TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas

2014/2015 Adriana Maria Pinto Gaspar da Rocha TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas março, 2015 Mestrado Integrado em Medicina Área: Patologia e Oncologia Tipologia: Dissertação Trabalho efetuado sob a Orientação de: Professora Doutora Ana Paula Soares Dias Ferreira Trabalho organizado de acordo com as normas da revista: The Journal of Clinical Endocrinology & Metabolism Adriana Maria Pinto Gaspar da Rocha TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas março, 2015 À minha mãe e ao meu pai. TERT Promoter Mutations Are a Major Indicator of Poor Outcome in Differentiated Thyroid Carcinomas Miguel Melo,* Adriana Gaspar da Rocha,* João Vinagre,* Rui Batista, Joana Peixoto, Catarina Tavares, Ricardo Celestino, Ana Almeida, Catarina Salgado, Catarina Eloy, Patrícia Castro, Hugo Prazeres, Jorge Lima, Teresina Amaro, Cláudia Lobo, Maria João Martins, Margarida Moura, Branca Cavaco, Valeriano Leite, José Manuel Cameselle-Teijeiro, Francisco Carrilho, Manuela Carvalheiro, Valdemar Máximo, Manuel Sobrinho-Simões, and Paula Soares† Context: Telomerase promoter mutations (TERT) were recently described in follicular cell-derived thyroid carcinomas (FCDTC) and seem to be more prevalent in aggressive cancers. Objectives: We aimed to evaluate the frequency of TERT promoter mutations in thyroid lesions and to investigate the prognostic significance of such mutations in a large cohort of patients with differentiated thyroid carcinomas (DTCs). Design: This was a retrospective observational study. Setting and Patients: We studied 647 tumors and tumor-like lesions. A total of 469 patients with FCDTC treated and followed in five university hospitals were included. Mean follow-up (⫾SD) was 7.8 ⫾5.8 years. Main Outcome Measures: Predictive value of TERT promoter mutations for distant metastasization, disease persistence at the end of follow-up, and disease-specific mortality. Results: TERT promoter mutations were found in 7.5% of papillary carcinomas (PTCs), 17.1% of follicular carcinomas, 29.0% of poorly differentiated carcinomas, and 33.3% of anaplastic thyroid carcinomas. Patients with TERT-mutated tumors were older (P⬍.001) and had larger tumors (P⫽ .002). In DTCs, TERT promoter mutations were significantly associated with distant metastases (P⬍ .001) and higher stage (P⬍.001). Patients with DTC harboring TERT promoter mutations were submitted to more radioiodine treatments (P⫽.009) with higher cumulative dose (P⫽.004) and to more treatment modalities (P⫽.001). At the end of follow-up, patients with TERT-mutated DTCs were more prone to have persistent disease (P⫽.001). TERT promoter mutations were significantly associated with disease-specific mortality [in the whole FCDTC (P⬍.001)] in DTCs (P⬍.001), PTCs (P⫽.001), and follicular carcinomas (P⬍.001). After adjusting for age at diagnosis and gender, the hazard ratio was 10.35 (95% confidence interval 2.01–53.24; P⫽.005) in DTC and 23.81 (95% confidence interval 1.36–415.76; P⫽.03) in PTCs. Conclusions: TERT promoter mutations are an indicator of clinically aggressive tumors, being correlated with worse outcome and disease-specific mortality in DTC. TERT promoter mutations have an independent prognostic value in DTC and, notably, in PTC. (J Clin Endocrinol Metab 99: E754–E765, 2014) ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received October 8, 2013. Accepted January 15, 2014. First Published Online January 29, 2014 † Author affiliations are shown at the bottom of the next page. * M.Me., A.G.d.R., and J.V. contributed equally to this work. Abbreviations: AJCC, American Joint Committee on Cancer; ATC, anaplastic thyroid carcinoma; CI, confidence interval; DTC, differentiated thyroid carcinoma; FCDTC, follicular cell-derived thyroid carcinoma; FTC, follicular thyroid carcinoma; HR, hazard ratio; OR, odds ratio; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid carcinoma. JCEM ONLINE Hot Topics in Translational