Genetic Landscape of Somatic Mutations in a Large Cohort of Sporadic Medullary Thyroid Carcinomas Studied by Next-Generation Targeted Sequencing

•We studied by NGS the largest cohort of sporadic MTC that has been studied so far•RET and RAS mutations have been confirmed as the major drivers in sporadic MTC•Allele frequency can be considered a new marker of bad prognosis in RET-mutated cases•Survival rate is significantly shorter in RET-mutated than in RAS-mutated cases Sporadic Medullary Thyroid Carcinoma (sMTC) is a rare but aggressive thyroid tumor. RET and RAS genes are present in about 50%–80% of cases, but most of the remaining cases are still orphan of a genetic driver. We studied the largest series of sMTC by deep sequencing to define the mutational landscape. With this methodology we greatly reduced the number of RET- or RAS-negative cases and we confirmed the central role of RET and RAS mutations. Moreover, we highlighted the bad prognostic role of RET mutations in sMTC and consolidated the favorable prognostic role of RAS mutations. For the first time, we showed that the variant allele frequency represents an additional prognostic marker inside the group of RET-mutated sMTC. Sporadic Medullary Thyroid Carcinoma (sMTC) is a rare but aggressive thyroid tumor. RET and RAS genes are present in about 50%–80% of cases, but most of the remaining cases are still orphan of a genetic driver. We studied the largest series of sMTC by deep sequencing to define the mutational landscape. With this methodology we greatly reduced the number of RET- or RAS-negative cases and we confirmed the central role of RET and RAS mutations. Moreover, we highlighted the bad prognostic role of RET mutations in sMTC and consolidated the favorable prognostic role of RAS mutations. For the first time, we showed that the variant allele frequency represents an additional prognostic marker inside the group of RET-mutated sMTC. Medullary Thyroid Carcinoma (MTC) originates from neural crest-derived parafollicular C-cells and can occur in hereditary (25%) or sporadic forms (75%) (Kouvaraki et al., 2005Kouvaraki M.A. Shapiro S.E. Perrier N.D. Cote G.J. Gagel R.F. Hoff A.O. Sherman S.I. Lee J.E. Evans D.B. RET proto-oncogene: a review and update of genotype-phenotype correlations in hereditary medullary thyroid cancer and associated endocrine tumors.Thyroid. 2005; 15: 531-544Crossref PubMed Scopus (225) Google Scholar). Germline-activating RET mutations are found in 95%–98% of hereditary MTC, whereas somatic RET mutations are present in 25%–40% of sporadic MTC (sMTC) (Cerrato et al., 2009Cerrato A. De Falco V. Santoro M. Molecular genetics of medullary thyroid carcinoma: the quest for novel therapeutic targets.J. Mol. Endocrinol. 2009; 43: 143-155Crossref PubMed Scopus (59) Google Scholar, Drosten and Putzer, 2006Drosten M. Putzer B.M. Mechanisms of Disease: cancer targeting and the impact of oncogenic RET for medullary thyroid carcinoma therapy.Nat. Clin. Pr. Oncol. 2006; 3: 564-574Crossref PubMed Scopus (76) Google Scholar, Kouvaraki et al., 2005Kouvaraki M.A. Shapiro S.E. Perrier N.D. Cote G.J. Gagel R.F. Hoff A.O. Sherman S.I. Lee J.E. Evans D.B. RET proto-oncogene: a review and update of genotype-phenotype correlations in hereditary medullary thyroid cancer and associated endocrine tumors.Thyroid. 2005; 15: 531-544Crossref PubMed Scopus (225) Google Scholar, Romei et al., 2011Romei C. Cosci B. Renzini G. Bottici V. Molinaro E. Agate L. Passannanti P. Viola D. Biagini A. Basolo F. et al.RET genetic screening of sporadic medullary thyroid cancer (MTC) allows the preclinical diagnosis of unsuspected gene carriers and the identification of a relevant percentage of hidden familial MTC (FMTC).Clin. Endocrinol. 