Prognostic Significance of TERT Promoter Mutations in Papillary Thyroid Carcinomas in a BRAF V600E Mutation

Abstract

Background:

The role of telomerase reverse transcriptase (TERT) promoter mutations in differentiated thyroid cancer has been well established. These mutations have a significantly higher prevalence in aggressive thyroid tumors, including widely invasive oncocytic carcinomas, poorly differentiated carcinomas, and anaplastic thyroid carcinomas. Interestingly, in some studies, TERT mutations were found to be more common in tumors with a BRAF^V600E mutation. However, mutational analysis of TERT promoter mutations in thyroid tumors has not been previously performed for patients in Korea, where the BRAF^V600E mutation in papillary thyroid carcinoma (PTC) is particularly prevalent. This study analyzed TERT promoter mutations in various thyroid tumors and examined their relationship with clinicopathologic factors and the BRAF^V600E mutation in PTC cases.

Methods:

Using 242 preoperative fine-needle aspiration biopsy specimens (including 207 PTCs) with confirmed histopathological diagnosis of the biopsied thyroid nodules, the TERT promoter status (C228T and C250T) was analyzed, and the relationship with clinicopathologic factors and the BRAF^V600E mutation in PTC cases was examined.

Results:

Of 242 patients, 14.5% (30/207), 26.7% (4/15), 50% (1/2), and 60% (2/5) of PTCs, follicular thyroid carcinomas, poorly differentiated carcinomas, and anaplastic thyroid carcinomas harbored a TERT^C228T mutation, respectively. The TERT^C228T mutation was associated with recurrence (p = 0.03). However, no association with other clinicopathologic factors in PTC was found. Coexistence of TERT^C228T and BRAF^V600E mutations was found in 13.0% of PTCs and was significantly associated with older age and advanced stage compared with the group negative for either mutation. The TERT^C228T mutation status was an independent prognostic factor for recurrence-free survival (hazard ratio = 3.08 [confidence interval 1.042–9.079]; p = 0.042) in patients with PTC in multivariate analysis.

Conclusions:

Identification of TERT promoter mutations in preoperative fine-needle aspiration biopsy specimens may help in better characterizing the prognosis and triaging thyroid cancer patients for appropriate treatment.

Introduction

Thyroid cancer typically presents as a thyroid nodule, commonly found incidentally and seen in up to 50% of patients >60 years of age (1,2). Most thyroid cancers are well-differentiated (WD) papillary carcinomas or follicular thyroid carcinomas (FTCs). Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer (3), and it accounts for 95% of thyroid cancer cases in Korea (4). PTC is associated with the best survival of all types of thyroid cancer, with five-year survival rates reaching 96% (5). Although the majority of PTC patients have an excellent survival prognosis, recurrence occurs in 15–35% of patients (6,7). Accurate identification of subsets of patients with risk factors for aggressive disease that have higher mortality rates can help guide management, and it can also prevent overtreatment of patients with low-risk disease. Several efforts have led to insights in how better to distinguish high-risk patients with aggressive PTC from the majority of low-risk patients (8 –11).

Thyroid cancer, like other human cancers, is genetically driven, and over the last several years, significant progress has been made in understanding underlying genetic mechanisms. The activating point mutation BRAF^V600E is the most common genetic alteration in PTCs and is present in approximately 29–77% of PTC cases (12 –14). The prevalence of the BRAF^V600E mutation in PTC is much higher in Korea (73–87%) than it is in Western countries (15 –17). Therefore, mutational analysis for the BRAF^V600E mutation in preoperative fine-needle aspiration biopsy (FNAB) specimens can increase the preoperative diagnostic accuracy for PTC in Korea (17,18). An increasing number of studies that include meta-analyses have been able to demonstrate an association between the BRAF status and aggressive tumor behavior (13,19 –21). Other studies, however, have failed to confirm these data, and this has resulted in uncertainty about the prognostic value of BRAF mutations in PTC (22 –27). The prognostic significance of the BRAF^V600E mutation has been especially controversial in Korea. Although some studies have reported an association between the BRAF^V600E mutation and extrathyroidal extension (ETE) and lymph node metastasis (4,28,29), most studies have failed to demonstrate an association with more aggressive clinicopathologic features in Korea (4,16,30 –33).