Endocrinology—Endocrine Care E754 jcem.endojournals.org J Clin Endocrinol Metab, May 2014, 99(5):E754–E765 doi: 10.1210/jc.2013-3734 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 February 2015. at 07:03 For personal use only. No other uses without permission. . All rights reserved. Telomerase activation is known to be a hallmark of cancer (1), being detected in up to 80% of malignant tumors (2, 3). Some tumors may maintain their telomeres by an alternative mechanism, which is telomerase independent, designated as alternative lengthening of telomeres, which appears to maintain telomeres through recombination-based interchromosomal exchange of sequence information (4, 5). The maintenance of telomere length in cancer cells is thought to result more frequently from telomerase reexpression than from alternative lengthening of telomeres, although the mechanisms underlying such process remain largely unknown. Thyroid tissue is a conditionally renewing tissue, which proliferates rarely in adult life (6). We have proposed that the embryonic remnants of the ultimobranchial body, the so-called solid cell nests of the thyroid, may represent the pool of thyroid stem cells because they express several stem cell markers, including telomerase (7). At variance withthis,normalthyroidtissueisthoughttobetelomerase negative, thus raising the possibility that the reactivation of telomerase may be a useful marker of tumor development (8). Several studies have examined telomerase activity in thyroid lesions and surrounding normal tissues using a PCR-based telomeric repeat amplification protocol assay for detection of telomerase activity and RT-PCR or quantitative real-time RT-PCR for the detection of TERT mRNA (for a review see reference 9). Thyroid carcinomas apparently display less frequent telomerase activation than other human carcinomas. Approximately 66% of all the thyroid carcinomas analyzed to date display telomerase activation that is more frequent in undifferentiated thyroid carcinomas than in differentiated carcinomas (9). Putting together the results obtained in the evaluation of telomerase activity by several authors, telomerase activity/ expression is reported in 48% of papillary thyroid carcinomas (PTCs) and 71% of follicular thyroid carcinomas (FTCs). TERT copy number gain was described in familial PTCs (10). Capezzone et al (11) observed telomerase activity in most sporadic and familial malignant thyroid tumors as well as in some adenomas. No telomerase activity was observed in hyperplastic nodules or in normal thyroid tissue of patients with sporadic PTCs (11). Altogether, the aforementioned findings suggest that telomerase activity may contribute to a more aggressive behavior of thyroid tumors (12–14). Recently highly frequent mutations in the promoter region of TERT were reported in melanomas, gliomas, and bladder and thyroid cancers (15–18). These mutations occur in two hot spot positions, located ⫺124 and ⫺146 bp upstream from the ATG start site (⫺124G⬎A and ⫺146G⬎A, C⬎T on the opposite strand) and confer enhanced TERT promoter activity (1, 2) putatively by generating a consensus binding site (GGAA) for ETS transcription factors within the TERT promoter region. In thyroid tumors, these mutations were shown to be associated with aggressive features and with the presence of BRAF or BRAF/RAS mutations (15, 19, 20). The role played by TERT promoter mutations in the clinical course of thyroid tumors, response to the therapy and survival of cancer patients, remains to be addressed. In the present study, we searched for the presence of telomerase promoter mutations in a large series of thyroid tumors and investigated the putative clinical significance of such somatic alterations. Materials and Methods All the procedures described in this study were in accordance with national and institutional ethical standards. Patients signed an informed consent form approved by the internal reviewing board. Patient tissue samples Six hundred forty-seven formalin-fixed, paraffin-embedded tissue samples from tumors and tumor-like lesions of the thyroid and from normal thyroid parenchyma localized at distance from the respective tumors were collected from the files of the Institute of Molecular Pathology and Immunology of the University of Porto(Porto,Portugal), correspondingtopatients followedupin five university hospitals in Portugal and Spain. The histology of all tumor samples was revised by three pathologists (C.E., J.M.C.