Molecular Profiling of Papillary Thyroid Carcinoma in Korea with a High Prevalence of BRAF V600E Mutation

Abstract

Background:

The BRAF^V600E mutation in papillary thyroid carcinoma (PTC) is particularly prevalent in Korea, and a considerable number of wild-type BRAF PTCs harbor RAS mutations. In addition, subsets of other genetic alterations clearly exist, but their prevalence in the Korean population has not been well studied. Recent increased insight into noninvasive encapsulated follicular variant PTC has prompted endocrine pathologists to reclassify this entity as “noninvasive follicular thyroid neoplasm with papillary-like nuclear features” (NIFTP). This study analyzed the genetic alterations among the histologic variants of PTC, including NIFTP.

Methods:

Mutations of the BRAF and RAS genes and rearrangement of the RET/PTC1, NTRK1, and ALK genes using 769 preoperative fine-needle aspiration specimens and resected PTCs were analyzed.

Results:

Molecular alterations were found in 687 (89.3%) of 769 PTCs. BRAF^V600E mutation (80.8%) was the most frequent alteration, followed by RAS mutation and RET/PTC1, NTRK1, and ALK rearrangements (5.6%, 2.1%, 0.4%, and 0%, respectively). The low prevalence of NTRK1 fusions and the absence of an ALK fusion detected in Korea may also be attributed to the higher prevalence of the BRAF^V600E mutation. There were significant differences in the frequency of the genetic alterations among the histologic variants of PTC. The prevalence of NIFTP in PTC was 2.7%, and among the NIFTPs, 28.6% and 57.1% harbored BRAF and RAS mutations, respectively. Clinicopathologic factors and mutational profiles between NIFTP and encapsulated follicular variant PTC with capsular invasion group were not significantly different.

Conclusions:

Genetic alterations in PTC vary among its different histologic variants and seem to be different in each ethnic group.

Introduction

Thyroid cancer, like other human cancers, is genetically driven, and over the last several years, significant progress has been made toward understanding the underlying genetic mechanisms. A majority of thyroid follicular cell-derived cancers harbor genetic and epigenetic alterations of the mitogen activated protein kinase (MAPK) signaling pathway, including RAS-RAF-MEK-MAPK-ERK. Activation of the MAPK signaling pathway plays a critical role in thyroid tumorigenesis (1). In papillary thyroid carcinoma (PTC), the MAPK signaling pathway is primarily altered by activating mutation of the BRAF and RAS genes and by rearrangements involving the RET and NTRK1 genes, in some cases (1). The BRAF^V600E mutation is the most common genetic alteration in PTC, as it is found in approximately 29–87% of PTC cases (2 –7). RAS mutations are the second most common mutation and are found in 10–20% of PTCs (8 –10). Although the prevalence of RET/PTC rearrangements is variable in the reported studies, this genetic alteration is found in a significant proportion of PTCs (11,12). RET/PTC rearrangements in PTC are more common in children and young adults and in patients with a history of radiation exposure (13). It is of note that these three genetic alterations are known to be mutually exclusive.

The Cancer Genome Atlas (TCGA) Research Network recently published the results of integrated genomic analyses from 496 patients with PTC, using combined analyses of genomic variants, gene expression, and methylation (14). This large-scale study enabled a thorough understanding of the molecular pathogenesis of PTC. PTC harbors a relatively low somatic mutation frequency, which may be the biological basis for the indolent clinical behavior of PTC (14). Besides, TCGA revealed novel oncogenic driver mutations in several genes, including E1F1AX, PPM1D, CHEK2, and gene fusions of unknown function (14). These efforts have led to a reduction of the fraction of PTC cases with unknown oncogenic drivers from 25% to 3.5% (14).

Genetic alterations that include BRAF, RAS, RET/PTC, and PAX8/PPARγ in PTC can be preoperatively detected in fine-needle aspiration (FNA) specimens from thyroid nodules. This preoperative mutational analysis could significantly improve the accuracy of cancer diagnosis in thyroid nodules and the management of patients with thyroid nodules (15). Testing of these mutations is especially helpful in cases of nodules with an indeterminate cytologic diagnosis, including atypia of undetermined significance or a follicular lesion of undetermined significance (AUS/FLUS), follicular neoplasm/ suspicious follicular neoplasm (FN/SFN), and suspicious for malignancy (SMC). Mutational testing improves the assessment of cancer risk for the patient.