Recently, the role of telomerase reverse transcriptase (TERT) promoter mutations, which contribute to aggressive behavior in differentiated thyroid cancer, has been documented (34 –38). TERT promoter mutations have been observed in 4.7–25% of differentiated thyroid cancers (34 –39). These mutations have a significantly higher prevalence in aggressive thyroid tumor types such as widely invasive oncocytic carcinomas, poorly differentiated (PD) carcinomas, and anaplastic thyroid carcinomas (ATC) (34 –36,40). TERT is the catalytic subunit of the telomerase complex and is a predominant determinant for controlling telomerase activity. Telomerase plays a key role in increasing the longevity of cells by maintaining the length of telomere caps at the end of chromosomes. Telomerase activation is involved in mechanisms of tumorigenesis and telomerase activity is upregulated in 85–90% of cancers (41). The mechanisms of telomerase reactivation in cancer have yet to be fully explored. It is known that activating mutations in the promoter of the TERT gene lead to increased telomerase expression. These TERT promoter mutations include two hot spots (C228T and C250T) that have been identified in various types of tumors. TERT promoter mutations were first reported in melanoma (42) and have subsequently been identified in urothelial carcinomas, gliomas, and hepatocellular carcinomas (43 –45). TERT promoter mutations have been associated with bladder cancer recurrence and poor survival of glioma patients (44,45).

In thyroid cancer, TERT promoter mutations are prevalent in advanced tumors with a higher recurrence (35). Interestingly, a significant association of a TERT^C228T with a BRAF mutation has been reported, and it has been proposed that their co-occurrence may define a more aggressive subgroup of PTCs (37). The TERT^C228T promoter mutation creates a consensus binding site for E-twenty-six (ETS)/ternary complex factors (TCFs) transcription factors, including GA-binding protein (GABP) (42,46,47), and the BRAF^V600E activated mitogen-activated protein kinase (MAPK) pathway induces expression of ETS transcription factor members (48). Hence, increased generation and interaction of ETS factors results in promoting the upregulation of the TERT promoter and subsequent telomerase activation. TERT promoter mutation analyses on thyroid FNAB specimens have not been performed in the Korean population, where the BRAF^V600E mutation in PTC is prevalent. This study analyzed various thyroid tumors for the presence of TERT promoter mutations and examined their relationship with clinicopathologic factors and co-occurrence with the BRAF^V600E mutation.

Materials and Methods

Patient selection and characteristics

Study approval was obtained from the Institutional Review Board (IRB) of Konkuk University Medical Center (KUH1210042). This study included 242 patients who underwent thyroidectomy for thyroid nodules between 2007 and 2014 at Konkuk University Medical Center in Seoul, Korea. Two hundred and forty-two FNAB specimens obtained preoperatively from the same number of patients with confirmed postoperative pathological diagnosis were evaluated. Also archival thyroid FNAB and resected pathologic specimens that had preoperative BRAF mutation analysis were blindly reevaluated according to the 2010 Bethesda System for Reporting Thyroid Cytopathology and 2004 World Health Organization (WHO) classification of thyroid neoplasm by two pathologists (T.S.H. [endocrine pathologist] and S.E.L.). In cases of a disagreement and to reach a consensus, another endocrine pathologist (C.K.J.) independently reviewed the cases. Among the 242 cases, six were classified as follicular adenomas (FA), 207 as PTC, 15 as FTC (six minimally invasive and nine widely invasive), two as PD carcinoma, five as ATC, six as medullary thyroid carcinomas (MTC), and one as squamous-cell carcinoma (SCC). Of 207 PTCs, 143 were classified as conventional, 24 as follicular variant, two as solid variant, two as Warthin-like variant, and one as oncocytic variant. Among 143 conventional PTCs (CPTCs), 18 were categorized as CPTC with ≥50% tall-cell component (TCC), 12 as CPTC with 10–50% TCC, two as CPTC with squamous-cell differentiation (CPTC with SD), and three as CPTC with ≥50% TCC and SD. The diagnosis of TCC was made on the basis of the definition in the 2004 WHO classification with tall columnar tumor cells having a height three times the width, having prominent cell borders, and a large amount of dense eosinophilic cytoplasm.

For PTCs, tumors >1 cm in diameter were selected. All samples of CPTC with ≥50% TCC, known as an aggressive variant of PTC, were selected in order to have a representative frequency of approximately 10% for the PTCs. All FTC, MTC, PD carcinomas, ATC, and some FA cases were analyzed. The total study population in the cohort consisted of 187 women (77.0%) and 56 men (23.0%), with a mean age of 48.4 ± 13.9 years.

DNA extraction

After slipping off the cover slips of FNAB cytology slides, atypical cells of interest were scraped, and DNA was extracted. Briefly, 20–50 μL of DNA extraction buffer solution (50 mM Tris buffer, pH 8.3; 1 mM EDTA, pH 8.0; 5% Tween 20 and 100 μg/mL proteinase K) with 10% resin was added to the scraped cells and incubated at 56.8°C for a minimum of 1 h. After incubation, the tubes were heated to 100°C for 10 min, followed by centrifugation to pellet the debris, and 5 μL of the supernatant was used in the polymerase chain reaction (PCR).

