Skip to main content
NCBI home page
Thyroid logoLink to Thyroid
. 2014 Aug 1;24(8):1256–1266. doi: 10.1089/thy.2013.0610

Concomitant RAS, RET/PTC, or BRAF Mutations in Advanced Stage of Papillary Thyroid Carcinoma

Minjing Zou 1, Essa Y Baitei 1, Ali S Alzahrani 2, Faisal S BinHumaid 1, Dania Alkhafaji 2, Roua A Al-Rijjal 1, Brian F Meyer 1, Yufei Shi 1,✉
PMCID: PMC4106383  PMID: 24798740

Abstract

Background: RET/PTC rearrangement, RAS, and BRAF mutations are considered to be mutually exclusive in papillary thyroid carcinoma (PTC). However, although concomitant mutations of RET/PTC, RAS, or BRAF have been reported recently, their significance for tumor progression and survival remains unclear. We sought to examine the prognostic value of concomitant mutations in PTC.

Methods: We investigated 88 PTC for concomitant mutations. Mutation in BRAF exon 15, KRAS, NRAS, and HRAS were studied by polymerase chain reaction (PCR)-sequencing of tumor DNA; RET/PTC rearrangement was determined by reverse transcription (RT)-PCR-sequencing of tumor cDNA.

Results: BRAFV600E was detected in 39 of 82 classic PTC (CPTC) and in all three tall-cell variants (49%, 42/85). KRAS mutation (p.Q61R and p.S65N) was detected in two CPTC (2%, 2/88) and NRASQ61R in one CPTC and two follicular variant PTC (FVPTC; 3%, 3/88). KRASS65N was identified for the first time in thyroid cancer and could activate mitogen-associated protein kinase (MAPK). RET/PTC-1 was detected in nine CPTC, one tall-cell variant, and two FVPTC. Concomitant BRAFV600E and KRAS, or BRAFV600E and RET/PTC-1 mutations were found in two CPTC, and six CPTC and one tall-cell variant, respectively. In total, 11 concomitant mutations were found in 88 PTC samples (13%), and most of them were in the advanced stage of disease (8/11, 73%; p<0.01).

Conclusions: Our data show that concomitant mutations are a frequent event in advanced PTC and are associated with poor prognosis. The concomitant mutations may represent intratumor heterogeneity and could exert a gene dosage effect to promote disease progression. KRASS65N can constitutively activate the MAPK pathway.

Introduction

Thyroid carcinoma is the most common type of endocrine malignancy and can be classified into papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC). PTC is the most common type of differentiated thyroid carcinoma, accounting for more than 80% of thyroid malignancies (1). Genetic alterations in the mitogen-associated protein kinase (MAPK) pathway play an important role in the initiation and progression of PTC. Notable among them are RAS point mutations, RET/PTC oncogene rearrangements, and BRAF point mutations. BRAF mutations are the most common genetic alteration in PTC from 28% to 83%, with an overall rate of 44% (2–4).

It is generally believed that genetic alterations in RET/PTC, RAS, and BRAF are mutually exclusive in PTC, and mutations at more than one of these genes are unlikely to provide an additional biological advantage (2,5,6). However, recent studies have shown concomitant mutations in PTC (7–10). Di Cristofaro et al. reported concomitant RAS and RET/PTC1 mutations in 1/24 (4%) follicular variant PTC (FVPTC), and BRAF and RET/PTC3 mutations in 1/26 (4%) classic PTC (CPTC) (8). Costa et al. showed concomitant BRAF and KRAS mutations in 4/35 PTC (11%) (10), whereas Henderson et al. reported concomitant BRAF and RET/PTC mutations in 5/54 (9.3%) recurrent PTC (7). Zhu et al. reported concomitant mutations of RET/PTC and RAS or BRAF in 8/15 (53%) subclonal or nonclonal PTC but none in clonal PTC (9). It is not clear whether concomitant mutations are associated with aggressive disease behavior and poor prognosis. In the present study, we investigated concomitant mutations in RET/PTC, RAS, and BRAF in 88 PTC from Saudi Arabia and its association with disease progression and prognosis.

Materials and Methods

Thyroid tumor specimens and cell lines

All tumor tissues were obtained at surgery with informed consent, and were immediately frozen in liquid nitrogen and stored at −70°C until processed. The clinical staging of thyroid cancer was based on the Tumor, Node, Metastasis (TNM) classification (11). Eighty-eight PTC diagnosed between 1987 and 2006 were selected randomly and included in the study: 82 CPTC, three tall-cell variants, and three FVPTC. The mean follow-up interval was 12 years. The following cell lines were studied to determine whether they have dual mutations in RET/PTC, RAS, or BRAF: K1, BCPAP, 8305C, 8505C, HTC-C3, BHT101, CAL62, TPC-1, and FRO. TPC-1 and FRO were kindly provided by Dr. James Fagin (Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY). Other cell lines were purchased from DSMZ (Braunschweig, Germany). ATC cell lines such as 8305C, 8505C, BHT101, FRO, and CAL62 were screened for concomitant mutations, since all of them carry either BRAF or RAS mutations, and it is not clear whether they have dual mutations in the MAPK pathway. The study was reviewed and approved by the Institutional Review Board.