2011; 74: 241-247Crossref PubMed Scopus (75) Google Scholar). Several types of somatic RET mutations have been reported in sMTC, with the most common mutation occurring in codon M918 within exon 16, which is present in up to 90% of RET-positive cases, followed by mutations in codon C634 within exon 11 (Elisei et al., 2014Elisei R. Molinaro E. Agate L. Bottici V. Viola D. Biagini A. Matrone A. Tacito A. Ciampi R. Vivaldi A. Romei C. Ret oncogene and thyroid carcinoma.J. Genet. Syndr. Gene Ther. 2014; 5: 10Google Scholar, Eng et al., 1994Eng C. Smith D.P. Mulligan L.M. Nagai M.A. Healey C.S. Ponder M.A. Gardner E. Scheumann G.F. Jackson C.E. Tunnacliffe A. et al.Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours.Hum. Mol. Genet. 1994; 3: 237-241Crossref PubMed Scopus (477) Google Scholar, Romei et al., 2016Romei C. Ciampi R. Elisei R. A comprehensive overview of the role of the RET proto-oncogene in thyroid carcinoma.Nat. Rev. Endocrinol. 2016; 12: 192-202Crossref PubMed Scopus (163) Google Scholar). The presence of RET somatic mutations in sMTC has been shown to have a negative prognostic value (Elisei et al., 2008Elisei R. Cosci B. Romei C. Bottici V. Renzini G. Molinaro E. Agate L. Vivaldi A. Faviana P. Basolo F. et al.Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study.J. Clin. Endocrinol. Metab. 2008; 93: 682-687Crossref PubMed Scopus (355) Google Scholar). In addition to point mutations, aneuploidy of chromosome 10 and RET gene amplification have been described in MTC cases, prevalently in cases with a somatic RET mutation (Ciampi et al., 2012Ciampi R. Romei C. Cosci B. Vivaldi A. Bottici V. Renzini G. Ugolini C. Tacito A. Basolo F. Pinchera A. Elisei R. Chromosome 10 and RET gene copy number alterations in hereditary and sporadic Medullary Thyroid Carcinoma.Mol. Cell. Endocrinol. 2012; 348: 176-182Crossref PubMed Scopus (16) Google Scholar). Recently, activating point mutations in RAS genes (H-, K-, and NRAS) has been described in RET-negative sMTC, with a variable percentage depending on the different series and screening techniques employed (Agrawal et al., 2013Agrawal N. Jiao Y. Sausen M. Leary R. Bettegowda C. Roberts N.J. Bhan S. Ho A.S. Khan Z. Bishop J. et al.Exomic sequencing of medullary thyroid cancer reveals dominant and mutually exclusive oncogenic mutations in RET and RAS.J. Clin. Endocrinol. Metab. 2013; 98: E364-E369Crossref PubMed Scopus (149) Google Scholar, Boichard et al., 2012Boichard A. Croux L. Al Ghuzlan A. Broutin S. Dupuy C. Leboulleux S. Schlumberger M. Bidart J.M. Lacroix L. Somatic RAS mutations occur in a large proportion of sporadic RET-negative medullary thyroid carcinomas and extend to a previously unidentified exon.J. Clin. Endocrinol. Metab. 2012; 97: E2031-E2035Crossref PubMed Scopus (95) Google Scholar, Ciampi et al., 2013Ciampi R. Mian C. Fugazzola L. Cosci B. Romei C. Barollo S. Cirello V. Bottici V. Marconcini G. Rosa P.M. et al.Evidence of a low prevalence of RAS mutations in a large medullary thyroid cancer series.Thyroid. 2013; 23: 50-57Crossref PubMed Scopus (118) Google Scholar, Moura et al., 2015Moura M.M. Cavaco B.M. Leite V. RAS proto-oncogene in medullary thyroid carcinoma.Endocr. Relat. Cancer. 2015; 22: R235-R252Crossref PubMed Scopus (61) Google Scholar, Moura et al., 2011Moura M.M. Cavaco B.M. Pinto A.E. Leite V. High prevalence of RAS mutations in RET-negative sporadic medullary thyroid carcinomas.J. Clin. Endocrinol. Metab. 