Concomitant RAS, RET/PTC, or BRAF mutations in advanced stage of papillary thyroid carcinoma

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: BRAF(V600E) 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 NRAS(Q61R) in one CPTC and two follicular variant PTC (FVPTC; 3%, 3/88). KRAS(S65N) 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 BRAF(V600E) and KRAS, or BRAF(V600E) 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. KRAS(S65N) can constitutively activate the MAPK pathway.

FIG. 1.

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.

FIG. 2.

Characterization of KRAS^S65N and NRAS^S65C on mitogen-associated protein kinase (MAPK) activity. (A) The mutant and wild-type constructs (KRAS^S65N, NRAS^S65C, and KRAS^WT) 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 KRAS^WT, KRAS^S65N, and NRAS^S65C 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 KRAS^S65N and NRAS ^S65C transfected cells following 48 h serum starvation. (C) Cell proliferation of HEK293 cells transfected with KRAS^S65N or KRAS^WT. 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 KRAS^S65N transfection. Both phospho-CREB and-ERK1/2 expressions were increased in CAL62 and BCPAP cell lines. HRAS^G12V 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.

FIG. 3.

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.

FIG. 4.

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).