cancers Article Genetic Determinants for Prediction of Outcome of Patients with Papillary Thyroid Carcinoma Antónia Afonso Póvoa 1,2,3,4, *,† , Elisabete Teixeira 2,3,4,† , Maria Rosa Bella-Cueto 5 , Rui Batista 2,3,4 , Ana Pestana 2,3,4 , Miguel Melo 2,3,6 , Thalita Alves 7 , Mafalda Pinto 2,3 , Manuel Sobrinho-Simões 2,3,4 , Jorge Maciel 1,8 and Paula Soares 2,3,4, * 1 Department of General Surgery, Centro Hospitalar de Vila Nova de Gaia/Espinho (CHVNG/E), 4434-502 Vila Nova de Gaia, Portugal; jpmaciel@ufp.edu.pt 2 IPATIMUP—Instituto de Patologia e Imunologia Molecular da Universidade do Porto, 4200-135 Porto, Portugal; eteixeira@ipatimup.pt (E.T.); rbatista@ipatimup.pt (R.B.); apestana@ipatimup.pt (A.P.); jmiguelmelo@live.com.pt (M.M.); mafaldap@ipatimup.pt (M.P.); ssimoes@ipatimup.pt (M.S.-S.) 3 Cancer Signaling and Metabolism, i3S—Instituto de Investigação e Inovação em Saúde, 4200-135 Porto, Portugal 4 Departament of Pathology, Faculdade de Medicina da Universidade do Porto, 4200-319, Porto, Portugal 5 Department of Pathology, Parc Taulí Sabadell Hospital Universitari—Institut d’Investigació i Innovació Parc Taulí—I3PT—Universitat Autònoma de Barcelona, 08208 Barcelona, Spain; rbella@tauli.cat 6 Department of Endocrinology, Centro Hospitalar Universitário de Coimbra,3000-075 Coimbra, Portugal 7 Laboratório de Endocrinologia Molecular e Translacional—Departamento de Medicina, Universidade Federal de São Paulo, São Paulo 04039-032, Brazil; tha.alves@gmail.com 8  Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, 4200-253 Porto, Portugal  * Correspondence: antonia.povoa@chvng.min-saude.pt (A.A.P.); psoares@ipatimup.pt (P.S.); Citation: Póvoa, A.A.; Teixeira, E.; Tel.: +35-19-6981-3884 (A.A.P.) † Both authors contributed equally for the work. Bella-Cueto, M.R.; Batista, R.; Pestana, A.; Melo, M.; Alves, T.; Pinto, M.; Sobrinho-Simões, M.; Maciel, J.; et al. Simple Summary: Aggressive metastatic disease is rare in papillary thyroid carcinoma (PTC), a Genetic Determinants for Prediction neoplasia that usually carries an excellent prognosis. BRAF, RAS, and TERT promoter (TERTp) genes of Outcome of Patients with Papillary are altered in PTC, and their impact on patient outcomes remains controversial. We performed Thyroid Carcinoma. Cancers 2021, 13, Sanger sequencing on a series of 241 PTCs to determine the role of genetic mutations (BRAF, RAS, 2048. https://doi.org/10.3390/ and TERTp) in PTC patient outcomes. The implication of RASmut tumors remain uncertain in clinical cancers13092048 terms. BRAFmut /TERTpwt tumors were prone to be associated with local aggressiveness (recurrent, persistent/disease), whereas TERTpmut tumors were predisposed to recurrent/persistent structural Academic Editor: Kennichi Kakudo disease, and disease-specific mortality. Our results indicate that different molecular markers play a distinct role in predicting PTC patient outcomes. Received: 12 March 2021 Accepted: 18 April 2021 Abstract: Papillary thyroid carcinoma (PTC) usually presents an excellent prognosis, but some pa- Published: 23 April 2021 tients present with aggressive metastatic disease. BRAF, RAS, and TERT promoter (TERTp) genes are altered in PTC, and their impact on patient outcomes remains controversial. We aimed to determine Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in the role of genetic alterations in PTC patient outcomes (recurrent/persistent disease, structural published maps and institutional affil- disease, and disease-specific mortality (DSM)). The series included 241 PTC patients submitted to iations. surgery, between 2002–2015, in a single hospital. DNA was extracted from tissue samples of 287 lesions (primary tumors and metastases). Molecular alterations were detected by Sanger sequenc- ing. Primary tumors presented 143 BRAF, 16 TERTp, and 13 RAS mutations. Isolated TERTpmut showed increased risk of structural disease (HR = 7.0, p < 0.001) and DSM (HR = 10.1, p = 0.001). Copyright: © 2021 by the authors. Combined genotypes, BRAFwt /TERTpmut (HR = 6.8, p = 0.003), BRAFmut /TERTpmut (HR = 3.2, Licensee MDPI, Basel, Switzerland. p = 0.056) and BRAFmut /TERTpwt (HR = 2.2, p = 0.023) showed increased risk of recurrent/persistent This article is an open access article disease. Patients with tumors BRAFwt /TERTpmut (HR = 24.2, p < 0.001) and BRAFmut /TERTpmut distributed under the terms and (HR = 11.5, p = 0.002) showed increased risk of structural disease. DSM was significantly increased conditions of the Creative Commons in patients with TERTpmut regardless of BRAF status (BRAFmut /TERTpmut , log-rank p < 0.001; Attribution (CC BY) license (https:// BRAFwt /TERTpmut , log-rank p < 0.001). Our results indicate that molecular markers may have a creativecommons.org/licenses/by/ role in predicting PTC patients’ outcome. BRAFmut /TERTpwt tumors were prone to associate with 4.0/). Cancers 2021, 13, 2048. https://doi.org/10.3390/cancers13092048 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 2048 2 of 16 local aggressiveness (recurrent/persistent disease), whereas TERTpmut tumors were predisposed to recurrent structural disease and DSM. Keywords: thyroid cancer; papillary thyroid carcinoma; prognosis; BRAF; RAS; TERT; patient outcome; recurrent/persistent disease; structural disease; PTC-specific mortality 1. Introduction Thyroid cancer (TC) is the most common endocrine malignancy worldwide, and its incidence has been remarkably increasing over the past three decades, particularly in developed countries [1–4]. According to GLOBOCAN 2018, TC accounts for 3.1% of all diagnosed cancer worldwide [5]. TC incidence was 18.6/100,000 in women and 4.1/100,000 in men in the same period [6], mainly due to one TC histotype, papillary thyroid carci- noma (PTC) [7,8]. The overall mortality rate for both genders was about 0.30/100,000 [6] (less than 5% at 10-year follow-up [9]), which remains low despite