Detection of the BRAF mutation by PCR amplification and pyrosequencing

Each PCR mixture contained forward and reverse primers (each 0.4 pmol), 0.2 mmoL of each dNTP, 1.5 mmol/L of MgCl₂, 1 × PCR buffer, 1.5 IU of Immolase DNA polymerase (Bioline, London, United Kingdom), and 5 μL of genomic DNA in a total volume of 50 μL. The PCR products were electrophoresed in a 2% agarose gel to confirm amplification of the PCR product. The biotinylated PCR product (20 μL) was attached to streptavidin–Sepharose beads (Amersham Biotechnology, Uppsala, Sweden) according to a standard protocol by incubating with shaking at room temperature for 10 min in binding buffer. The streptavidin–Sepharose beads were captured using a PSQ 96 sample prep tool with a 96 magnetic ejectable microcylinder (Biotage, Uppsala, Sweden). This tool was also used for a 1 min incubation of the biotin–streptavidin complex in 0.5 M of NaOH before washing in annealing buffer. Subsequently, the samples were hybridized to 15 pmol of sequencing primers in annealing buffer at 80°C for 2 min followed by cooling to room temperature. PCR primer and sequencing primer sequences were as follows: exon 15 (T1799A) forward, 5′-biotin-CTT CAT AAT GCT TGC TCT GAT AGG-3′; reverse, 5′-GGC CAA AAA TTT AAT CAG TGG AA-3′; sequencing reverse, 5′-CCA CTC CAT CGA GAT TT-3′. Pyrosequencing was performed using an SNP reagent kit (Biotage).

Identification of TERT promoter mutations by PCR amplification and direct sequencing

Standard PCR was carried out for sequencing to identify TERT promoter mutations. Briefly, a fragment of the TERT promoter was amplified by PCR using genomic DNA with the following primers: 5′-AGT GGA TTC GCG GGC ACA GA-3′ (sense) and 5′-CAG CGC TGC CTG AAA CTC-3′ (antisense). About 40–50 ng of genomic DNA was used in the PCR, which was carried out with an initial denaturation step at 95°C for 3 min, followed by 10 cycles of 95°C denaturation for 30 sec, 58–62°C annealing for 30 sec, and 68°C elongation for 1 min. This was then followed by 30 cycles of the same settings, except for elongation for an additional 5 sec in each cycle. The PCR was completed with a final elongation step at 68°C for 7 min. The PCR products were subsequently subjected to sequencing with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA), with the following PCR cycles: 35 cycles of denaturation for 60 sec at 96°C, annealing for 5 sec at 50°C, and elongation for 4 min at 60°C. PCR was performed at a final volume of 10 μL containing 2.5 μL of 70 ng/μL of PCR product, 0.5 μL of sequencing primer (3 picomoles), 5 × buffer, and AmpliTaq DNA polymerase. The sDNA sequence was then read on an ABI 3730XL DNA Analyzer (Applied Biosystems). Sequences were aligned using the ContigExpress alignment program (InforMax, Frederick, MD).

Statistical analysis

For analysis of the relationship between clinicopathologic factors, including sex, multiplicity, extrathyroidal extension (ETE), and presence of TERT^C228T/BRAF^V600E mutation, Pearson's chi-square test and Fisher's exact test were used. A linear-by-linear test was used to examine the association between T stage, N stage, AJCC stage, and TERT^C228T/BRAF^V600E mutations. The time to recurrence-free survival (RFS) was defined from the day of first surgery until recurrence. The survival rates were calculated using the Kaplan–Meier method, and the survival curves were compared with a log-rank test. The Cox proportional hazard model was used to evaluate the associations between clinicopathologic factors and RFS. The hazard ratio (HR) and its confidence interval (CI) were assessed for each factor. All tests were two-sided, with 0.05 serving as the level of significance. Statistical analysis was performed using SPSS software version 18.0 (SPSS, Inc., Chicago, IL).

Results

Prevalence of TERT^C228T/C250T and BRAF^V600E mutations in various thyroid tumors