-T., M.S.-S.) according to the World Health Organization criteria (21). Data on the histological characteristics of the 647 samples and the frequency of TERT promoter mutations in each group are summarized in Supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org. Twelve follicular adenomas Institute of Molecular Pathology and Immunology of the University of Porto (M.Me., A.G.d.R., J.V., R.B., J.P., C.T., R.C., A.A., C.S., C.E., P.C., H.P., J.L., V.M., M.S.-S., P.S.), 4200-465 Porto, Portugal; Medical Faculty, University of Porto (A.G.d.R., C.T.), 4200-139 Porto, Portugal; Institute of Biomedical Sciences of Abel Salazar, University of Porto (J.V., A.A.), 4050-313 Porto, Portugal; Department of Pathology and Oncology, Medical Faculty, University of Porto (J.L., V.M., M.S.-S., P.S.), 4200-139 Porto, Portugal; Departments of Endocrinology, Diabetes, and Metabolism (M.Me., F.C., M.C.) and Pathology (M.J.M.), University and Hospital Center of Coimbra, 3000-075 Coimbra, Portugal; Unit of Endocrinology (M.Me., M.C.), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal; School of Allied Health Sciences, ESTSP - Escola Superior de Tecnologia da Saúde do Porto (R.C.), Polytechnic of Porto, 4400-330 Vila Nova de Gaia, Portugal; Portuguese Institute of Oncology (H.P.), Coimbra Center, 3000-075 Coimbra, Portugal; Department of Pathology (T.A.), Hospital Pedro Hispano, 4464-513 Matosinhos, Portugal; Department of Pathology (C.L.), Portuguese Institute of Oncology, Porto Center, 4200-072 Porto, Portugal; Center for Investigation of Molecular Pathobiology (M.Mo., B.C., V.L.) and Department of Endocrinology (V.L.), Portuguese Institute of Oncology, Lisbon Center, 1099-023 Lisbon, Portugal; Center for the Study of Chronic Diseases (M.Mo., B.C., V.L.), Faculty of Medical Sciences, University of Lisbon, 1099-085 Lisbon, Portugal; Department of Pathology (J.M.C.-T.), Clinical University Hospital, Servicio Gallego de Salud - SERGAS, Medical Faculty, University of Santiago de Compostela, 15705 Santiago de Compostela, Spain; and Department of Pathology (M.S.-S.), Hospital S. João, 4200-319 Porto, Portugal doi: 10.1210/jc.2013-3734 jcem.endojournals.org E755 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 February 2015. at 07:03 For personal use only. No other uses without permission. . All rights reserved. and 27 PTCs from the Chernobyl-irradiated setting that we had previously studied (22) were also searched for the presence of TERT promoter mutations. Due to their etiopathogenic specificity (irradiation induced tumors) and lack of complete clinical information, they were not included in the subsequent clinicopathological analysis. DNA extraction DNA from formalin-fixed, paraffin-embedded tissues was retrieved from 10- ␮ m sections after careful microdissection. DNA extraction was performed using the Ultraprep tissue DNA kit (AHN Biotechnologie) following the manufacturer’s instructions. PCR and Sanger sequencing The genetic characterization of part of the tumors series regarding BRAF,NRAS,RET/PTC, and PAX8/PPARG had been previously reported; mutations were screened as previously described (23–25). To screen for TERT promoter mutations, we analyzed the hot spots previously identified by PCR followed by Sanger sequencing. TERT promoter mutation analysis was performed with the pair of primers forward TERT, CAGCGCTGCCTGAAACTC; and reverse TERT, GTCCTGCCCCTT CACCTT. Amplification of genomic DNA (25–100 ng) was performed by PCR using the QIAGEN multiplex PCR kit following the manufacturer’s instructions (further details on the amplification will be given upon request). Sequencing reaction was performed with the ABI Prism BigDye terminator kit (PerkinElmer), and the fragments were run in an ABI prism 3100 genetic analyzer (PerkinElmer). The sequencing reaction was performed in a forward direction, and all the detected mutations were further validated by a new independent analysis in both strands (Supplemental Figure 1). Patients’ follow-up Patients were treated and followed up in accordance with international protocols available at the time. Data regarding the number of radioiodine treatments and cumulative activity were retrieved from hospital records, together with other therapeutic procedures. For statistical analysis, we defined the category, additional treatments, in which we included other treatment modalities in addition to radioiodine, including surgery, external beam irradiation, and treatment with tyrosine kinase inhibitors (Supplemental Table 2). Patients were defined as being disease free at the end of follow-up if they had undetectable stimulated thyroglobulin (in the absence of thyroglobulin antibodies) and no imagiological evidence of disease. In the survival analysis, we have considered only deaths attributable to the thyroid carcinoma (disease-specific mortality). Statistical analysis Statistical analysis was conducted with SPSS version 20.0 (SPSS Inc). The results are expressed as a percentage or mean ⫾ SD. Statistical analysis was performed both on the whole series of follicular cell-derived thyroid carcinomas (FCDTCs) and consideringthedifferentgroupsoftumors.AFisher’sexacttest,ttest (unpaired, two tailed), and ANOVA were used when appropriate. The predictive value of TERT promoter mutations and other factors [age, gender, histologic category, extrathyroidal extension, vascular invasion, lymph node metastases, staging I–IV (Union for International Cancer Control/American Joint Committeeon Cancer [AJCC]), BRAFmutations, RAS mutations] for distant metastases and disease-free status at the end of follow-up were assessed using univariate and multivariate logistic regression models. Survival curves were plotted by the Kaplan-Meier method with the log-rank statistics. Multivariate survival analysis was performed using Cox regression. In the regression models, all the variables significantly associated with the specified outcome in the univariate model were included in the multivariate analysis. Mortality was expressed as a percentage and rate per person-year, the latter obtained dividing the number of thyroid cancer-specific deaths by the total follow-up time. Results were considered statistically significant at P⬍.05. Results TERT promoter mutations were not detected in normal parenchyma (n ⫽30) or in benign lesions (n ⫽81), such asa nodulargoiter (hyperplasticlesions),lymphocytic thyroiditis, or follicular adenomas. TERT promoter mutations were also not detected in medullary thyroid carcinomas (n ⫽28) or in any of the 39 tumors from the Chernobyl series (Supplemental Table 1). TERT promoter mutations were detected in 58 FCDTCs. The mutations were detected in 25 PTCs (7.5%), 12 FTCs (17.1%), nine poorly differentiated thyroid carcinomas (PDTCs; 29.0%), and 12 anaplastic thyroid carcinomas (ATCs; 33.3%) (Table 1). In differentiated thyroid carcinomas (DTCs), the overall prevalence of TERT promoter mutations was 9.2%. TERT promoter mutations were slightly more frequent in conventional PTCs (8.3%) than in cases of follicular variant of PTCs (6.8%).AllPTCsandFTCswithoncocyticfeaturesaswell as tall-cell PTCs and Warthin-like PTCs did not harbor TERT promoter mutations (Supplemental Table 1). Most of the mutated cases (48 of 58) presented the ⫺124G⬎A mutation and the remaining 10 cases presented the ⫺146 G⬎A. Because TERT promoter mutations were only detected in FCDTCs, the subsequent clinicopathological analysis was restricted to these tumors (Table 1). Relationship between TERT mutation and clinicopathological features In the group of