increasing TC inci- dence. While usually presenting an excellent prognosis, some PTC patients experience persistence/recurrence or metastatic disease [10,11], and patients with distant metastases (DM) display 45% disease-specific survival (DSS) at 10-year follow-up [12]. Presurgical prognostication is practically nonexistent [13]. Fine-needle aspiration biopsy (FNAB) is an important tool for TC diagnosis [13]. FNAB presents limitations, with 10–40% of cases be- ing classified as indeterminate without providing any prognostic indication [14,15]. Several factors (such as gender and age, tumor invasion, namely angioinvasion and extrathyroidal extension, presence of metastases, response to treatment) have proven prognostic value in PTC [16]. Over the last years, initiation and progression of TC have been steadily associated with genetic/epigenetic events that lead to the activation of cellular signaling pathways, namely MAPK and PI3K-AKT [17–19]. Several studies have advanced the detection of alterations of genes or gene panels for PTC diagnosis and/or prognosis evaluation and patient management [20]. Mutations in BRAF, RAS, and TERT promoter (TERTp) genes have been described as altered in PTC by our group [10,21,22] and by others [20,23,24]. Yet, discordance exists on how these genes impact tumor behavior and patient outcome. A BRAFV600E point mutation on exon 15 is frequently detected in PTC, accounting for more than 90% of all BRAFmut in TC. BRAFmut is present in about 45% PTCs [25] and has been associated, in some studies, with features of clinical aggressiveness such as older age, larger tumors, extrathyroidal extension (ETE), lymph node metastases (LNM), higher stage, and poorer prognosis [26,27]. The impact of BRAFmut in PTC DM, lack of response to radioiodine (RAI) therapy, and mortality are still controversial [10,26,28–30]. The genetic significance of RASmut in TC remains an open question since point mutations are detected in all subtypes of thyroid nodules, from benign to anaplastic lesions [31,32]. Studies on RASmut and TC prognostication have shown controversial results so far. Some studies showed no association of RASmut with poorer prognosis [33], while others have reported an association with DM and poorer survival [34,35]. TERTpmut was found in approximately 10% of PTC cases [4], being more frequent in aggressive PTC variants [22]. In TC, TERTpmut has been associated with older age, larger tumors, ETE, higher tumor stage, DM, RAI therapy resistance, and patient mortality, by our group [10,21,22] and by others [36–38]. Beyond their utility for diagnostic purposes, further data are needed in order to use molecular markers as prognostic tools in TC. In this study, we decided to evaluate BRAF, RAS (NRAS, HRAS, and KRAS), and TERTp molecular status in a consecutive series of PTC patients in an attempt to assess the impact of gene status on patients’ outcome, namely recurrent/persistent disease, structural disease, and patient survival. Cancers 2021, 13, 2048 3 of 16 2. Materials and Methods 2.1. Patient Samples The study was performed in a consecutive series of patients submitted to thyroid surgery at a single hospital (Centro Hospitalar de Vila Nova de Gaia e Espinho (CHVNG/E)), from January 2002 to December 2015 and diagnosed as PTC. Formalin-fixed, paraffin- embedded (FFPE) tissues were collected from institutional files. All tumor samples were reviewed by a single pathologist (MRB), according to the fourth edition of the World Health Organization classification of tumors of endocrine organs [39]. Inclusion criteria were PTC diagnosis in patients older than 18 years, followed for a minimum of two years (unless recurrence or disease-specific mortality (DSM) has occurred earlier), in which there were thyroid samples for histological re-evaluation. Following these criteria, 241 patients were included in the study. A total of 287 lesions were evaluated: besides 238 primary tumors, we reviewed 31 LNM at diagnosis, 16 locoregional recurrences (LR), and two DM, along with clinical information that was revised by a single physician (AAP). The histological diagnosis of the 241 patients was the following: classical PTC (CPTC), follicular variant PTC (FVPTC), oncocytic variant PTC (OVPTC: Warthin-like PTC, classical oncocytic PTC, and follicular patterned oncocytic PTC) and aggressive variants of PTC (APTC: tall cell, hobnail, diffuse sclerosing and solid trabecular). All procedures described in this study were in accordance with national and institu- tional ethical standards. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of CHVNG/E (Project investigation 30/2016, 28 January 2016, Comissão de Ética do Centro Hospitalar de Vila Nova de Gaia/Espinho). According to Portuguese law, informed consent is not required for retrospective studies. 2.2. Patient Follow-Up and Risk Stratification Patients were staged using the eighth edition of the American Joint Cancer Com- mittee/Union for International Cancer Control (AJCC/UICC) staging system [40]. Risk stratification at the second year and at the end of follow-up was evaluated according to the 2015 American Thyroid Association (ATA) guidelines [11] for patients submitted to total thyroidectomy followed by RAI therapy. For patients that were not submitted to RAI or less than total thyroidectomy, risk stratification was performed using the system published by Momesso et al. [41]. According to 2015 ATA guidelines, no clinical evidence of disease (NED) at final follow- up was established if patients had thyroglobulin (Tg) levels that fit excellent response [11], no detectable Tg antibodies (TgAb), and no structural evidence of disease. Patients were classified as persistent disease whenever Tg values fit indeterminate or incomplete response (elevated basal or stimulated Tg values alone, without structural correlation), or there was any evidence of disease