The status of two TERT promoter mutations along with the BRAF^V600E mutation were analyzed in 242 thyroid tumors using FNAB specimens obtained preoperatively. Mutations in the TERT promoter and BRAF^V600E were identified in 40 (16.5%) and 177 (73.1%) out of 242 tumor samples, respectively. All TERT promoter mutations were C228T mutations. None of the cases had a C250T mutation. The mutation frequency in each tumor type is shown in Table 1. The frequency of TERT^C228T and BRAF^V600E mutations was significantly different according to the histologic type of the thyroid tumor (p = 0.002 and p < 0.001, respectively). A TERT^C228T mutation was found in 1/6 (16.7%) FAs. Of 222 WD thyroid cancers (207 PTCs and 15 FTCs), 34 (15.3%) harbored a TERT^C228T mutation (14.5% of PTC and 26.7% of FTC). The mutation frequency in each PTC variant was as follows: 23/143 (16.1%) CPTCs without other component, 2/24 (8.3%) FVPTCs, and 1/2 (50%) Warthin-like variant PTCs. Among CPTCs with TCC, one case with 10–50% TCC (8.3%) harbored a TERT^C228T mutation. No TERT^C228T mutation was found in CPTC with ≥50% TCC, whereas all three CPTCs with ≥50% TCC and SD cases harbored a TERT^C228T mutation (Fig. 1). Collectively, from 33 cases that contained at least 10% TCC, four cases (12.1%) harbored a TERT^C228T mutation. Two CPTCs with SD, two solid variant PTCs, and one oncocytic variant PTC did not harbor a TERT^C228T mutation. Even though the numbers are small, 50% of PD carcinomas and 60% of ATCs showed a TERT^C228T mutation. One SCC, a rare primary thyroid epithelial tumor, had a TERT^C228T mutation. On the other hand, a TERT^C228T mutation was not observed in any of the six MTCs. The BRAF^V600E mutation was found in 84.1% of PTCs, 50% of PD carcinomas, and 20% of ATCs, whereas none of the FAs, FTCs, and MTCs harbored a BRAF^V600E mutation.

FIG. 1.

Representative case showing a papillary thyroid carcinoma histology with >50% of tall-cell component (A) and the associated electropherogram demonstrating a TERT^C228T mutation (B).

Table 1.

Frequency of TERT ^C228T and BRAF ^V600E Mutations in Thyroid Tumors

		TERT^C228T status		BRAF^V600E status
Histologic type	Total (n = 242)	Wild type (n = 202; 83.5%)	Mutant type (n = 40; 16.5%)	Wild type (n = 60; 24.8%)	Mutant type (n = 177; 73.1%)	N/A (n = 5; 2.1%)
FA	6 (2.5%)	5 (83.3%)	1 (16.7%)	6 (100.0%)	0	0
PTC	207 (85.5%)	177 (85.5%)	30 (14.5%)	32 (15.5%)	174 (84.1%)	1 (0.5%)
CPTC	178 (73.6%)	151 (84.8%)	27 (15.2%)	18 (10.1%)	159 (89.3%)	1 (0.6%)
Without other component	143 (59.1%)	120 (83.9%)	23 (16.1%)	16 (11.2%)	127 (88.8%)	0
With SD	2 (0.8%)	2 (100.0%)	0	0	2 (100.0%)	0
With TCC (10–50%)	12 (5.0%)	11 (91.7%)	1 (8.3%)	0	12 (100.0%)	0
With TCC (>50%)	18 (7.4%)	18 (100.0%)	0	2 (11.1%)	16 (88.9%)	0
With TCC (>50%) and SD	3 (1.2%)	0	3 (100.0%)	0	2 (66.7%)	1 (33.3%)
FVPTC	24 (9.9%)	22 (91.7%)	2 (8.3%)	13 (54.2%)	11 (45.8%)	0
FVPTC-E	13 (5.4%)	12 (92.3%)	1 (7.7%)	9 (69.2%)	4 (30.8%)	0
FVPTC-I	11 (4.5%)	10 (90.9%)	1 (9.1%)	4 (36.4%)	7 (63.6%)	0
Warthin-like variant	2 (0.8%)	1 (50.0%)	1 (50.0%)	0	2 (100.0%)	0
Oncocytic variant	1 (0.4%)	1 (100.0%)	0	0	1 (100.0%)	0
Solid variant	2 (0.8%)	2 (100.0%)	0	1 (50.0%)	1 (50.0%)	0
FTC	15 (6.2%)	11 (73.3%)	4 (26.7%)	15 (100.0%)	0	0
FTC-M	6 (2.5%)	4 (66.7%)	2 (33.3%)	6 (100.0%)	0	0
FTC-W	9 (3.7%)	7 (77.8%)	2 (22.2%)	9 (100.0%)	0	0
PD carcinoma	2 (0.8%)	1 (50.0%)	1 (50.0%)	1 (50.0%)	1 (50.0%)	0
ATC	5 (2.1%)	2 (40.0%)	3 (60.0%)	2 (40.0%)	1 (20.0%)	2 (40.0%)
MTC	6 (2.5%)	6 (100.0%)	0	4 (66.7%)	0	2 (33.3%)
SCC	1 (0.4%)	0	1 (100.0%)	0	1 (100.0%)	0

FA, follicular adenoma; CPTC, classical PTC; CPTC with SD, classical PTC with squamous differentiation; CPTC with TCC, classical PTC with tall-cell component; FVPTC-E, encapsulated follicular variant of PTC; FVPTC-I, infiltrative follicular variant of PTC; FTC-M, minimally invasive follicular carcinoma; FTC-W, widely invasive follicular carcinoma; PD, poorly differentiated; ATC, anaplastic thyroid carcinoma; MTC, medullary thyroid carcinoma; SCC, squamous cell carcinoma; N/A, not applicable.