Most recently, an international and multidisciplinary study proposed curbing overdiagnosis and overtreatment of indolent PTC, in particular the noninvasive encapsulated follicular variant PTC, and reclassifying these tumors as “noninvasive follicular thyroid neoplasm with papillary-like nuclear features” (NIFTP) (16). NIFTPs are defined by reproducible diagnostic criteria (16) and have been shown to represent low-risk tumors with very low to nonexistent metastatic potential and recurrence rates (17,18). For NIFTPs, they have been reported to be associated with mutation of RAS or rearrangements of PAX8/PPARγ but not the BRAF^V600E mutation (18 –22).

The V600E mutation of BRAF in PTC is highly prevalent in Korea, and a considerable number of wild-type BRAF PTCs harbor a RAS mutation. A subset of other genetic alterations also clearly exists. For example, TRK rearrangements involving the NTRK1 gene are identified in <5% of PTCs (23,24). ALK rearrangements have also been recently described in thyroid cancers, and their prevalence in PTCs has been reported to be up to 2.2% (25 –27). Previous genetic studies mainly concentrated on BRAF and RAS genes, and the prevalence of other genetic alterations have not been well studied in Korea (28,29). The present study aimed to determine the prevalence of other genetic alterations (RET/PTC1, NTRK1, and ALK gene rearrangements and RAS mutations) as well as the BRAF mutation and to define the molecular subtypes in PTC. In addition, the cases of encapsulated follicular variant of PTC (E-FVPTC) were classified into two groups according to the consensus diagnostic criteria (16), and their clinicopathologic and molecular features were investigated.

Materials and Methods

Patient selection and characteristics

This study was approved by the Institutional Review Board (IRB) of Konkuk University Medical Center, Seoul, Korea (KUH 1210043). This study included 779 patients who underwent thyroidectomy for thyroid nodules between 2010 and 2014 at Konkuk University Medical Center. The FNA specimens obtained preoperatively from the matched patients with confirmed postoperative pathological diagnosis were evaluated. The current Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) recognizes six diagnostic categories for reporting thyroid FNA samples. Using this reporting system, all preceding FNAs were categorized as benign, AUS/FLUS, FN/SFN, SMC, malignant, or nondiagnostic (ND) (30). Molecular analysis was performed after cytological diagnosis was established. RAS mutational analysis was performed when a wild-type (not mutated) result of the BRAF locus was obtained.

The archival slides obtained from resected pathologic specimens were blindly reevaluated according to the 2004 World Health Organization classification of thyroid neoplasm by two pathologists (T.S.H., an endocrine pathologist, and S.E.L.). Among 779 histologically confirmed cases, one (0.1%) was classified as nodular hyperplasia (NH), three (0.4%) as follicular adenoma (FA), 769 (98.7%) as PTC, two (0.3%) as minimally invasive follicular thyroid carcinoma (FTC), one (0.1%) as poorly differentiated carcinoma, one (0.1%) as anaplastic thyroid carcinoma, and two (0.3%) as medullary thyroid carcinoma.

For cases with multiple PTCs, only those PTCs having results of corresponding FNA were evaluated. Of 769 PTCs, 575 (74.8%) were classified as conventional (CPTC), 68 (8.9%) as E-FVPTC, 58 (7.5%) as infiltrative type of follicular variant (I-FVPTC), 39 (5.1%) as tall-cell variant (TVPTC), 12 (1.6%) as conventional with tall-cell features (CPTC with TF), three (0.4%) as solid variant, two (0.3%) as Warthin-like variant, four (0.5%) as oncocytic variant, and eight (1.0%) as PTC with squamous-cell differentiation (PTC with SD).