2011; 96: E863-E868Crossref PubMed Scopus (163) Google Scholar). RAS gene point mutations in MTC mainly occur in H- and KRAS, and they are usually mutually exclusive with RET mutations. In our previous study, we found that patients harboring RAS mutations showed a better prognosis than those harboring RET mutations or presenting no mutations (Ciampi et al., 2013Ciampi R. Mian C. Fugazzola L. Cosci B. Romei C. Barollo S. Cirello V. Bottici V. Marconcini G. Rosa P.M. et al.Evidence of a low prevalence of RAS mutations in a large medullary thyroid cancer series.Thyroid. 2013; 23: 50-57Crossref PubMed Scopus (118) Google Scholar). Despite the presence of RET and RAS somatic mutations, 20–50% sMTC are still orphans of a genetic driver. Assessing the mutational status, especially for the RET gene, is crucial for targeted therapies with tyrosine kinase inhibitors currently employed, such as vandetanib and cabozantinib (Elisei et al., 2013Elisei R. Schlumberger M.J. Müller S.P. Schöffski P. Brose M.S. Shah M.H. Licitra L. Jarzab B. Medvedev V. Kreissl M.C. et al.Cabozantinib in progressive medullary thyroid cancer.J. Clin. Oncol. 2013; 31: 3639-3646Crossref PubMed Scopus (691) Google Scholar, Wells et al., 2010Wells Jr., S.A. Gosnell J.E. Gagel R.F. Moley J. Pfister D. Sosa J.A. Skinner M. Krebs A. Vasselli J. Schlumberger M. Vandetanib for the treatment of patients with locally advanced or metastatic hereditary medullary thyroid cancer.J. Clin. Oncol. 2010; 28: 767-772Crossref PubMed Scopus (411) Google Scholar), and the discovery of new oncogene alterations remains crucial to individuate novel targets for this type of therapy. The recent advent of next-generation sequencing (NGS) techniques has dramatically changed our understanding of cancer genomics with the discovery of novel genetic alterations responsible for the pathogenesis of several cancer types (Berger and Mardis, 2018Berger M.F. Mardis E.R. The emerging clinical relevance of genomics in cancer medicine.Nat. Rev. Clin. Oncol. 2018; 15: 353-365Crossref PubMed Scopus (141) Google Scholar). In these recent years, NGS has been applied in endocrine research as well (Persani et al., 2018Persani L. de Filippis T. Colombo C. Gentilini D. GENETICS IN ENDOCRINOLOGY: genetic diagnosis of endocrine diseases by NGS: novel scenarios and unpredictable results and risks.Eur. J. Endocrinol. 2018; 179: R111-R123Crossref PubMed Scopus (15) Google Scholar), and several reports have been published for MTC, in some cases using a whole-exome approach (Agrawal et al., 2013Agrawal N. Jiao Y. Sausen M. Leary R. Bettegowda C. Roberts N.J. Bhan S. Ho A.S. Khan Z. Bishop J. et al.Exomic sequencing of medullary thyroid cancer reveals dominant and mutually exclusive oncogenic mutations in RET and RAS.J. Clin. Endocrinol. Metab. 2013; 98: E364-E369Crossref PubMed Scopus (149) Google Scholar, Chang et al., 2018Chang Y.-S. Chang C.-C. Huang H.-Y. Lin C.-Y. Yeh K.-T. Chang J.-G. Detection of molecular alterations in Taiwanese patients with medullary thyroid cancer using whole-exome sequencing.Endocr. Pathol. 2018; 29: 324-331Crossref PubMed Scopus (13) Google Scholar) but mainly targeted sequencing (Heilmann et al., 2016Heilmann A.M. Subbiah V. Wang K. Sun J.X. Elvin J.A. Chmielecki J. Sherman S.I. Murthy R. Busaidy N.L. Subbiah I. et al.Comprehensive genomic profiling of clinically advanced medullary thyroid carcinoma.Oncology. 2016; 90: 339-346Crossref PubMed Scopus (26) Google Scholar, Ji et al., 2015Ji J.H. Oh Y.L. Hong M. Yun J.W. Lee H.-W. Kim D. Ji Y. Kim D.-H. Park W.-Y. Shin H.-T. et al.Identification of driving ALK fusion genes and genomic landscape of medullary thyroid cancer.PLoS Genet. 