Clinical utility of TERT promoter mutations and ALK rearrangement in thyroid cancer patients with a high prevalence of the BRAF V600E mutation

Background Mutations in the TERT promoter, ALK rearrangement, and the BRAF V600E mutation are associated with aggressive clinicopathologic features in thyroid cancers. However, little is known about the impact of TERT promoter mutations and ALK rearrangement in thyroid cancer patients with a high prevalence of BRAF mutations. Methods We performed Sanger sequencing to detect BRAF V600E and TERT promoter mutations and both immunohistochemistry and fluorescence in situ hybridization to identify ALK rearrangement on 243 thyroid cancers. Results TERT promoter mutations were not present in 192 well-differentiated thyroid carcinomas (WDTC) without distant metastasis or in 9 medullary carcinomas. However, the mutations did occur in 40 % (12/30) of WDTC with distant metastasis, 29 % (2/7) of poorly differentiated carcinomas and 60 % (3/5) of anaplastic carcinomas. ALK rearrangement was not present in all thyroid cancers. The BRAF V600E mutation was more frequently found in WDTC without distant metastasis than in WDTC with distant metastasis (p = 0.007). In the cohort of WDTC with distant metastasis, patients with wild-type BRAF and TERT promoter had a significantly higher response rate after radioiodine therapy (p = 0.024), whereas the BRAF V600E mutation was significantly correlated with progressive disease (p = 0.025). Conclusions The TERT promoter mutation is an independent predictor for distant metastasis of WDTC, but ALK testing is not useful for clinical decision-making in Korean patients with a high prevalence of the BRAF V600E mutation. Radioiodine therapy for distant metastasis of WDTC is most effective in patients without BRAF V600E and TERT promoter mutations.


Background
Thyroid cancer is the most common type of endocrine tumor, with an incidence that has significantly increased in the last few decades [1,2]. Although well-differentiated thyroid carcinoma (WDTC) is one of the most curable of all cancers, approximately 10-20 % of patients with WDTC suffer from disease recurrence after surgery, and some eventually die from the disease [3][4][5]. Various risk stratification methods have been used for the proper management of patients with WDTC; however, none are completely accurate [6].
Molecular biomarkers have been used as an adjunct diagnostic marker of thyroid cancer and a predictor of patient prognosis [7,8]. The BRAF V600E mutation is the most common mutation in thyroid cancer, particularly in papillary thyroid carcinoma (PTC), and plays an important role in tumorigenesis and progression [9][10][11][12][13][14]. In Korea, PTC comprises 97.3 % of all thyroid cancers according to new data from the 2014 annual report of cancer statistics in Korea (http://www.cancer.go.kr/). The BRAF V600E mutation is highly prevalent in Korean PTC patients [11]. Currently, there is controversy regarding whether the BRAF V600E mutation is associated with poor prognosis and aggressive clinicopathologic features in Korean PTC patients; therefore, additional prognostic biomarkers to predict a more aggressive disease are needed [9,[15][16][17][18].
ALK gene rearrangements have recently been identified in thyroid cancer [26][27][28][29][30]. EML4, STRN, TFG, and GTF2IRD1 have been reported as ALK fusion partners [27,28,[30][31][32]. The prevalence of ALK-rearranged PTCs has been reported to be up to 2.2 %, although the number of study cases is limited [26]. A previous study reported that ALK rearrangements were more frequently found in aggressive thyroid cancer, while another study found mutations only in unselected consecutive PTC cases and not in aggressive disease, such as PTCs with distant metastasis, poorly differentiated carcinomas and anaplastic carcinomas [26,28].
We aimed to investigate the prevalence of TERT promoter and ALK mutations in thyroid cancer patients with a high prevalence of the BRAF V600E mutation and their potential contribution for the risk stratification of these patients. PTC was defined as a classic type with tall cell features if it consisted of less than 50 % tall cells and as a tall cell variant if it consisted of 50 % or more tall cells [33].