on cross-sectional imaging (ultrasonography (US), computed tomography (CT) scan), functional imaging (RAI scintigraphy or 2-[18 F]fluoro-2-deoxy-d- glucose positron-emission tomography (2-18 F-FDG-PET) scan), or biopsy-proven disease (cytology or histology). Several Tg assays were used with different functional sensitivities, reflecting the long study period. For the sake of simplification, the two patients who had positive TgAb, were included in the group of incomplete biochemical responses. Recurrence was defined if new biochemical, structural, and/or functional evidence of disease was detected following any period of NED. Patients were considered to have a positive structural disease status if any of the fol- lowing conditions were met: (1) positive cytology/histology, (2) highly suspicious lymph nodes or thyroid bed nodules in the neck US (hypervascularity, cystic areas, heterogeneous content, rounded shape, or enlargement on the follow-up), (3) findings in RAI scintigraphy, 18 F-FDG-PET scans, or other cross-sectional imaging highly suspicious for metastatic dis- ease. Recurrent/persistent disease (n = 57) was considered if patients had an indeterminate or incomplete response to treatment, whether it was only biochemical disease (n = 32) or biochemical and structural disease (n = 25). DSM was also an endpoint (patients dying Cancers 2021, 13, 2048 4 of 16 of unrelated conditions had the final status determined based on data available before their demise). 2.3. DNA Extraction DNA from FFPE tissues was retrieved from 10 µm sections after H&E guided care- ful microdissection. DNA extraction was performed using the GRS Genomic DNA Kit BroadRange (GRiSP Research Solutions, Porto, Portugal) following the manufacturer’s instructions. Quantitative and qualitative analysis of all samples was then performed by spectrophotometry using Nanodrop N-1000 Spectrophotometer for microvolume UV-Vis measurements (Thermo Scientific, Waltham, MA, USA). 2.4. PCR and Sanger Sequencing Analysis Genetic characterization of the series of tumors regarding BRAF, RAS (NRAS, HRAS, and KRAS), and TERTp mutations was performed as previously reported [9,19,27]. Primer design was performed accounting for the most frequent regions mutated in PTC, namely, BRAF codon 600, NRAS codon 61, HRAS and KRAS codons 12, 13, and 61, and TERTp-124, and −146 regions. Amplification of genomic DNA (25–50 ng) was achieved using the QIAGEN multiplex PCR kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The annealing temperature of 61 ◦ C was established after protocol optimization for BRAF, NRAS, and TERTp segments amplification in the same reaction. HRAS and KRAS were screened separately by a touchdown PCR using the MyTaq HS Mix 2× Bioline PCR Kit (Meridian Bioscience, Cincinnati, OH, USA) following the manufacturer’s instructions. PCR amplification was confirmed in 1–2% agarose gel electrophoresis (GRS Agarose LE, GRiSP, Oporto, Portugal) and followed by PCR product purification. All tested hotspot mutations were sequenced by Sanger sequencing using the ABI Prism Big Dye Termina- tor kit v3.1 Cycle Sequencing (Fisher Scientific Applied Biosystems® , Portsmouth, NH, USA). After sequencing product precipitation, fragments were analyzed by capillary elec- trophoresis using the Applied Biosystems 3130/3130 ×l Genetic Analyzers (Foster city, CA, USA). For all genes, all detected mutations were validated by performing a new and independent analysis. 2.5. Statistical Analysis Statistical analysis was performed with IBM SPSS Statistics version 25 (IBM, New York, NY, USA). Results were expressed in absolute frequency, percentage, mean ± standard de- viation (Std), and median ± interquartile range (IR). Distribution analyses were performed using crosstabs analyses. Unpaired t-test and Mann–Whitney tests were applied whenever adequate. Disease-free survival (DFS) and DSM were accessed by Kaplan–Meier and log-rank tests. Age- and gender-adjusted models were created to evaluate the mutational status impact of evaluated genes in the outcomes: recurrent/persistent disease, structural disease, and DSM. Hazard ratios (HR) were assessed by Cox proportional hazard models. Statistical significance was accepted with a two-tailed p-value < 0.05. 3. Results 3.1. Description of the Patients Out of 241 patients, 202 (83.8%) were female. The mean age at diagnosis was 52 ± 15.2 years (18–86 years), with 56.0% of the patients being <55 years. Median tu- mor size was 12 ± 10.0 mm (2–70 mm), with 97 (40.2%) primary tumors presenting a size ≤10 mm (microcarcinomas). The mean follow-up time was 7 ± 2.9 years (0.2–16.8 years). In three patients, the primary tumors were not available, and just their LNM was analyzed. Characteristics of the patients and tumors are summarized in Table 1. Cancers 2021, 13, 2048 5 of 16 Table 1. Series description: patient characteristics, tumor histology, staging, lymph node metastases, and patient outcome. Patient n n (%) n (Total) Staging n (%) Characteristics (Total) Age 241 T stage 241 <55 years 135 (56.0%) T1a 96 (39.8%) ≥55 years 106 (44.0%) T1b 97 (40.2%) Gender 241 T2 31 (12.9%) Female 202 (83.8%) T3a 7 (2.9%) Male 39 (16.2%) T3b 9 (3.7%) Histology n (%) n (total) T4a 1 (0.4%) Nodule size Median ± IR 12.0 ± 10.00 238 a N stage 241 Histological 238 a N0 207 (85.9%) subtype CPTC 156 (65.5%) N1 34 (14.1%) FVPTC 55 (23.1%) M stage 241 OVPTC 18 (7.6%) M0 237 (98.3%) AVPTC 9 (3.8%) M1 4 (1.7%) Necrosis 237 Stage 241 Absent 232 (97.9%) SI 225 (93.4%) Present 5 (2.1%) SII 12 (5.0%) Psammomas 237 SIV 4 (1.7%) Lymph node Absent 155 (65.4%) invasion and n (%) n (total) extranodal extension Present 82 (34.6%) Lateral compartment 241 Lymph node Focality 241 Absent 225 (93.4%) invasion Unifocal 151 (62.7%) Present 16 (6.6%) Multifocal 90 (37.3%) Lateral compartment 241 Extrathyroidal 240 Extranodal extension Absent 236 (97.9%) extension Absent/minimal 