patients with DTCs, the presence of TERT promoter mutations was significantly associated with older age (P⬍.001) and larger tumor size (P⫽.002) (Table 2). Patients with tumors harboring TERT mutations had more distant metastases (P⬍.001) and higher stage (P⬍.001). No association was found with the presence of vascular invasion, extrathyroidal extension, or lymph node metastases. A regression model was performed for factors associated with distant metastases in DTCs (Table 3). A total of E756 Melo et al TERT Promoter Mutations Predict Outcome in DTC J Clin Endocrinol Metab, May 2014, 99(5):E754–E765 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 February 2015. at 07:03 For personal use only. No other uses without permission. . All rights reserved. 44 patients (15.0%) with DTCs had distant metastases detected during follow-up; the metastases were located in the lung (n ⫽28), bone (n ⫽8), lung and bone (n ⫽6), brain (n ⫽1), and lung and kidney (n ⫽1). TERT promoter mutations [odds ratio (OR) 5.36; P⬍.001], gender (OR 2.31; P⫽.02), tumor size (OR 1.28; P⫽.009), and vascular invasion (OR 3.94; P⬍.001) were associated with distant metastases. TERT promoter mutations were found to be a predictor of distant metastases irrespectively of gender and vascular invasion. When all the features associated with distant metastases in the univariate model were introduced in the multivariate regression, vascular invasion became the only independent predictive factor of distant metastases. In patients with PTCs, the presence of TERT promoter mutations was associated with older age (P⬍.001), larger tumor size (P⫽.005), and higher stage (P⫽.02) (Table 2). Although patients with TERT mutation-positive PTCs had distant metastases more frequently than patients harboring TERT mutation-negative PTCs (27.8% vs 14.7%, respectively), the difference was not statistically significant (P⫽.07). In patients with FTCs, the presence of TERT promoter mutations was associated with older age (P⫽.004), higher stage (P⫽.007), and distant metastases (P⬍.001). In patients with PDTCs and ATCs, the presence of TERT promoter mutations was associated with older age (P⫽.003) and female gender (P⫽.02). Taken BRAF and RAS together, 60.3% of TERT-mutated tumors were also mutated for BRAF or RAS.In PTCs,there was a significant associationbetweenthe presence of TERT promoter mutations and the presence of BRAF mutation (P⫽.008) (Table 2). The BRAF mutation was not associated with increased lymph node or distant metastases or with patients’ outcome (survival) (see Figure 2H). In patients with TERT-mutated tumors, the coexistence of BRAF mutations was not associated with more aggressive clinicopathological features or worse outcome (Supplemental Table 3 and Supplemental Figure 2). No associations were found concerning RAS mutated tumors. None of the cases presenting RET/PTC (12 of 75) or PAX8/PPARG rearrangement (5 of 62) had TERT promoter mutations. Relationship between TERT promoter mutations, clinicopathological features, and outcome Mean follow-up time (⫾SD) of the patients was 7.8 ⫾ 5.8 years (range 0.1–38.9 y). The total follow-up for the whole of FCDTC patients was 2967.5 person-years, Table 1. Epidemiological, Histological, and Clinical Data of Patients With FCDTCs Included in the Study Total PTC FTC PDTC ATC Total number 469 332 70 31 36 Age at diagnosis, y, n 469 332 70 31 36 Mean, y 48.2 ⫾16.9 44.8 ⫾15.6 51.8 ⫾16.4 53.2 ⫾18.4 68.7 ⫾10.4 ⬍45 211 (45.0) 178 (53.6) 23 (32.9) 8 (25.8) 2 (5.6) ⱖ45 258 (55.0) 154 (46.4) 47 (67.1) 23 (74.2) 34 (94.4) Gender, n 459 327 68 29 35 Female 342 (74.5) 254 (77.7) 50 (73.5) 16 (55.2) 22 (62.9) Male 117 (25.5) 73 (22.3) 18 (26.5) 13 (44.8) 13 (37.1) Tumor size, cm, n 418 315 61 26 16 ⬍2 a 151 (36.1) 142 (45.0) 7 (11.5) 2 (7.7) 0 2–4 161 (38.5) 129 (41.0) 23 (37.7) 7 (26.9) 2 (12.5) ⬎4 106 (25.4) 44 (14.0) 31 (50.8) 17 (65.4) 14 (87.5) Extrathyroidal extension, n 373 268 49 20 36 Present 231 (61.9) 176 (65.7) 8 (16.3) 12 (60.0) 35 (97.2) Vascular invasion, n 