Detection of BRAF and RAS mutations

Mutational analysis for BRAF^V600E ^/K601E and NRAS, HRAS, and KRAS codons 12, 13 and 61, the most common sites of mutations, was performed. For FNA cytology slides, after slipping off the cover slips, atypical cells of interest were scraped, and DNA was extracted. Briefly, 20–50 μL of DNA extraction buffer solution (50 mM of Tris buffer, pH 8.3; 1 mM of EDTA, pH 8.0; 5% Tween 20; and 100 μg/mL of 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).

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 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 the 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 previously reported (28). Pyrosequencing was performed using a single-nucleotide polymorphism reagent kit (Biotage). Samples containing >10% mutation-positive cells were considered to be positive for BRAF and RAS gene mutations.

Detection of RET/PTC1 rearrangement

Total RNA from formalin-fixed paraffin-embedded (FFPE) samples was extracted using the High Pure FFPE RNA isolation kit (Roche Diagnostics, Mannheim, Germany). First-strand synthesis was performed with 2 μg of total RNA (1 μg as for the cell lysate) using the Tetro cDNA synthesis kit (Bioline International) according to the manufacturer's protocol. Negative controls were run in parallel with Tetro reverse transcriptase and DEPC water. cDNA reverse transcribed from the TPC1 cell line was used as a positive control for the RET/PTC1 rearrangement. Amplification was performed by real-time PCR using the Light Cycler 480 Instrument (Roche Diagnostics, Mannheim, Germany), and measurements were performed using the Light Cycler quantification software v1.5 (Roche Diagnostics). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping control gene.

Tissue microarrays

Tissue microarrays (TMAs) were constructed to include 3 mm cores of FFPE tumor tissue from 70 cases that were negative for RET/PTC1 rearrangement and BRAF and RAS mutations. The NTRK1 and ALK gene rearrangements were screened by immunohistochemistry (IHC) using TMAs.

TrkA IHC and NTRK1 fluorescence in situ hybridization

The TrkA IHC assay used mouse monoclonal TrkA antibody (ab76291; Abcam, Cambridge, MA) at a dilution of 1:400, treated, and incubated at room temperature for 1 h. TrkA immunostaining was scored as follows: negative (−), weak (+), moderate (++), and strong (+++). A moderate-to-strong signal was determined to be TrkA positive.

The analysis of NTRK1 gene rearrangements by fluorescence in situ hybridization (FISH) was performed only in cases of TrkA overexpression by IHC. A ZytoLight SPEC NTRK1 Dual Color Break Apart Probe was used for detection of translocations involving the NTRK1 gene at 1q23.1 (ZytoVision, Bremerhaven, Germany) and according to the operating instructions. With the use of appropriate filter sets, the interphases of normal cells or cells without a translocation involving the 1q23.1 band, two green/orange fusion signals appear. A 1q23.1 locus affected by a translocation is indicated by one separate green signal and one separate orange signal. A threshold of 15% nuclei positive for the separated green/orange signals was used to establish the cut-off for positive FISH.

ALK IHC and FISH

The ALK IHC assay used a mouse monoclonal ALK antibody (5A4; Novocastra, Newcastle, United Kingdom) at a dilution of 1:30, and the samples were treated and incubated at 42°C for 2 h. ALK IHC scores were assigned as follows: 0, no staining; 1+, faint or weak staining with >5% tumor cells or stronger staining intensity with ≤5% of tumor cells; 2+, moderate cytoplasmic reactivity for >5% tumor cells; and 3+, granular cytoplasmic reactivity of strong intensity in >5% of tumor cells (31). Cases that showed ALK-positive staining with a score of ≥1+ were analyzed by FISH using the Vysis ALK Break-Apart FISH Probe Kit (Abbott Laboratories, Abbott Park, IL). Samples were considered positive for ALK FISH if >15% of cells were positive or there was an isolated red signal in tumor cells.

Results

Patient demographics

The mutational status of the samples according to the histologic variant of PTC was evaluated. A total of 769 patients (616 women) with PTC were enrolled in this study. The mean age of patients with PTC was 48 years (range 17–84 years). Three hundred thirty-seven patients (43.8%) were <45 years of age, and 432 patients (56.2%) were >45 years of age. The mean size of PTC was 1.0 cm (range 0.1–5 cm).

