Accepted Manuscript TERT promoter mutations are associated with poor prognosis in cutaneous squamous cell carcinoma Manuel António Campos, MD, Sofia Macedo, MSc, Margarida Fernandes, MD, Ana Pestana, MSc, Joana Pardal, MD, Rui Batista, MSc, João Vinagre, PhD, Agostinho Sanches, MD, Armando Baptista, MD, José Manuel Lopes, MD, PhD, IFCAP, Paula Soares, PhD PII:
S0190-9622(18)32486-1
DOI:
10.1016/j.jaad.2018.08.032
Reference:
YMJD 12756
To appear in:
Journal of the American Academy of Dermatology
Received Date: 20 April 2018 Revised Date:
7 August 2018
Accepted Date: 16 August 2018
Please cite this article as: Campos MA, Macedo S, Fernandes M, Pestana A, Pardal J, Batista R, Vinagre J, Sanches A, Baptista A, Lopes JM, Soares P, TERT promoter mutations are associated with poor prognosis in cutaneous squamous cell carcinoma, Journal of the American Academy of Dermatology (2018), doi: 10.1016/j.jaad.2018.08.032. 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.
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Article type: Original article
2 Title: TERT promoter mutations are associated with poor prognosis in cutaneous squamous cell
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carcinoma
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Manuel António Campos, MD1,2,3,4; Sofia Macedo, MSc1,2; Margarida Fernandes, MD5; Ana Pestana,
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MSc,1,2,3; Joana Pardal, MD5; Rui Batista, MSc1,2,3; João Vinagre, PhD1,2,3; Agostinho Sanches, MD6;
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Armando Baptista, MD4; José Manuel Lopes, MD, PhD, IFCAP1,2,3,5†; and Paula Soares, PhD1,2,3, *†
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Porto, Portugal;
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Roberto Frias, s/n, 4200-465 Porto, Portugal;
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Vila Nova de Gaia, Portugal
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Portugal;
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Vila Nova de Gaia, Portugal;
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†
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*
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Paula Soares, PhD
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IPATIMUP
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Rua Roberto Frias s/n,
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4200-465, Porto, Portugal
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Tel: +351 225570700
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Fax: +351 225570799
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Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-135
Institute of Molecular Pathology and Immunology of the University of Porto (Ipatimup), Rua Dr.
Medical Faculty, University of Porto, Al. Prof. Hernâni Monteiro, 4200-139 Porto, Portugal;
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Dermatology Department, Centro Hospitalar Vila Nova de Gaia, Rua Conceição Fernandes, 4434-502
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Department of Pathology, Centro Hospitalar São João, Al. Prof. Hernâni Monteiro, 4200-139 Porto,
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Department of Pathology, Centro Hospitalar Vila Nova de Gaia, Rua Conceição Fernandes, 4434-502
These authors contributed equally to this work. Corresponding author:
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[email protected]
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Funding sources: Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020; Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01- 0145-FEDER-007274) and by the project “Advancing cancer research: from basic knowledge to application”; NORTE-01-0145-FEDER-000029; “Projetos Estruturados de I&D&I”, funded by Norte 2020-Programa Operacional Regional do Norte. Further funded by the European Regional Development Fund (ERDF) through the Operational Programme for Competitiveness and Internationalization – COMPETE 2020, and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, under project “POCI-01-0145-FEDER-016390: CANCEL STEM”. This work was also funded by the Portuguese Society of Dermatology and Venereology through a grant “Bolsa Juvenal Esteves 2016/Bolsa de Investigação LEO” to MAC.
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Conflicts of Interest: The authors have no conflict of interest to declare.
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Reprint requests: Paula Soares
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Abstract word count: 178 words
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Capsule summary word count: 45 words
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References: 35
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Figures: 1
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Supplementary figures: 0
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Tables: 3
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Supplementary tables: 1
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IRB approval status: Reviewed and approved by IRB.
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Keywords: TERT promoter mutation, TERT, prognostic biomarker, prognosis, biomarker, cutaneous
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squamous cell carcinoma, squamous cell carcinoma, recurrence, metastases, outcome.
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Abstract
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Background: TERT promoter (TERTp) mutations have been reported as potential predictors of poor prognosis in several
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cancers, but the prognostic value of TERTp mutations has not been determined for cutaneous squamous cell
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carcinoma (cSCC).
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Objective:
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outcome.
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Methods:
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To evaluate the frequency of TERTp mutations and correlate it with clinicopathological features and patient
We performed genetic profiling of TERTp mutations in a retrospective series of cSCC. The predictive value of
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TERTp mutations and clinicopathological parameters were assessed using logistic regression models.
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Results:
152 cSCC from 122 patients were analyzed for TERTp mutations: mutation rate was 31.6% (48/152) and was
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higher in invasive (42/121; 34.7%) than in in situ cSCC (6/31; 19.4%). Age > 75 years [OR 14.84; p = 0.013] and
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TERTp mutation [OR 8.11; p = 0.002] were independent predictors of local recurrence. TERTp mutation [OR 15.89;
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p = 0.022] was independently associated with higher risk of lymph node metastasis.
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Limitations: The restricted number of metastatic cases.
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Conclusion:
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TERTp mutations may prove to be a molecular biomarker with prognostic significance in invasive cSCC, but larger studies are needed.