Detection of BRAF and RAS mutations

Tumor tissue was obtained by standard sectioning, and DNA was extracted by standard proteinase-K treatment followed by phenol/chloroform extraction. BRAF exon 15 was amplified by PCR and directly sequenced as described previously (12). Mutations of KRAS, NRAS, and HRAS were analyzed by polymerase chain reaction (PCR) sequence analysis of exon 2 (for codon 12/13), exon 3 (for codon 61), and exon 4 in KRAS and NRAS; and exon 1 (codon 12/13), exon 2 (codon 61) and exon 3 in HRAS. Exon 4 was screened for non-hotspot mutations. PCR primers are listed in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/thy). The PCR conditions were 95°C for 5 min followed by 35 cycles of amplification (95°C for 40 s, 50–62°C for 40 s, and 72°C for 40 s) with final extension at 72°C for 5 min. The resulting PCR products were analyzed by gel electrophoresis. Each successfully amplified fragment was directly sequenced using the BigDye Terminator V3.1 Cycle Sequencing kit and ABI prism 3100 sequencer (Applied Biosystems, Foster City, CA). DNA from paraffin blocks of patients 27 and 78 was extracted as described previously (13).

Detection of RET/PTC rearrangements

Total RNA was extracted from tumor specimens and cell lines by the guanidine thiocyanate-phenol-chloroform method. The integrity of RNA was verified by denaturing gel electrophoresis. Two μg of total RNA were reverse-transcribed into cDNA using the Promega reverse transcription (RT) system (Promega, Madison, WI). Nested RT-PCR was used to amplify transcripts of RET/PTC rearrangements, using two sets of primers listed in Supplementary Table S1 and PCR conditions described above. The resulting PCR products were analyzed by gel electrophoresis and directly sequenced. GAPDH cDNA was used as an internal control for RNA quality. To rule out PCR contamination, positive samples were confirmed by repeating PCR using different batches of same specimen.

Screening for PAX8/PPARγ rearrangement in FVPTC

A fusion gene (PAX8/PPARγ) between the DNA binding domain of the thyroid-restricted transcription factor paired box gene 8 (PAX8) and the peroxisome proliferator-activated receptor γ gene (PPARγ) has been detected in about 10% of FVPTC. PAX8/PPARγ rearrangement was screened by RT-PCR in FVPTC as described previously (14).

Cloning and expression of mutant KRASS65N and NRASS65C

The full-length KRAS and NRAS cDNA clones were obtained from GenScript (Piscataway, NJ) and cloned into pcDNA3.1 under the control of the CMV promoter (Invitrogen, Carlsbad, CA). KRASS65N and NRASS65C mutants were obtained by site-directed mutagenesis and were verified by DNA sequencing. Wild-type KRAS and NRAS were used as controls. Equal amounts of the constructs were transfected into HEK293, BCPAP, and CAL62 cell lines using Lipofectamine (Invitrogen). Cells were collected 48 hours after transfection for MAPK and cAMP response element binding protein (CREB) activity.

Western blot analysis

Forty μg of protein from transfected cells was loaded onto a 12% SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene (PVDF) membrane and subject to Western blot analysis using anti-phospho-MEK1/2, anti-phospho-ERK 1/2 antibody, or anti-phospho-CREB (1:1000; Cell Signaling Technology, Inc., Danvers, MA).

Cell proliferation assay

Cell proliferation was measured by a non-radioactive MTS assay kit according to the manufacturer's procedure (Promega). Briefly, cells were plated in triplicate into 96-well plates (5×103 cells/well) containing 1% or 10% serum respectively for 48 h. At the final 4 h of incubation, 20 μL of CellTiter 96® AQueous One Solution reagent was added into each well for measurement of cell viability.

Statistical analysis

Fisher's exact test (two-tailed) was used. A p-value of ≤0.05 was considered significant.

Results

BRAF mutation

BRAFV600E mutation was detected in 39 of 82 classic PTC (48%) and in all three tall-cell variants (Fig. 1 and Table 1). No mutation was detected in FVPTC. The BRAFV600E mutation occurred more frequently in the advanced stages (stage III or IV) compared to the early stages (stage I or II) of CPTC: 17/26 (65%) versus 22/56 (39%) (Table 1; p<0.05, Fisher's exact test). The BRAFV600E mutation was also detected in 8305C, 8505C, HTC-C3, BHT101, BCPAP, K1, and FRO cells.

FIG. 1.

FIG. 1.

Open in a new tab

Detection of BRAF, RAS, and RET/PTC-1 mutations in papillary thyroid carcinoma (PTC). (A) RET/PTC-1 rearrangement was detected by reverse transcription polymerase chain reaction (RT-PCR) from patients. The PCR product was run on a 2% agarose gel. GAPDH cDNA was used as an internal control for RNA quality. (B) DNA sequence analysis of PCR products from three patients with concomitant mutations. The RAS and BRAF mutations are indicated by an arrow. The breakpoint of RET/PTC-1 rearrangement is indicated by a vertical line.

Table 1.