2015; 11: e1005467Crossref PubMed Scopus (73) Google Scholar, Simbolo et al., 2014Simbolo M. Mian C. Barollo S. Fassan M. Mafficini A. Neves D. Scardoni M. Pennelli G. Rugge M. Pelizzo M.R. et al.High-throughput mutation profiling improves diagnostic stratification of sporadic medullary thyroid carcinomas.Virchows Arch. 2014; 465: 73-78Crossref PubMed Scopus (49) Google Scholar, Wei et al., 2016Wei S. LiVolsi V.A. Montone K.T. Morrissette J.J.D. Baloch Z.W. Detection of molecular alterations in medullary thyroid carcinoma using next-generation sequencing: an institutional experience.Endocr. Pathol. 2016; 27: 359-362Crossref PubMed Scopus (18) Google Scholar). According to the results reported in these studies, despite the presence of some rare events present in a few cases, the common occurrence of mutually exclusive RET and RAS mutations has been confirmed to be the main pathogenic signature of sMTC. The few novel alterations found in these studies represent more likely a “private” mutation of that specific tumor than significantly recurrent genetic alterations. The major limits of these previous studies are the relative low number of cases analyzed and few data about the correlation between the mutations and clinical and pathological features of the tumors. We aimed to analyze a large series of sMTC by NGS targeted sequencing using a thyroid-specific gene mutation panel to delineate their mutational landscape and correlate the molecular data with the pathological characteristics of the tumors and with both the clinical features and outcome of patients affected with sMTC. Of 209 cases studied, 28 were excluded as not informative due to technical reasons or insufficient quality of data obtained. Informative sequencing data were then obtained for 181/209 (86.6%) sMTC. The mean value of the variant vertical coverage obtained was 2,038X (median, 2,049.5; range, 117.7–5,713), and the mean number of reads for the sample was 385,564.1 (median, 396,496.5; range, 21,198–1,744,507). In total, 166 genetic alterations were detected in 148 sMTC cases (Table S1). In particular, we found 152 single-nucleotide variations and 14 indels: 107/166 (64.5%) were found in the RET protooncogene, 48/166 (28.9%) in the three RAS genes (HRAS, KRAS, NRAS), 5/166 (3%) in the MET gene, 2/166 (1.2%) in the TP53 gene, 1/166 (0.6%) in the TSH receptor (TSHR) gene, 1/166 (0.6%) in the EIF1AX gene, 1/166 (0.6%) in the CHK2 gene, and 1/166 (0.6%) in thePPM1D gene. One hundred fifty-four gene alterations were validated by Sanger direct sequencing and confirmed to be somatic, five were found to be germline, one was confirmed in tissue DNA, but blood was not available for germline validation, and six were not validated by Sanger direct sequencing due to low Variation Allele Frequency (VAF) values or for other technical reasons. All the mutations that were previously detected by Sanger sequencing for somatic RET and RAS mutations were confirmed by NGS while in eight cases we observed a discrepancy with a previous negative result by Sanger and a positive one by NGS. This apparent discrepancy was due either to the low VAF that was under the detection limit of Sanger (<20%) or bad Sanger sequencing quality. As shown in Table S1, the number of cases harboring one or more genetic alterations was 148/181 (81.7%), whereas the remaining 33/181 (18.3%) did not carry any alteration targeted in our panel. In particular, 132/148 (89.2%) mutated cases harbored one single mutation, whereas 11/148 (7.4%) showed a heterogeneous pattern due to the presence of a somatic driver mutation coupled with one or more other somatic mutations and 5/148 (3.4%) harbored one somatic driver mutation coupled with a second germline mutation (Table 1).Table 1List of Cases Presenting Multiple Somatic Alterations and Somatic Coupled with a Germ-Line Alteration (in bold). Variant Allele Frequency (VAF) Values Are Reported in BracketsNoAlteration 