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Body of manuscript
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Introduction Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer in Caucasians.1 Ultraviolet
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radiation is the most common causal factor, and cSCC most frequently occurs in chronically sun-exposed areas,
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such as the face.2 cSCC carcinogenesis includes premalignant lesions, actinic keratosis and in situ squamous
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carcinoma/Bowen disease, invasive carcinoma, and metastatic cSCC carcinoma, although a multistep model is not
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always present.3 cSCC can recur and metastasize and metastatic cases have a poor prognosis with 25-35% five-year
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survival rate, and < 10% ten-year survival rate.4-6 Some clinicopathological prognostic markers have been proposed
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in cSCC for local recurrence and metastasis (tumor thickness > 6 mm, invasion beyond subcutaneous fat, perineural
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invasion, tumor size > 2 cm, poor differentiation, localization in the temple, lip, and ear).7
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Telomerase (TERT), a ribonucleoprotein complex that synthesizes telomeric DNA (TTAGGG hexamers), is
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responsible for maintaining telomere length.8 TERT promoter (TERTp) mutations create binding sites for
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transcription factors (ETS/TCF) that result in telomerase expression, increasing telomere length and stability,
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allowing cancer cells to divide, and preventing senescence or apoptosis. Recurrent somatic TERTp mutations were
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found in a high percentage of melanomas, cancers of the central nervous system, bladder cancers, and thyroid
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cancers (follicular cell-derived).9-13 In cutaneous carcinomas,
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attributed to TERTp mutations (cytidine to thymidine transitions at dipyrimidine motifs).9, 10 These TERTp mutations
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have been described as a potential biological predictor of metastasis and/or mortality in melanoma, glioblastoma,
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medulloblastoma, bladder, and thyroid cancers.12, 15-18 However, in cSCC, only small series of cases were evaluated
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and no information about the putative prognostic value of these changes is available in the literature.
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an UV light-induced damage signature has been
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In this study, we assessed TERTp mutations in a large series of cutaneous squamous cell carcinoma and
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correlated these mutations with clinicopathological features and patients outcome.
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Materials and methods
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Patient selection, sample selection, and clinicopathological characterization
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All the procedures described in this study were in accordance with national and institutional ethical standards
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and were previously approved by Local Ethical Review Committees. A more detailed methods description is
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available in supplementary file 1. A descriptive and statistical analysis of all consecutive cSCC surgically removed at Centro Hospitalar Vila
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Nova de Gaia e Espinho (CHVNGE) between January 2004 and December 2013 was performed. Inclusion criteria
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included immunocompetent patients with histologic diagnosis of cSCC and with available follow-up data. Exclusion
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criteria included lesions in patients with genetic diseases associated with increased risk of cSCC (e.g. xeroderma
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pigmentosum, epidermodysplasia verruciformis, and albinism). None of the cases of this retrospective series were
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treated with Mohs micrographic surgery. 184 histological specimens were revised by pathologists with experience in
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cutaneous neoplasms (JML, JP, and MF). Tumors were categorized based on the protocol for examination of
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specimens of the College of American Pathologists (CAP) and the American Joint Committee on Cancer (AJCC)
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guidelines.19 Representative tumor areas were marked by the pathologists on hematoxylin and eosin slides to
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perform manual microdissection.
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For statistical analysis, age at diagnosis was categorized into two groups according to the mean age (≤ 75 years
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vs >75 years), and topographic locations were classified according to International Statistical Classification of
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Diseases and Related Health Problems 10th Revision20 and according to sun-exposure. Specimens were classified
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into in situ cSCC and invasive cSCC according to histologic subtype. In addition to CAP protocol variables, other
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recorded categories were pattern of invasion, and presence of intra and peritumoral infiltrate. "T" of each tumor was
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classified according to the correlative TNM classification. As in other studies,21, 22 recurrence was defined as the
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development of a histologically confirmed cSCC in the same topographic area in addition to being identified by the
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assisting Dermatologist as recurrence. Progression-free survival was defined as the time until diagnosis of
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recurrence and/or metastasis. Progression-free survival and overall follow-up were recorded in months.
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DNA extraction and mutation analysis
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DNA was retrieved from 10-µm cuts of formalin-fixed paraffin-embedded tissues (FFPE) after careful
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microdissection. A DNA extraction kit (Citogene®, Citomed, Portugal) was used according to the manufacturer’s
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instructions. PCRs were performed with Qiagen Multiplex kit (Qiagen, Hilden, Germany) using the recommended
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settings.11 Direct sequencing reaction was performed with the BigDye Terminator Kit (Perkin-Elmer, Foster City,
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amplification/sequencing was performed for both positive and inconclusive (not confirmed as positive or negative)
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samples. Mutations were detected using the Mutation Surveyor DNA variant analysis software (Softgenetics,
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Pennsylvania, USA) and was matched with reference sequences from GenBank. Sequences were obtained from -270
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to – 50 base pairs (bps) upstream of the ATG codon, which include the recurrent TERTp mutations described in
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other cancers.9,
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125CC>TT, and tandem -138/139CC>TT.
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The described TERTp mutations evaluated included: -124C>T, -146C>T, tandem -124/-
Statistical analysis
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Statistical analysis was conducted with SPSS version 24.0 (SPSS Inc., USA). Descriptive statistics, Chi-
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square, Fisher´s exact test and student´s t-test (unpaired, two-tailed) were used when appropriate. The predictive
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value of TERTp and other variables for recurrence, metastasis, and progression-free survival was assessed using
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univariate and multivariate logistic regression models. In the regression models, all the variables that were
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significantly associated with the specified outcome in the univariate model were included in the multivariate
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analysis. Confidence intervals (CI) were calculated with coverage of 95%. Survival curves were plotted by Kaplan-
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Meier method with the log-rank statistics.