BRAF, RAS, or RET/PTC Mutation in Papillary Thyroid Carcinoma

Case # Age/sex Type/stage KRAS NRAS BRAF RET/PTC
1 18/M PTC/I ND ND V600E ND
2 21/F PTC/I ND ND ND ND
3 23/F PTC/I ND c.182 A>G, p.Q61R ND ND
4 24/M PTC/I ND ND ND ND
5 28/F PTC/I ND ND ND ND
6 36/F PTC/I ND ND ND PTC-1
7 38/F PTC/I ND ND ND ND
8 39/F PTC/I ND ND ND ND
9 40/M PTC/I ND ND ND PTC-1
10 40/F PTC/I ND ND ND ND
11 40/M PTC/I ND ND ND ND
12 43/F PTC/I ND ND ND ND
13 12/F MET PTC/I ND ND ND ND
14 18/F MET PTC/I ND ND ND ND
15 20/F MET PTC/I ND ND ND ND
16 21/F MET PTC/I ND ND ND ND
17 21/F MET PTC/I ND ND ND ND
18 22/F MET PTC/I ND ND ND ND
19 24/F MET PTC/I ND ND ND ND
20 24/F MET PTC/I ND ND ND ND
21 25/F MET PTC/I ND ND V600E ND
22 25/F MET PTC/I ND ND V600E ND
23 26/F MET PTC/I ND ND ND ND
24 26/F MET PTC/I ND ND V600E PTC-1
25 26/M MET PTC/I ND ND ND ND
26 27/F MET PTC/I ND ND ND ND
27 28/F Met PTC/I c.194 G>A, p.S65N ND V600E ND
28 29/F MET PTC/I ND ND V600E ND
29 30/F MET PTC/I ND ND V600E ND
30 32/F MET PTC/I ND ND V600E ND
31 33/F MET PTC/I ND ND ND ND
32 33/F MET PTC/I ND ND V600E ND
33 34/F MET PTC/I ND ND ND ND
34 34/F MET PTC/I ND ND V600E ND
35 35/F MET PTC/I ND ND V600E ND
36 36/F MET PTC/I ND ND ND ND
37 37/F MET PTC/I ND ND V600E ND
38 37/F MET PTC/I ND ND ND ND
39 39/M MET PTC/I ND ND ND ND
40 39/M MET PTC/I ND ND V600E ND
41 40/F MET PTC/I tall-cell variant ND ND V600E ND
42 41/F MET PTC/I ND ND V600E ND
43 41/F MET PTC/I ND ND V600E ND
44 41/F MET PTC/I ND ND V600E ND
45 42/F MET PTC/I ND ND ND ND
46 42/F MET PTC/I ND ND V600E ND
47 43/M FVPTC/I ND ND ND ND
48 44/F MET PTC/I ND ND V600E ND
49 27/F MET PTC/II ND ND ND ND
50 27/F MET PTC/II ND ND ND PTC-1
51 30/F MET PTC/II ND ND V600E ND
52 45/F FVPTC/II ND c.182 A>G, p.Q61R ND PTC-1
53 50/M PTC/II ND ND ND ND
54 50/F PTC/II ND ND ND ND
55 53/M PTC/II ND ND ND ND
56 57/F PTC/II ND ND V600E ND
57 60/F PTC/II ND ND V600E ND
58 62/F PTC/II ND ND V600E ND
59 76/F PTC/II ND ND ND ND
60 45/F PTC/III ND ND ND ND
61 46/M PTC/III ND ND ND ND
62 46/F PTC/III ND ND V600E ND
63 50/M PTC/III ND ND ND ND
64 52/M PTC/III ND ND V600E ND
65 55/F PTC/III ND ND V600E PTC-1
66 58/F PTC/III ND ND ND ND
67 59/F PTC/III ND ND V600E PTC-1
68 60/F PTC/III ND ND ND ND
69 60/M PTC/III ND ND V600E ND
70 65/M PTC/III ND ND V600E ND
71 66/M PTC/III ND ND V600E ND
72 66/M PTC/III ND ND ND ND
73 71/M PTC/III tall-cell variant ND ND V600E PTC-1
74 72/F PTC/III ND ND V600E ND
75 72/F PTC/III ND ND V600E PTC-1
76 73/F PTC/III ND ND V600E ND
77 78/M PTC/III tall-cell variant ND ND V600E ND
78 82/M PTC/III c.182A>G, p.Q61R ND V600E ND
79 48/M PTC/IVA ND ND ND ND
80 49/M PTC/IVB ND ND V600E PTC-1
81 52/M PTC/IVC ND ND V600E PTC-1
82 58/F PTC/IVA ND ND V600E ND
83 60/M PTC/IVC ND ND ND ND
84 62/M PTC/IVB ND ND ND ND
85 63/M PTC/IVC ND ND V600E ND
86 68/F FVPTC/IVC ND c.182 A>G, p.Q61R ND PTC-1
87 70/F PTC/IVB ND ND V600E ND
88 77/M PTC/IVB ND ND V600E ND
Cell lines
8305C   ATC ND ND V600E ND
8505C   ATC ND ND V600E ND
HTC-C3   ATC ND ND V600E ND
BHT101   ATC ND ND V600E ND
FRO   ATC ND ND V600E ND
CAL62   ATC KRASG12R ND ND ND
BCPAP   PTC ND ND V600E ND
K1   PTC ND ND V600E ND
TPC-1   PTC ND ND ND PTC-1
Open in a new tab

MET PTC refers to stage I or II tumors with local lymph node metastasis. Dual mutations are highlighted in bold.

PTC, papillary thyroid carcinoma; ND, not detected.

RAS mutation

KRAS mutation was detected in two CPTC: c.182A>G (p.Q61R) with stage III cancer, and c.194 G>A (p.S65N) with stage I cancer (Fig. 1). NRASQ61R was detected in one CPTC case and two FVPTC cases. No mutation was found in HRAS. The overall rate of RAS mutation was 6% (5/88) in the cohort studied (Table 2). KRASG12R was found in the CAL62 cell line. KRASS65N has not been reported before in thyroid cancer. The significance of the mutation was further investigated first in matched normal tissue from paraffin blocks. No mutation was found in normal tissue, suggesting that it is not a germ-line polymorphism (data not shown). Multiple protein sequence alignments around codon 65 showed that serine65 is highly conserved across different species (data not shown). Next, we used PolyPhen-2 to predict the possible effect of S65N substitution (http://genetics.bwh.harvard.edu/pph/). PolyPhen-2 predicted a possible damaging effect with a score of 0.919 of 1.0 (sensitivity: 0.81; specificity: 0.94). To confirm that KRASS65N or NRASS65C (reported previously in neuroendocrine lung cancer and melanoma but not characterized by functional studies) can activate the MAPK signaling pathway, we cloned and expressed KRASS65N and NRASS65C in HEK293 cells. As shown in Figure 2A, both KRASS65N and NRASS65C caused increased p-MEK and p-ERK expression in HEK293 cells. KRASS65N can also increase MAPK activity and cell proliferation in both low- and high-serum conditions (Fig. 2B and C). We further investigated the effect of KRASS65N on MAPK and CREB activity in the PTC cell line BCPAP and the ATC cell line CAL62. CREB is activated by phosphorylation at Ser133 by various signaling pathways, including the MAPK/ERK pathway. As shown in Figure 2B, the expression of both phospho-ERK and phospho-CREB was increased following KRASS65N expression. The basal level of phospho-ERK was also higher in BCPAP and CAL62 as compared to HEK293, probably due to the fact that BCPAP and CAL62 contain BRAFV600E and KRASG12R respectively.