1Alteration 2Alteration 3Alteration 441RET M918T (s) [47.3%]RET D925A (s) [46.7%]242RET M918T (s) [32.2%]RET R297H (s) [11.8%]196RET M918T (s) [12.3%]RET R833C (s) [25.0%]RET S891A (s) [20.3%]MET T1010I (n.v.) [18.6%]140RET M918T (s) [39.0%]KRAS K182E (n.v.) [20.0%]176RET C634W (s) [17.3%]NRAS A18V (s) [7.5%]253RET C634W (s) [11.6%]MET T1010I (s) [50.1%]132RET D898_E901del (s) [30.9%]RET S904P (s) [30.8%]169RET C620R (s) [41.5%]MET T1010I (n.v.) [6.7%]3RET C618G (n.v.) [40.5%]TP53 R283C (n.v.) [48.1%]251HRAS G13R (s) [23.3%]MET T1010I (s) [8.3%]39HRAS Q61R (s) [34.4%]RET M918T (s) [3.0%]88RET M918T (s) [19.7%]KRAS A130V (g) [44.9%]91RET C634Y (s) [37.3%]RET R215L (g) [49.9%]201RET D898_E901del (s) [26.6%]PPM1D K469E (g) [37.5%]20HRAS Q61R (s) [41.7%]MET T1010I (g) [51.4%]52TSHR I630L (s) [31.0]TP53 R158C (g) [52.8%](s), verified somatic; (n.v.), not detectable by direct sequencing. Open table in a new tab (s), verified somatic; (n.v.), not detectable by direct sequencing. Cases presenting RET somatic alterations as the driver were 101/181 (55.8%): in 88 cases as a single alteration and in 13 cases as multiple alterations. Cases presenting RAS mutations as the driver were 44/181 (24.3%): in 42 cases as a single mutation and 2 cases in association with either a somatic or germline MET T1010I mutation. Finally, 3/181 (1.6%) cases presented mutations in other genes (i.e., CHK2 W114*, EIF1AX G135A, and TSHR I630L) (Table S1). The remaining 33/181 (18.3%) were negative for all alterations targeted in our panel. As shown in Figure 1, 60/148 (40.5%) mutated sMTC harbored the RET M918T mutation. In 54 cases, it was present as a single mutation; and in 6 cases, it was associated with other RET (n = 3) or RAS mutations (n = 3). The details of associated mutations are reported in Table 1. The RET gene C634 codon was mutated in 18/148 (12.2%) cases with different aminoacidic alterations (Figure 1). In 15 cases, it was present as a single mutation, whereas, in 3 cases, it was associated with other alterations. A RET indel was present in 14/148 (9.5%) cases (Table S1 and Figure 1): in 12 cases, it was present as a single mutation, while in 2 cases it was associated with other alterations. Additionally, 3/148 (2%) cases presented the C620R mutation, and one of them harbored a simultaneous MET T1010I mutation. Another 2/148 (1.3%) cases showed a C618R mutation, and one of them had a simultaneous TP53 R283C mutation. Finally, 2/148 (1.3%) cases presented the RET S891A mutation, and 1/148 (0.7%) cases showed the RET C630R and 1/148 (0.7%) cases showed the RET S1024F mutation. Among cases harboring RET multiple mutations, cases n. 41 and 132 presented the RET M918T + D925A and RET D898_E902del + S904P mutations, respectively. The analysis of the specific sequencing reads associated with these mutations showed that they were very close and on the same allele (i.e., in –cis), likely consequent to a single mutational event (data not shown). A complete detailed description of these mutations is summarized in Table S1 and Figure 1. Alterations of the three RAS genes were present in 44/148 (29.7%) mutated cases. Of 148 sMTC cases, 31 (20.9%) were mutated in the HRAS gene and included 2 cases with a simultaneous somatic or germline MET T1010I mutation, respectively (Figure 1). Another 12/148 (8.2%) cases were positive in KRAS, and only 1/148 (0.7%) presented the NRAS Q61K mutation (Table S1 and Figure 1). Only 3/148 (2%) sMTC cases harbored single mutations in other genes belonging to our panel, such as CHK2 W114*, EIF1AX G135A, and TSHR I630L. The last case was also associated with a germline TP53 R158C mutation. Although the TSHR I630L mutation has been validated as