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significance level was adjusted by Bonferroni correction (with 0.05 divided by the number of performed
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comparisons for each dependent variable) when multiple comparisons were performed (results displayed in
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Supplementary Table 1).
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Results were considered statistically significant at p < 0.05. The
Results
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Of the 184 histologically characterized cases, we were unable to determine TERTp status in 32 cases due to
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small size and/or low quality of the samples. In total, 152 lesions from 122 patients were analyzed for TERTp
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mutations. Of these, 31 corresponded to in situ cSCC and 121 to invasive cSCC. The overall frequency of TERTp
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mutations was 31.6% (48 out of 152 cases). TERTp mutations were present in 6 out of 31 (19.4%) in situ cSCC and
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in 42 out of 121 (34.7%) invasive cSCC. The following mutations were detected: -124: G>A mutation in 26 of 48
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(54.2%) cases, -146: G>A mutation in 18 of 48 (37.5%) cases, and tandem mutation at position -124/-125 in 4 cases
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(8.3%). The mutations were mutually exclusive.
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Relationship between TERTp mutations and clinicopathological features
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Table 1 presents the clinicopathological features and the frequency of TERTp mutations in the series.
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Clinicopathological factors and their association with TERTp mutations are presented in Supplementary Table 1. We analyzed all cSCC and observed that TERTp mutations were present in the face, trunk, and upper and lower
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limb. TERTp mutations were more frequent in invasive compared to in situ cSCC, although there was no statistical
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significance.
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In in situ cSCC, men showed more TERTp mutation [5 out of 15 (33.3%) cases than women [1 out of 16 (6.3%)]. Three in situ cSCC recurred, but none of them had TERTp mutations.
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In invasive cSCC, TERTp mutation was associated with a larger maximum tumor thickness (4.8 ± 4.1 mm vs
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3.4 ± 2.2 mm; p = 0.050), and tumors with >6 mm thickness displayed a higher frequency of TERTp mutations [10
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out of 19 (52.6%) cases vs 29 out of 96 (30.2%) cases; p = 0.059)], although neither association reached statistical
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significance after adjusting p values with Bonferroni correction. Despite not reaching statistical significance, tumors
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with TERTp mutations had a larger size than tumors with wild-type TERTp (2.3 ± 1.8 cm vs 1.6 ± 1.2 respectively;
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p = 0.068). TERTp mutations were significantly more frequent in cases that recurred [13 out of 17 (76.5%) cases vs
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29 out of 104 (27.9%) cases; p < 0.001]. TERTp mutations were also more common in metastatic cases [7 out of 8
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(87.5%) cases) than in non-metastatic cases (35 out of 113 (31.0%) cases), but statistical analysis was precluded
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owing to a small number of wild-type cases with metastasis (n = 1).
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Relationship between TERTp mutation and outcome In this analysis, we only included invasive cSCC (n = 121). Mean follow-up time (± SD) of the patients was 43.7 ± 30.3 months (range 6-156 months).
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A total of 17 cases (14.0%) and 8 cases (6.6%) presented recurrence and metastasis (all were lymph node
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metastasis), respectively, during follow-up. Table 2 presents the main characteristics of the cases with adverse
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outcomes.
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in invasive cSCC (Table 3). When analyzing the factors associated with the risk of recurrence, age > 75 years [OR
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13.19; p = 0.014], absence of ulceration [OR 2.96; p = 0.048], and TERTp mutation [OR 8.41; p = 0.001] were
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identified to be predictors in the univariate analysis. When these aforementioned factors were included in the
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multivariate analysis, TERTp mutation [OR 8.11; p = 0.002] and age > 75 years [OR 14.84; p=0.013] were identified
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to be independent predictors of recurrence. As shown in Table 2, 82.4% of the cases that recurred were located on
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the face. When comparing facial and extra-facial lesions, no statistical difference was observed in the mean values
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of surgical margins (mean peripheral margins of 2.5 ± 3.5 vs 2.7 ± 1.8, respectively; mean deep margins of 2.6 ± 2.7
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vs 2.6 ± 2.1, respectively). Peripheral margins [OR 1.12; p = 0.121] and deep margins [OR 0.92; p = 0.924] were not
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associated with recurrence in the univariate analysis. Despite not achieving significance in the univariate analysis,
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we included margins in the multivariate analysis since lower margins may be a confounder in relation to TERTp
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mutation prognostic value in recurrence. After adjusting for margins, TERTp mutation [OR 6.75; p = 0.004] and age
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> 75 years [OR 13.00; p = 0.022] continued to be independent predictors of recurrence in the multivariate analysis.
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When we analyzed predictors of metastasis, univariate analysis demonstrated that few or absent peritumoral
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lymphocytes [OR 10.20: p = 0.033], subcutaneous tissue invasion [OR 5.46; p = 0.034], peripheral margins [OR
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1.16; p = 0.048], and TERTp mutation [OR 15.60; p = 0.012] were associated with a higher likelihood of metastasis.
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In the multivariate analysis, only TERTp mutation [15.89; p = 0.022] was independently associated with a higher
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risk of metastasis.
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The Kaplan-Meier survival analysis revealed that TERTp mutation (figure 1A and 1B) and age >75 years
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(figure 1C and 1D) were associated with a shorter time for recurrence (log rank p < 0.001 and p < 0.001) and a
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shorter time for metastasis to occur (log rank p = 0.002 and p = 0.007). Absent or few peritumoral lymphocytes
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(figure 1E) and subcutaneous tissue invasion (figure 1F) were associated with a shorter time for metastasis
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occurrence (log rank p = 0.007 and p = 0.014).
