Molecular Profile of Locally Aggressive Well Differentiated Thyroid Cancers.

Knowledge of the genetic landscape of aggressive well differentiated thyroid cancers (WDTC) is lacking. Retrospective review of institutional database was performed to identify locally-invasive thyroid carcinomas and a comparison cohort of low-risk WDTC. ThyroSeq v2 next-generation sequencing was performed on available tissue. Survival time was analyzed by Kaplan-Meier methods and compared between groups via the log-rank test. Time to recurrence, treating death as a competing risk, was analyzed by cumulative incidence and compared between groups. Of 80 T4 tumors, 29 (36%) were met inclusion criteria, of which, 25 had genetic and clinicopathologic data. Most (24/25, 96%) harbored at least one genetic alteration, most commonly BRAF V600E (19, 76%), followed by mutations in the promoter region of TERT (14, 56%). Co-occurrence of BRAF and TERT was identified in 12 (48%) and associated with significantly higher risk of recurrence (p < 0.05). Conversely, co-occurrence of BRAF and TERT was present in only 5 of 102 (5%) patients presenting with early-stage WDTC. Compared to early-stage WDTC, co-occurrence of BRAF and TERT mutations are common in locally advanced (T4) thyroid cancer and are associated with an increased risk of recurrence. This knowledge may help predict aggressive behavior pretreatment and inform perioperative decision-making.


Results
Among the roughly 180 patients with well-differentiated thyroid cancer seen at our institution per year from 1987 to 2013, 80 patients were managed surgically for well-differentiated T4 thyroid cancer, and 26 (36%) met criteria for inclusion into the study. Of the 26 tissue samples available for sequencing, one failed mutational analysis due to storage-related degradation of the genomic material, resulting in 25 patient samples with genomic sequencing evaluable (Fig. 1). Demographic and clinicopathological characteristics of the study cohort are illustrated in Table 1. There were 16 females and 9 males. Mean age at diagnosis was 58 years and patients were followed for a mean of 60 months . Histopathologic analysis of these tumors revealed conventional papillary thyroid carcinoma (PTC) in 12 (48%) cases, tall cell variant of papillary thyroid carcinoma (TCV-PTC) in 10 (40%), and Hürthle cell carcinoma (HCC) in 3 (12%). Mean tumor diameter measured 4.4 cm (0.7-11.8 cm), and 20 (95%) patients demonstrated regional lymph node involvement, with an average of 8 (1-26) pathological lymph nodes. Twenty patients were staged as T4a and five patients were staged as T4b. A total of 11 (44%) patients developed recurrent disease. There were 9 (36%) patients with recurrences in the neck and 3 of these patients also developed distant metastasis (two patients with pulmonary metastasis and one patient with widespread liver, brain, and spine metastasis). Two of these 11 patients without local recurrence developed pulmonary metastasis. Overall distant metastases were present in 5 (20%) patients at a range of 5 to 37 months after diagnosis. Of the 5 patients with distant metastasis four harbored a co-mutation with TERT c228T and BRAF. Six of the 25 (24%) patients died of disease.
Molecular analysis revealed alterations in 24/25 (96%) T4 tumors (Fig. 2). The most common mutation was BRAF V600E, present in 19 (76%) tumors. The second most frequently identified mutation involved the promoter region of the TERT gene and was found in 14 (56%) of T4 tumors. This included C228T TERT mutation in 9 tumors and C250T mutation in 5 tumors. Co-occurrence of TERT and BRAF V600E mutations was identified in 12 (48%) of stage IV WDTC, TERT was the only identified alteration in 2 (8%) of study group tumors. Other alterations included TP53 mutation, ETV6-NTRK3 fusion, ELE1/RET fusion, and RET/PTC3 fusion, all found in single cases.
Within our cohort of T4 tumors, those carrying BRAF V600E mutation revealed no significant univariable correlation with patient age, gender, tumor size, regional lymph node or distant metastasis. However, among patients with regional lymph node involvement, those with tumors that harbored BRAF V600E had a median of 8 positive lymph nodes compared to a median of 2 positive lymph nodes in tumors negative for this mutation, a difference that bordered statistical significance (p = 0.073). The mean number of lymph nodes examined in BRAF + and BRAF-cases were nearly equivalent at 25.8 and 24.5, respectively.