somatic, the other two mutations could not be, and consequently, we could not establish their potential driver role. As shown in Table 2, among the above-reported mutations, we found a series of 18 uncommon and/or novel alterations. With the exception of the somatic or germline MET T1010I mutation, which was present in five separate cases already harboring either RET or RAS alterations (Table 3), all the others were single mutations in single cases. Considering their rarity and according to the in silico analysis (i.e., Clinic Var and MutTaster prediction tests) and public database of known gene alterations (i.e., dbSNP and COSMIC and HGMD), we hypothesized that they could be private mutations whose driver role in the pathogenesis of the sMTC is unclear (Table 3).Table 2Details of Unconventional Alterations Found by NGS Targeted SequencingCaseGene MutationVAF (%)StatusdbSNP IDMAFCOSMIC ID α HGMD ID βClinVar Prediction γ MutTaster Prediction δNotes88KRAS c.389C > T; p.A130V44.9Germ liners730880473<0.01COSM4169153 αUncertain significance γSimultaneous RET M918T (s);reported as “neutral” (Wang et al., 2019Wang Q. Mehmood A. Wang H. Xu Q. Xiong Y. Wei D.Q. Computational screening and analysis of lung cancer related non-synonymous single nucleotide polymorphisms on the human kirsten rat sarcoma gene.Molecules. 2019; 24https://doi.org/10.3390/molecules24101951Google Scholar)41RET c.2774A > C; p.D925A46.7SomaticNovel–NovelDisease causing δOccurring in -cis with RET M918T (s)242RET c.890G > A; p.R297H11.8SomaticNovel–NovelPolymorphism δSimultaneous RET M918T (s)196RET c.2497C > T; p.R833C25Somaticrs377767422<0.01CM068590 βLikely pathogenic γSimultaneous RET M918T (s)20,169,196251,253MET c.3029C > T; p.T1010IVariousSomatic; germ liners56391007<0.01COSM707 αCM118113 βConflicting results δ176NRAS c.53C > T; p.A18V7.5SomaticNovel–NovelDisease causing δSimultaneous RET C634W (s)91RET c.644G > T; R215L49.9Germ liners748128929<0.01–Polymorphism δSimultaneous RET C634Y (s)201PPM1D c.1405A > G; p.K469E37.5Germ liners61756416<0.01–Disease causing δSimultaneous RET E898_E901del (s); reported as “benign” in breast and ovarian cancer (Ruark et al., 2013Ruark E. Snape K. Humburg P. Loveday C. Bajrami I. Brough R. Rodrigues D.N. Renwick A. Seal S. Ramsay E. et al.Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.Nature. 2013; 493: 406-410Crossref PubMed Scopus (181) Google Scholar)132RET c.2710T > C; p.S904P30.8SomaticNovel–NovelDisease causing δOccurring in -cis with RET E898_E901del (s)128RET c.1908_1909insTGCCGCACG; p.T636_V637delinsCRT35.4Somaticrs377767437–CI983210 βLikely pathogenic γDescribed germ-line in MEN2A (Höppner et al., 1998Höppner W. Dralle H. Brabant G. Duplication of 9 base pairs in the critical cysteine rich domain of the ret proto-oncogene causes multiple endocrine neoplasia type 2A.Hum. Mutat. 1998; : S128-S130https://doi.org/10.1002/humu.1380110143Crossref PubMed Scopus (36) Google Scholar)122RET c.1886_1891delTGTGCG; p.L629_D631delinsH38.4SomaticNA–COSM27040 α–Likely driver302RET c.1894_1902delGAGCTGTGC; p.E632_C634del42.9SomaticNovel–Novel–Likely driver252RET c.3071C > T; p.S1024F17.6SomaticNovel–NovelDisease causing δLikely driver3TP53 c.847C > T; p.R283C48.1n.v.rs149633775<0.01COSM10911 α/CM041458 βConflicting results δSimultaneous RET C618G (s)52TSHR c.1888A > C; p.I630L31Somatic––COSM26432 α/CM100952 βDisease causing δSimultaneous TP53 R158C (g)52TP53 c.472C > T; p.R158C52.8Germinalrs587780068<0.01COSM43848 α/CM121763 βPathogenic γSimultaneous TSHR I630L (s)196EIF1AX c.404G > C; G135A41.4n.v.NovelNovelDisease causing δ198CHK2 c.341G > A; W114*10.1n.v. in bloodNovelNovelDisease causing δn.v., not detectable by direct sequencing. Open table in a new tab Table 3List of Cases Presenting the MET T1010I Mutation in Association with RET or RAS Somatic MutationsN.Somatic MutationMET T1010I196RET M918T (s) [12.3%]RET S891A (s) [20.3%]RET R833C (s) [25.0%]MET T1010I (n.v.) [18.6%]253RET C634W (s) [11.6%]MET T1010I (s) [50.1%]169RET C620R (s) [41.5%]MET T1010I (n.v.) [6.7%]110HRAS Q61R (s) [41.7%]MET T1010I (g) [51.4%]251HRAS G13R (s) [23.2%]MET T1010I (s) [8.3%]Variant allele frequency (VAF) value is reported in brackets.