J. Hoffmann1 . 1 Heinrich-Heine-University, Urology, Dusseldorf, ¨ Germany, 2 University Hospital Essen, Urology, Essen, Germany, 3 Heinrich-Heine-University, Otolaryngology, Dusseldorf, ¨ Germany Background: Long non-coding RNAs (lncRNAs) comprise diverse RNA transcripts that are by definition longer than 200 nucleotides, but do not contain a significant open reading frame. Many of them show a tissuespecific expression pattern and undergo expression changes during tumor development and progression. In urothelial carcinoma (UC) tissues, several lncRNAs have been reported as overexpressed and proposed as candidate diagnostic or prognostic biomarkers, namely UCA1, MALAT1, H19, GAS5, TUG1, ncRAN and linc-UBC1. However, for most of these candidates, validation studies in independent sample cohorts and a detailed functional characterization in UC are lacking. Hence, we measured lncRNA expression by qRT-PCR in a large UC tissue set with complete clinical and histopathological data. These results were further compared with RNA-Seq data obtained via the TANRIC database (based on TCGA data) to validate differences between tumor and benign tissues and correlations with clinical outcome for the 7 candidates. Material and Method: LncRNA expression was measured by qRT-PCR in a tissue set comprising 106 UC samples and 10 normal tissue samples. p-Values for differential lncRNA-expression between UC and control samples were assessed by Wilcoxon rank sum test in R. TCGA expression data obtained from the TANRIC database for 252 tumor and 19 normal samples was analyzed with regard to differential expression in UC and clinical outcome by Kaplan– Meier analysis. Results: UCA1, GAS5, ncRAN and linc-UBC1 expression was increased in tumor tissues in both UC sample cohorts, but none of the differences were statistically significant. H19 and TUG1 displayed rather heterogeneous expression patterns. Contrary to previous reports, MALAT1 was found to be downregulated in both tissues sets. Kaplan–Meier analyses correlating overexpression with overall survival time did not reveal any significant association. Conclusions: The aim of this study was to validate the expression changes of 7 lncRNAs proposed as biomarkers in a large UC tissue sample cohort and by analysis of publicly available data sets. However, we could not reproduce any of the reported overexpressions. In addition, patient prognosis appeared to be unrelated to differential expression of candidate lncRNAs. Thus, UCA1, MALAT1, H19, GAS5, TUG1 and linc-UBC1 may not be suitable diagnostic biomarkers for urothelial bladder cancer. Limitations of our study are a strong bias towards invasive UC and the restriction to tumor tissues. No conflict of interest. 680 Searching for circulating miRNA markers in gastric cancer 1 ˙ J. Toton-Zura nska ´ , M. Sier˙ze˛ga2 , K.W. Maria1 , P. Radkowski1 , J. Kulig2 , P. Wołkow1 . 1 Jagiellonian University Medical College, Center for Medical Genomics − Omicron, Krakow, Poland, 2 Jagiellonian University Medical College, First Department of Surgery, Krakow, Poland

Background: MicroRNAs (miRNAs) are group of small noncoding RNAs, about 20−25 nucleotides in length, which regulate gene expression in diverse processes. It was proven that miRNA expression changes in response to various stimuli as well as in disease states. miRNA expression was shown to be regulated by miRNA promoters methylation changes what takes place in cancer. Up to date many microRNAs were connected with gastric cancer biology and were found also in circulation. As circulating microRNA are relatively stable and easy to obtain from liquid biopsy, they are good candidates for biomarkers of gastric cancer related processes. In the current, screeningphase of the study, we are searching broadly among all known microRNAs with the use of next generation sequencing, to find best candidates to monitor disease progression and predict long-term prognosis. Materials and Methods: In the first study phase serum samples from 40 patients and 16 controls pooled to 8, were analyzed. Small RNA extracted

from samples was quality controlled. Subsequently, small RNA libraries were prepared, size-selected to and sequenced on a next generation sequencer. Raw data was quality filtered, then normalized. Differential expression was calculated with DeSeq2. Expression data was analyzed in the context of demographic and pathological parameters (angioinvasion, perineural invasion, presence of distant metastases, etc.). Results and Discussion: Among down-regulated microRNAs we have found the let-7 family, which is known to target oncogenes like: c-Myc and Ras, JAK (Janus protein tyrosine kinase), STAT3 (signal transducer and activator of transcription 3), HMGA (high-mobility group A), which all are critical in tumorigenesis, proliferation and cancer cell invasion. Several previously unreported sequences were up-regulated, including mir-1285-5p, which targets ANKRD17 (Ankyrin Repeat Domain 17), miR-4642, which targets Cables2 (Cdk5 And Abl Enzyme Substrate 2) and TGFBR3 (Transforming Growth Factor Beta Receptor III) which proteins are down-regulated in stomach cancer. Conclusions: Expression of selected microRNAs will be further evaluated by qPCR experiments in a larger cohort of patients (n = 250) to validate their suitability for diagnostic, predictive, and prognostic applications. Finding relevant microRNA biomarkers could greatly improve clinical management by helping to choose treatment strategies and monitor disease progression. No conflict of interest. 682 Correlation of genetic polymorphism of TNFa and TGFb genes with protein level in patients with pancreatic and colorectal cancer in Polish population M. Zagozda1 , A. Sarnecka1 , Z. Staszczak1 , M. Nowak-Niezgoda2 , W.L. Olszewski1 , M. Durlik1 . 1 Mossakowski Medical Research Centre Polish Academy of Sciences- Warsaw- Poland, Department of Surgical Research & Transplantology, Warszawa, Poland, 2 Central Clinical Hospital- Ministry of Internal Affairs, Clinical Department of Gastroenterological Surgery and Transplantation, Warszawa, Poland Background: The signaling pathway of transforming growth factor beta (TGFb) and tumor necrosis factor a (TNFa) may be important in the development of pancreatic and colorectal cancer. TGFb is a ubiquitous growth factor that plays a key role in the regulation of growth and differentiation in a wide variety of cell types. Polymorphisms in these genes can cause functional inefficiency of their products and in consequence a tendency of some patients to the development of extensive inflammation and sepsis. The aim of the study was to research markers of bacterial infections, surgical wound healing disorders or fistula anastomosis in patients operated for pancreatic and colorectal cancer. Material and Methods: The study group consisted of 65 patients with pancreatic cancer and 41 patients with colorectal cancer. Detection of polymorphisms was performed using the PCR-RFLP or TaqMan Assays method. Levels of TNFa and TGFb proteins were measured using commercially available ELISA kits. A control group of 100 blood donors was ethnically matched to the study and randomly selected to comparing the study data. Results: There were not differences in GG and GA genotypes of TNFa G-308A in two groups of patients and control subjects. The mutated homozygote AA was higher in patients with colorectal cancer than in the second study group and controls (7.3% vs. 3.2% vs. 1.0%, p=NS). The average concentration of TNFa in patients with colorectal cancer (1.72±2.10 øg / ml) was significantly higher than the control group (0.29±0.52 øg/ml, p < 0.0001). No significant differences were found at TGFb T29C genotypes in all studied groups and controls. Comparing with the average level of proteins concentration, between the three genotypes, showed no statistically significant differences. It was also noted that TGFb protein concentration was significantly higher in patients with colorectal cancer than in patients with pancreatic cancer (p = 0.0353). Conclusions: Analyses showed that the presence of the polymorphic genotype −308AA of TNFa gene was not correlated with an increased level of that protein in two studied groups. In the case of TGFb gene polymorphism T29C we also did not observe increased TGFb concentration in the presence of a polymorphic genotype CC. It seems, therefore, that examined factors may not have a very strong impact on the ongoing process of cancer. No conflict of interest.



Prevention and Early Detection I 685 A novel detection method of metastatic cells in the cerebrospinal fluid of pediatric population with medulloblastoma using fluorescence lifetime imaging microscopy S. Gershanov1 , H. Toledano2 , S. Michowiz3 , G. Yahav4 , O. Barinfeld5 , A. Hirshberg6 , M. Salmon-Divon1 , D. Fixler4 , N. Goldenberg-Cohen5 . 1 Ariel University, Molecular Biology, Ariel, Israel, 2 Schneider Children’s Medical Center of Israel, Pediatric Oncology, Petach Tikva, Israel, 3 Schneider Children’s Medical Center of Israel, Pediatric Neurosurgery, Petach Tikva, Israel, 4 Bar Ilan University, Faculty of Engineering and Institute of Nanotechnology and Advanced Materials, Ramat Gan, Israel, 5 Tel Aviv University, The Krieger Eye Research Laboratory- Felsenstein Medical Research Center- Sakcler school of medicine, Tel Aviv, Israel, 6 Tel Aviv University, Oral Pathology and Oral Medicine- Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv, Israel Medulloblastoma (MB) is the most common malignant brain tumor in children, and one of the deadliest. About one third of children with MB already demonstrate metastatic spread at diagnosis and approximately 40% will experience metastatic tumor recurrence post treatment. Another highly malignant tumor of childhood is the atypical teratoid/rhabdoid tumor (ATRT) usually diagnosed in the first 2 years of life. Despite recent advances in treatment, patients with tumor dissemination have a poor survival rate. Moreover, early metastatic spread is often undetectable by imaging or cytology. The purpose of this study is to demonstrate the capabilities of a novel diagnostic method to improve early detection and monitoring of metastatic spread, using fluorescence lifetime imaging microscopy (FILM). Cells extracted from tumor at diagnosis and cerebrospinal fluid (CSF), 2 weeks after surgery and repeatedly during and after chemo/radiotherapy, of 15 children diagnosed with MB and 2 with ATRT. The cells obtained were subjected to nuclear staining by DAPI followed by fluorescence lifetime (FLT) measurement. FLT of DAPI was divided into 3 subgroups: prolonged, medium and short. Prolonged FLT was found predominantly in cells from the original tumor cells (median 4.27±0.28 ns) and in the CSF cells from metastatic children obtained before chemo/radiotherapy (median 6.28±0.22 ns); medium FLT in immune system cells from non-oncology pediatric patients (median 2.6±0.04 ns) who served as controls, also in CSF cells from non-metastatic children before chemo/radiotherapy (median 2.62±0.23 ns) and following treatment (median 2.62±0.21 ns). Short FLT predominated in CSF samples from metastatic children post treatment (median 2.4±0.29 ns), either chemotherapy or craniospinal radiation. FLIM is simple and fast assay with the potential to identify tumor spread in the CSF of children with malignant brain tumors. FLT measurements change according to treatment, hence may guide personalized therapy, improve outcome and increase survival. No conflict of interest. 686 Awareness and understanding of disease among hospitalized cancer patients in Pakistan