TBSRTC diagnostic categories

Among 769 histologically confirmed PTCs, one (0.1%) was classified as ND, seven (0.9%) as benign, 185 (24.1%) as AUS/FLUS, four (0.5%) as FN/SFN, 166 (21.6%) as SM, and 406 (52.8%) as malignant in preoperative FNA cytologies. The distribution of preoperative diagnostic categories according to the histologic variant of PTC is shown in Table 1. The majority of the PTCs except for follicular variant were classified as Bethesda category 5/6 in FNA. Of 68 E-FVPTCs, 66.2% (45/68) were diagnosed as Bethesda category 3/4 and 29.4% (20/68) as Bethesda category 5/6 in FNA. Among the 21 NIFTP cases, two (9.5%) were diagnosed as Bethesda category 2, 12 (57.1%) as category 3, four (19.0%) as category 5, and three (14.3%) as category 6. Of the 58 I-FVPTCs, 44.8% (26/58) were diagnosed as Bethesda category 3 but not 4.

Table 1.

Distribution of Preoperative Diagnostic Categories According to the Histologic Variant of Papillary Thyroid Carcinomas

	Bethesda category
Histology	1	2	3	4	5	6	Total
CPTC	1 (0.2%)	2 (0.3%)	113 (19.7%)	0	137 (23.9%)	322 (56.0%)	575 (74.8%)
E-FVPTC	0	3 (4.4%)	42 (61.8%)	3 (4.4%)	8 (11.8%)	12 (17.6%)	68 (9.5%)
NIFTP	0	2 (9.5%)	12 (57.1%)	0	4 (19.0%)	3 (14.3%)	21 (2.7%)
With capsular invasion	0	1 (2.2%)	30 (65.2%)	3 (6.5%)	4 (8.7%)	8 (17.4%)	46 (6.0%)
I-FVPTC	0	1 (1.7%)	26 (44.8%)	0	12 (20.7%)	19 (32.8%)	58 (7.5%)
TVPTC	0	1 (2.6%)	2 (5.1%)	0	7 (17.9%)	29 (74.4%)	39 (5.1%)
CPTC with TF	0	0	1 (8.3%)	0	1 (8.3%)	10 (83.3%)	12 (1.6%)
Oncocytic variant PTC	0	0	0	0	0	4 (100.0%)	4 (0.5%)
Warthin-like variant PTC	0	0	0	1 (50.0%)	0	1 (50.0%)	2 (0.3%)
Solid-variant PTC	0	0	0	0	0	3 (100.0%)	3 (0.4%)
PTC with SD	0	0	1 (12.5%)	0	1 (12.5%)	6 (75.0%)	8 (1.0%)
Total	1 (0.1%)	7 (0.9%)	185 (24.1%)	4 (0.5%)	166 (21.6%)	406 (52.8%)	769

PTC, papillary thyroid carcinoma; CPTC, classical PTC; E-FVPTC, encapsulated follicular variant of PTC; NIFTP, noninvasive follicular thyroid neoplasm with papillary-like nuclear features; I-FVPTC, infiltrative follicular variant of PTC; TVPTC, tall-cell variant of PTC; CPTC with TF, classical PTC with tall-cell features; PTC with SD, PTC with squamous differentiation.

Genetic alterations according to the histologic variants of PTC

Overall, molecular alterations were found in 687 (89.3%) of 769 PTCs. There was a significant difference in the frequency of genetic alterations according to the histologic variant of PTC (p < 0.001). The genetic alterations in PTC are summarized in Table 2. The frequency of BRAF mutations, RAS mutations, and arrangements for RET, NTRK1, and ALK was 81.3% (625/769), 5.6% (43/769), 2.1% (16/769), 0.4% (3/769), and 0% (0/769), respectively.

Table 2.