(s), verified somatic; (g), verified germinal; (n.v.), not detectable by direct sequencing. Open table in a new tab n.v., not detectable by direct sequencing. Variant allele frequency (VAF) value is reported in brackets. (s), verified somatic; (g), verified germinal; (n.v.), not detectable by direct sequencing. The sequencing data for the TERT promoter were available for 148/181 (81.8%) cases. Neither C228T nor C250T mutations were found in any of the studied cases. The whole-exome sequencing (WES), despite the wide and deep analysis, did not reveal any other recurrent somatic mutation either in the four sMTC negative at the targeted sequencing or in those already known to be RET mutated. The 175/209 (83.7%) sMTC cases, whose primary tumor was analyzed, were divided into four categories depending on the mutational status (RET M918T, RET other, RAS mutations, and not RET/not RAS) and were correlated with the clinical and pathological features of the patients (Table 4). A statistically significant correlation was found between the presence of RET mutations, both together and when considering M918T alone, and the advanced stage of the disease (p = 0.0025), higher T category (p < 0.0001), and the presence of both lymph-node (N) (p = 0.0021) and distant metastases (M) (p = 0.0073).Table 4Correlation between Mutational Status of RET and RAS Genes with Clinical and Pathological Features of the Primary sMTC CasesRET M918TRET OtherRASNot RET/Not RASp ValueSex0.1378aChi-squared test. Female51.2% (22/43)52.8% (19/36)72.5%(29/40)67.9% (19/28) Male48.8% (21/43)47.2% (17/36)27.5% (11/40)32.1% (9/28)Age at diagnosis (mean ± SD) (years)49.43 ± 13.9355.67 ± 14.3255.81 ± 15.4458.84 ± 15.700.0779bOne-way ANOVA test.Primary/metastases0.1609aChi-squared test. Primary74.1% (43/58)83.7% (36/43)90.9% (40/44)84.8% (28/33) Metastases25.9% (15/58)16.3% (7/43)9.1% (4/44)15.2% (5/33)Outcome<0.0001aChi-squared test. Disease-free26.3% (10/38)66.7% (20/30)61.8% (21/34)77.3% (17/22) Biochemical7.9% (3/38)6.7% (2/30)29.4% (10/34)9.1% (2/22) Metastatic/dead65.8% (25/38)26.6% (8/30)8.8% (3/34)13.6% (3/22)Stage0.0001 aChi-squared test. I18.9% (7/37)46.9% (15/32)62.5% (25/40)70.8% (17/24) III81.1% (30/37)53.1% (17/32)37.5% (15/40)29.2% (7/24)T Categories<0.0001aChi-squared test. T1+T237.1% (13/35)72.7% (24/33)90.0% (36/40)83.3% (20/24) T3+T462.9% (22/35)27.3% (9/33)10.0% (4/40)16.7% (4/24)Lymph-node metastasis (N)0.0021aChi-squared test. N030.6% (11/36)54.5% (18/33)66.7% (26/39)75.0% (18/24) N169.4% (25/36)45.5% (15/33)33.3% (13/39)25.0% (6/24)Distant metastasis (M)0.0073aChi-squared test. M077.8% (28/36)90.6% (29/32)97.5% (39/40)100.0% (24/24) M122.2% (8/36)9.4% (3/32)2.5% (1/40)0 (0/24)Unless stated, values are expressed in % (number/total number).a Chi-squared test.b One-way ANOVA test. Open table in a new tab Unless stated, values are expressed in % (number/total number). In contrast to RET-mutated cases, RAS-mutated sMTC cases were significantly associated with a better outcome (p = 0.001), a lower stage of disease (p = 0.0037), and lower T category (i.e., T1/T2) (p = 0.0015), but no correlation was observed between the presence of RAS mutation and other epidemiological and pathological features (Table 