230 (95.8%) Present 5 (2.1%) Major 10 (4.2%) Patient outcome n (%) n (total) Lymphatic 237 Recurrent/persistent 241 invasion Absent 190 (80.2%) disease Absent 184 (76.3%) Present 47 (19.8%) Present 57 (23.7%) Venous 239 Structural disease 241 invasion Absent 223 (93.3%) Absent 216 (89.6%) Present 16 (6.7%) Present 25 (10.4%) Resection 240 PTC related death 241 margins Alive or deceased R0 209 (87.1%) 232 (96.3%) by other causes Deceased R1 31 (12.9%) 9 (3.7%) of PTC CPTC: classical PTC; FVPTC: follicular variant PTC; OVPTC: oncocytic variant PTC (includes Warthin-like, classical oncocytic PTC, and follicular patterned oncocytic PTC); AVPTC: aggressive variants of PTC (includes tall cell, hobnail, diffuse sclerosing, and solid/trabecular variants). a Three primary tumors were not available, only lymph node metastases. Cancers 2021, 13, 2048 6 of 16 Two hundred twenty-five patients (93.4%) were at stage I, 12 patients (5.0%) were at stage II, and four patients (1.7%) were at stage IV. During follow-up, 57 patients (23.7%) had recurrent/persistent disease, 25 patients (10.4%) had structural disease (LNM and/or DM) and nine patients (3.7%) had DSM. 3.2. Primary Tumors’ Genetic Characterization The study of primary tumors demonstrated the presence of BRAFmut in 143 (62.7%), TERTpmut in 16 (7.3%), and RASmut in 13 (5.6%) patients. All BRAFmut cases presented with p.Val600Glu, which was present in all four histolog- ical subtypes, but more frequent in CPTC (Table 2). Three different TERTpmut were found, −124 G > A mutation was detected in 12 cases (5.5%) from all four histological variants (Table 2), −146 G > A mutation was found in three PTC (1.4%), all CPTC, and one tandem −124/−125 G > A mutation was detected in an FVPTC. Of the 16 TERTpmut tumors, 11 (5.1%) were concomitantly mutated for BRAF (BRAFmut /TERTpmut ), and four (1.9%) were only TERTpmut (BRAFwt /TERTpmut ). One hundred and twenty (56.1%) tumors were BRAFmut but not TERTp (BRAFmut /TERTpwt ). A total of 13 (5.6%) RASmut were detected in primary tumors. p.Gln61Arg mutation was detected in all RAS genes (NRAS, HRAS, and KRAS) with a frequency of 3.5%, 2.3%, and 1.7%, respectively, mostly in follicular patterned tumors. One KRAS p.Gln61Arg mutation was concomitantly present with a −124 G > A TERTpmut . Table 2. BRAF, TERTp, and RAS molecular status in primary tumors and distribution in the different PTC variants. Histological Variants of Primary Tumors All n (%) Gene(s) Status CPTC FVPTC OVPTC AVPTC n = 238 a n = 156 n = 55 n = 18 n=9 BRAF wt 85 39 33 9 4 p.Val600Glu 143 (62.7%) 110 (73.8%) 20 (37.7%) 8 (47.1%) 5 (55.6%) TERTp wt 202 133 47 14 8 −124 G > A 12 (5.5%) 8 (5.6%) 2 (4.0%) 1 (6.7%) 1 (11.1%) −146 G > A 3 (1.4%) 3 (2.1%) 0 (0.0%) 0 (0.0%) 0 (0.0%) −124/-125 G > A 1 (0.5%) 0 (0.0%) 1 (2.0%) 0 (0.0%) 0 (0.0%) BRAF/TERTp BRAFwt /TERTpwt 79 36 31 8 4 BRAFmut /TERTpwt 120 (56.1%) 94 (67.1%) 16 (32.0%) 6 (40.0%) 4 (44.4%) BRAFwt /TERTpmut 4 (1.9%) 2 (1.4%) 1 (2.0%) 1 (6.7%) 0 (0.0%) BRAFmut /TERTpmut 11 (5.1%) 8 (5.7%) 2 (4.0%) 0 (0.0%) 1 (11.1%) NRAS wt 223 152 47 15 9 p.Gln61Arg 8 (3.5%) 1 (0.7%) 5 (9.6%) 2 (11.8%) 0 (0.0%) HRAS wt 127 75 36 10 6 p.Gln61Arg 3 (2.3%) 0 (0.0%) 3 (7.7%) 0 (0.0%) 0 (0.0%) KRAS wt 117 67 35 10 5 p.Gln61Arg 2 (1.7%) 1 (1.5%) 1 (2.8%) 0 (0.0%) 0 (0.0%) RAS b wt 218 151 43 15 9 RASmut 13 (5.6%) 2 (1.3%) 9 (17.3%) 2 (11.8%) 0 (0.0%) CPTC: classical PTC; FVPTC: follicular variant PTC; OVPTC: oncocytic variant PTC (includes Warthin-like, classical oncocytic PTC, and follicular patterned oncocytic PTC); AVPTC: aggressive variants of PTC (includes tall cell, hobnail, diffuse sclerosing, and solid/trabecular variants). a Three primary tumors were not available, only lymph node metastases. b Represents RAS status if at least one of the RAS genes was sequenced; wt: wild-type; mut: mutated. The detailed univariate analysis of clinicopathological features in relation to combined gene mutations is presented in Supplementary Table S1. 3.3. Metastatic Lesions’ Genetic Characterization and Molecular Profile Concordance The study of 31 LNM that were present at diagnosis demonstrated the presence of BRAFmut in 17 LNM (54.8%) and TERTpmut in three LNM (10.0%). Of those, two LNM (6.7%) were concomitantly BRAFmut /TERTpmut . The study of 16 cervical recur- rences (14 recurrent LNM and two thyroid bed recurrences) revealed BRAFmut in 12 le- sions (75.0%), TERTpmut in five lesions (33.3%), and being in three lesions concomitant BRAFmut /TERTpmut (20.0%). The study of two DM demonstrated the presence of NRASwt Cancers 2021, 13, 2048 7 of 16 in one lesion and one concomitant BRAFmut /TERTpmut in the other lesion. Detailed information of metastatic lesions molecular profile is presented in Supplementary Table S2. 3.4. Molecular Alterations in Recurrent/Persistent Disease At 10-year evaluation, recurrent/persistent DFS was significantly lower in patients with TERTpmut tumors (41.7%, p = 0.034). Patients with TERTpmut tumors presented 2.3 times increased risk of recurrent/persistent disease (p = 0.04), but this association Cancers 2021, 13, x was lost when the model was adjusted for age and gender (p = 0.062) (Table 3). Recur- 7 of 16 rent/persistent DFS was not significantly different in patients with BRAF mut or RASmut wt wt tumors from patients with BRAF or RAS tumors (Figure 1A–C; Table 2). Considering concomitant mutations, tients with BRAF at 10-year wt or RAS evaluation, wt tumors recurrent/persistent (Figure 1A–C; DFS was Table 2). Considering significantly concomitant mu- lower in patients with BRAF mut /TERTpwt (log-rank p = 0.021), BRAFmut /TERTpmut (log- tations, at 10-year evaluation, recurrent/persistent DFS was significantly lower in patients wt /TERTpmut (log-rank p =mut withp BRAF rank = 0.035) and BRAF mut/TERTp wt (log-rank p = 0.021), BRAF 0.001) tumors /TERTp than in patients mut (log-rank with p = 0.035) and BRAF wt wt BRAFwt/TERTp /TERTpmuttumors (Figure (log-rank 1D). The p = 0.001) aforementioned tumors concomitant than in patients with BRAFmutations wt/TERTpsignifi- wt tu- cantly morsincreased the risk (Figure 1D). The of recurrent/persistent aforementioned diseasemutations concomitant in comparison to BRAFwt significantly /TERTpwt increased the tumors risk of(Table 3). recurrent/persistent disease in comparison to BRAFwt/TERTpwt tumors (Table 3). Figure Kaplan–Meier 1. 