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

We retrospectively enrolled 243 patients who underwent thyroid surgery for thyroid cancer at Seoul St. Mary’s Hospital of The Catholic University of Korea with the approval of the Institutional Review Board. Informed consent was obtained from every patient. The thyroid cancers studied included 192 consecutive WDTCs without distant metastasis (consisting of 127 classic PTCs, 11 classic PTCs with tall cell features, 9 encapsulated follicular variant PTCs, 7 infiltrative follicular variant PTCs, 16 tall cell variant PTCs, 1 oncocytic PTC, 1 Warthin-like PTC, and 20 minimally invasive FTCs), 30 consecutive WDTCs with distant metastasis (consisting of 14 classic PTCs, 4 classic PTCs with tall cell features, 1 encapsulated follicular variant PTC, 1 macrofollicular variant PTC, 5 tall cell variant PTCs, 1 columnar cell variant PTC, 1 diffuse sclerosing variant PTC, 2 minimally invasive FTCs, and 1 widely invasive FTC), 7 poorly differentiated carcinomas, 5 anaplastic carcinomas, and 9 medullary carcinomas. 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].

Genomic DNA was isolated from manually dissected 10-μm thick paraffin-embedded tissue sections using the RecoverAll™ Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA). Sanger sequencing was performed to detect the presence of BRAF V600E and TERT promoter mutations. Exon 15 of the BRAF gene was PCR-amplified as previously reported using the following forward primer (5′-TCATAATGCTTGCTCTGATAGGA-3′) and reverse primer (5′-GGCCAAAAATTTAATCAGTGGA-3′), resulting in a 224 bp PCR product [11, 33, 34]. A 193 bp fragment of the TERT promoter was amplified by PCR as previously reported using the following forward primer (5′-CACCCGTCCTGCCCCTTCACCTT-3′) and reverse primer (5′-GGCTTCCCACGTGCGCAGCAGGA-3′) [35]. All TERT promoter mutations were confirmed using another previously reported primer set that included the following forward primer (5′-AGTGGATTCGCGGGCACAGA-3′) and reverse primer (5′-CAGCGCTGCCTGAAACTC-3′) and resulted in a 235 bp PCR product [21].

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].

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].

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).

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 I² values were employed to assess statistical heterogeneity among studies. If significant heterogeneity was observed (p < 0.10 or I² > 50 %), the random effect model was used for meta-analysis. 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).

TERT promoter mutations were found in 12 (40 %) of 30 WDTCs with distant metastasis, 2 (29 %) of 7 poorly differentiated carcinomas, and 3 (60 %) of 5 anaplastic carcinomas. However, no such mutations were present in the 192 WDTCs without distant metastasis or the 9 medullar carcinomas (Table 1). Among TERT promoter mutations, the most common type was C228T (76 %), followed by C250T (18 %) and C250A (6 %) (Table 1) (Fig. 1). Among 12 WDTCs with TERT promoter mutations, the most frequent histologic subtype was the tall cell variant of PTC (Table 1).

The BRAF V600E mutation was found in 142 (83 %) of 172 PTCs without distant metastasis, 15 (56 %) of 27 PTCs with distant metastasis, 1 (14 %) of 7 poorly differentiated carcinomas and 4 (80 %) of 5 anaplastic carcinomas (Table 1). However, the BRAF V600E mutation was not found in 23 FTCs and 9 medullary carcinomas.

None of the 243 thyroid cancers had positive ALK immunohistochemistry or ALK break apart FISH (Table 1).

In 222 patients with WDTC, the presence of TERT promoter mutations was associated with older age (p = 0.017), larger tumor size (p = 0.043), aggressive histologic subtypes (p < 0.001), advanced pathologic T stage (p = 0.014), extrathyroidal extension (p = 0.035), lymph node metastasis (p = 0.011), lateral lymph node metastasis (p < 0.001), distant metastasis (p < 0.001), and advanced AJCC stage (p < 0.001) (Table 2). There was no association between TERT promoter mutations and the BRAF V600E mutation (Table 2).

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 stage (p < 0.001), extrathyroidal extension (p = 0.001), lymph node metastasis (p < 0.001), lateral lymph node metastasis (p < 0.001) and the TERT promoter mutation (p < 0.001). However, the BRAF V600E mutation was inversely associated with distant metastasis (p = 0.007).

In the multivariate analysis, the odds ratio (OR) for distant metastasis of WDTC in patients harboring tumors with TERT promoter mutations and lateral lymph node metastases was 155.298 (95 % confidence interval (CI) 3.362–999.990) and 11.159 (95 % CI 1.902–65.461), respectively (Table 3). The OR for distant metastases of WDTC in patients harboring tumors with the BRAF V600E mutation was 0.083 (95 % CI 0.021–0.327) (Table 3).

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 wild-type BRAF and TERT promoter genes (p = 0.024) (Fig. 2b). However, other combinations of genetic mutations were not correlated with RAI response.

Our study and 13 articles were included for the meta-analysis of TERT promoter mutation prevalence in various thyroid cancers [17, 18, 21, 24, 25, 32, 37‐43]. Significant heterogeneity was found in classic PTC, FTC, Hürthle cell carcinoma, and anaplastic carcinoma among the studies (Figs. 3 and 4). The mean frequencies of TERT promoter mutations in PTC, conventional FTC, Hürthle cell carcinoma, poorly differentiated carcinoma and anaplastic carcinoma were 11.3 % (95 % CI 9.3–13.5), 21.3 % (95 % CI 14.2–29.4), 6.7 % (95 % CI 0.2–21.4), 39.6 % (95 % CI 31.3–48.2), and 38.5 % (95 % CI 32.6–44.7), respectively (Figs. 3 and 4). When PTCs were stratified by histologic subtype, mean frequencies of TERT promoter mutations in classic, follicular, and tall cell variants were 8.8 % (95 % CI 6.8–11.1), 6.6 % (95 % CI 4.5–9.3), 27.5 % (95 % CI 21.0–34.7), respectively (Fig. 3). TERT promoter mutations were not found in a total of 132 medullary carcinoma patients including our case series [17, 21, 24, 25, 41].