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Discussion
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The most important added value of the present study is that we found that patients with mutated TERTp
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invasive cSCC have a substantially higher risk of adverse outcome (recurrence and metastasis). Our results are in
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accordance with those of previous studies in other cancers, where TERTp mutation was a significant predictor of
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poor prognosis.18, 23, 24 The usefulness of TERTp mutations as a prognostic marker is particularly relevant because
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invasive cSCC is highly frequent, a small percentage of such carcinomas behave aggressively, and there is a lack of
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good prognostic indicators in this setting. Concerning invasive cSCC, we report a lower mutation rate (34.7%) than those reported in most previously
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published studies (50.0% to 74.1%); however, those studies were conducted with a limited number of samples (5 to
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37 cases).13, 14, 25, 26 Since our study included a larger consecutive series of cases in a hospital, we consider that it
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represents a more accurate estimation of TERTp mutation in cSCC. Our study indicates that TERTp mutation may be
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more frequent in invasive than in situ cSCC, although studies with a larger number of in situ cases are necessary to
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confirm this premise.
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As in melanoma23, 24 mutated TERTp invasive cSCCs presented a larger maximum tumor thickness than wild-
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type TERTp cases, although there was no statistical significance in our study. Our results also showed a higher
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frequency, but not reaching significance, of TERTp mutations in tumors with > 6 mm thickness, a parameter that has
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been described to be associated with a higher risk of metastasis.21
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The role of TERTp mutation remains to be clarified in early cutaneous squamous cell carcinogenesis because
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our study revealed a higher rate of TERTp mutation in in situ cSCC (19.4%) than the previously reported rate
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(9.1%),13 even though recurrent in situ cSCC did not have this mutation in our series. Thus, studies comprising
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normal skin, AK, in situ, and invasive cSCCs are warranted to clarify the role of TERTp mutation in the putative
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pathogenic model (s) of cutaneous squamous cell carcinoma.
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Our recurrence rate (14.0%) in invasive cSCC is within the range of those reported in previous studies (3.0-
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16.0%).21, 22, 27-29 We observed a lymph node metastasis rate (6.6%) slightly higher than that reported in the literature
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(3.7-4.6%).21, 22 The fact that all cases were drawn from a hospital, which often assists patients with more advanced
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disease, may in part explain the differences in the aforementioned reported rates. Most of the recurrent invasive
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cSCC cases were located on the face, an anatomic region where larger clearance margins are difficult to attain.
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Despite this, facial location and mean margins were not associated with recurrence.
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Considering other prognostic factors in this series, age > 75 years was an independent predictor of recurrence
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in invasive cSCC, as identified in previous studies.22, 29-31 Our results also support that few or absent peritumoral
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infiltrate was associated with metastasis in the univariate analysis and with a shorter time until this adverse outcome
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occurred. In other tumor models (including melanoma), the absence or reduced number of lymphocytes (without
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prognosis.32-34 Invasion of the subcutaneous tissue, a classical risk factor for recurrence and metastasis of cSCC,7
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was a predictor of metastasis in the univariate analysis and was associated with a shorter time until metastization.
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Despite these results, when adjusted for other variables, few or absent peritumoral infiltrate and invasion of the
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subcutaneous tissue failed to be an independent prognostic predictor.
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We are aware that our series lacks a substantial number of metastatic cSCC samples with longer follow-up.
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Nevertheless, we evaluated a consecutive series and found that the prevalence of advanced cSCC in our cohort is
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actually slightly higher when compared to the frequency reported by others. Another limitation is that there could be
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a potential selection bias since not all initially selected cases had histological specimens available.
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We conclude that TERTp mutations may be potential markers for aggressive behavior in cSCC because they
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may be more frequently observed in invasive than in situ cSCC and were associated with recurrence and metastasis
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in invasive cSCC. Moreover, recurrence and metastasis in invasive cSCC were likely to occur sooner in cases with
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TERTp mutation. As in other cancers,35 the inclusion of TERTp mutation in management guidelines should be
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considered in cSCC.
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Abbreviation and acronym list
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Bps – base pairs
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CAP – College of American Pathologists
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cSCC – cutaneous squamous cell carcinoma
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FFPE – formalin-fixed paraffin-embedded
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TERT – telomerase
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TERTp – telomerase promoter
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ACCEPTED MANUSCRIPT 12 References
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ACCEPTED MANUSCRIPT 14 19. SB E. American Joint Committee on Cancer, American Cancer Society. AJCC Cancer Staging
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Handbook: From the AJCC Cancer Staging Manual. 7th ed. Springer 2010.
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20. Bramer GR. International statistical classification of diseases and related health problems. Tenth
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revision. World Health Stat Q 1988;41:32-6.
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21. Brantsch KD, Meisner C, Schonfisch B, Trilling B, Wehner-Caroli J, Rocken M et al. Analysis of risk
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factors determining prognosis of cutaneous squamous-cell carcinoma: a prospective study. Lancet Oncol
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2008;9:713-20.
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22. Schmults CD, Karia PS, Carter JB, Han J , Qureshi AA. Factors predictive of recurrence and death
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from cutaneous squamous cell carcinoma: a 10-year, single-institution cohort study. JAMA Dermatol
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2013;149:541-7.