In T4 tumors carrying both BRAF V600E and TERT mutations, no significant associations with patient age, gender, or clinicopathological parameters were identified. However, analysis of tumor recurrence by the Fine-Gray Test, adjusted for death as a competing risk, revealed that the presence of these two mutations was associated with a significantly increased risk of recurrence, with a 3-year cumulative incidence of 58.3% versus 18.3% (p = 0.04) ( Table 2, Fig. 3). Patients who harbored only the C228T TERT mutation experienced particularly high recurrence rates, with a 5-year cumulative incidence of 80% (p = 0.02). Kaplan-Meier survival analysis did not demonstrate significant differences in survival in patients with tumors carrying the BRAF V600E mutation or BRAF and TERT mutations. (2020) 10:8031 | https://doi.org/10.1038/s41598-020-64635-8 www.nature.com/scientificreports www.nature.com/scientificreports/ We next sought to determine if the mutational profile of T4 cancers was distinct from that of early stage cancers using a series of 102 consecutive T1-T2 cancers treated with surgical excision at our institution. PTC was the predominant diagnosis (91/102, 89%); follicular carcinomas comprised the remainder (11/102, 11%). The results of molecular analysis are shown in Table 3 and reveal, as expected, that mutations in BRAF and the RAS family of genes were the most commonly identified genetic alteration in early-stage WDTC, overall consistent with the findings of previous studies 6 . TERT mutations were found in 8 (8%) of the 102 T1-T2 tumors, and only 5 (5%) contained both BRAF V600E and TERT mutations. Overall, advanced T4 thyroid cancers more frequently harbored TERT mutations and had a 10-fold higher rate of co-occurring BRAF and TERT mutations (Fig. 2, Table 3).

Discussion
In this study, we provide genetic characterization of locally advanced T4 well differentiated thyroid cancer and demonstrate that the majority (19/25, 76%) are driven by BRAF V600E mutation and 12 (48%) also harbor co-existent TERT promoter mutations. Recurrence was particularly high in T4 WDTC with coexistent TERT C228T promoter mutations and thus, this genetic profile may allow further prognostication of WDTC.
Specifically, we found that the incidence of common driver mutations in locally advanced differentiated thyroid cancer is high, with 24 of 25 tumors (96%) harboring at least one mutation. Interestingly, two-thirds of T4 tumors harbored the BRAF V600E mutation and none showed RAS mutations, which is the second most common genetic event reported in PTC and FTC. RAS mutations are known to occur throughout the spectrum of thyroid cancers including encapsulated follicular variant thyroid cancers without nodal disease, as well as in  www.nature.com/scientificreports www.nature.com/scientificreports/ dedifferentiated or anaplastic cancers associated with distant metastasis 8,9 . The results of this study provide evidence that the majority of well differentiated T4 thyroid cancers have mutations in BRAF and/or TERT promoter regions.
Another difference in the molecular profile of T4 tumors as compared to the control cohort with T1/T2 tumors was that TERT mutations were observed in over half as compared to less than 10% of lower stage thyroid cancers (p < 0.05). The latter is like the incidence previously reported in the general population of WDTC 10,11 . In fact, the incidence of TERT mutations we observed in T4 well differentiated thyroid cancers was like that reported in poorly differentiated and anaplastic thyroid carcinomas. For example, Landa et al. identified TERT mutations in 50% of ATC and PDTC, 22% of PTC, and 25% of HCC 12 . A similar study by Liu et al. reported observed frequencies of TERT promoter mutations (C228T and C250T) in 50% of ATC, 25% of PTC, 22% of FTC, and 0% MTC 13 . However, our observed incidence of TERT promoter mutations is noted to be greater than that observed by Song et al., who noted 13% of patients in their cohort of stage III/IV patients possessed TERT promoter mutations. Interestingly, the incidence in stage I/II patients was also noted to be lower at 1.7% which perhaps reinforces the known geographic/ethnic variation in mutational landscapes 14 .