Table 2.

Frequency of BRAF, RAS, or RET/PTC Mutations in Different Stages of PTC

Stage Histology subtype Cases BRAF only KRAS only NRAS only RET/PTC-1 only BRAF and RAS BRAF and RET/PTC-1 RAS and RET/PTC-1
I CPTC 46 16 (35) 0 1 (2) 2 (4) 1 (2) 1 (2) 0
  Tall cell 1 1 (100) 0 0 0 0 0 0
  FVPTC 1 0 0 0 0 0 0 0
II CPTC 10 4 (40) 0 0 1 (10) 0 0 0
  FVPTC 1 0 0 0 0 0 0 1 (100)
III CPTC 17 7 (41) 0 0 0 1 (6) 3 (18) 0
  Tall cell 2 1 (50) 0 0 0 0 1 (50) 0
IV CPTC 9 4 (44) 0 0 0 0 2 (22) 0
  FVPTC 1 0 0 0 0 0 0 1 (100)
Total (%)   88 33 (38) 0 1 (1) 3 (3) 2 (2) 7 (8) 2 (2)
Open in a new tab

Numbers in parentheses indicate percentage of mutation in the histology subtype or group. HRAS mutation was not detected in the series.

FIG. 2.

FIG. 2.

Open in a new tab

Characterization of KRASS65N and NRASS65C on mitogen-associated protein kinase (MAPK) activity. (A) The mutant and wild-type constructs (KRASS65N, NRASS65C, and KRASWT) were transfected into HEK293 cells. Cells were collected 48 h after transfection for Western blot analysis. Increased pMEK1/2 and pERK 1/2 expression was detected by an anti-pMEK1/2 and anti-pERK 1/2 antibody respectively in cells transfected with mutant RAS constructs. The membrane was reprobed with total ERK1/2 and GAPDH (37Kd) antibody to monitor protein loading. Quantification of the Western blot results is shown in Supplementary Figure S1. (B) The KRASWT, KRASS65N, and NRASS65C were transfected into HEK293 cells. Cells were cultured in serum-free medium for an additional 2 days 24 h after transfection and collected for Western blot analysis. Increased pERK 1/2 and pMEK 1/2 expression was detected in both KRASS65N and NRAS S65C transfected cells following 48 h serum starvation. (C) Cell proliferation of HEK293 cells transfected with KRASS65N or KRASWT. The cells were plated into 96-well plates (5×103 cells/well) containing 1% or 10% serum respectively for 48 h. Data are expressed as mean±standard error of the mean (SEM) of triplicates and are representative of three different experiments (*p<0.01). (D) Increased MAPK and cAMP response element binding protein (CREB) activity in thyroid cancer cell lines following KRASS65N transfection. Both phospho-CREB and-ERK1/2 expressions were increased in CAL62 and BCPAP cell lines. HRASG12V was transfected into CAL62 and BCPAP cell lines as a positive control. The basal level of pEKR1/2 was also higher in CAL62 and BCPAP as compared to HEK293.

RET/PTC rearrangements

A RET/PTC-1 rearrangement was detected in nine CPTC (9/82, 11%), one tall-cell variant, two FVPTC, and the TPC-1 cell line (Fig. 1 and Table 2). The overall rate of RET/PTC-1 was 14% (12/88). RET/PTC-2 and -3 were not detected in this series of samples. RET/PTC-1 occurred more frequently in the late stage (7/29, 24%) than in the early stage of PTC (5/59, 9%; p=0.0548).

PAX8/PPARγ rearrangement

No rearrangement was detected in the three FVPTC cases (data not shown).

Concomitant BRAFV600E, RAS, or RET/PTC-1 mutations

Two CPTC samples had both BRAFV600E and KRASQ61R or KRASS65N. Six CPTC and one tall-cell variant contained both BRAFV600E and RET/PTC-1. Two FVPTC samples had both NRASQ61R and RET/PTC-1 (Table 1). None of the samples were found to have triple mutations. In total, concomitant mutations were found in 11/88 PTC samples (13%). Among them, three were found in early stages of PTC (3/59, 5%), and eight (8/29, 28%) in an advanced stage. The association of concomitant mutations with advanced stage was statistically significant (p<0.01). Eight cases with concomitant mutations were older than 45 years. Concomitant mutations were not found in nine cell lines studied. Co-existence of RET/PTC with RAS or BRAF has been reported in multiclonal PTC (9). We next examined different tumor sections of paraffin blocks for concomitant BRAF and RAS mutations found in our two cases. As shown in Table 3, the BRAF mutation was present in all tumor sections, whereas the RAS mutation was detected only in some sections. This may indicate intratumor heterogeneity. Histologic heterogeneity was not observed. Due to the limited sensitivity of Sanger sequencing to detect a low frequency of mutant alleles, we cloned the PCR fragments from tissue sections with negative RAS mutation into a TA vector, and screened 50 clones from each fragment for RAS mutation by direct Sanger sequencing. As shown in Table 3, a RAS mutation was detected in two sections that were negative by direct Sanger sequencing. We further evaluated the sensitivity of Sanger sequencing to detect KRAS mutations by mixing different amounts of wild-type and mutant KRAS DNA. As shown in Figure 3, about 20% mutant alleles are required in the sample to be reliably detected by Sanger sequencing (the detection limit is about 20%). In the lymph node metastases, the RAS mutation was significantly enriched and can be easily detected (Table 3).