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23. Griewank KG, Murali R, Puig-Butille JA, Schilling B, Livingstone E, Potrony M et al. TERT
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promoter mutation status as an independent prognostic factor in cutaneous melanoma. J Natl Cancer Inst
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2014;106.
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24. Populo H, Boaventura P, Vinagre J, Batista R, Mendes A, Caldas R et al. TERT promoter mutations
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in skin cancer: the effects of sun exposure and X-irradiation. J Invest Dermatol 2014;134:2251-7.
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25. Lin SY, Liao SL, Hong JB, Chu CY, Sheen YS, Jhuang JY et al. TERT promoter mutations in
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periocular carcinomas: implications of ultraviolet light in pathogenesis. Br J Ophthalmol 2016;100:274-7.
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26. Cheng KA, Kurtis B, Babayeva S, Zhuge J, Tantchou I, Cai D et al. Heterogeneity of TERT promoter
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mutations status in squamous cell carcinomas of different anatomical sites. Ann Diagn Pathol
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2015;19:146-8.
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27. Clayman GL, Lee JJ, Holsinger FC, Zhou X, Duvic M, El-Naggar AK et al. Mortality risk from
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squamous cell skin cancer. J Clin Oncol 2005;23:759-65.
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28. Leibovitch I, Huilgol SC, Selva D, Hill D, Richards S , Paver R. Cutaneous squamous cell carcinoma
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treated with Mohs micrographic surgery in Australia I. Experience over 10 years. J Am Acad Dermatol
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2005;53:253-60.
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ACCEPTED MANUSCRIPT 15 29. Eroglu A, Berberoglu U , Berreroglu S. Risk factors related to locoregional recurrence in squamous
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cell carcinoma of the skin. J Surg Oncol 1996;61:124-30.
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30. Diaz-Corpas T, Morales-Suarez-Varela M, Rausell Fontestad N, Fuertes Prosper A, Marquina-Vila A
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, Jorda-Cuevas E. Squamous Cell Carcinoma: Clinical and Pathological Features and Associated Risk
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Factors in an Observational Study of 118 Patients. Actas Dermosifiliogr 2015;106:806-15.
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31. Kyrgidis A, Tzellos TG, Kechagias N, Patrikidou A, Xirou P, Kitikidou K et al. Cutaneous squamous
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cell carcinoma (SCC) of the head and neck: risk factors of overall and recurrence-free survival. Eur J
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Cancer 2010;46:1563-72.
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32. Senovilla L, Vacchelli E, Galon J, Adjemian S, Eggermont A, Fridman WH et al. Trial watch:
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Prognostic and predictive value of the immune infiltrate in cancer. Oncoimmunology 2012;1:1323-43.
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33. Azimi F, Scolyer RA, Rumcheva P, Moncrieff M, Murali R, McCarthy SW et al. Tumor-infiltrating
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lymphocyte grade is an independent predictor of sentinel lymph node status and survival in patients with
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cutaneous melanoma. J Clin Oncol 2012;30:2678-83.
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34. Sondergaard K , Schou G. Survival with primary cutaneous malignant melanoma, evaluated from
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2012 cases. A multivariate regression analysis. Virchows Arch A Pathol Anat Histopathol 1985;406:179-
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95.
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35. Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE et al. 2015 American
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Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated
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Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and