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illness. Although the patients were more satisfied with care than the information they had received, awareness was not related to satisfaction (p > 0.05). Most of the patients (71.0%) were not satisfied with the quantity and quality of the information they had received from their health care provider. Conclusions: Our findings suggest that although cancer patients want and need to have adequate information regarding their disease, the amount and quality of information they receive is not optimal leading to adoption of passive information seeking strategy causing misconceptions about disease. No conflict of interest. 687 Inflammatory potential of diet and risk of laryngeal cancer in a case–control study from Italy N. Shivappa1 , J. Hebert1 , V. Rosato2 , D. Serraino3 , C. La Vecchia2 . 1 University of South Carolina, Epidemiology and Biostatistics, Columbia, USA, 2 Universita‘ degli Studi di Milano, Department of Clinical Sciences and Community Health, Milan, Italy, 3 Centro di Riferimento Oncologico, Unit of Epidemiology and Biostatistics, Aviano, Italy Background: Besides tobacco and alcohol, diet and inflammation have been suggested to be important risk factors for laryngeal cancer. Materials and Methods: In this study, we examined the role of a dietary inflammatory index (DII) to predict laryngeal cancer in a multicentre case– control study conducted between 1992 and 2000 in Italy. This study included 460 cases with incident, histologically confirmed laryngeal cancer, and 1088 controls hospitalized for acute non-neoplastic diseases unrelated to tobacco and alcohol consumption. The DII was computed based on dietary intake assessed by a validated 78-item food frequency questionnaire. Logistic regression models were used to estimate odds ratios (ORs), and the corresponding 95% confidence intervals (CIs), adjusted for age, sex, study center, education, body mass index, tobacco smoking, alcohol drinking, and non-alcohol energy intake. Results: Subjects with higher DII scores (i.e., with a more pro-inflammatory diet) had a higher risk of laryngeal cancer. The OR was 3.30 (95% CI 2.06, 5.28; p for trend 80 TRACERx patient blood samples have been processed and CTCs biobanked and single CTCs were isolated using a DEPArray system for 18 patients. Whole genome amplification (WGA) was performed on >70 CTCs and White Blood Cell controls. A bioinformatics pipeline has been developed to design efficiently WGA compatible PCR amplicons spanning target mutations identified in the matched tumour. CNA profiles have been generated from the isolated CTCs to investigate tumour heterogeneity and further molecular analysis is currently on-going. Conclusion: A robust workflow for enrichment, enumeration, isolation and molecular analysis of CTCs from early stage NSCLC patients on the TRACERx study has been developed. Bioinformatics pipelines are now in place to facilitate efficient comparison of the genetic status of patients CTCs, resected tumours and for relapsing patients, metastatic biopsies, to better understand the role of tumour heterogeneity and mutational status on disease recurrence. No conflict of interest. 813 The prognostic potential of the long non-coding RNA MALAT1 in prostate cancer K. Hiew1 , S.M. Bokobza2 , C.A. Hart1 , T. Elliott3 , N.R. Smith2 , M. Brown1 , N.W. Clarke4 . 1 Faculty Institute of Cancer Sciences, Genito-urinary cancer research group, Manchester, United Kingdom, 2 Astrazeneca, Oncology iMed, Macclesfield, United Kingdom, 3 Christie Hospital NHS Foundation Trust, Oncology, Manchester, United Kingdom, 4 Christie Hospital NHS Foundation Trust, Urology, Manchester, United Kingdom Background: Prostate cancer is the most common male cancer in the UK (47,300 new cases) resulting in >10,800 deaths per year. Currently there are no biomarkers at the point of initial diagnosis which robustly identify patients who will develop metastatic disease. MALAT1 (metastasis associated lung

adenocarcinoma transcript 1) a long non-coding RNA, originally identified in lung adenocarcinoma, has been shown to modulate genes regulating cell motility. MALAT1 has been associated tumour cell survival, growth and metastatic behaviour in both renal and urothelial carcinomas but its role in prostate cancer is unknown. Methods: Regions of tumour from archival FFPE TURP specimens with up to 21 years of clinical follow data, were macro-dissected for RNA extraction and subsequent 405 gene expression analysis using the NanoString© platform. Principle component analysis was conducted using Qlucore Omics Explorer 3.2 with specific correlations with MALAT1 analysed through Pearson’s Coefficient Correlation. Pathway modelling was performed using DAVID Functional Annotation Clustering and statistical analysis conducted in SPSS. mRNA expression levels were assessed in cell lines of defined characteristic by qRT-PCR. Results: PCA analysis identified a 31 gene signature, including MALAT1, which differentiates between M0 and M1 at diagnosis (p = 0.0126, q = 0.1044). Over expression of MALAT1 correlated with time to progression (47 months vs 120 months; p < 0.05) and shorter overall survival in M0 patients at diagnosis (95 months vs 130 months; p = 0.026) but did not predict time to progression or overall survival in M1 patients. Pathway analysis suggests a link between MALAT1 and both Hedgehog and PIK3CA/Akt signalling pathways. Conclusion: MALAT1 expression in diagnostic biopsies has prognostic potential in prostate cancer patients presenting with M0 disease. No conflict of interest. 814 A novel PCR error correction algorithm for cell-free DNA next generation sequencing data using high performance computing C.S. Kim1 , S. Gulati2 , M. Ayub3 , D.G. Rothwell2 , S. Mohan2 , C. Dive3 , G. Brady3 , C. Miller4 . 1 Cancer Research UK Manchester Institute, Clinical and Experimental Pharmacology Group and Medical Research Council Manchester Single Cell Research Centre and RNA Biology Group, Manchester, United Kingdom, 2 Cancer Research UK Manchester Institute, Clinical and Experimental Pharmacology Group, Manchester, United Kingdom, 3 Cancer Research UK Manchester Institute, Clinical and Experimental Pharmacology Group and Medical Research Council Manchester Single Cell Research Centre, Manchester, United Kingdom, 4 Cancer Research UK Manchester Institute, RNA Biology Group and Medical Research Council Manchester Single Cell Research Centre, Manchester, United Kingdom Background: Cell-free DNA (cfDNA) profiling has emerged as an importantminimally invasive means to profile and monitor the mutation status of solid tumours in cancer patients. Since the concentration of cfDNA available from blood plasma/serum samples is relatively small, cfDNA sample libraries are heavily dependent upon PCR amplification prior to high-throughput DNA sequencing. As a consequence, a major challenge arising with the cfDNA mutation profiling is to identify and eliminate the errors introduced during PCR amplification. In this study, we present a novel, computationally efficient algorithm designed to distinguish PCR amplification-related errors from genuine mutations. Material and Method: The algorithm consists of three main modules. It first identifies and clusters input reads that are duplicated from the same cfDNA template. It then generates consensus sequences using a de novo assembly algorithm. Finally, reads are compared to the consensus sequence to identify PCR amplification-related errors. The algorithm provides summary statistics that include estimates of PCR error rate, the PCR amplification efficiency and a statistical significance score for error detection. The algorithm is implemented within the MapReduce framework for parallel computation using compute clusters. Results: Performance was assessed using simulated data generated with known values for PCR-efficiency and error rate. These data confirmed that the algorithm was able to significantly improve the accuracy of cfDNA sequencing data, with a True Positive rate for error detection and correction of >98%, and a False Negative rate of 100mM) showed only modest activity. Combinations of pitavastatin with zoledronic acid, and to lesser extend risedronate, displayed synergistic activity in A2780, CisA2780, Ovcar-4, Igrov-1 and Skov-3 cells or additivity in Cov-318, Cov-362, Ovcar-5 and Ovcar-8 cells.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Antagonism was observed in Ovcar-3 cells. The combination of pitavastatin and zoledronic acid also potentiated the cytotoxic activity measured by trypan blue and ATP cell viability assays. Caspase activity was significantly higher than that expected for the combination estimated from the Bliss independence criterion. Immunoblot analysis demonstrated that the combination resulted in significant accumulation of cleaved PARP which was greater than that observed with each single agent. Conclusion: These data demonstrate that zoledronic acid interacted synergistically with pitavastatin in several assays and raise the possibility that BP may be combined with pitavastatin for the treatment of ovarian cancer. No conflict of interest. 830 Whole exome analysis of HER-2 positive human breast cancers: molecular mechanisms underlying response to neoadjuvant therapy with trastuzumab M. La Ferla1 , P. Aretini1 , C. Scatena2 , M. Menicagli1 , F. Lessi1 , S. Franceschi1 , L. Cantini3 , G. Bevilacqua1 , A.G. Naccarato2 , A. Fontana3 , C.M. Mazzanti1 . 1 Fondazione Pisana per la Scienza − ONLUS, Pisa, Italy, 2 Division of Pathology, University Hospital of Pisa, Pisa, Italy, 3 University Medical Oncology Unit II, Azienda Ospedaliero-Universitaria Pisana AOUP, Pisa, Italy Introduction: HER2 receptor is overexpressed in 20−30% of BC and it has been chosen to be an effective therapeutic target. Trastuzumab (Herceptin), a humanized monoclonal antibody, has been approved by FDA to be anti-HER2 therapy, but the mechanism by which trastuzumab exerts its antitumor activity is not fully understood. Despite that almost 50% of HER2 Positive BC patients do not respond to trastuzumab based therapy or become resistant during the therapy. An understanding of trastuzumab resistance mechanisms would be a helpful tool in the development of rational drug combinations to circumvent resistance and allow better selection of patients likely to respond. The aim of this study is the correlation of the mutational profile of HER-2 positive patients with complete response and partial response to the neoadjuvant therapy with trastuzumab and evaluation of possible molecular factors predictive of response. Materials and Methods: We performed, so far, whole-exome DNA sequencing (Ion Proton system and NextSeq™500-Illumina) to analyze primary FFPE biopsy and primary surgical removed tumors of HER2 positive BC patients specifically selected for different response to the neoadjuvant therapy with trastuzumab and all ER and PR negative: 6 Full Responders (FR) with a complete disappearance of the tumor mass after the therapy and 6 Partial Responders (PR) with a partial shrinkage of the tumor mass, which is then removed by surgery. More patient samples are in the process to be analyzed by whole exome analysis for a total of 20 patients (10 each group). Results and Discussions: We identified 23 genes that are mutated in the PR patients or in the FR patients that discriminate selectively the two groups of patients statistically significative. From this panel of genes we focus our attention on a missense mutation in ANKRD44 gene present only in 3 PR patients. This mutation could be involved in a constitutive loop of activation of NF-Kb pathway that could result in a mechanism of resistance for PR patients. Thanks to literature analysis we found that some of these variants are already associated with trastuzumab resistance and sensitivity. Conclusions: Our findings indicate a possible involvement of a combination of gene mutations associated with resistance to the trastuzumab therapy for the PR patients. The final gold is the creation of a gene panel implicated in resistance and/or in the complete response to trastuzumab. Moreover other genes which resulted differentially expressed between PR and FR HER2+ patients are in the process of being investigated. However functional studies, as well as association studies in a larger patient dataset are needed to explore our hypothesis. No conflict of interest. 831 Similarities between the Marfan syndrome and cancer: Implications of the Fibrillin−TGFb axis on cancer biology and treatment ´ 1, J.L. Parra1 , R. Mayor1 , I. Huber1 , I. Cuartas1 , A. Arias1 , C. Raventos J. Seoane1 . 1 Vall d’Hebron Institute of Oncology, Translational Research, Barcelona, Spain Exome sequencing of a cohort of lung tumours revealed the presence of somatic mutations in FBN2. Interestingly, the identified mutations consisted in the same base substitutions that cause Marfan-like syndromes. Fibrillins act as a reservoirof TGFb, controlling its physiological activity. Marfan-like syndromes are caused by mutations in FBNs and are characterized by a hyperactive TGFb activity due to the impairment of fibrillins to sequester TGFb ligands in the extracellular matrix. The fact that the mutations identified in lung tumours are identical to the germinal mutations that cause Marfan-like syndromes lead us to hypothesize that lung tumours with Marfan-like mutations in FBN2 will present a hyperactive TGFb pathway. The aberrantly high TGFb signal confers a selective advantage through the induction of immunosuppression, cell invasion, angiogenesis and metastasis suggesting that in tumours with Marfan-like mutations, TGFb might be a good therapeutic target.