Genetic Alterations According to the Histologic Variant of PTC

	CPTC	NIFTP	E-FVPTC with CI	I-FVPTC	TVPTC	CPTC with TF	PTC with SD	Oncocytic variant PTC	Warthin-like variant PTC	Solid variant PTC	Total
Wild type	55 (9.6%)	3 (13.6%)	7 (15.2%)	14 (24.1%)	1 (2.5%)	0	0	1 (25.0%)	0	1 (33.3%)	82 (10.6%)
BRAF mutation	499 (86.8%)	7 (31.8%)	18 (39.1%)	38 (65.5%)	37 (92.5%)	12 (100%)	7 (87.5%)	3 (75.0%)	2 (100%)	2 (66.7%)	625 (81.3%)
RAS mutation	4 (0.7%)	12 (54.5%)	21 (45.7%)	6 (10.3%)	0	0	0	0	0	0	43 (5.6%)
RET/PTC1 rearrangement	15 (2.6%)	0	0	0	0	0	1 (12.5%)	0	0	0	16 (2.1%)
NTRK1 rearrangements	2 (0.3%)	0	0	0	1 (2.5%)	0	0	0	0	0	3 (0.4%)
Total	575 (74.8%)	21 (2.7%)	47 (6.1%)	58 (7.5%)	39 (5.1%)	12 (1.6%)	8 (1.0%)	4 (0.5%)	2 (0.3%)	3 (0.4%)	769

CI, capsular invasion.

BRAF mutation

A BRAF^V600E mutation was identified in 499 (86.9%) CPTCs, 19 (27.9%) E-FVPTCs, 35 (60.3%) I-FVPTCs, 37 (92.5%) TVPTCs, 12 (100%) CPTCs with TF, three (75.0%) oncocytic variant PTCs, two (100%) Warthin-like variant PTCs, two (66.7%) solid variant PTCs, and seven (87.5%) PTCs with SD. A BRAF^K601E mutation was identified exclusively in six (8.8%) E-FVPTCs and three (5.2%) I-FVPTCs.

RAS mutation

The total mutation rate of the RAS gene mutations (two KRAS Q61K and one HRAS Q61R) in CPTC was 0.7% (4/575). In I-FVPTC, the RAS mutation rate was 10.3% (6/58), and the most common RAS mutation was NRAS Q61R (n = 4), followed by HRAS Q61R (n = 1), and KRAS Q61K (n = 1). In E-FVPTC, the RAS mutation rate was 48.5% (33/68), and the most common RAS mutation was NRAS Q61R (n = 22) followed by HRAS Q61R (n = 10) and KRAS Q61K (n = 1). On the other hand, no RAS mutation was observed in the remaining histologic variants of PTC.

RET/PTC1 rearrangement

Fifteen (2.6%) of 575 CPTCs harbored the RET/PTC1 rearrangement. Of eight PTCs with SD, only one (12.5%) case harbored the RET/PTC1 rearrangement. None of the remaining histologic variants harbored a RET/PTC1 rearrangement.

NTRK1 rearrangements

The screening for NTRK1 gene rearrangements by IHC was performed in 70 cases that were negative for RET/PTC1 rearrangements and BRAF and RAS mutations. The screen revealed six TrkA positive cases. FISH study using the “break-apart” probe was performed in six IHC-positive cases. Finally, of 769 PTCs, three cases were identified that had NTRK1 gene rearrangements in this study (0.4% prevalence). The clinicopathologic data of the three cases with NTRK1 gene rearrangements are described in Table 3. All three patients with NTRK1 gene rearrangements were female with a median age of 47 years (range 31–59 years). Of these, two cases had CPTC and one had a TVPTC. Extrathyroidal extension was noted in two patients. Among three cases, two showed lymph node metastasis, but recurrence was not identified in any cases.

Table 3.

Clinicopathologic Data of the Three Cases with NTRK1 Gene Rearrangements

Number	Sex	Age	Bethesda category	Histology	Tumor multiplicity	Tumor size	ETE	LN metastasis	AJCC stage	Recurrence
1	F	31	6	CPTC	0	0.4	1	1	I	No
2	F	59	6	CPTC	1	1.5	0	0	I	No
3	F	47	3	TVPTC	0	1.1	1	1	III	No

ETE, extrathyroidal extension; LN, lymph node; RM, resection margin.

