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Ross Camidge, Heather Wakelee, Maurice Perol, Geoffrey R. Oxnard, Karena Kosco,. Peter Croucher, Errin Samuelsz, Cecile Rose Vibat, Shiloh Guerrero, ...

Accepted Manuscript A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma Karen L. Reckamp, Vladislava O. Melnikova, Chris Karlovich, Lecia V. Sequist, D. Ross Camidge, Heather Wakelee, Maurice Perol, Geoffrey R. Oxnard, Karena Kosco, Peter Croucher, Errin Samuelsz, Cecile Rose Vibat, Shiloh Guerrero, Jennifer Geis, David Berz, Elaina Mann, Shannon Matheny, Lindsey Rolfe, Mitch Raponi, Mark G. Erlander, Shirish Gadgeel PII:

S1556-0864(16)30587-1

DOI:

10.1016/j.jtho.2016.05.035

Reference:

JTHO 247

To appear in:

Journal of Thoracic Oncology

Received Date: 20 April 2016 Accepted Date: 24 May 2016

Please cite this article as: Reckamp KL, Melnikova VO, Karlovich C, Sequist LV, Camidge DR, Wakelee H, Perol M, Oxnard GR, Kosco K, Croucher P, Samuelsz E, Vibat CR, Guerrero S, Geis J, Berz D, Mann E, Matheny S, Rolfe L, Raponi M, Erlander MG, Gadgeel S, A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma, Journal of Thoracic Oncology (2016), doi: 10.1016/j.jtho.2016.05.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma Karen L. Reckamp,1* Vladislava O. Melnikova,2* Chris Karlovich,3* Lecia V. Sequist,4 D. Ross

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Camidge,5 Heather Wakelee,6 Maurice Perol,7 Geoffrey R. Oxnard,8 Karena Kosco,2 Peter Croucher,2 Errin Samuelsz,2 Cecile Rose Vibat,2 Shiloh Guerrero,2 Jennifer Geis,3 David Berz,9 Elaina Mann,3 Shannon Matheny,3 Lindsey Rolfe,3 Mitch Raponi,3 Mark G. Erlander,2 and Shirish Gadgeel10

City of Hope Comprehensive Cancer Center, Duarte, CA; 2Trovagene, Inc., San Diego, CA;

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1 3

Clovis Oncology, Inc., San Francisco, CA; 4Massachusetts General Hospital, Boston, MA; University of Colorado, Denver, CO; 6Stanford University Medical Center, Stanford, CA;

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Hospices Civils de Lyon, Lyon, France; 8Dana Farber Cancer Institute, Boston, MA; 9Beverly

Hills Cancer Center, Beverly Hills, CA; City of Hope, Duarte, CA; 10Barbara Karmanos Cancer Institute, Detroit, MI

*, K.L. Reckamp, V.O. Melnikova and C. Karlovich contributed equally to this article.

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Address for correspondence: Karen L. Reckamp, MD, City of Hope Comprehensive Cancer Center, 1500 E Duarte Rd, Duarte, CA 91010; email: [email protected] Dr. Reckamp reports personal fees from Trovagene, and Institutional clinical trial support from Clovis during the

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conduct of the study. V.O. Melnikova, K. Kosco, P. Chroucher, E. Samuelsz, C.R. Vibat, J.Geiss, and M. Erlander are employees of Trovagene. C. Karlovich, E. Mann, L.Rolfe, and M.Raponi are employees of Clovis Oncology and hold stock in Clovis Oncology. L.V. Sequist reports Institutional trial support from Novartis, during the conduct of the study;

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noncompensated consulting for Boehringer Ingelheim, Clovis Oncology, Novartis, Merrimack Pharmaceuticals, and Taiho; compensated consulting for AstraZeneca and Ariad. Dr. Camidge reports personal fees from Clovis, outside the submitted work. Dr. Wakelee reports personal fees from Peregrine, grants from Novartis, personal fees from ACEA, grants and personal fees from Pfizer, grants from BMS, grants from XCovery, grants from Celgene, grants from Roche/Genentech , grants from MedImmune, grants from Gilead, grants from AstraZeneca, grants from Lilly, outside the submitted work. Dr. PEROL reports personal fees from Clovis Oncology, personal fees from AstraZeneca, personal fees from Roche, personal fees from Boehringer-Ingelheim, outside the submitted work. Dr. Oxnard reports personal fees from AstraZeneca, personal fees from Clovis, personal fees from Sysmex, personal fees from Boehringer-Ingelheim, personal fees from Inivata, outside the submitted work. In addition, Dr. Oxnard has a patent Development and application of NSCLC plasma genotyping using digital PCR pending. Dr. Berz has nothing to disclose. Dr. Gadgeel reports personal fees from Roche/Genentech, personal fees from Astra-Zeneca, outside the submitted work.