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Materials and Methods: 1. Bioinformatics approximations to study the incidence of Marfan-like mutations. 2. Characterization of lung cancer cell lines, patient-derived tumouroids and patient-derived xenografts (PDX) with FBN mutations. 3. CRISPR/Cas9 technology to reverse FBN1 and FBN2 mutations to study the biology of FBN in cancer. Results: We have generated a bioinformatic tool to identify functional mutations in FBNs based on the Marfan-like mutations public data bases. 1. We have identified Marfan-like mutations in a wide variety of tumors. In particular, we focus in lung tumors since these mutations are present in 5% of the cases. 2. Tumors with Marfan-like mutations showed high TGFb pathway activity, enrichment of the TGFb gene signature, alterations in tumor microenvironment and the prevalence of M2 phenotype immune cells. 3. Marfan-like mutations confer tumor sensitivity to anti-TGFb treatments in preclinical models. 4. Repair of Marfan-like mutations by CRISPR reduces tumor growth and fibrosis, polarizes the immune cell infiltration towards an M1 phenotype and desensitizes tumors to TGFb treatments in vivo. Discussion: We have identified Marfan-like mutations as driver mutations in lung cancer which potentially can be used as a new therapeutic biomarker. To assess their role in tumor biology, we have selectively reverted the FBN2 mutations in PDX lung tumor models using CRISPR. We observed that repair of the FBN2 mutations induces changes in tumor growth, tumor microenvironment and in the immune system phenotype. We have observed that orthotopic xenografts models of lung cancer with Marfan-like mutations are sensitive to TGFBR inhibitors and, importantly, these tumors become resistant to the inhibitor once the FBN mutations are repaired. Finally, we propose the use of anti-TGFb treatments as a therapeutic tool in tumors with Marfan-like mutations. No conflict of interest. 832 NSCLC − multiplex immunohistochemical staining for diagnosis in small biopsies B.S. Nielsen1 , K. Holmstrøm1 , T. Møller1 , H. Stender2 , M. Kristensson3 , E. Santoni-Rugiu4 . 1 Bioneer A/S, Diagnostics, Hørsholm, Denmark, 2 Stender Diagnotics, Diagnostics, Gentofte, Denmark, 3 Visiopharm, Technical Sales, Horsholm, Denmark, 4 Rigshospitalet- Copenhagen University Hospital, Pathology, Copenhagen, Denmark Background: Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer diagnoses and in 75% of cases is inoperable and diagnosed on small biopsies. The histological subtyping of NSCLC, especially the differentiation between adenocarcinomas (AC) and squamous cell carcinomas (SCC), is important because of different treatment. Histopathological evaluation of small samples to discriminate AC from SCC subtypes is often challenging because of poor morphology and insufficient tissue. Thus, confirmatory immunostainings to support the diagnosis are necessary. Multiple immunostainings in NSCLC help discriminating AC from SCC including cytokeratin 5 and 7 (CK5 and CK7) and the transcriptional factors TTF1 and P40, and in addition ALK-staining is required for selecting patients to targeted therapy. We have developed an immunochemical diagnostic analysis that allows the detection of the 5 biomarkers on a single tissue section using clinically implemented primary antibodies. Material and Methods: Surgical samples and needle biopsies from NSCLC patients of AC and SCC subtypes were obtained from Rigshospitalet. Unmodified primary antibodies for the detection of CK5, CK7, TTF1, P40 and ALK were used. Four out of 5 antibodies were of mouse origin and had the same isotype (IgG1). Multiplex immunofluorescent sequential staining was performed using Cy3-labelled secondary antibodies and intermediate antibody elution steps following imaging of each individual biomarker immunoassay. Digital images of each individual biomarker analysis were processed and shown as a ‘virtual DAB’ staining or as a combined superimposed single fluorescence image. Results: Multiplex immunohistochemical staining of tissue sections with primary antibodies of the same origin and isotype required designing a sequential assay format that allows each biomarker assay to be performed individually followed by imaging the result and subsequently ‘deleting’ the signal before moving on with the next biomarker assay. We show that this can be done on one single tissue section, by providing evidence for the efficiency of eluting antibodies (primary and secondary) between the assays, and the ability to combine and superimpose individual images of biomarker assays after the sequential analysis. We demonstrate that this protocol is also applicable to small biopsies. Conclusion: We have developed a sequential immunofluorescent diagnostic assay that can detect up to five different biomarkers relevant for NSCLCdiagnostics applicable to both surgical and bioptic samples. The assay format is independent of the origin and isotype of primary antibodies. The tissue

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section remains intact throughout the sequence of assays enabling perfect superimposition and co-localization of different signals. Conflict of interest: Other Substantive Relationships: BSN, KHO, TRM are full time employees at Bioneer A/S. HS is founder and owner of Stender Diagnostics. MK is full time employee at Visiopharm A/S. 833 Population analysis of patients diagnosed with renal carcinoma − A retrospective single center study in Argentina M.N. Gandur Quiroga1 , M.D.N. Juarez Rusjan2 , M.A. Krasnapolski2 , ´ C. Pulero3 , L.B. Todaro4 , P.J. Azurmendi5 . 1 Institute of Oncology Angel H ´ Roffo. UBA, Clinical Oncology, Ciudad Autonoma de Buenos Aires, Argentina, 2 ´ Institute of Oncology Angel H Roffo. UBA, Molecular Pathology, Ciudad ´ ´ Autonoma de Buenos Aires, Argentina, 3 Institute of Oncology Angel H Roffo. ´ UBA, Pathology, Ciudad Autonoma de Buenos Aires, Argentina, 4 Institute ´ ´ of Oncology Angel H Roffo. UBA, Research Area, Ciudad Autonoma de Buenos Aires, Argentina, 5 Intitute of Medical Research Alfredo Lanari. UBA, ´ Experimental Nephrology and Molecular Biochemistry, Ciudad Autonoma de Buenos Aires, Argentina Introduction: Our institution admits an average of 50 new patients with Renal Cancer per year. In our country, this pathology is the 9th leading cause of death from cancer in men and the 13th in women (ratio 1.5: 1), being the most lethal urological cancer. Clear cell renal cell carcinoma (ccRCC) is the most common histological type and comprises a heterogeneous group of cancers with variable clinical evolution. VHL-driven pathway over-activation is associated with 60−80% of sporadic cases of ccRCC. They trigger the expression of angiogenesis-related genes and lead to the deregulation of several metabolic pathways. Background: Previously, on a smaller population we have observed statistically significant associations between high expression of molecules related to angiogenic and metabolic pathways (such as Ang-2, VEGFR2, VEGFR-3, PDGFA and CAIX) with different disease manifestations such as the presence of metastases, hypercholesterolemia, hypercalcemia, hypertension (HT) and survival parameters. We have also found correlations among the expression of the aforementioned molecular markers. Objective: The objective of this study was to, retrospectively, analyze the clinicopathological characteristics and prognostic factors evaluating the medical charts of all renal cancer patients of our institute since 2002, in order to expand our previous study related to the search of diagnostic, prognostic and/or predictive biomarkers. Patients and Methods: Medical records of patients diagnosed with renal cancer were identified retrospectively between 2002 and 2014. Out of 285 renal tumors 271 were renal carcinomas, 96 (61 men and 35 women) of which were available for analysis, follow-up time 4.1±2.1 years (yr). Patient clinicopathological characteristics, risk and prognostic factors as well as OS and PFS were reviewed. Results: The median age at diagnosis (Dx) was 57 [IQR 25–75: 51−66] yr, being lower in men than in women (54 [49−63] vs 62 [52−68], respectively, p = 0.028). The 25% of patients died at the age of 66±2.5 yr. Patients with metastasis at Dx showed a 6.5 fold (95% CI: 2.9–14.7, p < 0.0001) increase in death risk than those who did not have metastasis. Multiple regression analysis showed that OS depends on Fuhrman grade, gender and HT (unadjusted R = 0.4, adjusted R = 0.32, p = 0.042). The Spearman Correlation analysis showed that death was associated with: OS (ø = −0.28, p = 0.01), PFS (ø = −0.51, p = 0.004), performance status (ø = 0.28, p = 0.01), metastasis at Dx (ø = 0.56, p = 0.000001) and low hemoglobin (ø = 0.29, p = 0.02). Conclusions: Following this analysis, population could be stratified into good and poor prognosis. Accordingly, data obtained from the previously analyzed molecules will be re-analyzed following the main objective of this study: to define biomarkers for prognostic and/or predictive for this disease. No conflict of interest. 834 Monitoring circulating tumour DNA in patients receiving selective internal radiation therapy for liver metastases and intrahepatic cholangiocarcinoma H. Winter1 , P. Kaisaki2 , A. Cutts3 , R. Carter1 , T. Greenhalgh1 , A. Schuh1 , J. Taylor2 , R. Sharma1 . 1 University of Oxford, Department of Oncology, Oxford, United Kingdom, 2 Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, Oxford, United Kingdom, 3 John Radcliffe Hospital, Oncology, Oxford, United Kingdom Background: Precision medicine mandates accurate histological and molecular diagnostics. Temporal and spatial tumour heterogeneity has been reported as a key determinant of cancer resistance. The accurate detection of evolving somatic mutations and the early assessment of response to new therapies are emerging challenges. Monitoring cancers following exposure to new therapies such as selective internal radiation therapy (SIRT) require new biomarkers of response. The standard techniques of RECIST imaging at three months were developed as biomarkers of response to chemotherapy and may not reflect tumour changes to these newer therapeutic strategies.