ALK rearrangements

The presence of ALK gene rearrangements was also screened for in 70 cases that were negative for RET/PTC1 rearrangement and BRAF and RAS mutations by IHC and FISH. No ALK rearrangements were identified in any of the 70 PTCs.

E-FVPTC including noninvasive encapsulated follicular variant of PTC (Table 4)

The 68 E-FVPTC cases were further classified into two groups according to the consensus diagnostic criteria (16). Of 68 E-FVPTC cases, 21 (30.9%) were NIFTP and 47 (69.1%) were E-FVPTC with capsular invasion. Among 769 PTCs, the prevalence of NIFTP was 2.7%. There was no significant difference in clinicopathologic factors between the NIFTP subgroup and the E-FVPTC with capsular invasion subgroup. In the NIFTP subgroup, there were 16 (76.2%) women and five (23.8%) men, with a mean age at diagnosis of 49 years. Of these, 11 (52.4%) patients underwent total thyroidectomy and 10 (47.6%) underwent lobectomy. Of the 21 NIFTP cases, only one (4.8%) case had lymph node metastasis, but recurrence was not identified in any of the patients. One NIFTP case with lymph node metastasis harbored a BRAF^V600E mutation. There was no significant difference in the mutational profile between the NIFTP subgroup and the E-FVPTC with capsular invasion subgroup. BRAF and RAS mutations were detected in 25 (36.8%) and 33 (48.5%) of the 68 E-FVPTC cases, respectively, and in six (28.6%) and 12 (57.1%) of the 21 NIFTP cases, respectively. RAS mutations included six NRAS Q61R (50%) and six HRAS Q61R (50%) mutations. However, RET/PTC1 and NTRK1 rearrangements were not observed in E-FVPTC cases.

Table 4.

Relationship of Clinicopathologic Factors and Two Subgroups of 68 Encapsulated Follicular Variant PTCs

	Total (n = 68)	NIFTP (n = 21; 30.9%)	E-FVPTC with capsular invasion (n = 47; 69.1%)	p-Value
Sex
Female	54 (79.4%)	16 (76.2%)	38 (80.9%)	0.749
Male	14 (20.6%)	5 (23.8%)	9 (19.1%)
Age, years (range)		49 (29–66)	50 (19–71)	0.730
Tumor size, cm (range)		1.3 (0.4–3.0)	1.7 (0.4–4.3)	0.095
Bethesda category
2	3 (4.4%)	2 (9.5%)	1 (2.1%)	0.317
3	41 (60.3%)	11 (52.4%)	30 (63.8%)
4	3 (4.4%)	0	3 (6.4%)
5	9 (13.2%)	5 (23.8%)	4 (8.5%)
6	12 (17.6%)	3 (14.3%)	9 (19.1%)
LN metastasis
Yes	8 (14.5%)	1 (5.3%)	7 (19.4%)	0.239
No	47 (85.5%)	18 (94.7%)	29 (80.6%)
AJCC stage
1	47 (81.0%)	18 (90.0%)	29 (76.3%)	0.331
2	6 (10.3%)	2 (10.0%)	4 (10.5%)
3	5 (8.6%)	0	5 (13.2%)
Extent of surgery
Lobectomy	37 (54.4%)	10 (47.6%)	27 (57.4%)	0.215
Total thyroidectomy	31 (45.6%)	11 (52.4%)	20 (42.6%)
Recurrence
Yes	1 (1.5%)	0	1 (2.1%)	1
No	67 (95.8%)	21 (100.0%)	46 (97.9%)
BRAF mutation
Wild	43 (63.2%)	15 (71.4%)	28 (59.6%)	0.659
V600E	19 (27.9%)	5 (23.8%)	14 (29.8%)
K601E	6 (8.8%)	1 (4.8%)	5 (10.6%)
RAS mutation
Wild	35 (51.5%)	9 (42.9%)	26 (55.3%)	0.433
Mutant	33 (48.5%)	12 (57.1%)	21 (44.7%)