1. Figure Kaplan–Meier curves of PTC curves of recurrent/persistent of PTC disease-free of recurrent/persistent survival disease-free by tumor survival molecular by tumor status molecular in comparison status in compar- with isonwild withtype wildtumors; (A) BRAF type tumors; status, (A) BRAF (B) TERTp status, status, (B) TERTp (C) RAS status, status, (C) RAS (D)(D) status, combined BRAF combined and BRAF TERTp and TERTpstatus inin status comparison comparison toto wild-type wild-type tumors; tumors; wt: wt: wild-type; wild-type; mut: mut: mutated. mutated. Table 3. Analysis of molecular status in the risk of recurrent/persistent disease. Recurrent/Persistent Recurrent/Persistent Unadjusted HR Adjusted HR Molecular Status n p-Value p-Value * Disease Absent Disease Present (95% CI) (95% CI) BRAF status 228 BRAFwt 85 70 (39.8%) 15 (28.8%) 1 (Reference) 1 (Reference) BRAFmut 143 106 (60.2%) 37 (71.2%) 1.6 (0.88–2.91) 0.128 − − TERTp status 218 TERTpwt 202 157 (94.6%) 45 (86.5%) 1 1 TERTpmut 16 9 (5.4%) 7 (13.5%) 2.3 (1.04–5.12) 0.040 2.2 (0.96–5.01) 0.062 RAS status 231 RASwt 218 168 (93.9%) 50 (96.2%) 1 1 RASmut 13 11 (6.1%) 2 (3.8%) 0.6 (0.16–2.61) 0.530 − − BRAF and TERTp status 214 BRAFwt/TERTpwt 79 68 (41.7%) 11 (21.6%) 1 1 BRAFmut/TERTpwt 120 87 (53.4%) 33 (64.7%) 2.2 (1.11–4.34) 0.024 2.2 (1.11–4.36) 0.023 BRAFwt/TERTpmut 4 1 (0.6%) 3 (5.9%) 6.5 (1.82–23.42) 0.004 6.8 (1.89–24.55) 0.003 Cancers 2021, 13, 2048 8 of 16 Table 3. Analysis of molecular status in the risk of recurrent/persistent disease. Recurrent/ Recurrent/ Unadjusted Adjusted HR Molecular Status n Persistent Persistent p-Value p-Value * HR (95% CI) (95% CI) Disease Absent Disease Present BRAF status 228 BRAFwt 85 70 (39.8%) 15 (28.8%) 1 (Reference) 1 (Reference) BRAFmut 143 106 (60.2%) 37 (71.2%) 1.6 (0.88–2.91) 0.128 − − TERTp status 218 TERTpwt 202 157 (94.6%) 45 (86.5%) 1 1 TERTpmut 16 9 (5.4%) 7 (13.5%) 2.3 (1.04–5.12) 0.040 2.2 (0.96–5.01) 0.062 RAS status 231 RASwt 218 168 (93.9%) 50 (96.2%) 1 1 RASmut 13 11 (6.1%) 2 (3.8%) 0.6 (0.16–2.61) 0.530 − − BRAF and TERTp status 214 BRAFwt /TERTpwt 79 68 (41.7%) 11 (21.6%) 1 1 BRAFmut /TERTpwt 120 87 (53.4%) 33 (64.7%) 2.2 (1.11–4.34) 0.024 2.2 (1.11–4.36) 0.023 BRAFwt /TERTpmut 4 1 (0.6%) 3 (5.9%) 6.5 (1.82–23.42) 0.004 6.8 (1.89–24.55) 0.003 BRAFmut /TERTpmut 11 7 (4.3%) 4 (7.8%) 3.4 (1.10–10.82) 0.043 3.2 (0.97–10.33) 0.056 n: number of cases; HR: Hazard ratio calculated by Cox Regression analysis; CI: Confidence interval; * Age- and gender-adjusted; p-value; wt: wild-type; mut: mutated. 3.5. Molecular Alterations in Structural Disease Patients with TERTpmut tumors had significantly lower structural disease-free survival (SDFS) (log-rank p < 0.001) than patients without TERTpmut (Figure 2B) and presented significantly increased risk of structural disease, even when adjusted for age and gender (HR = 7.0, p < 0.001) (Table 4). SDFS was not significantly influenced by BRAFmut or RASmut tumors (Figure 2A,C). Considering concomitant mutations, at 10-year evaluation, SDFS was significantly lower in patients with BRAFmut /TERTpmut (log-rank p < 0.001) and BRAFwt /TERTpmut (log-rank p < 0.001) tumors than in patients with BRAFwt /TERTpwt tumors (Figure 2D). The aforementioned concomitant mutations significantly increased the risk of structural disease status both in unadjusted and adjusted models (Table 4). Patients with BRAFmut /TERTpwt tumors did not display a significantly lower SDFS than BRAFwt /TERTpwt tumors (log-rank p = 0.103) (Figure 2D). Table 4. Analysis of molecular status in the risk of structural disease. Structural Structural Unadjusted HR Adjusted HR Molecular Status n Disease Disease p-Value p-Value * (95% CI) (95% CI) Absent Present BRAF status 228 BRAFwt 85 79 (38.3%) 6 (27.3%) 1 (Reference) 1 (Reference) BRAFmut 143 127 (61.7%) 16 (72.7%) 1.7 (0.65–4.26) 0.286 − − TERTp status 218 TERTpwt 202 187 (95.4%) 15 (68.2%) 1 1 TERTpmut 16 9 (4.6%) 7 (31.8%) 7.4 (3.01–18.23) <0.001 7.0 (2.67–18.54) <0.001 RAS status 231 RASwt 218 198 (94.3%) 20 (95.2%) 1 1 RASmut 13 12 (5.7%) 1 (4.8%) 0.9 (0.11–6.35) 0.875 − − BRAF and TERTp status 214 BRAFwt /TERTpwt 79 76 (39.6%) 3 (13.6%) 1 1 BRAFmut /TERTpwt 120 108 (56.3%) 12 (54.5%) 2.8 (0.79–9.90) 0.112 − − BRAFwt /TERTpmut 4 1 (0.5%) 3 (13.6%) 24.3 (4.89–120.80) <0.001 24.2 (4.80–122.05) <0.001 BRAFmut /TERTpmut 11 7 (3.6%) 4 (18.2%) 13.2 (2.93–59.05) 0.001 11.5 (2.40–55.60) 0.002 n: number of cases; HR: Hazard ratio calculated by Cox Regression analysis; CI: Confidence interval; * Age- and gender-adjusted; p-value; wt: wild-type; mut: mutated. gender (HR = 7.0, p < 0.001) (Table 4). SDFS was not significantly influenced by BRAFmut or RASmut tumors (Figure 2A,C). Considering concomitant mutations, at 10-year evalua- tion, SDFS was significantly lower in patients with BRAFmut/TERTpmut (log-rank p < 0.001) and BRAFwt/TERTpmut (log-rank p < 0.001) tumors than in patients with BRAFwt/TERTpwt tumors (Figure 2D). The aforementioned concomitant mutations significantly increased Cancers 2021, 13, 2048 the risk of structural disease status both in unadjusted and adjusted models (Table 94). of 16 Patients with BRAF /TERTp tumors did not display a significantly lower SDFS than mut wt BRAFwt/TERTpwt tumors (log-rank p = 0.103) (Figure 2D). Kaplan–Meier Figure 2. Figure survival 2. Kaplan–Meier curves survival of PTC curves structural of PTC structuraldisease-free survival disease-free survival (SDFS) (SDFS) by tumor by tumor molecular molecular status status in com- in parison with wild type tumors; (A) BRAF status, (B) TERTp status, (C) RAS status, (D) combined BRAF comparison with wild type tumors; (A) BRAF status, (B) TERTp status, (C) RAS status, (D) combined BRAF and TERTp and TERTp status in comparison to wild-type tumors; wt: wild-type; mut: mutated. status in comparison to wild-type tumors; wt: wild-type; mut: mutated. 