Table 3.

BRAF and RAS Mutation in Different Tumor Sections

Case # Age/sex Type/stage Tumor sections BRAF RAS Frequency of mutant RAS allele in 50 clones (%)
27 28/F PTC/I PT-A V600E NT 0/50
      PT-B V600E NT 3/50 (6)
      PT-C V600E NT 4/50 (8)
      LNM-A V600E KRASS65N 17/50 (34)
      LNM-B V600E KRASS65N 14/50 (28)
78 82/M PTC/III PT-A V600E NT 6/50 (12)
      PT-B V600E KRASQ61R 12/50 (24)
      PT-C V600E KRASQ61R Not done
      PT-D V600E KRASQ61R Not done
Open in a new tab

PT, primary tumor; LNM, lymph node metastasis; NT, not detected by direct Sanger sequencing.

FIG. 3.

FIG. 3.

Open in a new tab

Sensitivity of Sanger sequencing to detect KRAS mutations. Different amounts of wild-type and mutant KRAS DNA were mixed together in a total concentration of 100 ng DNA for PCR. PCR products were directly sequenced. At least 20% mutant alleles need to be present in the sample to be reliably detected by Sanger sequencing.

To determine whether concomitant mutations were associated with a poor clinical outcome, we analyzed disease-free survival of the patients who had concomitant mutations versus those without mutation or those who had only one mutation. As shown in Figure 4, the Kaplan–Meier curve shows a significant difference in the recurrence-free probability over a 10-year period among patients with no mutation, a single mutation, and double mutations (log-rank test χ2=18.2, p<0.0001), and between patients with single and double mutations (log-rank test χ2=4.2, p<0.041). These data indicate that patients with concomitant mutations had much poorer clinical outcomes than those with a single or no mutation.

FIG. 4.

FIG. 4.

Open in a new tab

Kaplan–Meier analysis for recurrence-free probability. The Kaplan–Meier curves shows a significant difference in the recurrence-free probability over a 10-year period after the diagnosis among patients with no mutation, a single mutation, and double mutations (log-rank test χ2=18.2, p<0.0001), and also between patients with single mutation and double mutations (log-rank test χ2=4.2, p<0.041).

Discussion

In the present study, we found 11 cases of PTC (13%) with concomitant BRAF, RAS, or RET/PTC-1 mutations. Concomitant mutations occurred more frequently in advanced stages of disease. Long-term follow-up showed that patients with concomitant mutations had a poor response to treatment and shorter disease-free survival.

We also report the first case of KRASS65N in PTC (1%). Mutation in KRAS codon 65 has been reported in one case of cervical cancer (1/107, 1%) (15). A codon 65 mutation in NRAS has been reported in 2/12 (17%) neuroendocrine lung cancers and 1/35 (3%) melanomas (16). The melanoma with NRASS65C also had BRAFV600E (16). The significance of codon 65 mutations in RAS has not been investigated previously, although the residue is highly conserved across different species. We demonstrate in the present study that codon 65 mutations in both KRAS and NRAS can constitutively activate the MAPK pathway. To our knowledge, this is the first report showing the functional significance of a RAS codon 65 mutation. The prevalence of a codon 65 mutation appears to be low in human cancers. However, many studies have only screened RAS mutations at codon 12/13 and 61, and may miss this mutation. Two of three FVPTC (66.7%) in our study have NRAS mutations. A NRAS mutation is more frequent in FTC (17) and FVPTC (8), supporting the notion that FVPTC shares many molecular features of FTC.

The overall rate of RAS mutation is 6% (5/88) in the cohort studied. The frequency of RAS mutation in PTC is variable depending on the cohort studied, ranging from 0% to 16% (2,8,18), with an overall rate of 11% (17). Higher frequencies have been reported in poorly differentiated thyroid carcinomas (23%) (19) and FTC (20%) (17). In the study by Wang et al. (18), a RAS mutation was found in 3 of 31 FTC (10%) and none of the 141 PTC in a Chinese population. Similar results were also reported by Di Cristofaro et al. in a French population (8). In contrast, Kimura et al. reported a RAS mutation in 11 of 67 PTC (16%). As discussed by Vasco et al. (17), multiple factors may contribute to such a wide range of variations, including the methodology used to detect mutations (the overall rate of mutations was significantly lower when estimated with direct sequencing than without: 12.3% vs. 17%), environmental factors (iodine deficiency, radiation exposure, and food-borne carcinogens), geographic variation, and tumor heterogeneity. Based on the pooled literature analysis by Vasco et al. (17), the most common mutation in PTC is NRASQ61R (5%), whereas the KRAS and HRAS mutation rate is about 3% and 2.6% respectively.

