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

Summary 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.

The presence of RET somatic mutations in sMTC has been shown to have a negative prognostic value (Elisei et al., 2008). 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 .
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., 2013;Boichard et al., 2012;Moura et al., 2015Moura et al., , 2011. 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 . 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 Wells et al., 2010), 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, 2018).
In these recent years, NGS has been applied in endocrine research as well (Persani et al., 2018), and several reports have been published for MTC, in some cases using a whole-exome approach (Agrawal et al., 2013;Chang et al., 2018) but mainly targeted sequencing (Heilmann et al., 2016;Ji et al., 2015;Simbolo et al., 2014;Wei et al., 2016). 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.

Sequencing Metrics and Overview of Gene Alterations Detected by NGS Targeted Sequencing
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).
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.

General Distribution of Mutations
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).

Types of Mutations
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.
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.

Other Unconventional Mutations: Prevalence, Types, and Associations
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.

Rare and Uncommon Mutations In the Analyzed Genes
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).

TERT Promoter C228 and C250 Mutational Status
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.

Whole-Exome Sequencing
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.

Correlation of the Mutational Status of Primary sMTC with the Clinical and Pathological Features of the Patients
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).
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).
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).

Correlation of the Variant Allele Frequency Value with the Tumor Size and Outcome of the Patients
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 .
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- 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) (