325 257 53 8 7 Present 157 (48.3) 103 (40.1) 39 (73.6) 8 (100.0) 7 (100.0) Lymph node metastasis, n 391 298 47 24 22 Present 234 (59.8) 202 (67.8) 8 (17.0) 14 (58.3) 10 (45.5) Distant metastasis, n 337 263 31 20 23 Present 76 (22.6) 36 (13.7) 8 (25.8) 14 (70.0) 18 (78.3) Stage (sixth UICC/AJCC), n 310 225 29 20 36 I 134 (43.2) 124 (55.1) 8 (27.6) 2 (10.0) 0 II 36 (11.6) 25 (11.1) 8 (27.6) 3 (15.0) 0 III 45 (14.5) 36 (16.0) 5 (17.2) 4 (20.0) 0 IV 95 (30.7) 40 (17.8) 8 (27.6) 11 (55.0) 36 (100.0) TERT promoter, n 469 332 70 31 36 Wild-type 411 (87.6) 307 (92.5) 58 (82.9) 22 (71.0) 24 (66.7) Mutation 58 (12.4) 25 (7.5) 12 (17.1) 9 (29.0) 12 (33.3) Abbreviations: n, number of patients with available data for each feature; UICC, Union for International Cancer Control. Numbers in parentheses represent percentages within each category. a Number of microcarcinomas (ⱕ1.0 cm): total, 47 (11.2%); PTC, 43 (13.7%); FTC, 4 (6.6%); PDTC, 0, ATC, 0. doi: 10.1210/jc.2013-3734 jcem.endojournals.org E757 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 February 2015. at 07:03 For personal use only. No other uses without permission. . All rights reserved. moter mutations may serve as a marker of aggressive disease and poor outcome in DTCs. Our data show that DTC patients with TERT promotermutated tumors were less prone to be free of disease at the end of follow-up and were submitted to more radioiodine treatments with higher cumulative activities as well as to a greater number of other treatment modalities. Combining this information with the worse outcome of the patients, it is likely that, in the future, TERT promoter mutational status may be used to individualize treatment decisions, namely the type of surgery (if the molecular study proves to be feasible in fine needle aspiration cytology), the decision to perform radioiodine ablation, and the amount of 131 I to administer to patients. The latter issue is particularly relevant due to the current trend to reduce the number of patients submitted to radioiodine ablation and to use lower doses (36–38). Internationalconsensusand guidelinesmayalsotakeinto account TERT promoter mutational status to establish the best follow-up strategy for the individual patient. Considering that DNA or RNA of TERT has been used as vaccines to induce T-helper cells and specific cytotoxic T lymphocyte responses against TERT-positive tumors (39, 40), it seems possible that, in the future, TERT-based immunotherapies may be tailored for patients with TERT-mutated carcinomas because these tumors carry a poor outcome, possibly because of a worse response to current therapies. Further studies are necessary to address this issue. All the institutions involved in the present study followed international guidelines, but there were discrepancies regarding prophylactic central lymph node dissection. Taking into consideration that the role of such dissection is currently under debate and the existing differences from institution to institution of the present study, we did not investigate the implications of prophylactic lymph node dissection on the outcome. TERT mutations were associated with BRAF mutations, highlighting the coexistence of activation of BRAF and of TERT genes, previously reported in melanoma (16) and thyroid (15). Horn et al (16) advanced that the mutation creates newly consensus binding sites for TCF subfamily transcription factors (Elk1 and Elk4) that can be activated by BRAF. Both in thyroid carcinoma and in melanoma, it seems that a background status of activated BRAF enhances the effects of TERT promoter mutation. Our previous results in TERT mRNA expression corroborated this assumption showing an increased TERT expression in tumors harboring BRAF and TERT mutation (15). However, the concurrence or coexistence in the current study of TERT and BRAF mutations was not associatedwith increasedaggressiveness andworseoutcome in comparison with the presence of TERT mutations alone (Supplemental Table 3 and Supplemental Figure 