Concomitant RAS and BRAF mutations have been reported previously in PTC, poorly differentiated and anaplastic thyroid carcinomas (10), colorectal cancers (20), and cell lines (21). By using pyrosequencing to quantitate BRAF and RAS mutations, Guerra et al. demonstrated simultaneous subclonal BRAF and RAS mutations in two PTC (3%) (22).The frequency of concomitant KRAS and BRAF mutations increased along progression of colorectal cancers, suggesting that activation of both genes may have a synergistic effect for disease progression (20). In our two cases with concomitant RAS and BRAF mutations (one of them was a stage I PTC at diagnosis), they responded poorly to treatment. The disease relapsed within five years after surgery and progressed aggressively. Given that the BRAF mutation is present in all tumor sections and the RAS in some sections, it is likely that the RAS mutation occurred later than the BRAF and may play a role in disease progression. In the four cases of concomitant RAS and BRAF mutations reported by Costa et al. (10), two cases show a negative RAS and a positive BRAF mutation in some primary tumor sections, but concomitant RAS and BRAF mutations are present in all sections of lymph node metastasis, supporting the role of RAS in disease progression. RAS can promote thyroid tumorigenesis through both the MAKP and PIK3CA/AKT pathways. Concomitant RAS and BRAF mutations may allow simultaneous activation of both signaling pathways in cancer cells to gain growth advantage. Indeed, activation of AKT has frequently been found in PTC and is associated with cancer progression (23,24). PIK3CA and AKT mutations also frequently coexist with RAS and BRAF mutations in patients with advanced cancers (10,25,26). Although occurring less frequently, AKT1, AKT3, and PIK3CA mutations have been reported in PTC (27,28).

BRAF inhibitors have been undergoing clinical trials for thyroid cancer with mixed results (29,30). Recent studies have demonstrated that BRAF inhibitors may paradoxically increase MAPK activation in the presence of mutated RAS or wild-type BRAF (31–33). Therefore, patients with concomitant RAS and BRAF mutations may not be suitable for BRAF inhibitor treatment. Reactivation of MAPK or activation of PIK3CA/AKT (34) plays an important role in BRAF inhibitor resistance (35). MAPK reactivation can occur via NRAS mutations, COT overexpression, BRAFV600E alternative splicing or amplification, and MEK1 mutation, whereas PIK3CA/AKT can be activated through overexpression of receptor tyrosine kinases (35). RET/PTC can stimulate both PIK3CA/AKT and MAPK pathways (6,36,37). For patients with concomitant RET/PTC and BRAF mutations, it remains to be determined whether RET/PTC plays any role in BRAF inhibitor resistance, since cancer cells may escape inhibition via PIK3CA/AKT survival pathway. Targeting both MAPK and PIK3CA/AKT pathways may offer better long-term therapeutic effects. Selection of patients based on their mutation profile for clinical trials or treatment strategies may be warranted.

RET/PTC-1 was detected in 12 cases of PTC (14%). The presence of a RET/PTC rearrangement is not an indication of malignancy, as it occurs in benign thyroid nodules and is associated with a high growth rate of these nodules (38). Papillary carcinomas are often multifocal, and individual tumor foci in patients with multifocal PTC often arise from independent clonal origins (39). Based on the methodology used in the current study, we cannot distinguish whether dual mutations are present in the same cell or in different cells of the same tumor. We found concomitant mutations of RET/PTC-1 and BRAF or RAS in nine cases of PTC but none in the studied cell lines, suggesting intratumor heterogeneity or multiclonal origin in tumor samples and monoclonal origin of cell lines. The mutual exclusivity of gene mutations in the MAPK pathway may still hold if tumors are of monoclonal origin. A recent study of a single kidney cancer has shown remarkable diversity in the genetic changes that have taken place in different parts of the tumor (40). In advanced thyroid carcinoma, multiple genetic changes may occur in different parts of the tumor, resulting in concomitant mutations. Among nine cases with concomitant RET/PTC-1 and BRAF or RAS mutations, seven cases (78%) are in the late stage of cancer, indicating that RET/PTC-1 may occur late in subclonal PTC and is involved in tumor progression, as suggested previously by Unger et al. (41). Recently, Guerra et al. also reported concomitant subclonal BRAF and RET/PTC mutations in 19.4% (14/72) case of PTC (42). RAS mutations may have been underestimated in thyroid cancer. Using sensitive allele-specific competitive blocker-PCR (ACB-PCR), which can detect less than 1% mutant alleles, Myers et al. detected KRAS mutations in 30% of PTC (43). Although Sanger sequencing is less sensitive than pyrosequencing (detection limit is about 5%) or ACB-PCR to detect RAS mutations, mutations detected by Sanger sequencing indicate that at least 20% mutant alleles are present in the tumor sample, which may play a more significant role in promoting tumor progression. The oncogenic effects of RET/PTC-1 are mediated via RAS, which allows growth promoting signals through both the MAPK and PIK3CA/AKT pathways, and concomitant mutations of RET/PTC-1 and BRAF or RET/PTC-1 and RAS in subclonal/multiclonal PTC may provide a synergistic effect for disease progression.

Supplementary Material

Supplemental data
Supp_Table1.pdf (26.6KB, pdf)
Supplemental data
Supp_Fig1.pdf (75.9KB, pdf)

Acknowledgment

The study is supported by a Biotechnology grant #10-BIO957-20 from King Abdulaziz City for Science and Technology.

Author Disclosure Statement

All authors have nothing to declare.