Clinicopathologic demographics of the patients with PTC

The relationship with the TERT^C228T mutation and the BRAF^V600E mutation in 207 PTC cases was also analyzed. The study population consisted of 161 women (77.8%) and 46 men (22.2%), with a mean age of 47.5 ± 13.4 years. Eighty-eight patients (42.5%) were <45 years old, and 119 patients (57.5%) were >45 years old. Of these, 158 patients (90.8%) underwent total thyroidectomy, and 19 patients (9.2%) underwent lobectomy. Median duration of follow-up was 48 months (range 0–95 months) after surgery. Of 207 patients, recurrence occurred in 18 (8.7%) patients. Tumor recurrence was present in 10/143 (7.0%) CPTCs, 1/13 (7.7%) encapsulated FVPTCs, 1/11 (9.1%) infiltrative FVPTCs, 1/18 (5.6%) CPTCs with ≥50% TCC, 1/12 (8.3%) CPTCs with 10–50% TCC, 1/2 (50%) CPTCs with SD, 2/3 (66.7%) CPTCs with ≥50% TCC and SD, and 1/2 (50%) Warthin-like variant PTCs. No recurrence was identified in one solid and one oncocytic variant PTC. The recurrence rate according to the histologic type was statistically different (p = 0.022).

Association between TERT^C228T/BRAF^V600E mutations and clinicopathologic features of PTC

Of 207 patients with PTC, 30 (14.5%) and 175 (84.5%) harbored either a TERT^C228T or a BRAF^V600E mutation, respectively. The association of a TERT^C228T or a BRAF^V600E mutation and clinicopathologic factors in PTC patients was evaluated (Table 2). Patients with the TERT^C228T mutation were more likely to have a recurrence (p = 0.029). However, there was no significant difference in TERT^C228T mutation status regarding sex, age at diagnosis, size, tumor multiplicity, ETE, T stage, N stage, and AJCC stage.

Table 2.

Relationship of TERT ^C228T/BRAF^V600E Mutation and Clinicopathologic Factors in 207 Papillary Thyroid Carcinomas

		TERT^C228T status			BRAF^V600E status
	Total (n = 207)	Wild type (n = 177; 85.5%)	Mutant type (n = 30; 14.5%)	p-Value	Wild type (n = 32; 15.5%)	Mutant type (n = 175, 84.5%)	p-Value
Sex
Male	46 (22.2%)	37 (20.9%)	9 (30.0%)	0.268	5 (15.6%)	41 (23.4%)	0.329
Female	161 (77.8%)	140 (79.1%)	21 (70.0%)		27 (84.4%)	134 (76.6%)
Age at diagnosis (years)	46 ± 13.4	47 ± 13.0	49 ± 15.4	0.647	40 ± 12.4	49 ± 13.2	0.001
Size	1.76 ± 0.9	1.75 ± 0.9	1.82 ± 0.9	0.69	1.9 ± 1.1	1.7 ± 0.9	0.364
Multiplicity
No	131 (63.3%)	109 (61.6%)	22 (73.3%)	0.217	28 (87.5%)	103 (58.9%)	0.002
Yes	76 (36.7%)	68 (38.4%)	8 (26.7%)		4 (12.5%)	72 (41.1%)
ETE
No	107 (51.7%)	94 (53.1%)	13 (43.3%)	0.322	21 (65.6%)	86 (49.1%)	0.086
Yes	100 (48.3%)	83 (46.9%)	17 (56.7%)		11 (34.4%)	89 (50.9%)
T stage
1	83 (40.1%)	75 (42.4%)	8 (26.7%)	0.231	16 (50.0%)	67 (38.3%)	0.178
2	23 (11.1%)	19 (10.7%)	4 (13.3%)		5 (15.6%)	18 (10.3%)
3	101 (48.8%)	83 (46.9%)	18 (60.0%)		11 (34.4%)	90 (51.4%)
N stage
0	103 (49.8%)	89 (50.3%)	14 (46.7%)	0.43	21 (65.6%)	82 (46.9%)	0.126
1a	66 (31.9%)	58 (32.8%)	8 (26.7%)		8 (25.0%)	58 (33.1%)
1b	38 (18.4%)	30 (16.9%)	8 (26.7%)		3 (9.4%)	35 (20.0%)
AJCC stage
I	114 (55.1%)	100 (56.5%)	14 (46.7%)	0.699	26 (81.3%)	88 (50.3%)	0.009
II	9 (4.3%)	8 (4.5%)	1 (3.3%)		0	9 (5.1%)
III	68 (32.9%)	56 (31.6%)	12 (40.0%)		6 (18.8%)	62 (35.4%)
IVA	16 (7.7%)	13 (7.3%)	3 (10.0%)		0	16 (9.1%)
Recurrence
No	189 (91.3%)	165 (93.2%)	24 (80.0%)	0.029	29 (90.6%)	160 (91.4%)	1
Yes	18 (8.7%)	12 (6.8%)	6 (20.0%)		3 (9.4%)	15 (8.6%)

Statistically significant values are shown in bold.