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Differentiated Thyroid Cancer. Thyroid 2016;26:1-133.
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ACCEPTED MANUSCRIPT 16 Figure legend
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Figure 1. Kaplan-Meier curves for recurrence-free survival and metastasis-free survival of invasive
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cSCC, respectively, according to TERTp status (1A and 1B) and age (1C and 1D). Kaplan-Meier curves
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for metastasis-free survival of invasive cSCC according to peritumoral lymphocytic infiltration (1E) and
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level of invasion (1F).
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ACCEPTED MANUSCRIPT 17 Table legends
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Table 1. “Clinicopathological features and the frequency of TERT promoter mutations”
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Table 2. “Features of invasive cSCC in patients with recurrence and/or lymph node metastasis”
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Table 3. “Predictive factors for recurrence and lymph node metastasis”
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Supplementary Table 1. “Clinicopathological and molecular associations with TERT promoter mutations
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in cSCC”
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ACCEPTED MANUSCRIPT 18 Table 1. “Clinicopathological features and the frequency of TERT promoter mutations” all lesions
121 76.5 ± 12.7 73.0 ± 12.3 81.6 ± 11.6
87 (57.2) 65 (42.8)
15 (48.4) 16 (51.6)
103 (67.8) 43 (28.3) 6 (3.9)
11 (35.5) 18 (58.1) 2 (6.5)
97 (63.8) 8 (5.3) 21 (13.8) 20 (13.2) 6 (3.9) 42.9 ± 28.8 39.8 ± 29.3
9 (29.0) 4 (12.9) 6 (19.4) 10 (32.3) 2 (6.5) 39.7 ± 22.0 38.4 ± 22.2
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31 78.5 ± 7.0 77.8 ± 6.8 78.4 ± 7.8
72 (59.5) 49 (40.5)
92 (76.0) 25 (20.7) 4 (3.3)
88 (72.7) 4 (3.3) 15 (12.4) 10 (8.3) 4 (3.3) 43.7 ± 30.3 40.1 ± 30.9
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Histological features
invasive cSCC
152
129 (84.9) 23 (15.1)
28 (90.3) 3 (9.7)
101 (83.5) 20 (16.5)
132 (86.8) 20 (13.2)
28 (90.3) 3 (9.7)
104 (86.0) 17 (14.0)
144 (94.7) 8 (5.3)
31 (100) 0
113 (93.4) 8 (6.6)
104 (68.4) 48 (31.6)
25 (80.6) 6 (19.4)
79 (65.3) 42 (34.7)
34 (22.4) 118 (77.6) 1.9 ± 1.5
6 (19.4) 25 (80.6) 1.9 ± 1.2
28 (23.1) 93 (76.9) 1.9 ± 1.5
69 (45.4) 39 (25.7) 44 (28.9) 2.3 ± 2.9 2.6±2.4
10 (32.3) 9 (29.0) 12 (38.7) 1.6 ± 1.5 3.0 ± 1.7
59 (48.8) 30 (24.8) 32 (26.4) 2.5 ± 3.1 2.5 ± 2.5
53 (34.9) 91 (59.9) 8 (5.3)
14 (45.2) 15 (48.4) 2 (6.5)
39 (32.2) 76 (62.8) 6 (5.0)
50 (32.9) 92 (60.5) 10 (6.6)
6 (19.4) 25 (80.6)
44 (36.4) 67 (55.4) 10 (8.3)
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Clinical and molecular features
Age at diagnosis (mean (±SD)) Male Female Gender (n (%)) Male Female Sun exposure (n (%)) Chronic Intermittent Undetermined Localization (n (%)) Face Trunk Upper limb Lower limb Undetermined Follow-up (months) Progression free survival (months) Adverse outcome No Yes Recurrence No Yes Metastases No Yes TERTp mutations Wild-type Mutation Procedure (n (%)) Biopsy Excision Maximum tumor size (cm) Maximum tumor size 75 Ulceration Yes No Level of invasion Dermis Subcutaneous tissue Peritumoral infiltrate Moderate-intense Few-absent Peripheral margins*
Multivariate Analysis
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21 Supplementary Table 1. “Clinicopathological and molecular associations with TERT promoter mutations in cSCC” in situ cSCC
Clinical pathological features
Total
TERTp WT
p
Number of cases
152
104 (68.4%)
48 (31.6%)
Age at diagnosis (mean (±SD)) Male Female
76.9 ± 11.7 73.9 ± 11.6 80.8 ± 10.7
77.2 ± 11.4 73.1 ± 11.7 81.8 ± 9.3
75.9 ± 12.6 75.1 ± 11.8 77.7 ± 14.5
Gender (n (%)) Male Female
87 (57.2) 65 (42.8)
55 (52.9) 49 (47.1)
32 (66.7) 16 (33.3)
Sun exposure (n (%)) Chronic Intermittent Undetermined
103 (67.8) 43 (28.3) 6 (3.9)
67 (64.4) 32 (30.8) 5 (4.8)
36 (75.0) 11 (22.9) 1 (2.1)
Localization Face Trunk Upper limb Lower limb Undetermined
97 (63.8) 8 (5.3) 21 (13.8) 20 (13.2) 6 (3.9)
64 (61.5) 3 (2.9) 14 (13.5) 18 (17.3) 5 (4.8)
33 (68.8) 5 (10.4) 7 (14.6) 2 (4.2) 1 (2.1)
Localization* Extra-facial Face
49 (33.6) 97 (66.4)
35 (35.4) 64 (64.6)
14 (29.8) 33 (70.2)
0.506
34 (22.4) 118 (77.6)
26 (25.0) 78 (75.0)
8 (16.7) 40 (83.3)
0.252
Maximum tumor size (cm)
1.9 ± 1.5
1.7 ± 1.2
2.2 ± 1.8
Maximum tumor size < 2 cm >= 2 cm Cannot be assessed