Immunohistochemistry for ALK overexpression
Immunohistochemistry was performed on paraffinembedded whole tissue sections of surgical specimens using the ALK antibody (clone p80, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) and the Polink-2 HRP plus anti-rabbit DAB detection kit (GBI Labs, Mukilteo, WA, USA). As a positive control, we used paraffin-embedded tissue sections from two lung adenocarcinomas with previously confirmed ALK rearrangement by fluorescence in situ hybridization (FISH).

FISH for ALK rearrangement
We performed FISH to detect ALK rearrangement using a ZytoLight SPEC ALK Dual Color Break Apart Probe and Kit (ZytoVision GmbH, Bremerhaven, Germany) according to the manufacturer's protocol [29]. The positive criterion for ALK rearrangement was defined as > 15 % of split signal separation and/or isolated red signal in at least 100 tumor cells as previously described [26,29].
Evaluation of response to radioiodine therapy All 30 WDTC patients with distant metastasis underwent radioactive iodine (RAI) therapy. The response to RAI ablation was evaluated with a whole body iodine -131 scan, evaluation of serum thyroglobulin levels, and a computerized tomography scan. Clinical outcomes to RAI therapy were classified as complete remission (CR), partial response (PR), stable disease (SD), and progressive disease (PD) according to previously described criteria [36].

Statistical analysis
The Pearson's chi-square test or Fisher's exact test was used to assess the relationship between two nominal variables. The Student's t-test and Mann-Whitney test were used to compare two different groups of continuous parametric or nonparametric data, respectively. For the multivariate analysis, parameters that were significant at p < 0.25 in the univariate analysis were included in a multiple logistic regression test. Two-sided tests with p < 0.05 were considered to be statistically significant. Statistical analysis was performed with SPSS ver. 21.0 software (SPSS Inc., Chicago, IL, USA) and SAS ver. 9.3 software (SAS Institute Inc., Cary, NC, USA).

Meta-analysis of the proportion of TERT promoter mutations
We searched the literature for TERT promoter mutations in thyroid cancer using PubMed and Google up to November 2015, and selected eligible articles. We then conducted a meta-analysis of the proportion of TERT promoter mutations according to the histologic types of thyroid cancers. Cochran Q test and I 2 values were employed to assess statistical heterogeneity among studies. If significant heterogeneity was observed (p < 0.10 or I 2 > 50 %), the random effect model was used for metaanalysis. Otherwise, we used a fixed-effect model for the meta-analysis. Meta-analyses were performed using done using MedCalc version 13.0.2 software (MedCalc, Ostend, Belgium).
None of the 243 thyroid cancers had positive ALK immunohistochemistry or ALK break apart FISH (Table 1).

Relationship between clinicopathologic and molecular features and distant metastases of WDTCs
The mean follow-up period of the patients with WDTC was 36.1 months. In 14 patients, distant metastases were found within 6 months of first diagnosis. Distant metastases occurred in the lung (n = 24), bone (n = 3), lung and bone (n = 2), and brain (n = 1). Distant metastasis was associated with larger tumor size (p = 0.001), aggressive histologic subtype (p = 0.003), advanced pT    Table 3).

Impact of molecular genotypes on response of metastatic WDTCs to RAI therapy
Of the 30 WDTCs with distant metastasis, 9 (30 %) had coexisting BRAF V600E and TERT promoter mutations and 12 (40 %), including follicular and diffuse sclerosing variants of PTC, had no mutations (Fig. 2a).
The structural response of distant metastatic disease to RAI was evaluated at least 6 months after RAI therapy. Of the 30 WDTC patients with distant metastasis, six (20 %) patients had PR and six (20 %) had SD after RAI ablation whereas none achieved CR and 18 (60 %) had PD. There was a significant correlation between tumors with the BRAF V600E mutation alone and the progression of distant metastatic disease after RAI therapy (p = 0.025), but TERT promoter mutations alone were not associated with PD (Fig. 2b). PR or SD after RAI therapy was significantly more likely in patients with wildtype BRAF and TERT promoter genes (p = 0.024) (Fig. 2b). However, other combinations of genetic mutations were not correlated with RAI response.