Discussion

The variability in the prevalence of each mutation according to the different histological variants suggests that different driver oncogenic groups represent distinct phenotypic and biologic properties for each PTC variant. TCGA described that the majority of CPTCs clustered with BRAF^V600E -mutated tumors, without overlapping with the RAS, RET/PTC, and PAX8/PPARγ alterations (14). On the other hand, RAS and PAX8/PPARγ alterations were identified in follicular-patterned thyroid tumors, including FA, follicular carcinoma, and FVPTC (32). However, the prevalence of each genetic alteration has been variously reported in different geographic areas. In the present study, at least one of the genetic alterations tested was found in 89.3% of 769 PTCs, which is similar to the prevalence reported in previous studies (33 –35). The most common genetic alteration was a BRAF mutation (81.3%), followed by RAS mutations (5.6%). The prevalence of BRAF mutations was higher and the one of RAS mutations was lower compared with previous studies (33 –35). These findings could be explained by the fact that the prevalence of the BRAF^V600E mutation in PTC is much higher in Korea (73–87%) compared with Western countries (2 –4).

In the present study, the association between histologic variant of PTC and genetic alterations was evaluated, and the frequency of each genetic alteration was found to be significantly different according to the histologic variant of PTC. The BRAF^V600E mutation was found in the majority of all the histologic variants, except for E-FVPTC. On the other hand, RAS mutations were mainly identified in the follicular variant, especially in the E-FVPTC cases. In thyroid nodules with AUS/FLUS FNA category, a sequential algorithmic approach was recently proposed with BRAF mutational analysis, followed by N, H, and KRAS codons 12, 13, and 61 mutational analyses (28). In practice, this approach may help in the management of patients with a cytological diagnosis of AUS/FLUS. Histological analysis of RAS mutation–positive AUS/FLUS aspirates confirmed that RAS positivity had a 96% positive predictive value for predicting FVPTC in this series (28).

PTCs with a RET/PTC1 rearrangement were almost exclusively CPTCs. These findings are in agreement with most published studies (8,9,36).

NTRK1 gene rearrangements were identified in 3/769 (0.4%) PTC cases. Even though TRK rearrangements involving the NTRK1 gene have been identified in <5% of PTCs (23,24), the prevalence was much lower in the present study (0.4%). This may be attributed to the higher prevalence of the BRAF^V600E mutation in Korea. This is the first report of the frequency of NTRK1 rearrangements in PTC of the Korean population where the BRAF^V600E mutation is prevalent. To date, three fusion partner genes have been identified in PTC, and all these fusion types lead to expression and activation of the tyrosine kinase domain of NTRK1. The fusions are also tumorigenic for the thyroid follicular epithelial cells. It has been reported that the presence of NTRK1 rearrangements correlated with a higher rate of local recurrence and tumor-related mortality but not with the tumor subtypes (23,37). Brzezianska et al. reported that NTRK1 rearrangements were identified in one CPTC and three FVPTC out of 33 PTCs (23). NTRK1 rearrangements were identified in two CPTCs and one TVPTC from the total of 769 PTCs, but recurrence was not identified in any of the cases. The number of the NTRK1 rearrangements cases was too small to analyze for clinicopathologic features in this study. Therefore, a large cohort study with PTCs harboring NTRK1 rearrangements is needed to validate a histologic association and prognostic impact of NTRK1 rearrangements. Of six TrkA IHC-positive cases, three cases were finally confirmed to have NTRK1 rearrangements by FISH. Park et al. recently reported that the expression level of TrkA was significantly correlated with NTRK1 rearrangements in colon cancer (38). In their study, only three out of seven TrKA IHC cases turned out to have NTRK1 rearrangements in FISH analysis, whereas all TrkA IHC negative cases did not show NTRK1 rearrangements in FISH analysis (38). From these data, IHC for TrkA can be a viable screening strategy for NTRK1 rearrangements.