69 (45.4) 39 (25.7) 44 (28.9)
47 (66.2) 24 (33.8)
22 (59.5) 15 (40.5)
0.489
Peripheral margins (mm)
2.3 ± 2.9
2.0 ± 2.5
2.9 ± 3.4
Deep margins (mm)
2.6±2.4
2.5 ± 2.3
53 (34.9) 91 (59.9)
32 (33.0) 65 (67.0)
TERTp WT
TERTp Mut
31
25 (80.6%)
6 (19.4%)
0.544 0.451 0.189
78.5 ± 7.0 77.8 ± 6.8 78.4 ± 7.8
78.6 ± 7.4 78.7 ± 6.6 78.5 ± 8.1
76.2 ± 6.9 76.0 ± 7.7 77.0
0.119
15 (48.4) 16 (51.6)
10 (40.0) 15 (60.0)
11 (35.5) 18 (58.1) 2 (6.5)
9 (36.0) 14 (56.0) 2 (8.0)
9 (29.0) 4 (12.9) 6 (19.4) 10 (32.3) 2 (6.5)
7 (28.0) 1 (4.0) 6 (24.0) 9 (36.0) 2 (8.0)
5 (83.3) 1 (16.7)
2 (33.3) 4 (66.7) 0
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NA
NA
p
Total
2 (33.3) 3 (50.0) 0 1 (16.7) 0
0.476 0.490 0.863 NA
NA
NA
TERTp WT
TERTp Mut
p
121
79 (65.3%)
42 (34.7%)
76.5 ±12.7 73.0 ± 12.3 81.6 ± 11.6
76.8 ± 12.5 71.8 ± 12.3 83.3 ± 9.5
75.9 ± 13.3 74.9 ± 12.5 77.7 ± 15.0
0.726 0.314 0.124
72 (59.5) 49 (40.5)
45 (57.0) 34 (43.0)
27 (64.3) 15 (35.7)
0.435
92 (76.0) 25 (20.7) 4 (3.3)
58 (73.4) 18 (22.8) 3 (3.8)
34 (81.0) 7 (16.7) 1 (2.4)
88 (72.7) 4 (3.3) 15 (12.4) 10 (8.3) 4 (3.3)
57 (72.2) 2 (2.5) 8 (10.1) 9 (11.4) 3 (3.8)
31 (73.8) 2 (4.8) 7 (16.7) 1 (2.4) 1 (2.4)
NA
NA
20 (69.0) 9 (31.0)
16 (69.6) 7 (30.4)
4 (66.7) 2 (33.3)
NA
29 (24.8) 88 (75.2)
19 (25.0) 57 (75.0)
10 (24.4) 31 (75.6)
0.942
6 (19.4) 25 (80.6)
4 (16.0) 21 (84.0)
2 (33.3) 4 (66.7)
NA
28 (23.1) 93 (76.9)
22 (27.8) 57 (72.2)
6 (14.3) 36 (85.7)
0.092
1.9 ± 1.2
2.0±1.2
1.8 ± 1.5
0.765
1.9 ± 1.5
1.6 ± 1.2
2.3 ± 1.8
0.068
10 (32.3) 9 (29.0) 12 (38.7)
8 (53.3) 7 (46.7)
2 (50.0) 2 (50.0)
NA
59 (48.8) 30 (24.8) 32 (26.4)
39 (69.6) 17 (30.4)
20 (60.6) 13 (39.4)
0.384
0.125
1.6 ± 1.5
1.6 ± 1.5
1.5 ± 1.3
0.900
2.5 ± 3.1
2.2 ± 2.8
3.0 ± 3.6
0.191
2.7 ± 2.5
0.627
3.0 ± 1.7
2.6 ± 1.5
4.8 ± 1.0
0.017
2.5 ± 2.5
2.5 ± 2.5
2.5 ± 2.5
0.963
21 (44.7) 26 (55.3)
0.173
14 (45.2) 15 (48.4)
9 (39.1) 14 (60.9)
5 (83.3) 1 (16.7)
NA
39 (32.2) 76 (62.8)
23 (31.1) 51 (68.9)
16 (39.0) 25 (61.0)
0.389
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Procedure
0.149
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Biopsy Excision
Total
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TERTp Mut
Invasive cSCC
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All lesions
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395
Ulceration No Yes
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Maximum tumor thickness Maximum tumor thickness < 6 mm ≥ 6 mm Cannot be assessed Intratumoral infiltrate Moderate-intense Few-absent
15 (33.3) 30 (66.7)
0.750
31 (20.4) 121 (79.6)
25 (24.0) 79 (76.0)
6 (12.5) 42 (87.5)
0.101
6 (19.4) 25 (80.6)
5 (20.0) 20 (80.0)
1 (16.7) 5 (83.3)
NA
44 (36.4) 67 (55.4) 10 (8.3)
30 (41.7) 42 (58.3)
14 (35.9) 25 (64.1)
9 (7.4) 2 (1.7) 110 (90.9)
6 (7.6) 2 (2.5) 71 (89.9)
3 (7.1) 0 39 (92.9)
43 (35.5) 74 (61.2)
31 (40.3) 46 (59.7)
12 (30.0) 28 (70.0)
106 (87.6)
70 (90.9)
36 (90.0)
11 (9.1) 4 (3.3)
7 (9.1)
4 (10.0)
66 (54.5) 51 (42.1) 4 (3.3)
43 (56.6) 33 (43.4)
23 (56.1) 18 (43.9)
0.960
36 (29.8) 53 (43.8) 25 (20.7) 7 (5.8)
23 (30.7) 37 (49.3) 15 (20.0)
13 (33.3) 16 (41.0) 10 (25.6)
0.667
89 (73.6) 25 (20.7) 7 (5.8)
60 (80.0) 15 (20.0)
29 (74.4) 10 (25.6)
0.490
3.8±3.1
3.4±2.2
4.8±4.1
0.050
96 (79.3) 19 (15.7) 6 (5.0)
67 (88.2) 9 (11.8)
29 (74.4) 10 (25.6)
0.059
13 (10.7) 108 (89.3)
8 (10.1) 71 (89.9)
5 (11.9) 37 (88.1)
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35 (36.1) 62 (63.9)
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50 (32.9) 92 (60.5) 10 (6.6)
6 (5.0)
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Histologic grade Well-moderately differentiated Poorly differentiated Cannot be assessed Pattern of invasion Expansive Infiltrative Cannot be assessed Level of invasion Papillary dermis Reticular dermis Subcutaneous tissue Cannot be assessed Level of invasion Dermis Subcutaneous tissue Cannot be assessed
2 (6.5)
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Actinic Keratosis No Yes Undetermined Invasion Non-invasive Invasive Histologic type Acantholytic Verrucous NOS Histologic grade Well-differentiated Moderately-Poorly differentiated Cannot be assessed
8 (5.3)
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Undetermined
0.553
NA
0.275
4 (3.3)
NA
NA
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Follow-up (months)
68 (56.2) 53 (43.8)
45 (57.0) 34 (43.0)
23 (54.8) 19 (45.2)
0.816
116 (95.9) 5 (4.1)
75 (94.9) 4 (5.1)
41 (97.6) 1 (2.4)
NA
118 (97.5) 3 (2.5)
78 (98.7) 1 (1.3)
40 (95.2) 2 (4.8)
NA
104 (86.0) 17 (14.0)
75 (94.9) 4 (5.1)
29 (69.0) 13 (31.0)
T, tandem -124/-125CC>TT and tandem -138/139CC>TT.