Discussion
In our study, the BRAF V600E mutation was significantly associated with low response rate of metastatic WDTCs to RAI therapy. These results are consistent with previous studies that have demonstrated high frequency of BRAFV600E mutation in RAI-refractory metastatic thyroid cancers [1,47]. However, there was no significant effect of TERT promoter mutations on distant metastasis of WDTCs. The most likely mechanism of resistance to RAI therapy is the impaired iodidehandling machinery in metastatic thyroid cancer [1].
Many studies have reported that BRAFV600E mutation reduces the expression of thyroid iodine-handling genes (sodium iodide symporter, thyroid-stimulating hormone receptor, thyroglobulin, and thyroperoxidase) in thyroid cancer [1,47,48]. However, mechanism underlying the RAI therapy resistance associated with TERT promoter mutations remains uncertain. Xing et al reported that coexisting BRAF V600E and TERT C228T mutations defined the most aggressive subgroup of PTC when analyzed in terms of clinicopathologic features, tumor recurrence and disease-free survival rate [18]. We did not observe this trend in our study (data not shown).
Two TERT C228T and C250T mutations create consensus binding motifs for the E-twenty-six (ETS)/ternary complex transcription factor (TCF) and increase the transcriptional activity of the TERT promoter [19,23]. TERT promoter mutations in thyroid cancer and glioma were associated with increased mRNA expression and telomerase activity [17,49]. BRAF V600E and TERT promoter mutations can activate the mitogen-activated protein kinase (MAPK) signaling pathway in thyroid cancer [21]. In previous studies, TERT promoter mutations were more frequently found in BRAF V600E mutation-positive PTCs, suggesting an incremental and synergistic effect of the coexisting two mutations in   [18,21]. In our study, the TERT promoter mutation status was not associated with the incidence of the BRAF V600E mutation. These discrepancies may be associated with ethnic differences given that there is a higher prevalence of the BRAF V600E mutation and lower occurrence of TERT promoter mutations in Korean patients than in Western patients. Therefore, our study results cannot be generalized to other populations. We found no TERT promoter mutations in medullary carcinoma. This finding is consistent with previous reports [21,24]. Moreover, TERT promoter mutations were not found in benign thyroid nodules, whereas they were more prevalent in poorly differentiated or anaplastic carcinomas than in WDTCs [21,24]. Therefore, it is suggested that TERT promoter mutations are involved only in the tumorigenesis of follicular-cell derived thyroid cancers, particularly in aggressive subtypes, and may occur as a late molecular-genetic event that induces dedifferentiation of WDTCs [21].
ALK gene rearrangements are mutually exclusive with all other known thyroid cancer driver mutations and have been reported in up to 2.2 % of PTCs, 4 % of poorly differentiated carcinomas, and 4 % of anaplastic carcinomas [26,28,32]. In our study, ALK rearrangement was not identified in any thyroid cancers.
The main limitations of our study were the relatively small sample size of metastatic cancers and the short follow-up period. Although the analyses for disease recurrence and survival of patients were not available, we could evaluate the therapeutic response to RAI based on the distant metastatic disease genotypes. We report for the first time the clinical impact of TERT promoter mutations on thyroid cancers that occur in a BRAF V600E prevalent area.

Conclusions
Our study demonstrated that Korean patients have a higher BRAF V600E prevalence and lower prevalence of the TERT promoter mutation and ALK rearrangement in thyroid cancers than do Western patients. TERT promoter mutation is associated with aggressive clinicopathologic features and is a strong predictor of distant metastasis of WDTC. In Korea, the BRAF V600E-negative WDTCs more frequently develop distant metastasis than BRAF V600E-positive tumors. When WDTC patients develop distant metastases, RAI therapy is most effective in patients without BRAF V600E and TERT promoter mutations. Further prospective evaluation that includes a larger sample size is needed.