The analysis of circulating tumour (ct)DNA to detect recurrence and to predict response is an emerging research priority. The objective of this study was to explore the feasibility of the quantification and sequencing of serial ctDNA measurements in patients with liver predominant metastatic disease receiving SIRT. A secondary aim was the analysis of ctDNA to detect the evolution of somatic mutations following high dose liver radiotherapy. Methods: A prospective imaging biomarker study was performed in patients with intrahepatic cholangiocarcinoma and liver metastases from colorectal cancer. All patients received SIRT for metastatic liver-dominant disease. Plasma was extracted from 30 ml blood samples and frozen within 4 hours from patients at 3 time points: baseline (before SIRT), 4 weeks and 10 weeks after SIRT. Quantification of ctDNA and buffy coat germline DNA was assessed by the Qubit dsDNA fluorometer. Ion Torrent Amplicon sequencing was performed with the cancer hotspot panel v.2. Results of sequencing of primary tumours were obtained from pyrosequencing. Results: Twenty three patients were recruited from March to December 2015. Ion Torrent amplicon sequencing detected somatic mutations in ctDNA analysed at baseline, including KRAS G13D, KRAS G12V and a rare AKT1 E17K mutation. There was high concordance with formalin fixed paraffin embedded (ffpe) historical samples, sequenced up to 1100 days previously. Changes from baseline to one month after SIRT in quantification of ctDNA will be presented at the conference. Conclusions: ctDNA is emerging as a biomarker of metastatic colorectal cancer. Here we demonstrate that quantification of ctDNA is feasible at serial time points in patients receiving SIRT. Comparison of mutational status measured in ctDNA and ffpe samples shows high concordance, despite long intervals between the original biopsy and the blood sample. Future work is planned to look at ctDNA as a tool to detect evolution of somatic mutations following high dose radiation. No conflict of interest. 835 Pitavastatin is a potential treatment for drug-resistant ovarian cancer E. Robinson1 , S. Jones1 , K. Menezes2 , M. Abdullah1 , E. Stronach2 , C. Hoskins1 , A. Richardson1 . 1 Keele University, Institute for science and technology in medicine, Stoke on Trent, United Kingdom, 2 Imperial College, Institute of Reproductive and Developmental Biology, London, United Kingdom Background: Most patients with ovarian cancer respond to chemotherapy but only 40% with advanced disease survive beyond 5 years. The emergence of drug resistance limits the effect of further rounds of chemotherapy. New therapies are needed which are effective in chemotherapy-resistant disease. Statins inhibit the production of mevalonate by HMG CoA reducatase (HMGCR), the rate-limiting step in the synthesis of cholesterol. This pathway also supplies isoprenoids such as geranylgeraniol which are necessary for the membrane localization and activity of many small G-protein oncogenes. HMGCR has been recognized as a “metabolic oncogene” and mutation of TP53, an almost invariant event in ovarian cancer, increases the expression of HMGCR. We have recently shown that statins are a potential treatment for ovarian cancer. Lipophilic statins are the most potent at causing cell death but these have a short half-life in plasma. This is critical because many clinical trials have evaluated short half-life statins administered at an inadequate frequency to ensure the continual inhibition of HMGCR which we have shown is necessary to cause cancer cell death. Pitavastatin is unique among statins because it is both lipophilic and has a long half-life in plasma. This has prompted us to evaluate pitavastatin as a potential treatment for ovarian cancer. Methods: The activity of pitavastatin on ovarian cancer cell lines was assessed by measuring cell growth, trypan blue exclusion, caspase activity, PARP cleavage and inhibition of xenograft growth. Results and Discussion: HMGCR was found to be over-expressed in 8 ovarian cancer cell lines compared to normal epithelial cell lines. Pitavastatin inhibited the growth of each of these cell lines in both monolayer (IC50 = 0.2−8 mM) and spheroid cultures (IC50 = 0.6−4 mM) irrespective of their sensitivity to conventional chemotherapy. Furthermore, pitavastatin inhibited with equal potency the growth of paired cell lines (PEA1 v. PEA2, PEO1 v. PEO4) derived from patients before and after the onset of clinical drug resistance. This suggests that pitavastatin has the potential to treat ovarian cancer that has become resistant to chemotherapy. Pitavastatin caused accumulation of a sub G1 population, increased caspase activity and PARP cleavage. The activity of pitavastatin in these assays was suppressed by the addition of geranylgeraniol as well as by organic solvent extracts from several foodstuffs including sunflower oil. Ovcar-4 xenografts in nude mice regressed when treated with pitavastatin when animals were maintained on a controlled diet lacking geranylgeraniol but not in mice receiving a diet containing fat. Conclusion: These data suggest that pitavastatin warrants clinical evaluation in ovarian cancer but patients’ diet should be controlled to limit ingestion of isoprenoids which suppress the activity of statins. Conflict of interest: Other Substantive Relationships: Euan Stronach is an employee of GSK.

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 836 Application of sequencing, liquid biopsies and patient-derived xenografts for personalized medicine in melanoma M.R. Girotti1 , G. Gremel1 , R. Lee1 , E. Galvani1 , D. Rothwell2 , A.K. Mandal1 , K.H.J. Lim1 , G. Saturno1 , S.J. Furney1 , F. Baenke1 , M. Pedersen3 , M. Smith1 , A. Fusi4 , N. Dhomen1 , G. Brady2 , P. Lorigan4 , C. Dive2 , R. Marais1 . 1 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 Cancer Research UK Manchester Institute, Clinical and Experimental Pharmacology Team, Manchester, United Kingdom, 3 The Institute of Cancer Research, Targeted Therapy Team, London, United Kingdom, 4 The University of Manchester, The Christie NHS Foundation Trust, Manchester, United Kingdom Background: Targeted and immunotherapies have transformed melanoma care, extending median survival from ~9 to over 25 months but nevertheless, most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end we aimed to develop protocols to facilitate individualized treatment decisions for advanced melanoma patients. Material and Methods: We have analyzed 364 samples from 214 patients by Whole exome sequencing (WES), circulating tumor DNA (ctDNA), establishment of patient-derived xenografts (PDXs) and circulating tumor cellderived xenografts (CDX). Results: WES and targeted sequencing of ctDNA allowed us to monitor responses to therapy and to identify and then monitor mechanisms of resistance and we propose that longitudinal monitoring of BRAF and NRAS driver mutations should be used to prospectively follow patient responses to targeted therapy and immunotherapy. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and BRAFinhibitor-resistant BRAF mutant tumors, which were then validated in PDXs. For one of our patients, WES revealed that trametinib plus paclitaxel may have been an effective therapeutic option and we validated this combination in the patient’s PDX. We therefore propose that integration of WES and ctDNA sequencing with functional studies in PDXs provides a powerful combination that can change clinical practice and improve patient outcomes. We also developed CDXs as an alternative to PDXs when tumors are inaccessible or difficult to biopsy. We establish a proof-of-principle for this approach in melanoma and show that the CDXs resemble patient tumors and their response to therapies. Our data show that melanoma CTCs are tumorigenic and that in mice they have similar tropism to the tumors in the patients. Conclusions: Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses, and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments. No conflict of interest.

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microenvironment in larger cohorts is needed to identify robust determinants of response to immune checkpoint inhibitors. No conflict of interest. 839 Clinical significance of non-coding RNAs expression in human hepatocellular carcinoma K. Hur1 , S.Y. Jang2 , S.Y. Park2 , G. Kim1 , Y.H. Choi1 , Y.R. Lee2 , S.H. Lee2 , S.K. Jang2 , W.Y. Tak2 , Y.O. Kweon2 . 1 Kyungpook National University School of Medicine, Department of Biochemistry and Cell Biology, Daegu, South Korea, 2 Kyungpook National University Hospital, Department of Internal Medicine, Daegu, Korea Background: Hepatocellular carcinoma (HCC) is one of the most leading causes of cancer-related death worldwide. It is therefore important to understand the mechanistic roles of biomolecules involved in HCC development. Recently, non-coding RNAs (ncRNAs) including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have emerged as novel gene-regulators. Many studies have indicated that miRNAs and lncRNAs are frequently aberrantly expressed in various human cancers, which have potential roles in both oncogenes and tumor-suppressors. The aim of this study was to determine the clinical relevancies of expression of 2 lncRNAs (lncRNA-ATB and MEG3) and 2 miRNAs (miR-21 and miR-31) in HCC. Material and Methods: We analyzed clinical specimens from 100 pairs of HCC tissues and matched normal liver (NL) tissues. MiRNAs and lncRNAs expression levels were evaluated by quantitative real-time PCR (qRT-PCR) and their expression was normalized relative to U6 and GAPDH expression, respectively. In addition, we determined the clinical significance of the miR-21 and lncRNA-ATB expression in matching tissue and serum samples from HCC patients. Results: MiRNA-21 significantly increased in HCC (P < 0.01), whereas miRNA-31 decreased in HCC (P < 0.001) compared to corresponding NL tissues. LncRNA-ATB was significantly up-regulated in HCC (P < 0.05); however, lncRNA-MEG3 was down-regulated in HCC (P < 0.05) compared to corresponding NL tissues. In addition, expression of microRNA-21 and lncRNA-ATB was significantly associated with the tumor size (T stage; P = 0.0092) and the tumor size (T stage; P = 0.0013) and poor prognosis (BCLC stage; P = 0.0039) of HCC patients, respectively. More significantly, high expression of miR-21 and lncRNA-ATB was significantly associated with poor survival of HCC patients. Conclusions: We conclude that non-coding RNAs (lncRNA-ATB and miR-21) expression has the potential to serve as biomarkers for prognosis and targeted therapy in HCC patients. No conflict of interest. 840 Dickkopf-1 (DKK-1) expression is a novel lymph node metastasis predictor in early gastric cancer