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ABSTRACT Introduction: Approximately 60% of NSCLC patients receiving EGFR tyrosine kinase inhibitors develop resistance through the acquisition of EGFR T790M mutation. We aimed to demonstrate that a highly sensitive and quantitative next-generation sequencing analysis of EGFR mutations

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is feasible from urine and plasma. Methods: Short footprint mutation enrichment NGS assays were used to interrogate EGFR activating mutations and the T790M resistance mutation in urine or plasma from patients

enrolled in TIGER-X (NCT01526928), a phase 1/2 clinical study of rociletinib in previously treated patients with EGFR mutant-positive advanced NSCLC.

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Results: Of 63 patients, 60 had evaluable tissue specimens. Using the tissue result as

reference, the sensitivity of EGFR mutation detection in urine was 72% (34/47) for T790M, 75%

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(12/16) for L858R, and 67% (28/42) for exon 19 deletions. With specimens that met a recommended volume of 90-100 mL, the sensitivity was 93% (13/14) for T790M, 80% (4/5) for L858R, and 83% (10/12) for exon 19 deletions. A comparable sensitivity of EGFR mutation detection was observed in plasma: 93% (38/41) for T790M, 100% (17/17) for L858R, and 87% (34/39) for exon 19 deletions. Together, urine and plasma testing identified 12 additional T790M-positive cases that were either undetectable or inadequate by tissue test. In 9 patients

observed by day 21.

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monitored while on treatment with rociletinib, a rapid decrease in urine T790M levels was

Conclusions: DNA derived from NSCLC tumors can be detected with high sensitivity in urine and plasma, enabling diagnostic detection and monitoring of therapeutic response from these

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non-invasive “liquid biopsy” samples.

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Key Words: urine, circulating tumor DNA, NSCLC, EGFR mutations, T790M Abbreviations: ctDNA, circulating tumor DNA; NSCLC, non-small cell lung cancer; EGFR, epidermal growth factor receptor; WT, wild-type; NGS, next-generation sequencing; CV%, coefficient of variation percent; GEq, genome equivalents; PCR, polymerase chain reaction; EBV, Epstein-Bar Virus; H&E, Hematoxylin and Eosin; FFPE, formalin-fixed paraffin-embedded; RECIST, response evaluation criteria in solid tumors; PR, partial response; SD, stable disease; PD, progressive disease.

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INTRODUCTION A major challenge for assessing EGFR mutation status in advanced non-small cell lung cancer (NSCLC) is the availability of suitable biopsy tissue for molecular testing. Clinical studies suggest 10-20% of all NSCLC biopsies are inadequate for molecular analysis because of a lack

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of either sufficient tumor cells or amplifiable DNA.1, 2 Biopsies also pose an economic burden and health risk to patients, with biopsy-associated patient morbidity (e.g., pneumothorax)

observed in 12 to 21% of image-guided transthoracic needle tissue biopsies.3 Moreover, despite guidelines recommending EGFR testing at diagnosis for guiding first-line treatment decisions4, 5,

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up to 25% of lung cancer patients receive treatment prior to EGFR mutation assessment.6

Physicians cite tumor histology (i.e. squamous), insufficient tumor samples, poor health status of the patient, long turnaround times for tests and patient’s desire to initiate therapy as reasons

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for failure to undergo timely molecular testing.6

NSCLC patients receiving first-line tyrosine kinase inhibitors (TKIs) targeting EGFR mutation-positive tumors (i.e. erlotinib, gefitinib, afatinib) develop resistance to therapy through the emergence of a second mutation in EGFR, T790M, in approximately 60% of cases.7 Rebiopsy of these patients is still an emerging standard of care, and up to 25% of cases may be medically ineligible due to comorbidities or the lack of an accessible lesion.8 False negative

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results could occur with tissue biopsies, likely due to the underlying intra- and inter-tumoral heterogeneity often associated with resistance mechanisms such as T790M.9, 10 Detection and monitoring of cancer-specific genomic alterations in blood, specifically through the assessment of circulating tumor DNA (ctDNA), is a minimally-invasive alternative to

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a tissue biopsy that has shown promise in overcoming some of the challenges associated with sampling from tissue.11 However, ctDNA presents its own challenges for clinical diagnostics. It is highly fragmented, may be very rare (27.5 copies per 100,000 GEq), with 7 experiencing PR or SD as best overall confirmed response and 2 with PD as best overall confirmed response. For all 9 patients, there was a significant decrease in T790M levels in urine after cycle 1 relative to baseline irrespective of best overall confirmed response (range −51% to −100%; p=0.0091, two-sided Wilcoxon test; p

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