3.6. Molecular Alterations in Disease-Specific Mortality DSM was significantly increased in patients with TERTpmut tumors (log-rank p < 0.001) in comparison with patients without TERTpmut (Figure 3B). When adjusted for gender and age at diagnosis, TERTpmut significantly increased the risk of DSM (HR = 10.1, p = 0.010) (Table 5). DSM was not significantly increased in patients with BRAFmut or RASmut tumors (Figure 3A,C). DSM was increased in patients with BRAFmut /TERTpmut (log-rank p < 0.001) and BRAFwt /TERTpmut (log-rank p = 0.035) tumors in comparison with patients with BRAFwt /TERTpwt tumors (Figure 3D). There were no DSM events in patients with BRAFmut /TERTpwt tumors (Table 5). Table 5. Analysis of molecular status in the risk of disease-specific mortality. Disease- Disease- Specific Specific Unadjusted HR Adjusted HR Molecular Status n p-Value p-Value * Mortality Mortality (95% CI) (95% CI) Absent Present BRAF status 228 BRAFwt 85 81 (36.8%) 4 (50.0%) 1 (Reference) 1 (Reference) BRAFmut 143 139 (63.2%) 4 (50.0%) 0.6 (0.15–2.39) 0.466 - - TERTp status 218 TERTpwt 202 199 (94.8%) 3 (37.5%) 1 1 TERTpmut 16 11 (5.2%) 5 (62.5%) 23.9 (5.70–100.23) <0.001 10.1 (1.76–58.41) 0.010 RAS status 231 RASwt 218 212 (94.6%) 6 (85.7%) 1 1 RASmut 13 12 (5.4%) 1 (14.3%) 2.4 (0.28–20.36) 0.429 - - BRAF and TERTp status 214 BRAFwt /TERTpwt 79 76 (36.9%) 3 (37.5%) 1 1 BRAFmut /TERTpwt 120 120 (58.3%) 0 (0.0%) n.a. n.a. - - BRAFwt /TERTpmut 4 3 (1.5%) 1 (25.0%) 6.5 (0.67–63.31) 0.109 - - BRAFmut /TERTpmut 11 7 (3.4%) 4 (50.0%) 11.6 (2.59–52.09) 0.001 n.s. n.s. n: number of cases; HR (Hazard ratio) calculated by Cox Regression analysis; CI: Confidence interval; * Age- and gender-adjusted; p-value; wt: wild-type; mut: mutated. Cancers Cancers 2021, 2021, 13,13, x 2048 1010 of of 1616 Figure Kaplan–Meier 3. 3. Figure Kaplan–Meier survival curves survival ofof curves PTCPTCdisease-specific survival disease-specific (DSS) survival (DSS) byby tumor molecular tumor status molecular inin status comparison comparison with wild with wild type tumors; type tumors;(A) BRAF (A) BRAF status, (B)(B) status, TERTp TERTp status, (C) status, RAS (C) RASstatus, (D) status, combined (D) BRAF combined BRAF and andTERTp TERTp status inin status comparison comparison toto wild-type wild-type tumors; tumors; wt:wt: wild-type; wild-type; mut: mut: mutated. mutated. Table 5. Analysis of molecular status in the risk of disease-specific mortality. 4. Discussion Molecular Status In Disease-Specific n the present study, we aimed to Disease-Specific investigate Unadjusted the role HR (95% playedAdjusted p-Value by point HRmutations p-Value * Mortality Absent Mortality Present CI) of BRAF, TERTp, and RAS (NRAS, HRAS, and KRAS) in the outcome of PTC patients (95% CI) BRAF status 228 (recurrent/persistent disease, structural disease status, and DSM). BRAFwt 85 81 (36.8%) 4 (50.0%) 1 (Reference) 1 (Reference) BRAFmut 143 The most frequent 139 (63.2%) alteration 4 (50.0%) in our PTC series was, as0.466 0.6 (0.15–2.39) expected, mutation - of BRAF- gene. BRAF mut was also frequent in LNM at diagnosis and often detected in locoregional TERTp status 218 TERTpwt recurrences. 202 199 All three RAS3 genes (94.8%) (37.5%) were found mutated 1 in primary tumors, 1 being overall TERTpmut the 16 second11 most (5.2%)common 5alteration (62.5%) in PTC. The occurrence 23.9 (5.70–100.23) of TERTp <0.001 mut , although0.010 10.1 (1.76–58.41) less RAS status common than alterations of the other genes, was higher in recurrent and metastatic lesions, 231 RASwt 218 namely 212 (94.6%) distant metastases,6than(85.7%)in primary tumors, 1 as hitherto shown by1our group [10]. RASmut 13 12 (5.4%) mut 1 (14.3%) 2.4 (0.28–20.36) 0.429 - - The role of BRAF in the outcome of patients is still an open question. In our BRAF and TERTp status 214 mut BRAFwt/TERTpwt study, 79 patients whose tumors 76 (36.9%) had BRAF , regardless 3 (37.5%) 1 of the status of the other 1 mutations, BRAF /TERTp mut wt did 120 not present a 120 (58.3%) higher risk for 0 (0.0%) any of the outcomes n.a. evaluated n.a. (recurrent/persistent - - BRAFwt/TERTpmut disease, 4 structural 3 (1.5%) disease1 status, (25.0%) or DSM). In 2015, George 6.5 (0.67–63.31) 0.109et al. [42] -reported a 92% - BRAFmut/TERTpmut frequency 11 of BRAFmut in a4series 7 (3.4%) (50.0%)of recurrent/persistent 11.6 (2.59–52.09) tumors.0.001 The increased n.s. incidence n.s. n: number of cases; HR (Hazard of BRAF mutcalculated ratio) and the lackby Cox of Regression a control groupanalysis; inCI: Confidence this study did interval; * Age- not allow toand gender-any establish adjusted; p-value; wt: wild-type; mut: mutated. association with recurrent/persistent disease [42]. In 2019, de Castro et al. [43] also did not find an association between BRAFmut and recurrent/persistent disease, although their 4. Discussion series contained 48% BRAFmut tumors. This lack of association was corroborated by other studiesIn[44–46]. Somestudy, the present studieswehaveaimed shown that older the to investigate age role and played male gender by point are mutations strong andof independent risk factors for PTC—specific mortality in patients with BRAF mut tumors, but BRAF, TERTp, and RAS (NRAS, HRAS, and KRAS) in the outcome of PTC patients (re- not current/persistent BRAFwtstructural in patients with disease, tumors [27,47]. diseaseWe did not status, andfind DSM).similar results in our series. The strength of BRAF