837 Modelling anti-PD-1 treatment in a transgenic murine model of BRAFV600E-driven melanoma E. Galvani1 , K. Hogan1 , L. Boon2 , G. Gremel1 , A. Viros1 , A. Mandal1 , M. Smith1 , J. Swan3 , A. Banyard3 , G. Ashton3 , N. Dhomen1 , R. Marais1 . 1 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 EPIRUS Biopharmaceuticals Netherlands, CLG- Upstream PD & Bioassay Development, Utrecht, Netherlands, 3 Cancer Research UK Manchester Institute, Research Services, Manchester, United Kingdom Introduction: Immune checkpoint inhibitors have recently revolutionised the field of cancer treatment providing for the first time durable responses in metastatic melanoma and non-small cell lung cancer patients. It is imperative, moving this area of treatment forward, to understand the basis of such responses and be able to identify the patients most likely to benefit from these therapies. Material and Methods: Melanomas derived from a BRAFV600E transgenic mouse model in which chronic exposure to ultraviolet radiation (UVR) was used to simulate the effect of sunburn, were compared for their response to programmed cell death-1 (PD-1) blockade. Tumours were subsequently subjected to comprehensive molecular and immune profiling using whole exome sequencing, immunohistochemistry, and flow cytometry. Results and Discussion: BRAFV600E-driven melanomas developed in mice exposed to UVR presented with significant increase in single nucleotide variants (SNVs), and thus higher mutational load, as compared to non-UVR treated tumours. Responses to anti-PD-1 treatment were compared between tumours having low (~10 total missense SNVs) and high (>100 total missense SNVs) non-synonymous mutational load. No significant association between tumour mutation burden and response to PD-1 blockade was observed in our BRAFV600E-driven melanoma model. Detailed analysis of low- and highmutation burden tumours in light of their response to treatment will be presented. Conclusions: Consistent with previous studies, our data highlight that integrated molecular characterisation of tumour tissue and associated

Y.J. Huh1 , H.M. Lee1 , M.S. Cho2 , J.H. Lee1 . 1 Ewha Womans University Mokdong hospital, Surgery, Seoul, Korea, 2 Ewha Womans University Mokdong hospital, Pathology, Seoul, Korea Background: In early gastric cancer (EGC), predicting the lymph node metastasis (LNM) is important to increase the efficacy of surgical treatment and to select the minimally invasive endoscopic treatment. Therefore, novel method for predicting LNM is required. Although radiologic modalities such as CT and MRI are widely used for LNM diagnosis, it has several limitations including insufficient resolution and sensitivity. Dickkopf-1 (DKK-1), an inhibitor of Wnt signaling pathway, has been reported to promote cancer cell invasion and migration. However, the clinical significance of DKK-1 for predicting LNM has not been previously investigated in GC patients. The aim of this study was to determine the potential ability of DKK-1 expression to predict LNM in EGC. Material and Methods: We analyzed expression of DKK-1 in 126 EGC tissue specimens, which included LNM-positive (LN(+), n = 42) and LNMnegative (LN(-), n = 84). Expression status of DKK-1 protein was determined by immunohistochemical staining using formalin-fixed, paraffin-embedded tissue blocks from the patients underwent surgery due to the EGC from 2002 to 2013 at Ewha Womans University Mokdong Hospital. The area of staining was quantified according to the proportion of stained cells and categorized as low; 0−30% and high; >30%. In clinicopathological analyses, the tumor size and depth of invasion were matched between two groups (LN(+) and LN (-)) to exclude the effect to LNM of those factors. Results: We observed significant up-regulation of DKK-1 in EGC tissues compared to corresponding normal gastric mucosa tissues. In addition, DKK-1 was more frequently elevated in LN(+) EGC patient group than in LN(-) EGC patient group (40.7% vs. 26.9%, P = 0.073). In further subgroup analyses, male gender (P = 0.036) and undifferentiated histologic type (P = 0.038) were significantly correlated with LN metastasis in EGC. More importantly, high DKK-1 protein expression was an independent predictor for lymph node metastasis (odds ratio: 2.55, 95% confidence interval: 1.12–5.83, P = 0.027).

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Conclusions: We concluded that high expression of DKK-1 in tissue has the potential to serve as a novel biomarker for predicting LNM in EGC patients. No conflict of interest. 841 Selective inhibition of notch signalling and cancer stem cells by an antibody targeting active ADAM10 L. Atapattu1 , N. Saha2 , C. Chheang1 , M. Vail1 , M. Eissman3 , M. Ernst3 , A. Scott4 , D. Nikolov2 , P. Janes1 . 1 Monash University, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Clayton, Australia, 2 Memorial Sloan-Kettering Cancer Center, Structural Biology Program, New York, USA, 3 Olivia Newton-John Cancer Research Institute, Cancer and Inflammation Laboratory, Heidelberg, Australia, 4 Olivia Newton-John Cancer Research Institute, Tumour targeting program, Heidelberg, Australia Background: The transmembrane metalloprotease ADAM10 cleaves ligands and receptors of the Notch, Eph and erbB families, among other targets, thereby activating key signalling pathways of critical importance in tumour proliferation and maintenance, neo-angiogenesis and metastasis. ADAM10 is also over-expressed in a variety of cancers in which these pathways are deregulated, suggesting it as a promising target for therapy. Materials and Methods: We raised monoclonal antibodies (mAbs) against the non-catalytic Cys-rich domain of ADAM10, which we previously identified to specify substrate cleavage, and tested our lead mAb in vitro and in vivo as potential tumour inhibitor. We also used structural and functional analysis to investigate the mechanism of action. Results: Lead ADAM10 mAb 8C7 displays novel selectivity for an active conformation of ADAM10 preferentially found in tumours compared to normal tissues. 8C7 particularly targets cancer stem-like cells (CSCs) with high Notch signalling, which it inhibits. It also inhibits tumour growth in mouse models of gastric and colorectal cancer, most effectively following chemotherapy, in keeping with CSC involvement in chemoresistance. Conclusions: Our results indicate mAb 8C7 is a potential therapeutic to target ADAM10-dependent tumour growth and drug resistance. No conflict of interest.


Translational Research II 844 Treating malignant glioma and brain metastasis with nanoparticles: Challenges of a peptide-based targeting and passage through the blood–brain-barrier 1 ¨ , E. Casals2 , P. Filppu1 , J. Tanjore Ramanathan1 , V. Le Joncour1 , M. Hyvonen A. Ayo1 , J. Westermarck3 , J. Rosenholm2 , P. Laakkonen1 . 1 Research Progam Unit- Translational Cancer Biology, Biomedicum Helsinki- University ˚ of Helsinki, Helsinki, Finland, 2 BioNanoMaterials Research Group, Abo Academy University, Turku, Finland, 3 Finnish Cancer Institute, Turku Center for Biotechnology, Turku, Finland

High grade malignant gliomas (GBM) represent the most common and aggressive brain tumors in adults. Brain metastasis of peripheral cancer are considered as the terminal stage of the disease and are detected only shortly before patient death. They are commonly characterized by a highly invasive tumor cell population and a dense but disorganized vascularization causing hypoxia/necrosis and intracerebral hemorrhages. Current treatment strategies suffer of modest efficiency and fail to prevent tumor metastasis and relapse. In addition, poor permeability through the Blood–Brain-Barrier (BBB) and their inability to target infiltrative cells and metastatic nodes reduces the efficacy. Moreover, most of the therapeutics are cytotoxic for both tumor and healthy cells, causing numerous and severe adverse effects. Nanotherapeutics (NT) represent promising candidates to treat brain neoplasms. Beyond their protection and controlled release of drugs, thus limiting adverse effects in patients, the great flexibility in the synthesis and external decoration of nanoparticles is offering outstanding tumor cell targeting properties and greatly favor the passage through the BBB. We used a novel in vitro model that recreates the gliovascular niche to dissect the NT capability to cross BBB and to efficiently target neoplastic cells. Selected NT were then tested in 2 animal models for their capability to diffuse within the brain parenchyma and efficiently kill the tumor cell populations. Intracranial graft of patient derived GBM and intracardiac injection of breast cancer cells, mimicking the terminal metastatic colonization of the brain, were conducted in immunocompromised mice. From 12 different NT constructs we demonstrated that two in particular exhibited interesting properties in vitro. The smallest NT was the most effective to cross the multicellular barrier whereas the largest PEGylated NT exhibited better resistance to lysosomal degradation. We also showed that the targeting peptide present at the NT surface was specifically recognizing and binding to