DISCUSSION
In recent years, the introduction of NGS techniques has revolutionized research and the diagnosis of many diseases, including endocrine diseases (Persani et al., 2018). In particular, cancer research was hugely improved by this high-throughput techniques, being able to sequence large genome and transcript portions and increasing the probability of discovering novel mutations, especially in less frequently studied genes (Berger and Mardis, 2018;Kamps et al., 2017).
Several studies have been performed in MTC, both employing WES (Agrawal et al., 2013;Chang et al., 2018) and targeted sequencing with specific panels of gene mutations (Heilmann et al., 2016;Ji et al., 2015;Simbolo et al., 2014;Wei et al., 2016). In summary, these studies confirmed the role of RET and RAS somatic mutations as main drivers in the pathogenesis of sMTC; on the other hand, very few alternative genetic alterations have been discovered, still leaving a rather large portion of cases negative for common somatic gene alterations.
In the present study, we characterized 181 sMTCs by NGS targeted sequencing, and this series represents, to our knowledge, the largest analyzed so far. For this purpose, we designed a custom gene mutational panel that includes all amplicons covering the entire coding region of the RET, HRAS, KRAS, and NRAS genes that are known to be involved in C-cell tumorigenesis  and all the known gene alterations involved in follicular thyroid cancer tumorigenesis Nikiforova et al., 2013).
Our data showed that 55.8% of cases harbored RET genetic alterations, confirming that RET, particularly the M918T mutation, is the main driver oncogene in sMTC. The second main driver oncogene has been confirmed to be RAS, particularly HRAS and KRAS genes, which were altered in 24.3% of sMTC cases.  whose driver role remains unclear. According to these results, the prevalence of complete negative cases in our series was narrowed to 18.3% of cases (Figure 1), which increased up to 19.9% if we include the subgroup with the uncommon mutations. In agreement with the results of other two studies (Agrawal et al., 2013;Chang et al., 2018), also in our hands the WES was unable to identify other recurrence mutations that could further reduce this subgroup of negative cases. It is conceivable that for these cases other types of genetic and/or epigenetic alterations can play a role.
Compared with previous data obtained analyzing sMTC by Sanger direct sequencing Elisei et al., 2008), we obtained an overall higher number of mutated cases, especially for RAS positive cases and, at the same time, we reduced the negative or ''mutational orphan'' sMTC cases. These results strongly support the use, whenever possible, of deep-sequencing techniques whose sensitivity is significantly higher than that of Sanger sequencing; this is a very important aspect to address, especially in metastatic patients who could benefit from targeted therapy whose choice largely depends on the knowledge of mutational status (Viola et al., 2016).
In our series, most cases harbored single mutually exclusive alterations (89.2%), whereas only a small portion of cases (7.4%) harbored multiple somatic mutations (  noteworthy that, in two of four cases with two simultaneous RET mutations (Table 1, cases 41 and 132), the VAF was the same. This finding, combined with the observation that the mutations were closely mapping in -cis within the same sequencing read, indicates that they might be the result of a single simultaneous mutational event in a single allele of the same cell and not truly heterogeneous mutations, thus reducing the percentage of ''true'' heterogeneous cases to 6.5% of sMTC.
In some cases, the VAF observed in additional mutations was rather low and, in several cases, they represented ''novel'' mutations never described before and with an uncertain pathogenic role ( Table 2). The meaning of these mutations is unknown, but being aware of their presence can be relevant for the follow-up of the patients because, if selected, these mutations could be responsible for acquired resistance during therapy with kinase inhibitors (Camidge et al., 2014;Chen and Fu, 2011), and their presence should not be overlooked.
We also found five cases in which a germinal mutation of key genes in thyroid tumorigenesis was also present and whose real pathogenic role is unclear (Table 1). These five alterations have been already reported in the dbSNP database, although VAF <0.01 excludes them as common SNPs (Table 2). The rarity of these alterations in the normal population suggests some pathogenic role for the tumor , although in silico predictive scores describe only some of them as pathogenic. In particular, two of them (i.e, KRAS A130V and PPM1D K469E) have been described as non-pathogenic/neutral alterations (Ruark et al., 2013;Wang et al., 2019). Even less is known about the new somatic indel mutation (i.e., RET c.1894_1902del-GAGCTGTGC; p.E632_C634del) that has never been described previously (Table 2).
Among cases presenting multiple mutations we did not find any difference in terms of number and type of mutations when comparing cases in which we analyzed the primary tumor tissues with in which we analyzed the metastatic lesions. The most recurrent additional mutation (5/181, 2.8%) observed in our series was the MET T1010I point mutation that was present at either the somatic or the germinal level (Table 3), as demonstrated also by the VAF (i.e., approximately 50% when germinal and less than 20% when somatic). In our series, MET T1010I was always associated to a driver mutation either in RET or RAS gene. The T1010I mutation is located in exon 14 of the MET gene encoding for the juxtamembrane portion of the tyrosine kinase (Comoglio et al., 2018) and exon 14 alterations, which are described in different tumor types, might be crucial for activation of the MET oncogene (Vigna et al., 1999). Germinal and/or somatic MET T1010I mutations have been described in several tumor types (Comoglio et al., 2018), including thyroid tumors (Wasenius et al., 2005). In this last study, 104 thyroid tumors were analyzed and somatic or germline MET T1010I mutations were found in 7% of samples: particularly, 1/12 (8.3%) sMTC harbored a germline MET T1010I. We also found either somatic (4/5) or germline (1/5) METT1010I-mutated cases, but all of them were characterized by the presence of another mutation likely driving the tumoral transformation. In general, the role of this mutation in cancer is not fully understood, and conflicting results exist, especially in other human tumors. As suggested for other human cancers (Neklason et al., 2011), a predisposing role of MET T1010I, especially when present at the germline level, might be hypothesized also for sMTC.
Other than describing the oncogene mutations involved in the pathogenesis of sMTC, we also compared the mutational status of these tumors with the clinical and pathological features of patients (Table 4). The presence of RET mutations and, in particular, the M918T was confirmed to be significantly associated with a worse outcome, a higher tumoral staging, a higher T category, and the presence of lymph-node and distant metastases. Moreover, when we compared the survival Kaplan-Meier curves of patients with sMTC with RET or RAS somatic mutations, a significantly lower percentage of surviving patients in the RET-positive cases was found. These results confirmed in a much larger series our previously reported data Elisei et al., 2008) and those recently collected in a meta-analysis summarizing the clinical significance of the mutational status in MTC (Vuong et al., 2018).
Simultaneously, we observed that RAS-positive cases were significantly associated with a better outcome, a lower tumor staging, and a lower rate of T categories than RET-positive cases, and, also when compared with the RAS negative cases, independent from the presence of RET mutation (Table 5). With these results, we confirmed that sMTC with RAS mutations have, in general, a less aggressive phenotype and a better prognosis as we previously observed  and herein, as definitively demonstrated by the virtually 100% rate of survival of patients harboring RAS mutations (Figure 2).
Compared with previous studies, using the NGS approach, we could also evaluate the VAF value, adding a parameter that it is not possible to evaluate using Sanger direct sequencing. The mutated allele abundance has been demonstrated to be a prognostic factor itself in tumors, including PTC, in which a higher BRAF V600E VAF predicts a poorer outcome (Guerra et al., 2012). Our results demonstrated that, also in this series, larger tumors not only are more frequently RET mutated but also have a higher VAF that corresponds to a higher percentage of mutated cells. This observation suggests that the presence of a RET-mutated allele, particularly M918T, confers a growth advantage and results in larger clonal tumors. These findings are also in line with our previous observation of a lower rate of RET M918T mutation in micro-MTC that, when mutated, likely harbored a much lower VAF of the RET mutation not detectable with the traditional Sanger sequencing . Moreover, we demonstrated that a higher VAF correlated with a poorer prognosis, both when considering all mutations and when considering only RET mutations. According to these findings, VAF may be included in the list of bad prognostic markers in the subgroup of RET-mutated sMTC cases.
By contrast, RAS-mutated tumors appear to be clonal also in smaller tumors and no difference in the outcome of patients was observed compared with the RAS-mutated allele abundance. Based on this observation, we can hypothesize that two different sMTC types, in terms of both development and aggressiveness, might exist (i.e., RET-like and RAS-like) and further studies are needed to confirm this hypothesis.
Finally, we studied most of the sMTC for the presence of mutations in the TERT gene promoter hotspots (C228, C250), and, conversely, regarding what happens to other thyroid neoplasms (Alzahrani et al., 2016), TERT promoter mutations in C228 and C250, at least in our study, do not play any role in the pathogenesis and/or progression of sMTC.
In conclusion, in the present study, that included the largest series of sMTC studied so far by NGS, we confirmed that RET and RAS gene alterations are the main actors in driving the development of this rare human tumor. Moreover, we reinforced the concept that RET-mutated cases have a more aggressive phenotype and a poorer prognosis, particularly when the VAF is higher, so that this parameter can be considered a new marker of a worse prognosis in RET-mutated sMTC. At the same time, we demonstrated that RAS-mutated cases have a better outcome than RET-mutated cases. Finally, a lower-than-expected percentage of sMTC cases are still orphans of a recognized genetic driver, and further studies for alternative mechanisms of tumor transformation need to be performed.