mut The most frequenton patient outcomes alteration in our PTC may be overestimated series was, as expected,in themutation aforementioned of BRAF studies mut gene. [27,47] BRAFmut since wasinalso them, no other frequent in molecular alterations, LNM at diagnosis andnamely often TERTp detected in, were tested. locoregional Asrecurrences. discussed below, our RAS All three results indicate genes werethatfoundthe mutated presence in TERTpmuttumors, of primary in PTC,being and not of overall BRAF mut , indeed plays a major role in the outcome. the second most common alteration in PTC. The occurrence of TERTp , although less mut Cancers 2021, 13, 2048 11 of 16 Several studies have demonstrated BRAFmut association with PTC local invasive- ness, recurrent/persistent disease, and LNM [30,48–51]; detection of BRAFmut has led to advancing the idea that it may predict recurrence in low-risk CPTC patients [52]. Other studies suggested that BRAFmut was associated with poorer outcomes, RAI treatment resistance, and DSM [26,29]. In contrast with this, it was suggested that isolated BRAF status has a limited role in guiding patient management [11], and other studies did not validate BRAFmut impact on the outcome of patients [28,44,53,54]. In our analysis, isolated evaluation of BRAFmut , regardless of the status of the other mutations, converged with the limited role of this mutation in ascertain recurrent/persistent disease, structural disease, or DSM. We detected mutations in all RAS genes. The most commonly described p.Gln61Arg mutation [55] was present in 13 primary tumors. It was shown that RASmut is present in all stages of thyroid neoplasia [55] ruling out the utilization of RASmut as a marker of malig- nancy. In a few studies, RASmut has been correlated with DM and poorer outcomes [34,56]. In our series, RASmut was not associated with a poorer prognosis; patients with RASmut had a stage I diagnosis, usually presenting excellent responses to therapy and were free of disease at the end of follow-up, except for one patient who had concomitant TERTpmut and died from PTC. We can hypothesize that, in this patient, TERTpmut was determinant in the outcome rather than the presence of KRASmut . Of note, no RASmut was detected in locoregional metastases nor DM (Supplementary Table S2). TERTpmut has been consistently associated with poorer outcomes in PTC patients [57]. Some authors showed that recurrent/persistent disease was four times more frequent in patients with TERTpmut than in patients with TERTpwt tumors [58,59]. Whereas in another study, no significant increased risk of recurrent/persistent disease in patients with TERTpmut tumors was reported [60]. The former finding fits with our own results; in the unadjusted analysis, we observed that patients with TERTpmut tumors had twice the risk of recurrent/persistent disease; upon age and gender adjustment, there is still a suggested association (p = 0.062; Table 3). In our study, TERTpmut tumors were associated with significantly lower SDFS and significantly increased DSM. Furthermore, patients with TERTpmut tumors presented a significantly increased risk of structural disease and of DSM, after adjustment for age and gender, suggesting that TERTpmut may contribute to worse prognosis in PTC patients, as previously shown by our group [21]. Several studies have consistently associated TERTpmut with DSM [21,22,42,46,57–59,61,62]. Given previous studies showing a frequent concomitant BRAFmut and TERTpmut in thyroid cancer [13,37,46,63–65], we tested the possible relationship of molecular status com- binations and different outcomes. This analysis also aimed to evaluate a more trustworthy effect of each mutation separately. Comparing with BRAFwt /TERTpwt tumors, we observed that BRAFmut /TERTpwt tumors were associated with an increased risk of recurrent/persistent disease. BRAFwt /TERTpmut tumors were associated with an increased risk of recurrent/persistent disease, structural disease, and DSM. We are aware that the latter group (DSM) had few cases, and therefore these results should be interpreted with caution. Nevertheless, taking into account that TERTpmut is far less common than BRAFmut in PTC (less than 10% vs. 45%) [4,25] and that TERTpmut is consistently associated with more aggressive forms of PTC and with increased risk of disease [22,53], we think that our results support the determinant role TERTpmut in the outcome of PTC patients. In our series, 11/16 TERTpmut cases presented concomitant BRAFmut . We observed that BRAFmut /TERTpmut significantly increased the risk of recurrent/persistent disease, structural disease, and DSM in comparison to BRAFwt /TERTpwt tumors. In accordance with this, Kim et al. [61] showed that the presence of TERTpmut was associated with increased mortality in PTC patients and further demonstrated that BRAFmut /TERTpmut tumors were associated with worsened DSM in PTC patients in comparison to patients with isolated BRAFmut tumors. Cancers 2021, 13, 2048 12 of 16 It was suggested that BRAFmut /TERTpmut tumors presented a higher risk of worse out- comes in comparison with BRAFwt /TERTpmut tumors [24,66]. In our study, we observed that DSM was similar in cases with BRAFwt /TERTpmut tumors and BRAFmut /TERTpmut tumors. Our results differ from those reported by Vuong et al. [67]; in the latter metanalysis, tumor aggressiveness varies according to isolated BRAFmut , isolated TERTpmut , or con- comitant BRAFmut /TERTpmut . Vuong et al. [67] stratified PTC tumors’ aggressiveness in four groups with decreasing aggressiveness: BRAFmut /TERTpmut > BRAFwt /TERTpmut = BRAFmut /TERTpwt > BRAFwt /TERTpwt . Xing et al. [24] proposed a synergistic role of con- comitant BRAFmut /TERTpmut on patients’ prognosis; in their series, BRAFmut /TERTpmut tumors were associated with increased disease recurrence, higher than the sum of the two mutations alone, even