its target-receptor, the latter being largely expressed in GBM and prometastatic breast cancer cells. After reducing the selected number of NT candidates designed for in vivo studies, focus will on their capability to cross the actual BBB, and whether the administration via the intravenous route or the nasal ways could enhance the delivery to the brain and ultimately reach the tumor masses. At this date, experiments are ongoing. We demonstrate that NT constructs containing a targeting peptide can efficiently bind to tumor cells, with modest to none endothelial cell and astrocyte retention and toxicity. By enriching the NT with conventional cytotoxic chemotherapies and/or therapeutic-designed shRNA we could thus efficiently target and kill the tumor cells with minimal toxicity for the surrounding brain tissue. No conflict of interest. 845 Development of a new workflow to identify novel marker in normal and adenoma tissue of CRC patients N. Lange1 , M. Schoeppler1 , A. Tiedemann1 , M. Spyra2 , F.T. Unger3 , H. Juhl4 , K.A. David4 . 1 Indivumed GmbH, Genomics, Hamburg, Germany, 2 Indivumed GmbH, Immunohistochemistry, Hamburg, Germany, 3 Indivumed GmbH, Proteomics, Hamburg, Germany, 4 Indivumed GmbH, Indivumed GmbH, Hamburg, Germany Background: Although colorectal cancer (CRC) is well diagnosable and highly treatable in early stages, CRC still belongs to the second most common cause of cancer related deaths in Europe. Colorectal cancer development underwent a step-wise transformation from normal epithelium into adenoma and can end up as an invasive and metastatic tumor. In particular, the first step of CRC development is not fully understood. Identification of deregulated targets within this step not only leads to a better understanding but also opens the possibility of treatment to suppress transformation and avoid surgery. Therefore, the aim of this study was the establishment of a new workflow to identify novel marker within the normal to adenoma transformation process. Material and Methods: For the experiments, biospecimen of high quality were collected in a highly standardized manner with ischemia times below 15 minutes. Epithelial cells from adenoma and corresponding normal tissue of a small cohort of patients that were diagnosed with stage 1 colorectal adenocarcinoma were isolated by Laser Capture Microdissection (LCM). Following microdissection, RNA was isolated and whole transcriptome amplification was performed. Afterwards amplified samples were used for the simultaneous analysis of 44 selected genes by quantitative real-time PCR (qPCR). Results: Within this study, we developed a LCM-qPCR based workflow for the detection of gene expression of multiple targets from low amounts of starting material. An input of 1 ng of high quality total RNA into the whole transcriptome amplification process resulted in reliable and reproducible gene expression data. Integrated controls showed that neither inhibitory effects or contaminations nor RNA integrity affected gene expression results. Using this new workflow, several differentially expressed genes involved in diverse signaling pathways were identified in normal and adenoma epithelial samples. This newly established workflow allows simultaneous gene expression analysis of multiple targets of accurate defined low occurring starting material. Conclusion: Beside the use of high quality biospecimen, the isolation of epithelial cells from normal and adenoma tissue by LCM is an essential requirement to obtain reliable data. The elucidation of pathway relationships, interactions and mechanisms underlying transformation of normal into adenoma cells will greatly improve our knowledge of cancer development and may lead to the identification of new marker and potentially new treatable targets. No conflict of interest. 846 The utilisation of analytical chemistry techniques in detecting novel biomarkers in oral cancer N. Sinevici1 , O. Jeffrey1 . 1 Trinity College Dublin, Dental Science, Dublin, Ireland Background: Protein glycosylation is a diverse and crucial post translational modification involved in physiological and pathophisiological conditions. The biological importance of glycosylation as well as its biomarker potential has attracted the development of more efficient separation and more sensitive detection methods. Alterations in the glycome have been highlighted in many pathologies. In respect to cancers of the oral cavity very few early diagnostic and prognostic biomarkers exist and their clinical utility remains minimal. The morbidity and mortality of such cancers remains a concern to modern society. Inability of early diagnosis challenges the scientific community to find reliable biomarkers in a suitable biofluid. The aim of this study was to develop and optimize analytical methods enabling the detection of glycans and determining their potential usefulness in biomarker discovery. Materials and Methods: Due to the complexity of glycosylation, an orthogonal analytical approach was necessary for structural characterization. The release of N-glycans from the protein backbone was achieved using

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 the endoglycosidase, PNGase F followed by glycan derivatization with the 2-aminobenzamide (2-AB) fluorophore. Glycans were analysed using hydrophilic interaction liquid chromatography (HILIC) coupled with Ultra low Performance Chromatography (UPLC). Exoglycosidase enzymatic digestions and comparison of pre and post enzymatic digestion chromatographic profiles were used to identify peak shifts in order to determine the presence of a specific residue(s). Weak anion exchange (WAX) Chromatography and Mass Spectrometry (MS) were used to assist in the identification of glycan structures. Results: Oral biofluids were found to contain a heavily glycosylated protein composition. All types of glycans complex, hybrid and high mannonse structures were present. The main glycans were neutral glycans with smaller levels of mono, di- and tri-sialylated glycans. Furthermore glycans were heavily fucosylated. Conclusion: Late diagnosis remains one of the major drawbacks in the treatment of oral cancer followed by chemo-resistance and recurrence. Glycosylation changes are the most frequent biochemical alterations associated with cancer, both in terms of specific hypo- and hyper-glycosylation events. Among the most frequently seen alterations in cancer cells and secreted glycoproteins is an aberrant pattern of N-linked oligosaccharide modifications. Sensitive and reliable diagnostic markers for oral cancers and recurrence of oral cancers remain unavailable. This study demonstrates the applicability of the developed methodology and its potential identification of alterations in the glycoproteome to facilitate the detection of cancers within the oral cavity. No conflict of interest. 847 Validation of a model of drug-induced aggressive liver cancer: gene expression and tumor recurrence J. Van Pelt1 , J. Dekervel2 , A. Bulle2 , H. Van Malenstein2 , P. Windmolders2 , E. Van Cutsem1 , C. Verslype1 . 1 KU Leuven, Hepatology and Digestive Oncology, Leuven, Belgium, 2 KU Leuven, Hepatology, Leuven, Belgium Background: For a long time, Epithelial-to-Mesenchymal Transition (EMT) was considered the driving mechanism of the epithelial cancer cell to metastasize to distant organs, this paradigm was very recently challenged by the finding that cells can spread without undergoing EMT. EMT has been shown to be important for drug-resistance that almost always develops during systemic cancer therapy. There has been extensive research on gene expression signatures in HCC, classifying cancer patients with the aim to predict patient survival or disease recurrence in view of treatment options. Aim: We wanted to explore the clinical importance of an EMT phenotype in liver cancer for recurrence of a tumor after resection. Methods: We present a novel translational approach using human sorafenib resistant hepatoma cell line in which we studied morphology, gene expression by microarray and invasive potential. We used gene set enrichment analysis (GSEA) and pathway analysis for the evaluation of the signature. Clinical validation was done using 5 large patient data sets submitted to Gene Expression Omnibus (GEO) against clinical outcome. Results: The resistant cells changed their appearance, lost E-cadherin and KRT19 and showed high expression of vimentin, indicating epithelial-tomesenchymal transition. Resistant cells showed reduced adherent growth, became more invasive and lost liver-specific gene expression. We determined differentially expressed genes in the drug-resistant cells. Using GSEA, resistant cells showed loss of expression of genes included in the good survival signature proposed by Lee et al. [1] and changes concordant with poor prognostic HCC such as the proliferation subclass of Chiang et al. [2] or the G3 subtype described by Boyault et al. [3]. We validated these genes in 3 large published data sets of hepatocellular carcinoma and developed a gene score. This score was applied on 2 independent patient cohorts were it could predict the recurrence risk at 3 years after resection and when applied on the surrounding liver tissue the same genes also correlated with late recurrence. Four patient classes with each different time patterns and rates of recurrence could be identified based on combining tumor and liver scores. In a multivariate Cox regression analysis our gene score was an independent risk factor for recurrence (P = 0.007). Conclusions: The in vitro sorafenib drug resistance model in HCC shows strong similarities with the more aggressive molecular subclasses identified by several groups. Using this gene expression we developed a small 7 gene risk score that is able to simultaneously predict the risk of early and late tumor recurrence demonstrating the clinical relevance of this EMT model. Reference(s) [1] Lee JS et al. Hepatology (2004) 40: 667. [2] Chiang DY et al. Cancer Res (2008) 68, 6779. [3] Boyault S et al. Hepatology (2007) 45: 42. No conflict of interest.

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848 A computational approach to discriminate between human and mouse reads in sequencing data from Patient Derived Tumour Xenografts M. Callari1 , A. Sati Batra1 , R.N. Batra1 , H. Clifford1 , W. Greenwood1 , S.J. Sammut1 , S.F. Chin1 , A. Bruna1 , O.M. Rueda1 , C. Caldas1 . 1 University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom Background: Patient Derived Tumour Xenografts (PDTXs) are emerging pre-clinical models that better represent clinical tumours and intra-tumour heterogeneity compared with cell lines. In most of the studies involving PDTXs, their molecular characterization is central; however, the analysis of sequencing data from PDTXs is hampered by the presence of mouse stroma contamination. Due to the high homology between the two organisms, a proportion of mouse reads can still map to homologous regions of the human genome, likely with some mismatch, heavily affecting downstream analysis. Material and Methods: We carried out controlled experiments where fixed amounts of human and mouse DNA (or RNA) were mixed, ranging from a pure human sample to a pure mouse sample. Whole Exome Sequencing (WES), RNA-seq and Reduced Representation Bisulfite Sequencing (RRBS) were performed. A bioinformatic pipeline was developed to distinguish human from mouse reads by aligning raw data against both human and mouse genomes. The validity of the approach was confirmed in sequencing data from PDTXs and matched primary tumours. Results: The developed method was able to correctly classify human and mouse reads with an accuracy >99.9%. Using the controlled experiment, we also derived a calibration curve linking the percentage of human DNA in the sample and the percentage of reads classified as human. This calibration curve can then be used to estimate the percentage of mouse contamination in the sample. We verified that the alignment of PDTX WES data using the human reference genome only can generate up to thousands of false positive calls in samples with a mouse contamination as low as 10%. By applying our approach, we were able to prevent the calling of these artefacts increasing the correlation between each PDTX and the matched primary tumour. Also in RNA-seq and RRBS data, distinguishing between human and mouse reads gave more accurate estimates of the tumour transcriptome and methylome. Importantly, it also allowed analysing separately the microenvironment contribution. Conclusions: An ad-hoc bioinformatic pipeline is needed for the analysis of PDTX sequencing data. Here we present a simple and fast solution to accurately discriminate reads having human or mouse origin. No conflict of interest. 849 High throughput capture and profiling of CTCs using innovative technologies for gene expression C. Jarman1 , A. Lejeune-Dodge1 , S. Peter2 , K. Hull2 , D. Donnelly1 . 1 Labcyte, Applications, Sunnyvale, USA, 2 Angle PLC, R&D, Guildford, United Kingdom Introduction: The use of molecular diagnostics in oncology is rapidly evolving. Recent advancements have led to the production of “live” data for translational medicine and clinical guidance. Capturing intra tumoral and clonal diversity using liquid biopsies is an example of such an advancement. This precious tool enables the representation of cancer heterogeneity using molecular profiling of circulating tumour cells (CTCs) at gene expression level. Here we present a robust workflow for the capture of CTCs from patient-blood samples using Angle PLC’s Parsortix system along with CTC enrichment using the Echo Acoustic Liquid Handler from Labcyte for contactless cell transfer. Following enrichment, expression profiling reactions were set up by dispensing miniaturised volumes of reagents for high throughput qPCR read out − also using the Echo system. This approach was implemented at Angle PLC to address the need of increased throughput for molecular profiling by qPCR at the single cell level. Methods: Linearity of cell transfer: Patient blood samples were spiked with fluorescently labelled CaOV3 cells. CTCs were harvested using the Parsortix system. Harvested CTCs were transferred using the Echo system into 384-well microplates for visual inspection and counting. Gene expression profiling: Three positive controls were added to the plate, alongside three no template controls (NTC), using the Echo system. The Echo system was also used to transfer reagents from Ambion’s Single Cell-to-CT kit directly onto the CTCs and controls to perform reverse transcription, preamplification and qPCR with in a miniaturized reaction (10-fold reduction). qPCR was performed using the Roche LightCycler 480. Results: Accurate transfer of cells using the Echo system was validated. This enabled an enrichment of CTCs over white blood cells from Parsortixprocessed patient blood samples. Miniaturisation of the ‘Single Cell-to-CT’ kit for EpCAM gene expression in CaCOV3 cells & qPCR reactions using the Echo system established a robust method for flexible high throughput profiling investigations with a major impact on cost reduction. Absence of amplification in NTC & the expected differential of 3Ct between dilutions of positive controls