Statistical analysis Statistical analysis was conducted using SPSS version 24.0 (SPSS Inc.). The results are expressed as a percentage or mean ± SD. Statistical analysis was performed on both the whole series of cSCC and two different
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groups of cSCC (in situ and invasive cSCC). Chi-square, Fisher´s exact test, and student´s t-test (unpaired, twotailed) were used when appropriate. The predictive value of TERT promoter mutations and other variables (age, gender, sun exposure, localization, presence of ulceration, presence of actinic keratosis, tumor size, histologic type,
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histologic grade, pattern of invasion, level of invasion, maximum tumor thickness, intratumoral infiltrate, peritumoral infiltrate, lymphovascular invasion, and perineural invasion) for recurrence, metastasis, and progressionfree survival were assessed using univariate and multivariate logistic regression models. Survival curves were plotted by the Kaplan-Meier method with the log-rank statistics. In the regression models, all the variables that were
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significantly associated with the specified outcome in the univariate model were included in the multivariate analysis. Confidence intervals (CI) were calculated with coverage of 95%. Results were considered statistically
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significant at p < 0.05. The significance level was adjusted by Bonferroni correction (with 0.05 divided by the number of performed comparisons for each dependent variable) when assessing TERTp mutations according to different clinicopathological and molecular characteristics (results displayed in supplementary table 1), as multiple
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comparisons were performed.
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1. Farasat S, Yu SS, Neel VA, Nehal KS, Lardaro T, Mihm MC et al. A new American Joint Committee on Cancer staging system for cutaneous squamous cell carcinoma: creation and rationale for inclusion of
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tumor (T) characteristics. J Am Acad Dermatol 2011;64:1051-9. 2. SB E. American Joint Committee on Cancer, American Cancer Society. AJCC Cancer Staging Handbook: From the AJCC Cancer Staging Manual. 7th ed. Springer 2010. 3. Bramer GR. International statistical classification of diseases and related health problems. Tenth revision. World Health Stat Q 1988;41:32-6.
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4. Brantsch KD, Meisner C, Schonfisch B, Trilling B, Wehner-Caroli J, Rocken M et al. Analysis of risk factors determining prognosis of cutaneous squamous-cell carcinoma: a prospective study. Lancet Oncol 2008;9:713-20.
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5. Schmults CD, Karia PS, Carter JB, Han J , Qureshi AA. Factors predictive of recurrence and death from cutaneous squamous cell carcinoma: a 10-year, single-institution cohort study. JAMA Dermatol 2013;149:541-7.
in human cancers. Nat Commun 2013;4:2185.
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6. Vinagre J, Almeida A, Populo H, Batista R, Lyra J, Pinto V et al. Frequency of TERT promoter mutations
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7. Horn S, Figl A, Rachakonda PS, Fischer C, Sucker A, Gast A et al. TERT promoter mutations in familial and sporadic melanoma. Science 2013;339:959-61.
8. Huang FW, Hodis E, Xu MJ, Kryukov GV, Chin L , Garraway LA. Highly recurrent TERT promoter
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mutations in human melanoma. Science 2013;339:957-9.
ACCEPTED MANUSCRIPT Capsule summary •
Patients with TERTp mutated cutaneous squamous cell carcinoma (cSCC) have higher risks for local recurrence and lymph node metastases. In the future, TERTp mutation may be included in the prognostic assessment of patients with cSCC.
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•
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Statistical Analyses Plan (SAP)
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carcinoma
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TERT promoter mutations are associated with poor prognosis in cutaneous squamous cell
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Study Objectives
cutaneous squamous cell carcinoma (cSCC).
Primary Objective
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Assess TERTp mutations, clinicopathological features and outcome in a series of
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Assess TERTp mutations in a large series of cutaneous squamous cell carcinoma and
(recurrence or metastasis).
Study design
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correlate these mutations with clinicopathological features and patient´s outcome
Retrospective study - Genetic profiling of TERTp mutations in a retrospective series of
Study population
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cSCC
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Consecutive cutaneous squamous cell carcinoma (cSCC) surgically removed at Centro Hospitalar Vila Nova de Gaia and Espinho (CHVNGE) between January 2004 and
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December 2013.
Definition of Population for Analysis The study population included cSCC surgically removed at Centro Hospitalar Vila Nova de Gaia and Espinho (CHVNGE).
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Statistical Methodology Statistical Procedures Continuous variables were summarized by mean and standard deviation. Categorical
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data were summarized as the number and percentage of subjects in each category.
The comparison of groups was performed by using parametric test such as t-tests for
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or equivalent non-parametric test as appropriate.
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continuous variables and Chi-square test (or Fisher exact test) for categorical variables
A logistic regression model was used to find associations between clinicopathological features, TERTp status and adverse outcome, controlling for possible confounding variables. In the regression models, all the variables significantly associated with the
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specified outcome in the univariate model were included in the multivariate analysis.
Kaplan-Meier survival curves are presented to investigate the effect of TERTp mutation
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and other variables in progression free survival.
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All statistical tests were performed under a two-sided significance level of 5%. The significance level was adjusted by Bonferroni correction (with 0.05 divided by the number of performed comparisons for each dependent variable) when assessing TERTp mutations according to different clinicopathological and molecular characteristics (results displayed in supplementary table 1), as multiple comparisons were performed.