References

  • 1.Hundahl SA, Fleming ID, Fremgen AM, Menck HR.1998A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985–1995 [see comments] [sic.]. Cancer 83:2638–2648 [DOI] [PubMed] [Google Scholar]
  • 2.Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA.2003High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 63:1454–1457 [PubMed] [Google Scholar]
  • 3.Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B, Beller U, Westra WH, Ladenson PW, Sidransky D.2003BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst 95:625–627 [DOI] [PubMed] [Google Scholar]
  • 4.Xing M.2005BRAF mutation in thyroid cancer. Endocr Relat Cancer 12:245–262 [DOI] [PubMed] [Google Scholar]
  • 5.Xing M.2007BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev 28:742–762 [DOI] [PubMed] [Google Scholar]
  • 6.Melillo RM, Castellone MD, Guarino V, De Falco V, Cirafici AM, Salvatore G, Caiazzo F, Basolo F, Giannini R, Kruhoffer M, Orntoft T, Fusco A, Santoro M.2005The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells. J Clin Invest 115:1068–1081 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 7.Henderson YC, Shellenberger TD, Williams MD, El-Naggar AK, Fredrick MJ, Cieply KM, Clayman GL.2009High rate of BRAF and RET/PTC dual mutations associated with recurrent papillary thyroid carcinoma. Clin Cancer Res 15:485–491 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Di Cristofaro J, Marcy M, Vasko V, Sebag F, Fakhry N, Wynford-Thomas D, De Micco C.2006Molecular genetic study comparing follicular variant versus classic papillary thyroid carcinomas: association of N-ras mutation in codon 61 with follicular variant. Hum Pathol 37:824–830 [DOI] [PubMed] [Google Scholar]
  • 9.Zhu Z, Ciampi R, Nikiforova MN, Gandhi M, Nikiforov YE.2006Prevalence of RET/PTC rearrangements in thyroid papillary carcinomas: effects of the detection methods and genetic heterogeneity. J Clin Endocrinol Metab 91:3603–3610 [DOI] [PubMed] [Google Scholar]
  • 10.Costa AM, Herrero A, Fresno MF, Heymann J, Alvarez JA, Cameselle-Teijeiro J, Garcia-Rostan G.2008BRAF mutation associated with other genetic events identifies a subset of aggressive papillary thyroid carcinoma. Clin Endocrinol (Oxf) 68:618–634 [DOI] [PubMed] [Google Scholar]
  • 11.Thyroid 2010. In: Edge SB, Byrd DR, Compton CC, et al. (eds): AJCC Cancer Staging Manual. Seventh edition. Springer, New York, NY, pp. 87–96 [Google Scholar]
  • 12.Baitei EY, Zou M, Al-Mohanna F, Collison K, Alzahrani AS, Farid NR, Meyer B, Shi Y.2009Aberrant BRAF splicing as an alternative mechanism for oncogenic B-Raf activation in thyroid carcinoma. J Pathol 217:707–715 [DOI] [PubMed] [Google Scholar]
  • 13.Shi YF, Zou MJ, Schmidt H, Juhasz F, Stensky V, Robb D, Farid NR.1991High rates of ras codon 61 mutation in thyroid tumors in an iodide-deficient area. Cancer Res 51:2690–2693 [PubMed] [Google Scholar]
  • 14.Dwight T, Thoppe SR, Foukakis T, Lui WO, Wallin G, Hoog A, Frisk T, Larsson C, Zedenius J.2003Involvement of the PAX8/peroxisome proliferator-activated receptor gamma rearrangement in follicular thyroid tumors. J Clin Endocrinol Metab 88:4440–4445 [DOI] [PubMed] [Google Scholar]
  • 15.Wegman P, Ahlin C, Sorbe B.2011Genetic alterations in the K-Ras gene influence the prognosis in patients with cervical cancer treated by radiotherapy. Int J Gynecol Cancer 21:86–91 [DOI] [PubMed] [Google Scholar]
  • 16.Brose MS, Volpe P, Feldman M, Kumar M, Rishi I, Gerrero R, Einhorn E, Herlyn M, Minna J, Nicholson A, Roth JA, Albelda SM, Davies H, Cox C, Brignell G, Stephens P, Futreal PA, Wooster R, Stratton MR, Weber BL.2002BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res 62:6997–7000 [PubMed] [Google Scholar]
  • 17.Vasko V, Ferrand M, Di Cristofaro J, Carayon P, Henry JF, de Micco C.2003Specific pattern of RAS oncogene mutations in follicular thyroid tumors. J Clin Endocrinol Metab 88:2745–2752 [DOI] [PubMed] [Google Scholar]
  • 18.Wang Y, Hou P, Yu H, Wang W, Ji M, Zhao S, Yan S, Sun X, Liu D, Shi B, Zhu G, Condouris S, Xing M.2007High prevalence and mutual exclusivity of genetic alterations in the phosphatidylinositol-3-kinase/akt pathway in thyroid tumors. J Clin Endocrinol Metab 92:2387–2390 [DOI] [PubMed] [Google Scholar]
  • 19.Volante M, Rapa I, Gandhi M, Bussolati G, Giachino D, Papotti M, Nikiforov YE.2009RAS mutations are the predominant molecular alteration in poorly differentiated thyroid carcinomas and bear prognostic impact. J Clin Endocrinol Metab 94:4735–4741 [DOI] [PubMed] [Google Scholar]
  • 20.Oliveira C, Velho S, Moutinho C, Ferreira A, Preto A, Domingo E, Capelinha AF, Duval A, Hamelin R, Machado JC, Schwartz S, Jr., Carneiro F, Seruca R.2007KRAS and BRAF oncogenic mutations in MSS colorectal carcinoma progression. Oncogene 26:158–163 [DOI] [PubMed] [Google Scholar]
  • 21.Seth R, Crook S, Ibrahem S, Fadhil W, Jackson D, Ilyas M.2009Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in advanced colorectal cancer. Gut 58:1234–1241 [DOI] [PubMed] [Google Scholar]
  • 22.Guerra A, Sapio MR, Marotta V, Campanile E, Rossi S, Forno I, Fugazzola L, Budillon A, Moccia T, Fenzi G, Vitale M.2012The primary occurrence of BRAF(V600E) is a rare clonal event in papillary thyroid carcinoma. J Clin Endocrinol Metab 97:517–524 [DOI] [PubMed] [Google Scholar]