5).Table 5Association of RAS-Mutated sMTC Cases and Clinical and Pathological FeaturesRAS+RAS-p ValueSex0.089aChi-squared test. Female72.5% (29/40)56.4% (62/110) Male27.5% (11/40)43.6% (48/110)Age at diagnosis (mean ± SD)55.81 ± 15.4458.84 ± 15.700.571bStudent Unpaired t test.Outcome0.0003aChi-squared test. Disease-free61.8% (21/34)52.2% (47/90) Biochemical29.4% (10/34)7.8% (7/90) Metastatic/dead8.8% (3/34)40% (36/90)Stage0.0037 aChi-squared test. I + II62.5% (25/40)41.5% (39/94) III + IV37.5% (15/40)58.5% (55/94)T Categories0.0015aChi-squared test. T1+T290.0% (36/40)62.4% (58/93) T3+T410.0% (4/40)37.6% (35/93)Lymph-node metastasis (N)0.0857aChi-squared test. N066.7% (26/39)49.5% (46/93) N133.3% (13/39)50.5% (47/93)Distant metastasis (M)0.107aChi-squared test. M097.5% (39/40)88.2% (82/93) M12.5% (1/40)11.8 (11/93)Unless stated, values are expressed in % (number/total number).a Chi-squared test.b Student Unpaired t test. Open table in a new tab Unless stated, values are expressed in % (number/total number). A strong correlation was also found between the presence of RET mutations and a worse patient outcome (p < 0.0001), and the survival of Kaplan-Meier curves confirmed that patients with sMTC harboring the RET mutation had a higher rate of cancer-related deaths than patients harboring RAS mutations (log rank = 4.41; p = 0.035) (Figure 2). The overall mean VAF of the mutations found was 35.1% (median, 30.2; range, 4.4–95.2). However, a big difference in VAF was observed among different cases with the lowest VAF observed in the rare and uncommon alterations (Table S1). According to the VAF, we could hypothesize the role of the mutations, especially in those cases with more than one alteration: the mutation with the greatest VAF would likely be the driver mutation (Li et al., 2017Li M.M. Datto M. Duncavage E.J. Kulkarni S. Lindeman N.I. Roy S. Tsimberidou A.M. Vnencak-Jones C.L. Wolff D.J. Younes A. Nikiforova M.N. Standards and guidelines for the interpretation and reporting of sequence variants in cancer.J. Mol. Diagn. 2017; 19: 4-23Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). As shown in Figure 3, panel A1, when we compared in 95 patients with primary tumor the VAF value of the driver mutation, any type, with the sMTC tumor size (in centimeters), we observed that larger tumors harbored mutations with a higher VAF value (p < 0.0001). However, when we performed the same analysis in subgroups according to the type of mutation, the correlation was confirmed in the subgroups of RET-mutated cases, either when all RET (Figure 3, panel A2) or only RET M918T-mutated cases were considered (Figure 3, panel A3) (p < 0.0001 and p = 0.0013, respectively) but not in the subgroup harboring RAS mutations (Figure 3, panel A4). Analyzing 103 patients, a higher VAF value of the driver mutation was also correlated with a worse outcome of the patients, as demonstrated by a significantly higher VAF value in patients with metastatic disease with respect to disease-free patients, both when considering all cases with any type of mutation (p = 0.003) (Figure 3, panel B1) and when analyzing the subgroup with only RET mutations (p = 0.047) (Figure 3, panel B2). By contrast, this correlation was not found in the subgroup with only RAS-positive cases (Figure 3, panel B3). In recent years, the introduction of NGS techniques has revolutionized research and the diagnosis of many diseases, including endocrine diseases (Persani et al., 2018Persani L. de Filippis T. Colombo C. Gentilini D. GENETICS IN ENDOCRINOLOGY: genetic diagnosis of endocrine diseases by NGS: novel scenarios and unpredictable results and risks.Eur. J. Endocrinol. 2018; 179: R111-R123Crossref Pub