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validated acceptance criteria. Amplification profile for EpCAM gene expression identified the presence of CTCs in specific wells in qualitative manner. Conclusions: Following capture of CTCs using the Parsortix system, gentle contactless cell transfer using the Echo liquid handler enabled standardisation of CTC enrichment allowing detection by qPCR. Miniaturisation of reagents volumes greatly reduced the cost of the full workflow. This work provided a proof of concept that used the Parsortix system and Echo liquid handler as an end-to-end platform to capture, enrich and profile CTCs. No conflict of interest. 851 Association of angiopoietin-2 and Ki-67 expression with vascular density and sunitinib response in metastatic renal cell carcinoma A. Lampinen1 , J. Rautiola2 , T. Mirtti3 , A. Ristimaki ¨ 4 , H. Joensuu2 , P. Bono2 , P. Saharinen5 . 1 University of Helsinki, Research Programs Unit- Translational Cancer Biology Program, Helsinki, Finland, 2 Helsinki University Central Hospital, Comprehensive Cancer Center, Helsinki, Finland, 3 University of Helsinki, Institute for Molecular Medicine Finland, Helsinki, Finland, 4 Helsinki University Central Hospital, Department of Pathology, Helsinki, Finland, 5 University of Helsinki, Translational Cancer Biology Program and Wihuri Research Institute, Helsinki, Finland Background: The angiopoietin-2 (Ang2, Angpt2) growth factor is a contextdependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase known to promote tumour angiogenesis and metastasis. In clinical cancer trials, angiopoietin antagonists are combined with VEGF-based antiangiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. The purpose of this study was to correlate the baseline expression of endothelial angiopoietin-2 (Ang2), CD31 and the cell proliferation marker Ki-67 to the outcome of first-line sunitinib therapy in mRCC. Materials and Methods: We evaluated using immunohistochemistry pretherapeutic Ang2, CD31 and Ki67 expression in formalin-fixed paraffinembedded tumor samples from 136 patients with metastatic RCC, who were subsequently treated with first-line sunitinib. Results: Ang2 was exclusively expressed in the endothelial cells of tumor blood vessels. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS 15.7 vs. 28.5 months, P = 0.015). Conclusions: In summary, high cancer Ang2 expression was associated with high CBR, but not with PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS in mRCC patients treated with first-line sunitinib. No conflict of interest. 852 Combined anti-MET/EGFR treatment results in complete tumor regression and prevents resistance onset in a MET-amplified gastroesophageal xenopatient cohort S. Giordano1 , M. Apicella1 , C. Migliore1 , T. Capeloa2 , S. Menegon2 , M. Cargnelutti1 , A. Sapino3 , P. Cassoni4 , S. Marsoni5 , S. Corso1 . 1 University of Torino- Candiolo Cancer Institute- FPO-IRCCS- Candiolo- Italy, Department of Oncology, Candiolo, Italy, 2 University of Torino- Candiolo Cancer InstituteFPO-IRCCS- Candiolo- Italy, Department of clinical and biological sciences, Candiolo, Italy, 3 University of Torino- Candiolo Cancer Institute- FPO-IRCCSCandiolo- Italy, Department of Medical Sciences, Candiolo, Italy, 4 University of Torino- Italy, Department of Medical Sciences, Candiolo, Italy, 5 Candiolo Cancer Institute- FPO-IRCCS- Candiolo- Italy, clinical trials, Candiolo, Italy Introduction: Gastric cancer is the world third leading cause of cancer mortality. In spite of the significant therapeutic advances, the overall clinical outcome for patients with advanced gastric cancer is poor, with 5−20% 5-year survival. The only targeted therapy approved so far are trastuzumab, and Ramucirumab which have given unsatisfactory results. Around 50% of gastric tumors bear genetic alterations affecting tyrosine kinase pathways (mainly HER2, EGFR, HER3, FGFR2 and MET pathways) but their clinical validation as tumor drivers is missing. The need for new therapeutic options and the possible presence of ‘druggable’ targets prompted us to investigate potential targeted therapies for this disease. Material and Methods: We generated a platform of gastroesophageal tumor patient-derived xenografts (PDXs), in which tumor surgical specimens are directly transferred into mice. Upon engraftment, the tumor is split and reimplanted in a cohort of mice, allowing the simultaneous testing of different drugs on the same tumor. Thanks to the establishment of a network of 15 Italian centers for samples collection, we generated around 80 gastric PDXs and successfully derived cell lines and organoids from engrafted tumors.

Results and Discussion: Among the tumors collected so far, we found HER2, EGFR, FGFR2, MET and KRAS amplifications. We exploited this gastric PDX platform for: (1) Validation of candidate oncogenes as relevant targets and identification of efficient therapeutic strategies; (2) identification of novel molecular targets; (3) identification of genetic predictors of response/resistance. In this PDX platform we identified one tumor bearing high level of MET gene amplification (26 copies). We found report that despite the high MET amplification level, in the absence of qualitative or quantitative alterations of EGFR, MET inhibitors induced only tumor growth inhibition, while dual MET/EGFR inhibition led to complete tumor regression. Importantly, the combo treatment completely prevented the onset of resistance, which quite rapidly appeared in tumors treated with anti-MET monotherapy. We found that this secondary resistance was due to EGFR activation and could be overcome by dual MET/EGFR inhibition. In vitro experiments performed on tumor-derived primary cells confirmed that MET inhibitors were not able to abrogate the activation of downstream transducers and that only the combined anti-MET/EGFR treatment completely shut off the signaling. Conclusion: Previously reported cases, as well as the one described here, showed only partial and transient sensitivity to anti-MET therapy. The finding that combined anti-MET/EGFR therapy − even in the absence of EGFR genetic alterations − induced complete and durable response, represents a proof of concept and guarantees further investigations, opening a new perspective of treatment for these patients. No conflict of interest. 853 Monitoring metastatic melanoma treatment resistance using circulating tumour DNA S. Murphy1 , J. Wan1 , D. Gale1 , J. Morris1 , F. Mouliere1 , G. Bignell2 , C. Alifrangis2 , C. Parkinson3 , A. Durrani4 , U. McDermott2 , C. Massie1 , P. Corrie3 , N. Rosenfeld1 . 1 Cancer Research UK Cambridge Research Institute, Rosenfeld Group, Cambridge, United Kingdom, 2 Wellcome Trust Sanger Institute, Hinxton, United Kingdom, 3 Addenbrooke’s Hospital, Department of Oncology, Cambridge, United Kingdom, 4 Addenbrooke’s Hospital, Department of Plastic Surgery, Cambridge, United Kingdom Background: Metastatic melanoma is an advanced skin cancer with a life expectancy of 2−7 months. Although patients may initially respond to novel molecular therapies, patients inevitably relapse due to the emergence of drug resistance. Non-invasive methods to monitor treatment response and resistance are needed, which may facilitate personalised treatment decisions. Short DNA fragments are continuously released by cancer cells into the bloodstream, termed as circulating tumour DNA (ctDNA). ctDNA levels have been shown to reflect tumour burden at the time of sampling. Here, ctDNA analysis was carried out to monitor for treatment resistance on a molecular level via serial blood tests. Materials and Methods: Tumour and plasma samples were collected from AJCC Stage III or IV melanoma patients enroled on MelResist, a translational, multi-centre research study. Exome sequencing was carried out on melanoma tumour samples at baseline and disease progression. Variants identified by exome sequencing were used to design patient-specific panels for TaggedAmplicon sequencing (TAm-seq). TAm-seq was carried out on serial plasma DNA samples, enabling the longitudinal monitoring of multiple mutations. Results: To date, 72 patients have been recruited. Individualised Tam-Seq panels were run on plasma from 8 patients, tracking ~50 mutations per patient. Using this approach, tumour dynamics across multiple mutations were monitored, providing insight into the processes underlying the emergence of treatment resistance. ctDNA dynamics correlate with clinical progression data, and precede LDH increases in a number of cases. Conclusion: Monitoring multiple mutations per patient in ctDNA may provide insight into clonal evolution during treatment, and may have utility for identifying treatment resistance earlier than conventional markers. Conflict of interest: Board of Directors: Nitzan Rosenfeld is the CSO of Inivata. Other Substantive Relationships: Nitzan Rosenfeld and Davina Gale are cofounders of Inivata. Nitzan Rosenfeld is a coinventor of patent applications describing methods for analysis of rare DNA fragments. 854 Development of a tool inhibitor of GCN5 towards the treatment of ccRCC S. Walker1 , K. Duffell1 , J. Downs2 , S. Ward1 . 1 University of Sussex, Sussex Drug Discovery Centre, Brighton- East Sussex, United Kingdom, 2 University of Sussex, Genome Damage and Stability Centre, Brighton- East Sussex, United Kingdom BAF180 (BRG1-associated factor 180) is a subunit of PBAF (Polybromo BRG1-Associated Factor), a chromatin remodelling complex. Recently it was identified as a major ccRCC (clear cell Renal Cell Carcinoma) cancer gene, exhibiting inactivating mutations in 41% of samples of primary ccRCC, and thus represents an opportunity to target ccRCC via a synthetic lethality approach. In unpublished work (Hopkins, S. and Downs, J.), GCN5 (general control of amino acid synthesis protein 5) has been identified as synthetically lethal with