  • 23.Miyakawa M, Tsushima T, Murakami H, Wakai K, Isozaki O, Takano K.2003Increased expression of phosphorylated p70S6 kinase and Akt in papillary thyroid cancer tissues. Endocr J 50:77–83 [DOI] [PubMed] [Google Scholar]
  • 24.Vasko V, Saji M, Hardy E, Kruhlak M, Larin A, Savchenko V, Miyakawa M, Isozaki O, Murakami H, Tsushima T, Burman KD, De Micco C, Ringel MD.2004Akt activation and localisation correlate with tumour invasion and oncogene expression in thyroid cancer. J Med Genet 41:161–170 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Janku F, Lee JJ, Tsimberidou AM, Hong DS, Naing A, Falchook GS, Fu S, Luthra R, Garrido-Laguna I, Kurzrock R.2011PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers. PLoS One 6:e22769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ricarte-Filho JC, Ryder M, Chitale DA, Rivera M, Heguy A, Ladanyi M, Janakiraman M, Solit D, Knauf JA, Tuttle RM, Ghossein RA, Fagin JA.2009Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1. Cancer Res 69:4885–4893 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Sozopoulos E, Litsiou H, Voutsinas G, Mitsiades N, Anagnostakis N, Tseva T, Patsouris E, Tseleni-Balafouta S.2010Mutational and immunohistochemical study of the PI3K/Akt pathway in papillary thyroid carcinoma in Greece. Endocr Pathol 21:90–100 [DOI] [PubMed] [Google Scholar]
  • 28.Abubaker J, Jehan Z, Bavi P, Sultana M, Al-Harbi S, Ibrahim M, Al-Nuaim A, Ahmed M, Amin T, Al-Fehaily M, Al-Sanea O, Al-Dayel F, Uddin S, Al-Kuraya KS.2008Clinicopathological analysis of papillary thyroid cancer with PIK3CA alterations in a Middle Eastern population. J Clin Endocrinol Metab 93:611–618 [DOI] [PubMed] [Google Scholar]
  • 29.Gupta-Abramson V, Troxel AB, Nellore A, Puttaswamy K, Redlinger M, Ransone K, Mandel SJ, Flaherty KT, Loevner LA, O'Dwyer PJ, Brose MS.2008Phase II trial of sorafenib in advanced thyroid cancer. J Clin Oncol 26:4714–4719 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ho AL, Sherman E.2011Clinical development of kinase inhibitors for the treatment of differentiated thyroid cancer. Clin Adv Hematol Oncol 9:32–41 [PubMed] [Google Scholar]
  • 31.Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, Hussain J, Reis-Filho JS, Springer CJ, Pritchard C, Marais R.2010Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140:209–221 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, Alvarado R, Ludlam MJ, Stokoe D, Gloor SL, Vigers G, Morales T, Aliagas I, Liu B, Sideris S, Hoeflich KP, Jaiswal BS, Seshagiri S, Koeppen H, Belvin M, Friedman LS, Malek S.2010RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464:431–435 [DOI] [PubMed] [Google Scholar]
  • 33.Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N.2010RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464:427–430 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis M, Cipolla AK, Wubbenhorst B, Xu X, Gimotty PA, Kee D, Santiago-Walker AE, Letrero R, D'Andrea K, Pushparajan A, Hayden JE, Brown KD, Laquerre S, McArthur GA, Sosman JA, Nathanson KL, Herlyn M.2010Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 18:683–695 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lo RS.2012Receptor tyrosine kinases in cancer escape from BRAF inhibitors. Cell Res 22:945–947 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Miyagi E, Braga-Basaria M, Hardy E, Vasko V, Burman KD, Jhiang S, Saji M, Ringel MD.2004Chronic expression of RET/PTC 3 enhances basal and insulin-stimulated PI3 kinase/AKT signaling and increases IRS-2 expression in FRTL-5 thyroid cells. Mol Carcinog 41:98–107 [DOI] [PubMed] [Google Scholar]
  • 37.Segouffin-Cariou C, Billaud M.2000Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. J Biol Chem 275:3568–3576 [DOI] [PubMed] [Google Scholar]
  • 38.Sapio MR, Guerra A, Marotta V, Campanile E, Formisano R, Deandrea M, Motta M, Limone PP, Fenzi G, Rossi G, Vitale M.2011High growth rate of benign thyroid nodules bearing RET/PTC rearrangements. J Clin Endocrinol Metab 96:E916–919 [DOI] [PubMed] [Google Scholar]
  • 39.Shattuck TM, Westra WH, Ladenson PW, Arnold A.2005Independent clonal origins of distinct tumor foci in multifocal papillary thyroid carcinoma. N Engl J Med 352:2406–2412 [DOI] [PubMed] [Google Scholar]
  • 40.Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S, McDonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA, Swanton C.2012Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366:883–892 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Unger K, Zitzelsberger H, Salvatore G, Santoro M, Bogdanova T, Braselmann H, Kastner P, Zurnadzhy L, Tronko N, Hutzler P, Thomas G.2004Heterogeneity in the distribution of RET/PTC rearrangements within individual post-Chernobyl papillary thyroid carcinomas. J Clin Endocrinol Metab 89:4272–4279 [DOI] [PubMed] [Google Scholar]
  • 42.Guerra A, Zeppa P, Bifulco M, Vitale M.2014Concomitant BRAF(V600E) mutation and RET/PTC rearrangement is a frequent occurrence in papillary thyroid carcinoma. Thyroid 24:254–259 [DOI] [PubMed] [Google Scholar]
  • 43.Myers MB, McKim KL, Parsons BL.2014A subset of papillary thyroid carcinomas contain KRAS mutant subpopulations at levels above normal thyroid. Mol Carcinog 53:159–167 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental data
Supp_Table1.pdf (26.6KB, pdf)
Supplemental data
Supp_Fig1.pdf (75.9KB, pdf)

Articles from Thyroid are provided here courtesy of Mary Ann Liebert, Inc.

RESOURCES