ETE, extrathyroidal extension.

The BRAF^V600E mutation was significantly associated with age at diagnosis, multiplicity, and AJCC stage. Patients with a BRAF^V600E mutation were older than those with wild-type BRAF (p = 0.001). The mutation was found more frequently in advanced stages, whereas wild-type BRAF was more prevalent in stage I cases (p = 0.009). However, recurrence rate was not different according to BRAF^V600E mutation status (p = 1.000).

Association between TERT^C228T and BRAF^V600E mutations

A TERT^C228T mutation was found in 15.4% (27/175) and 9.4% (3/32) of BRAF^V600E mutant and wild-type BRAF cases, respectively. Conversely, the BRAF^V600E mutation was found in 90.0% (27/30) and 83.6% (148/177) of TERT^C228T mutant and wild-type TERT tumors, respectively. The TERT^C228T mutation was not significantly associated with BRAF^V600E mutation (p = 0.584).

The cases were also classified into four groups according to BRAF/TERT mutation status as follows: BRAF–/TERT– (n = 29; 14%); BRAF+/TERT– (n = 148; 71.5%); BRAF–/TERT+ (n = 3; 1.4%); and BRAF+/TERT+ (n = 27; 13.0%). The clinicopathologic characteristics among these four groups were also evaluated in Table 3. Both the TERT^C228T and BRAF^V600E mutant group was significantly associated with older age and advanced stage compared with the BRAF–/TERT– group.

Table 3.

Association of BRAF/TERT Mutation Status and Clinicopathologic Factors in 207 Papillary Thyroid Carcinomas

	Total (n = 207)	BRAF–/TERT– (n = 29, 14%)	BRAF+/TERT– (n = 148, 71.5%)	p-Value	BRAF–/TERT+ (n = 3, 1.4%)	p-Value	BRAF+/TERT+ (n = 27, 13.0%)	p-Value
Sex
Male	46 (22.2%)	4 (13.8%)	33 (22.3%)	0.303	1 (33.3%)	0.41	8 (29.6%)	0.149
Female	161 (77.8%)	25 (86.2%)	115 (77.7%)		2 (66.7%)		19 (70.4%)
Age at diagnosis (years)	46 ± 13.4	40 ± 11.42	49 ± 12.96	0.001	41001.96	0.967	49967.96	0.012
Size	1.762.96	1.962.96	1.762.96	0.386	2.086.96	0.862	1.862.96	0.767
Multiplicity
No	131 (63.3%)	25 (86.2%)	84 (56.8%)	0.003	3 (100%)	1	19 (70.4%)	0.244
Yes	76 (36.7%)	4 (13.8%)	64 (43.2%)		0		8 (29.6%)
ETE
No	107 (51.7%)	18 (62.1%)	76 (51.4%)	0.29	3 (100%)	0.534	10 (37.0%)	0.061
Yes	100 (48.3%)	11 (37.9%)	72 (48.6%)		0		17 (63.0%)
T stage
1	83 (40.1%)	14 (48.3%)	61 (41.2%)	0.514	2 (66.7%)	0.379	6 (22.2%)	0.076
2	23 (11.1%)	4 (13.8%)	15 (10.1%)		1 (33.3%)		3 (11.1%)
3	101 (48.8%)	11 (37.9%)	72 (48.6%)		0		18 (66.7%)
N stage
0	103 (49.8%)	20 (69.0%)	69 (46.6%)	0.11	1 (33.3%)	0.266	13 (48.1%)	0.158
1a	66 (31.9%)	6 (20.7%)	52 (35.1%)		2 (66.7%)		6 (22.2%)
1b	38 (18.4%)	3 (10.3%)	27 (18.2%)		0		8 (29.6%)
AJCC stage
I	114 (55.1%)	24 (82.8%)	76 (51.4%)	0.017	2 (66.7%)	0.476	12 (44.4%)	0.006
II	9 (4.3%)	0	8 (5.4%)		0		1 (3.7%)
III	68 (32.9%)	5 (17.2%)	51 (34.5%)		1 (33.3%)		11 (40.7%)
IVA	16 (7.7%)	0	13 (8.8%)		0		3 (11.1%)
Recurrence
no	189 (91.3%)	27 (93.1%)	138 (93.2%)	1	2 (66.7%)	0.263	22 (81.5%)	0.244
yes	18 (8.7%)	2 (6.9%)	10 (6.8%)		1 (33.3%)		5 (18.5%)

p-Values are from the comparison of the indicated genetic group in the column immediately left of the p-value column with the no mutation group. Statistically significant values are shown in bold.