Limitations of the Study
The main limitation of this study is that we used a custom targeting sequencing panel including a large series of genes involved in thyroid cancer pathogenesis that failed in providing evidence of strong alternative mechanisms of pathogenesis besides RET and RAS mutations. Although in some cases WES analysis was also performed, the possibility that other alterations may be involved cannot be completely ruled out with this study.

METHODS
All methods can be found in the accompanying Transparent Methods supplemental file.

DATA AND CODE AVAILABILITY
For the following novel mutation a submission to COSMIC DATABASE has been requested and an identifier COSP47106 has been generated by COSMIC as a proof of successful data submission.

AUTHOR CONTRIBUTIONS
R.C. and C.R. were responsible for the major part of the project, its design, and analysis of data. T.R. provided major technical support in collecting samples and nucleic acid extraction. A.P. provided clinical and pathological data from the patients and statistical assistance. A.T. performed the TERT promoter sequencing. V.C., D.V., and V.B. were responsible for recruiting the patients and collecting blood samples and informed consents. L.T., C.U., and F.B. provided FFPE tumor samples and were the pathologists responsible for all the histology classification of tumors. R.C. and R.E. wrote the manuscript. R.E. is the senior author and responsible for the supervision of the project. sequenced exons 2, 3, 4 of HRAS, KRAS and NRAS mutations following standard screening procedures in our laboratory .

DECLARATION OF INTERESTS
Tissue samples were collected at surgery and snap-frozen at -80° when patients were operated in Pisa; for the others, formalin-fixed paraffin-embedded (FFPE) tissues were used. Blood samples were collected in EDTA and were available for most of the cases studied.  Twenty-five nanograms of tumoral genomic DNA were amplified with specific primers for the Applied Biosystem, Calsbad, CA, USA) following the manufacturer's instructions.

NGS Data analysis
Raw sequencing data analysis was analyzed using Torrent Suite Software v.5.6 (Life Technologies) and included alignment to the hg19 human reference genome, quality score assignment, variant calling and coverage analysis. The data were further analyzed by Ion Reporter v.5.6 (https://ionreporter.thermofisher.com) with a bioinformatics workflow that calculated the Phred Quality score and performed annotation to dbSNP, COSMIC, and ExAC databases; data were finally filtered to several in silico prediction tools of pathogenicity (PhyloP, SIFT, Grantham, PolyPhen and FATHMM).
The system could evaluate the variant allele frequency (VAF) of the mutated allele within the sample. The VAF value for the heterozygous germline variations was 50% while, for somatic variations, it was variable depending on the abundance of the tumor cells with respect to normal cells in the analyzed sample, as well as on the heterogeneity of mutations in tumoral cells . We arbitrarily set the clinical sensitivity of VAF > 10% for variations already known to be drivers (i.e., RET, HRAS, KRAS); clinical sensitivity was lowered to VAF ≥ 5% for putative additional mutations as previously suggested . Nevertheless, driver alterations in typical hotspots (such as RET M918T mutation) with a lower VAF value were considered positive if validated by Sanger direct sequencing.
With a few exceptions, all variants detected were validated in the same tissue DNA by direct sequencing using specific conditions and primers, while novel and unconventional variations were tested on blood genomic DNA to assess their somatic or germline origin.

Whole exome sequencing
Six sMTC, 4 negative at the targeted sequencing analysis for any mutation and 2 positive only for RET somatic mutation were further analyzed by whole exome sequencing (WES) through an external service (IGA Technology Services Srl, Udine, Italy) to look for any other possible gene alteration not detected with our gene mutation panel.

Statistical analysis
Statistical analysis and graphs were generated using Prism GraphPad (version 8) (https://www.graphpad.com/). The different prevalence values of categorical data within the different mutational status were analyzed by Chi-squared test. The differences in the outcome and mutational status categories were evaluated by 1-way ANOVA and unpaired Student's t-test with Welch's correction in the case of significant differences between variances. The correlation between the VAF and tumor size was evaluated by a linear regression curve, while differences in survival between RET-and RAS-mutated sMTC cases were analyzed by Kaplan-Meier curves with the log-rank test. Differences were considered statistically significant when the P value was less than 0.05.