ALK gene rearrangements have been identified as an oncogenic driver and to be mutually exclusive with other oncogenic alterations found in PTC (14,26,27,39,40). With this background, the presence of ALK fusion transcripts was screened for in 70 cases that were negative for BRAF, RAS, and RET alterations. In this study, no ALK rearrangements were identified. The prevalence and prognostic impact of the ALK rearrangements in PTC has been rather controversial. It was reported that ALK rearrangements were more common in aggressive thyroid tumor, including PTC with distant metastasis, poorly differentiated carcinoma, and anaplastic carcinoma (25,27). However, another study by Bae et al. on 243 thyroid tumors including 42 aggressive tumors (30 well-differentiated thyroid carcinomas with distant metastasis, 7 PD carcinomas, and 5 anaplastic carcinomas) found no ALK rearrangements in aggressive thyroid tumors (41). To date, only a handful of PTC cases have been found to harbor ALK rearrangements. A large cohort study therefore needs to be performed to address these conflicting findings.

After the report by Nikiforov et al. regarding NIFTP, the concern about NIFTP became higher worldwide, and the existing treatment protocol for FVPTC was challenged in Korea. Therefore, an analysis of the prevalence, clinical behavior, and molecular alterations of each subgroup of FVPTC is needed. In the present study, NIFTP comprised 2.7% of all PTC cases (16.7% of FVPTC and 30.9% of E-FVPTC). Other studies reported that NIFTP accounted for 22.9% of PTC and 71.4% of FVPTC (42,43). This discrepancy might be attributed to the differences in the ethnic backgrounds and definitions for capsular invasion. As such, strict histopathological criteria should be applied to diagnose NIFTP. Of 21 NIFTP, only one case showed lymph node metastasis, which is different from a previous study (16). RAS mutations were the most common mutation in NIFTP, which is in agreement with previous studies (18 –22). However, NIFTPs in the present study had an appreciable percentage (23.8%) of BRAF^V600E mutations, albeit at a much lower frequency than CPTC. At the authors' institution, surgery is recommended for BRAF-positive thyroid nodules with a preoperative cytological diagnosis of AUS/FLUS and FN/SFN. This has led to the detection of more BRAF-mutated PTCs, especially BRAF-mutated FVPTCs, since a considerable number of FVPTCs are categorized as AUS/FLUS or FN/SFN in preoperative FNA studies. According to a previous report, thyroid nodules with a preoperative AUS/FLUS and FN/SFN diagnosis comprised 42.7% of the FVPTCs (44). In fact, one of the six NIFTPs harboring a BRAF^V600E mutation was diagnosed as AUS/FLUS preoperatively. Therefore, the prevalence of BRAF mutations in the NIFTP cases at the authors' institution would be higher than that of other institutions where most of the thyroid nodules in the AUS/FLUS and FN/SFN categories are not resected. At any rate, the frequency of BRAF^V600E mutations in E-FVPTC remains controversial, showing a varying prevalence of 0–24% (45 –49). Whether the differences in the prevalence of BRAF mutations in NIFTP can be attributed to ethnic differences remains unclear and should be confirmed by larger cohort studies in Korea where somatic BRAF^V600E mutations are prevalent. Another reason for this discrepancy could be associated with differences in diagnosing FVPTC.

In conclusion, to the authors' knowledge, this is the first study to perform comprehensive molecular profiling of histologic variants of PTC in a large Korean cohort, a population with a high prevalence of the BRAF^V600E mutation. Genetic alterations involving thyroid tumorigenesis vary according to the histologic variants of PTC, and it is likely that a given ethnic group with different genetic and environmental backgrounds will have a different prevalence. The proportion of NIFTP in the Korean population appears to be lower than that in Western populations, whereas the clinicopathologic factors and mutational profiles between NIFTP and the encapsulated FVPTC with capsular invasion group were not significantly different. Therefore, a better understanding of the prevalence, clinical behaviors, and genetic alterations involved in the pathogenesis of PTC in each ethnic population is required.

Footnotes

Acknowledgments

This paper was written as part of Konkuk University's research support program for its faculty on sabbatical leave in 2014.

Author Disclosure Statement

The authors declare that there is no conflict of interest regarding the publication of this article.

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