Relationship of clinicopathologic factors with RFS

When the patients were divided into two groups based on TERT^C228T mutation status, the TERT^C228T mutant group showed significantly lower RFS (p = 0.014) than the wild-type group did. Kaplan–Meier survival curves and corresponding p-values are shown in Figure 2.

FIG. 2.

Kaplan–Meier survival curves with log-rank test of recurrence-free survival after classification into two groups based on the presence of a TERT^C228T mutation (A) or a BRAF^V600E mutation (B).

The prognostic relevance of clinicopathologic and biologic parameters on RFS of the patients with PTC was analyzed. RFS was significantly associated with tumor size (p = 0.001), ETE (p = 0.036), nodal stage (p = 0.004), and TERT^C228T mutation status (p = 0.020) in univariate analysis. In multivariate analysis, tumor size (p = 0.019) and TERT^C228T mutation status (p = 0.042) remained as an independent predictor of tumor recurrence when other clinicopathologic cofactors including BRAF^V600E mutation were adjusted (Table 4).

Table 4.

Multivariate Analysis of Clinicopathologic Factors for Recurrence in Papillary Thyroid Carcinoma

	Univariate		Multivariate
	HR [CI]	p-Value	HR [CI]	p-Value
Age
<45 years	1	0.804
≥45 years	1.128 [0.437–2.911]
Size	1.849 [1.298–2.635]	0.001	1.554 [1.075–2.247]	0.019
Tumor multiplicity	1.087 [0.421–2.807]	0.863
ETE	3.02 [1.075–8.466]	0.036	1.798 [0.575–5.617]	0.313
N stage
0	1	0.004	1	0.096
1a	1.32 [0.355–4.926]	0.677	1.141 [0.297–4.381]	0.848
1b	5.43 [1.818–16.20]	0.002	3.312 [0.976–11.242]	0.055
BRAF^V600E status	0.74 [0.210–2.593]	0.635	0.567 [0.154–2.085]	0.393
TERT^C228T status	3.197 [1.199–8.522]	0.020	3.076 [1.042–9.079]	0.042

Statistically significant values are shown in bold.

HR, hazard ratio; CI, confidence interval.

Discussion

The frequency of TERT promoter mutations was evaluated using 242 FNAB specimens of thyroid tumors. The frequency of TERT promoter mutations has been reported to vary between 7.5% and 25% in WD thyroid cancers, and it was found in up to 50% of oncocytic carcinomas, PD carcinomas, and ATCs (34 –36,40). In the present study, 14.5% (30/207) of PTCs, 26.7% (4/15) of FTCs, 50.0% (1/2) of PD carcinomas, and 60.0% (3/5) of ATCs harbored a TERT^C228T mutation. Although the frequency of TERT promoter mutations in FTC was slightly higher than those in previous studies, this stepwise increase from differentiated to PD and ATC is in agreement with previous data (34 –37). The tall-cell variant of PTC (TVPTC) is known to be associated with a less favorable prognosis (49,50). Liu et al. (35) found that the prevalence of TERT mutations in TVPTC (30.8%) was higher than that in CPTC (35). In the present study, out of 33 PTCs containing at least 10% TCC, four (12.1%) cases harbored the TERT^C228T mutation, which is lower than the prevalence reported in a previous study (35). However, Dettmer et al. recently identified that TERT mutations were found in 15.9% (7/44) of PTCs with a TCC component >10%, and the majority of TVPTCs with adverse outcome did not harbor TERT mutations (51). In the present study, the majority of patients with PTCs harboring a TCC component of >10% also did not develop recurrences. Because results on the prevalence of TERT mutations in Korean TVPTC cases are not available for comparison, a multicenter larger cohort study with PTCs harboring TCC needs to be performed. TERT promoter mutations were identified in 16.7% (1/6) of FAs. The identification of TERT promoter mutations in benign thyroid neoplasms was previously reported, and the authors suggested that TERT promoter mutations may occur as an early genetic event in thyroid follicular tumors (52).

Most of the TERT mutation analysis in thyroid neoplasm was performed with formalin-fixed paraffin-embedded (FFPE) specimens. One recent TERT promotor mutation analysis performed on thyroid FNAB revealed that 3.6% of the PTCs harbored a mutation, and the authors suggested that the lower observed prevalence might be attributed to the low sensitivity of the direct sequencing method on FNAB specimens due to sparse cancer cells (53). However, the prevalence of TERT promoter mutations in thyroid FNAB samples in the present study does not differ from that of the several recent studies performed on FFPE specimens (34 –38). Even though we did not compare the prevalence of TERT mutations between FNAB and matched FFPE specimens, BRAF mutation analysis using FNAB and matched FFPE specimens in the laboratory showed a 97.5% concordance rate (not published). Mutational analysis using FNAB specimens is more sensitive and specific when target cells are dissected properly. Moreover, FNAB specimens with alcohol fixation can preserve DNA better than FFPE specimens can.