when performing multivariable adjustments for the classical clinicopathologic risk factors. Xing et al. [24] hypothesized that a possible synergistic effect between the two mutations might occur, advancing that coexistence of the two mutations might be associated with increased expression of the TERT mRNA in PTC [24,64–66,68]. We observed that BRAFwt /TERTpmut tumors had an increased risk of recurrent/persistent disease and structural disease in comparison with BRAFmut /TERTpmut tumors. These results may indicate that TERTpmut , rather than BRAFmut , is the molecular alteration that confers higher aggressiveness to PTC. These results fit with our previous studies, in which we showed that PTC patients with TERTpmut tumors had a significantly lower survival [10,21] at variance with the results obtained regarding BRAFmut . Similar findings were reported by Vuong et al. [53], who concluded that TERTpmut was associated with unfavorable DSS and DFS, whereas BRAFmut revealed an association with increased risk of recurrence but not with mortality, thus concluding that BRAFmut usefulness to evaluate patient prognosis must be cautiously considered [53]. Gandolfi et al. [37] reported de- creased survival in tumors TERTpmut and BRAFmut /TERTpmut , but no association between BRAFmut and DM or decreased survival was detected. BRAFmut appears associated with local aggressiveness, while TERTpmut was associated with distant metastasis that confers a dismal prognosis [10]. In accordance with our series, no significant differences in recur- rent/persistent disease, structural disease, or DSM were observed when comparing patients with BRAFmut /TERTpmut tumors and patients whose tumors were BRAFwt /TERTpmut (Supplementary Figures S1–S3). The low number of tumors with BRAFwt /TERTpmut genotype in our series indicates that a larger series is necessary to confirm our results. 5. Conclusions We realize that our series has advantages and disadvantages; the positive aspect resides in the fact that it is a consecutive real-life series obtained from a single hospital and not a selected series. On the other hand, we are aware of the consequent limitations: few patients with DM and few patients with PTC-related death. As an example, having only nine patients dying from PTC disease makes the multivariate analysis for such output rather limited. These results should be evaluated with caution also due to the low number of patients with some genotypes. Summing up, our results indicate that molecular markers can play a role in predict- ing the outcome of PTC patients. We have data supporting the importance of search- ing BRAFmut /TERTpwt in terms of putative association with local aggressiveness (recur- rent/persistent disease). Furthermore, we have observed a much more important result regarding the prognosis of patients with tumors presenting TERTp point mutations, given their increased risk to develop structural disease and DSM. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cancers13092048/s1, Figure S1: Kaplan–Meier curves of recurrent/persistent disease-free survival by concomitant BRAF and TERT promoter mutations in comparison to tumors mutated only for BRAF (E) and only for TERTp (F). Figure S2: Kaplan–Meier curves of structural disease-free survival by concomitant BRAF and TERT promoter mutations in comparison to tumors mutated only for BRAF (E) and only for TERTp (F). Figure S3: Kaplan–Meier curves of disease-specific survival by concomitant BRAF and TERT promoter mutations in comparison to tumors mutated only Cancers 2021, 13, 2048 13 of 16 for BRAF (E) and only for TERTp (F). Table S1: Univariate analysis of clinicopathological features and combined gene mutations. Table S2: BRAF, TERTp, and RAS molecular status in lymph node metastases at diagnosis, locoregional recurrences, and distant metastases and distribution in the different PTC variants. Author Contributions: Conceptualization, A.A.P. and P.S.; methodology, A.A.P., E.T., and M.R.B.-C.; software, A.A.P. and E.T.; validation, A.A.P., E.T., and P.S.; formal analysis, A.A.P., M.R.B.-C., and E.T.; investigation, A.A.P., E.T., R.B., A.P., T.A., and M.P.; resources, P.S.; data curation, A.A.P. and E.T.; writing—original draft preparation, A.A.P. and E.T.; writing—review and editing, M.M., M.S.-S., J.M., and P.S.; supervision, P.S. and J.M.; project administration, P.S.; funding acquisition, P.S. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by FCT, the Portuguese Foundation for Science and Technology, through a PhD grant to E.T. SFRH/BD/143458/2019. This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020. Additional funding by the European Regional Development Fund (ERDF) through the Operational Programme for Competi- tiveness and Internationalization—COMPETE2020, and Portuguese national funds via FCT, under project POCI-01-0145-FEDER-016390: CANCEL STEM and from the FCT under the project POCI- 01-0145-FEDER-031438: The other faces of telomerase: Looking beyond tumor immortalization (PDTC/MED_ONC/31438/2017). Additional funding through the Sociedade Portuguesa de En- docrinologia, Diabetes e Metabolismo-BOLSA SPEDM PARA PROJECTO DE INVESTIGAÇÃO-2017. Institutional Review Board Statement: The study was conducted in accordance with the Declara- tion of Helsinki, and the protocol was approved by the Ethics Committee of CHVNG/E (Project investigation 30/2016, 28 January 2016, Comissão de Ética do Centro Hospitalar de Vila Nova de Gaia/Espinho). Informed Consent Statement: It was an anonymized retrospective study that was exempted from the informed consent according to DR n.º 18/2005, Série I-A de 2005-01-26. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Raposo, L.; Morais, S.; Oliveira, M.J.; Marques, A.P.; Bento, M.J.; Lunet, N. Trends in thyroid cancer incidence and mortality in Portugal. Eur. J. 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