High rate of BRAF and RET/PTC dual mutations associated with recurrent papillary thyroid carcinoma

Abstract

Purpose: Papillary thyroid carcinoma (PTC), the most common thyroid malignancy, usually possesses BRAF mutation or rearranged in translation (RET)/PTC rearrangements. PTC usually possesses BRAF mutation or RET/PTC rearrangements. The mutation status of patients with recurrent PTC has never been characterized in a large population.

Experimental design: Mutation status was determined in a cohort of 54 patients with recurrent PTC and analyzed for clinicopathologic relationships. BRAF and ras mutations were determined by PCR and sequencing of genomic DNA. RET/PTC rearrangements were analyzed by reverse transcription-PCR.

Results: BRAF mutation in exon 15 (V600E) was found in 42/54 (77.8%) recurrent PTC patients. The RET/PTC rearrangements were detected in 9 of 54 (16.7%) patients. In addition, 5 of 54 (9.3%) recurrent PTC patients had both a BRAF mutation and a RET/PTC rearrangement. The prevalence of tumors with dual mutations found in the recurrent population far exceeds the frequency historically reported for patients with primary PTC. Patients with dual mutations were significantly older (80% older than 45 years) than patients with a BRAF mutation alone (38% older than 45 years).

Conclusions: Recurrent PTC is significantly associated with a predominant BRAF mutation. RET/PTC rearrangements, although commonly associated with primary PTCs in younger patients, are uncommonly found in recurrent PTC patients. In addition, the incidence of dual mutations was higher in patients with recurrent PTC than in those primary PTC, as reported by others.

Fig. 1

Detection of RET/PTC rearrangements and BRAF mutation in recurrent PTC. A, RET/PTC1 rearrangement from patient #39 was detected in total RNA. The positive control (+control) was cDNA made from TPC-1 cells (positive for RET/PTC1 rearrangement), and negative control (−control) was cDNA made from NPA87 cells (BRAF mutated and no RET/PTC rearrangements). B, total RNA was prepared from paraffin-embedded tissues (from patients #10, #18, #25, #28, #42, #47, #48, and #57) and RT-PCR was done to detect RET/PTC 2 or RET/PTC3 rearrangement (arrow). The PCR product was visualized on a 2% agarose gel stained with ethidium bromide. Actin was used as a control. The positive controls for RET/PTC2 and RET/PTC3 were plasmids containing the cDNAs of each gene and negative controls were buffer only without any cDNA added. C, PCR product of exon 15 of BRAF from patients with dual mutations (#18, #25, #28, #42, and #57) was sequenced with forward primer (left) and reverse primer (right). Arrows, the transition from T to A by forward primer and A to T by reverse primer.

Fig. 2

Age of recurrent PTC patients in each mutation group. Fifty-four recurrent PTC patients were separated into five groups by their mutation status, including BRAF mutation only (BRAF), RET/PTC rearrangements only (RET/PTC), dual mutations with a BRAF mutation and a RET/PTC rearrangement (B & R), K-ras mutation only (K-ras), or no detectable mutation (none). Solid line, age at 45 y as discussed in the Results. The graph was generated by Prism software.