Next, the relationships of TERT promoter and BRAF^V600E mutation status and clinicopathologic factors in PTC specimens were evaluated. Among clinicopathologic factors, only recurrence rate was related with the presence of a TERT^C228T mutation. The BRAF^V600E mutation was not associated with recurrence, even though it was more frequently found in advanced stages. These findings are in agreement with most published studies (4,16,30 –33). As previously described, the prognostic significance of the BRAF^V600E mutation remains controversial in Korea. One reason could be that prevalence of BRAF mutations in PTC is much higher in Korea compared with other countries and that the numbers for the remaining wild-type BRAF cases are not powered for analysis. The reason(s) for the higher prevalence of BRAF mutations in PTC of Korean patients remain elusive. A further reason for the controversy concerning the predictive value of a BRAF mutation could be that adverse patient outcomes with multiple tumor relapses and eventual death resulting from PTC are rare events (54).

Although a statistically significant association was not observed between the presence of TERT and BRAF mutations, the TERT^C228T mutation was highly prevalent in tumors harboring the BRAF^V600E mutation. The coexistence of a TERT^C228T and a BRAF^V600E mutation was found in 13.0% (27/207) of all PTCs, which is higher than the prevalence observed in previous studies (37,38). This may perhaps be explained by the higher prevalence of the BRAF^V600E mutation in Korea. The coexistence of a TERT^C228T and a BRAF^V600E mutation was significantly associated with older age and advanced stage compared with the group negative for either mutation.

Patients with the TERT^C228T mutation had significantly lower RFS than those with wild-type TERT did. This also remained an independent predictor of tumor recurrence when other clinicopathologic cofactors, including the BRAF^V600E mutation, were adjusted. The findings concerning the risk factor associated with the presence of a TERT mutation are in line with the 2015 American Thyroid Association (ATA) Guidelines for Thyroid Nodules and Cancer that propose a number of hazard factors for persistence/recurrence of the tumor and death (55). For the risk categories provided, the presence of a TERT mutation is listed as placing patients at a high possibility of recurrence, regardless of the presence of a BRAF^V600E mutation. Therefore, preoperative testing for TERT promoter mutations on FNAB samples could help in predicting the prognosis of PTC patients and triaging the patient to receive an appropriate treatment protocol at initial surgery.

The main limitation of this study is having a relatively small number of patients with short follow-up periods. Even though the median follow-up duration was 48 months after operation, for some patients, this is too short a time to evaluate for recurrence. Since nearly all TERT mutation cases (27/30) also harbored the BRAF^V600E mutation, the present study is statistically unable to conclude on the relationship between the two mutations. Therefore, a large cohort study with a longer duration of follow-up is needed to validate the prognostic impact of TERT promoter and BRAF mutations.

In conclusion, among thyroid cancer patients in Korea, to the authors' knowledge, this is the first study performing a TERT promotor mutation analysis in combination with a BRAF^V600E analysis on FNAB specimens. The prevalence of these mutations in various thyroid tumors and the prognostic impact of the TERT^C228T mutation among patients where the BRAF^V600E mutation is frequent were determined. The results provide evidence to adopt TERT promoter mutation analysis as a preoperative adjunctive diagnostic test, thereby providing a risk stratification tool to guide a tailored approach to patient treatment.

Footnotes

Acknowledgments

The authors thank Dr. Chan-Kwon Jung (Department of Pathology, College of Medicine, The Catholic University, Seoul, Korea) for reviewing thyroid tumor slides.

Author Disclosure Statement

The authors have nothing to disclose.

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Prognostic Significance of TERT Promoter Mutations in Papillary Thyroid Carcinomas in a BRAF V600E Mutation–Prevalent Population

Abstract

Background:

Methods:

Results:

Conclusions:

Introduction

Materials and Methods

Patient selection and characteristics

DNA extraction

Detection of the BRAF mutation by PCR amplification and pyrosequencing

Identification of TERT promoter mutations by PCR amplification and direct sequencing

Statistical analysis

Results

Prevalence of TERTC228T/C250T and BRAFV600E mutations in various thyroid tumors

Clinicopathologic demographics of the patients with PTC

Association between TERTC228T/BRAFV600E mutations and clinicopathologic features of PTC

Association between TERTC228T and BRAFV600E mutations

Relationship of clinicopathologic factors with RFS

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Prevalence of TERT^C228T/C250T and BRAF^V600E mutations in various thyroid tumors

Association between TERT^C228T/BRAF^V600E mutations and clinicopathologic features of PTC

Association between TERT^C228T and BRAF^V600E mutations