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 BAF180. GCN5 is a histone acetyltransferase (HAT) responsible for promoting transcriptional activation. GCN5 contains a HAT domain and a bromodomain, both of which were predicted as druggable by assessment with Fpocket 2.0. Here focus is on the bromodomain. In this work it was shown that the bromodomain represents a promising target for tool inhibitor development. A structure guided approach was used to design chemical libraries of compounds which might offer improved selectivity and potency compared to micromolar fragments from unpublished work (Structural Genomics Consortium) for the closely related homolog PCAF (P300/CBPassociated factor). These libraries were synthesised and screened in house using a thermal shift assay and hits were tested by Isothermal titration calorimetry to confirm any actives. No conflict of interest. 855 Evaluating the efficiency of hyaluronic acid for specific tumour targeting A. Spadea1 , J.M. Rios de la Rosa1 , M. Mehibel1 , A. Marianne2 , N. Tirelli3 , I.J. Stratford1 . 1 University of Manchester, School of Pharmacy, Manchester, United Kingdom, 2 Astrazeneca, Drug Targeting, Manchester, United Kingdom, 3 University of Manchester, Inflammation and Repair and School of Materials, Manchester, United Kingdom Background: The success of cancer treatment has primarily been hindered by the non-specific effects of conventional anticancer therapies. The development of a delivery system capable of specifically targeting the tumours could be a promising strategy to overcome this issue. One of the currently most investigated cancer targets is the hyaluronic acid (HA) membrane receptor CD44, which is overexpressed in most cancers. CD44 is also considered a reliable marker of cancer-initiating cells and its expression levels are associated with tumour relapse after therapy and poor prognosis. The aim of this project was to evaluate the efficiency of HA to specifically bind CD44 and thereby, selectively target tumour cells. In order to achieve this, a panel of cancer cell lines and normal fibroblasts were screened for CD44 expression and for their ability to binding and internalise HA. Material and Methods: Human dermal fibroblasts (HDF) and a wide panel of cancer cell lines selected from breast, prostatic, head and neck, pancreatic, ovarian, colorectal, thyroid and endometrial cancer were evaluated for their CD44 expression (total and different variants) using western blotting, flow cytometry and immunofluorescence techniques. The uptake of HA was evaluated with flow cytometry and microscopy techniques. Results: The analysis of CD44 expression showed distinct levels and isoform profiles in the different cancer cell lines. Overall, the level of cellular uptake of HA correlated, in most cell lines, with the level of CD44 expression. Further, all the cells expressing CD44 were found able to bind and internalise HA. The lowest HA internalisation was observed in Ishikawa endometrial cancer cells, which have a low and non-homogeneous expression of the receptor. Interestingly, HDF did not internalise large quantities of HA despite their high CD44 expression, and this could be due to receptor inactivation. Surprisingly, LNCaP, CD44 negative prostate cancer cells, showed unspecific uptake of HA. Thus, part of our future work will be to investigate the specificity of this CD44 targeting, analyzing the HA uptake into cells where the receptor has been blocked with anti-CD44 antibodies or transiently knocked down with anti-CD44 siRNA. Conclusions: In order to effectively use hyaluronic acid as a selective targeting agent for tumours, we need to understand the interplay between CD44 expression and functionality, and the underlying mechanism(s) for unspecific uptake. The experiments described here are a first step to help us achieve this understanding. No conflict of interest. 856 High HMGN5 expression confers resistance to tamoxifen and its expression is predictive of response to tamoxifen in ER+ breast cancer patients D. Elias1 , L. Suntharalingam1 , H. Vever1 , A. Lykkesfeldt2 , M. Bak3 , H. Ditzel1,4 . 1 University of Southern Denmark, Cancer and Inflammation Research, Odense C, Denmark, 2 The Danish Cancer Society, Breast Cancer Group- Cell Death and Metabolism, Copenhagen, Denmark, 3 Odense University Hospital, Department of Pathology, Odense, Denmark, 4 Odense University Hospital, Department of Oncology, Odense, Denmark Introduction: A significant proportion of ER+ breast cancers do not respond to first line anti-estrogen treatments leading to patients suffering from the unnecessary side effects of treatments they do not benefit from. This underlines the urgent need for biomarkers capable of accurate separation of patients into treatment categories for optimal patient management. This project is aimed at identifying biomarkers capable of classifying ER+ breast cancer patients that are likely to respond to anti-estrogen therapy. Materials and Methods: Gene array was conducted on tamoxifen-resistant breast cancer cell lines using Affymetrix gene chip. The expression levels of selected genes at mRNA level were further evaluated in two cohorts

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of breast cancer patients. Pathway analysis was conducted using Ingenuity Pathway Analysis software. Gene knockdown was performed using siRNA transfection on MCF7-derived tamoxifen-resistant and -sensitive breast cancer cell lines. Cell number and proliferation was measured using crystal violetbased colorimetric and BrdU incorporation methods, while apoptosis was measured using a cell death detection kit. Protein expression was measured in tissue microarrays of 312 ER+ breast cancer patients treated with tamoxifen as adjuvant therapy by immunohistochemistry and correlated with clinical outcome. Results: Approximately 1100 genes showed marked alteration in expression in tamoxifen-resistant breast cancer cell lines versus the parental breast cancer cell lines. Among these genes, 400 also exhibited similar alteration in 2 cohorts of 316 and 201 ER+ primary breast tumors when patients who experienced recurrence within 5 years of surgery compared to those who were disease free for 7 years following surgery. Among these genes, High Mobility Group Nucleosome Binding Domain 5 (HMGN5), a chromatin binding protein affecting histone modifications, was found to be a central molecule in pathways associated with cell proliferation and death. Knockdown of HMGN5 in tamoxifen-resistant cell lines resulted in increased sensitivity to tamoxifen (p = 0.011) as determined by crystal violet-based colorimetric assay, enhanced tamoxifen induced cell death (p = 0.041), while no significant effect on proliferation (p = 0.071) was observed. Knockdown of HMGN5 did not significantly affect the proliferation (p = 0.12) or apoptotic death (p = 0.065) of the parental tamoxifen-sensitive cell line. Evaluation of the expression of HMGN5 using immunohistochemistry in a cohort of ER+ breast cancer patients (n = 312) treated with tamoxifen showed that increased expression of HMGN5 correlated significantly (p < 0.04) with shorter disease free survival. Conclusion: HMGN5 may play a significant role in tamoxifen induced cell death and its expression may serve as a prognostic marker for tamoxifen response in ER+ breast cancer. No conflict of interest. 857 Identifying changes in mutational dynamics in patients with early breast cancer undergoing neoadjuvant chemotherapy S.J. Sammut1 , S.F. Chin1 , O.M. Rueda1 , S.J. Dawson2 , M. Callari1 , E. Provenzano3 , J. Abraham4 , L. Hughes-Davies4 , H. Earl4 , C. Caldas1 . 1 University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom, 2 Peter MacCallum Cancer Centre, Molecular Biomarkers and Translational Genomics Laboratory, Victoria, Australia, 3 Cambridge University Hospitals NHS Foundation Trust, Department of Histopathology, Cambridge, United Kingdom, 4 Cambridge University Hospitals NHS Foundation Trust, Department of Oncology, Cambridge, United Kingdom Background: Neoadjuvant chemotherapy has become standard practice in patients with high-risk early breast cancer. Not only does it improve rates of breast conservation surgery, but also enables prediction of local recurrence and survival by using tumour response to treatment as a surrogate. In addition, the neoadjuvant setting provides a unique opportunity to study in-vivo the impact of cytotoxic systemic therapy on the biology of treatment na¨ıve breast cancer. Our aim was to identify the change in clonal dynamics and mutational profile of treatment na¨ıve early breast cancer while subjected to the selection pressures of chemotherapy and targeted therapies. Materials and Methods: 74 patients with Stage I-III breast cancer receiving neoadjuvant chemotherapy were recruited to the Trans-NEO translational study. Sequential tumour biopsies were taken, when possible, prior to commencing chemotherapy, midway through treatment and on completion of chemotherapy. Somatic variants and copy number alterations were characterised by whole exome (minimum 80x coverage) and shallow whole genome sequencing of tumour and peripheral blood leucocytes at all available time points to generate a comprehensive landscape of changes in genomic alterations during chemotherapy. Using Bayesian clustering methods, the change in clonal composition of tumours during the duration of chemotherapy was characterised, allowing the genomic identification of tumour clones that were susceptible to chemotherapy, as well as others that had not been eliminated by treatment. Results: Of all patients analysed, 22 (30%) had complete pathological response, 19 (26%) had an excellent response (1.5 fold) and wild type EGFR/KRAS was treated with the corresponding targeted therapy. Tumor growth inhibition (TGI) was highest in the Cisplatin (95%), followed by Sirolimus (83%) and Cisplatin+ATRA+Nimotuzumab (69%), indicating a correlation between the markers and the treatment response. Cisplatin arm showed the highest loss of body weight (25%), while the mortality rates were high in the Celecoxib (4/5) and Sirolimus arms (3/5); drug treatment is currently undergoing for other PDXs. In parallel, microarray series (n = 92) involving targeted therapy studies are currently under analysis for marker identification. These markers will be validated in the PDX samples after drug treatment. Conclusion: This preliminary study indicated a response that correlated to the molecular profile with the highest TGI and low mortality/minimal body weight loss being in the combination arm as compared to the standard therapy (high TGI and body weight loss). No conflict of interest. 866 Differential methylation of a CpG site in the CD95-ligand promoter predicts the response to therapy with APG101 in glioblastoma 1

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C. Gieffers , C. Merz , J. Sykora , C. Kunz , M. Thiemann , B. Wiestler , W. Wick3 , H. Fricke1 . 1 Apogenix AG, Analytics/Protein Chemistry, Heidelberg, Germany, 2 Deutsches Krebsforschungszentrum, Klinische ¨ Kooperationseinheit Neuroonkologie, Heidelberg, Germany, 3 Universitatsklinik Heidelberg, Neuroonkologie G370, Heidelberg, Germany Introduction: CD95 (APO-1/Fas) is a member of the Tumour Necrosis Factor Receptor Super Family. Binding of CD95-ligand (CD95L) to CD95 triggers intracellular signal transduction that is critically involved in the invasive growth of glioblastoma cells. Invasion of malignant glioblastoma cells into the brain parenchyma is responsible for poor therapeutic outcome of currently available treatments. The inhibition of CD95/CD95L mediated invasive growth of glioblastoma cells represents an attractive novel therapeutic concept. Apogenix has developed APG101, a fully human fusion protein consisting of the extracellular domain of CD95 and the Fc-domain of an IgG. APG101 has been confirmed as a potent inhibitor of CD95L induced invasion of glioblastoma cells in vitro. In a randomized phase 2 study in glioblastoma patients with 1st or 2nd relapse the combined therapy of APG101 plus radiotherapy (RT) was found to be superior to RT alone in a clinically relevant order of magnitude in all efficacy endpoints (i.e. PFS-6, PFS and OS). At the same time APG101 exhibited an excellent safety profile and was well tolerated. Materials and Methods: For the identification of potential biomarkers we used available tissue sections originating from archived primary tumour samples of study patients and analyzed them for the expression of CD95L by IHC as well as for DNA methylation status. Results: A genome-wide assessment of DNA methylation identified a single CpG-site (CpG2) upstream of the CD95L-promotor that showed differential methylation between APG101 responders (PFS >5 months) and non-responders (PFS