The 48% frequency of co-occurrence of TERT and BRAF mutations in our cohort of T4 appears to be higher than the observed frequency described in papillary, poorly differentiated, and anaplastic thyroid carcinoma 11,12,15 as well as analyses completed in other geographic populations 14 . The TERT/BRAF genotype in this cohort of T4 was nearly 7-fold higher than that seen in the lower risk PTC and CPTC cohort described by Xing et al 15 . These studies all provided initial evidence that TERT mutations may be associated with aggressive disease. Our findings provide further support to the notion that the development of aggressive thyroid carcinoma is driven by the acquisition of multiple mutations which cooperate to enhance tumor growth and spread.
In this cohort of T4 cases, the presence of BRAF mutations alone and BRAF and TERT mutations were associated with an increased risk of disease recurrence. This is consistent with findings in other series of well differentiated thyroid cancer, where BRAF mutations in tumors>1 cm in size and particularly co-occurring BRAF and TERT mutations were associated with a significantly higher risk of disease recurrence 16,17 . A subset of BRAF + thyroid cancers will present with aggressive T4 tumors and or recur, but we do not know why. In our study groups BRAF V600E, was present in 19/25 (76%) of T4 tumors and 45/102 (44%) of T1/T2 WDTC tumors. Co-occurrence of TERT/BRAFV600E mutations were present in 12/25 (48%) of T4 and 5/102 (5%) of T1/ T2 thyroid carcinomas. There is a building body of evidence that co-mutations of BRAF/TERT signify a highly aggressive form of thyroid cancer that is more likely to recur. We did not find a significant correlation between the presence of these mutations and mortality in this cohort though it has been noted in other studies 14 . This may be due to the relatively small number of deaths observed in this patient cohort. Further, though others 14 have shown synergy between TERT promoter mutations and RAS mutations, the lack of RAS mutations in our cohort precludes any such interpretation of the data.
Out of the four cases in this cohort that did not have BRAF or TERT mutations, there were tumors with a TP53 mutation, ETV6-NTRK3 fusion, ELE1/RET fusion, and RET/PTC3 fusion. TP53 mutations are known to occur more frequently in de-differentiated thyroid cancer, but they have also been found in well differentiated cancers with a tendency to be more aggressive 18 .
This study has a few limitations including the small sample size and retrospective nature. Fortunately, locally advanced WDTC is rare, but this provides few opportunities to study this cohort prospectively. Another limitation is the study group of T4 tumors had a much higher incidence of TCV-PTC, a more aggressive histologic variant, than the control group. Also, there was a low number of WDTC-specific deaths limiting the survival analysis. A prospective multicenter study on the molecular phenotype and survival of advanced WDTC is needed. However, it must be noted that studies of over 388 patients with WDTC in the TCGA by Shen et al. supported a model that predicted poor clinical outcomes based on mutational status 19 . There they posit that possessing BRAF V600E or RAS mutations in conjunction with TERT promoter mutations portend higher risk than BRAF V600E  www.nature.com/scientificreports www.nature.com/scientificreports/ or TERT promoter mutations alone and that these mutations are poorer prognosticators as compared to RAS mutations alone.
In summary, the results of this study show that T4 well differentiated thyroid cancer develops primarily via BRAF V600E-initiated pathway, and more than half of them also carry TERT mutations. Furthermore, we show that co-occurrence of BRAF and TERT may serve as a marker for highly aggressive locally invasive thyroid carcinoma with an increased risk for recurrence particularly if associated with C228T and, if found preoperatively, such genetic profile could influence patient management. Further work is needed to determine if the co-occurrence of these mutations is also associated with a risk of recurrence and mortality in T4 thyroid cancer in a larger cohort of patients. In addition, characterization of recurrent/metastatic lesions to identify possible transformation and/or differences in molecular profile in these well-differentiated thyroid cancers is needed.