2). Nonetheless, these results should be viewed with caution due to the low number of patients in each of the two groups. It remains to be fully clarified the biological meaning of the association between TERT promoter mutations and the metastatic nature of thyroid tumors. Being one of the hallmarks of cancer and enabling replicative immortality of cancer cells, TERT activation appears to be independent of the tumor microenvironment because there is no substantive evidence for stromal contributions to telomere stabilization in cancer cells (41). Our findings suggest that a link between telomerase activity and metastatic capacity may exist. We can speculate that this link may be inherent to the extended survival of TERT-mutated cells during the circulation phase and/or the homing in distant places, rather than to the increased invasiveness of such cells, but the discussion of this issue is beyond the scope of the present study. We conclude that TERT promoter mutations are an indicator of clinical aggressiveness of follicular cell-derived thyroid carcinomas, being associated with distant metastases, worse response to treatment, and poor outcome. The detection of such mutations appears to be, per se, a promising prognostic indicator in DTCs and PTCs. Acknowledgments We thank Margarida Marques (Department of Technology and Information of the University Hospital of Coimbra) for the valuable review of the statistical analysis. We acknowledge GENZYME for funding our work through a research project. Addressall correspondence and requests forreprints to: Paula Soares, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal. E-mail: [email protected]. This study was supported by the Portuguese Foundation for Science and Technology through PhD Grant SFRH/BD/81940/ 2011 (to J.V.); PhD Grant SFRH/BD/87887/2012 (to C.T.); PhD Grant SFRH/BD/79135/2011 (to A.A.); and the Scientific Investigation Project PIC/IC/83037/2007. Further funding was obtained from the project “Microenvironment, Metabolism and Cancer,” partially supported by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referência Estratégico Nacional, and through the European Regional Development Fund. The work of J.M.C.-T. was supported by Grant PI12/00749-FEDER from the Instituto de Salud Carlos III, the Ministry of Economy and Competitiveness (Madrid, Spain). The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) is an associate laboratory of the Portuguese Ministry of Science, Technology, and Higher Education, which is partially supported by the Foundation for Science and Technology. Disclosure Summary: The authors have nothing to disclose. E764 Melo et al TERT Promoter Mutations Predict Outcome in DTC J Clin Endocrinol Metab, May 2014, 99(5):E754–E765 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 February 2015. at 07:03 For personal use only. No other uses without permission. . All rights reserved. References 1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000; 100:57–70. 2. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994;266:2011–2015. 3. Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer. 1997;33:787–791. 4. Bryan TM, Englezou A, Dalla-Pozza L, Dunham MA, Reddel RR. Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat Med. 1997;3:1271–1274. 5. Bryan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR. 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Cancer Cell. 2012; 21:309–322. doi: 10.1210/jc.2013-3734 jcem.endojournals.org E765 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 February 2015. at 07:03 For personal use only. No other uses without permission. . All rights reserved. Supplemental Figure 1 - Sequencing chromatographs of the TERT promoter locus in genomic tumor DNA obtained by Sanger sequencing (-124 normal, -124 G>A mutation, -146 normal, - 146 G>A mutation) Supplemental Figure 2 – Kaplan-Meier disease-specific survival curve for both groups (TERT mutated / BRAF wild-type and TERT mutated / BRAF mutated) p=NS (0.70) Supplemental Table 1 – Histological diagnoses of the 647 thyroid tissue samples searched for the presence of TERT promoter mutations Histotypes No. of cases TERT n (%) Normal thyroid 30 0 (0.0) Lymphocytic thyroiditis 9 0 (0.0) Nodular goiter 12 0 (0.0) Follicular adenoma Conventional 53 0 (0.0) Oncocytic variant 7 0 (0.0) Follicular carcinoma Conventional 43 12 (27.9) Oncocytic variant 27 0 (0.0) Papillary carcinoma Conventional 230 19 (8.3) Follicular variant 74 5 (6.8) Oncocytic variant Papillary architecture 6 0 (0.0) Follicular architecture 3 0 (0.0) Solid variant 7 0 (0.0) Warthin-like variant 6 0 (0.0) Tall-cell variant 2 0 (0.0) Mucoepidermoid carcinoma 4 1 (25.0) Poorly differentiated carcinoma 31 9 (29.0) Anaplastic carcinoma 36 12 (33.3) Medullary carcinoma 28 0 (0.0) Post-Chernobyl series Follicular adenomas 12 0 (0.0) Papillary carcinoma 27 0 (0.0) Supplemental Table 2 - Summary of treatment procedures in differentiated thyroid carcinomas All patients TERT wild type TERT mutated p value Number of radioiodine therapies 1.96±1.29 1.81±1.16 2.69±1.78 0.009 Cumulative activity (GBq/mCi) 9.8±8.9/ 264.1±240.4 9.0±8.1/ 242.8±218.1 15.8±12.4/ 425.8±334.8 0.004 Number of additional treatments* 1.36±1.73 1.13±1.52 2.56±2.45 0.001 *Number of additional treatments includes surgical procedures, external beam irradiation and treatment with tyrosine kinase inhibitors. Each surgical procedure was counted as one «additional treatment». Treatment with tyrosine kinase inhibitors was counted as one «additional treatment» if the patient took the drug for more than six months, and regardless of the utilization of more than one drug of the same class. Supplemental Table 3 - Clinico-pathological data of patients with PTC harboring TERT promoter mutations and wild-type or mutated BRAF All patients TERT mut / BRAF wt TERT mut / BRAF mut p value Clinico-pathological Age at diagnosis, y (n=24) 58.4±13.2 52.2±8.0 60.0±14.4 NS (0.22)* Gender (n=24) Female 16 (66.7) 5 (83.3) 11 (61.1) NS (0.32) Male 8 (33.3) 1 (16.7%) 7 (38.9) Mean tumor size, cm (n=23) 3.2±2.2 2.9±2.1 3.3±2.3 NS (0.66)* Extrathyroidal extension (n=18) Present 14 (77.8) 3 (60.0) 11 (84.6) NS (0.26) Vascular invasion (n=16) Present 5 (31.3) 2 (66.7) 3 (23.1) NS (0.14) LN metastases (n=23) Present 18 (78.3) 5 (83.3) 13 (76.5) NS (0.73) Distant metastases (n=18) Present 5 (27.8) 3 (50.0) 2 (16.7) NS (0.14) Stage (n=14) I 3 (21.4) 0 3 (37.5) NS (0.29) II 1 (7.1) 1 (16.7) 0 III 4 (28.6) 2 (33.3) 2 (25.0) IV 6 (42.9) 3 (50.0) 3 (37.5) Clinical status at the end of follow-up (n=12) Disease persistence 6 (50.0) 4 (66.7) 2 (33.3) NS (0.25) Disease-specific mortality (n=19) 2 (10.5) 1 (16.7) 1 (7.7) NS (0.70)** * Due to low numbers in each group, Mann-Whitney U test was used ** Kaplan-Meier log-rank p values Agradecimentos À Professora Paula Soares, pelo convite para integrar esta equipa de investigação, pelos desafios e pelo apoio constante. Foi a Professora que me possibilitou trabalhar neste “mundo” da Ciência, uma experiência profissional incomparável… a curiosidade viciante! Ao Professor Sobrinho Simões, pelo exemplo de educação, humanismo e excelência. É possível ser-se grande e humilde; é possível ser-se superior sem presunção ou arrogância. A simpatia… A todo o grupo Cancer Biology, aos que cá estão e aos que cá estiveram, pelos ensinamentos, pela colaboração, pela ajuda… e pelas brincadeiras! Ao Ipatimup e seus membros, por me terem ensinado o que é uma grande casa, o que é “vestir a camisola”. Ao Miguel, por ter aparecido quando menos esperava, mas quando mais precisava… por me ter mostrado que se pode fazer boa medicina e boa ciência ao mesmo tempo… por me ter mostrado que o nosso verdadeiro valor está no nosso carácter e não nas nossas mãos… Por fim, à minha mãe e ao meu pai, aos meus irmãos, à minha restante e enorme família, que sempre me apoiaram em todo o meu percurso académico e profissional… a frase “já sabes, nós estamos aqui para o que for preciso” é um tesouro que nos protege e que se guarda no coração. Anexos