Comparison of Three BRAF Mutation Tests in Formalin-Fixed Paraffin Embedded Clinical Samples

Abstract

Background: Recently, BRAF inhibitors showed dramatic treatment outcomes in BRAF V600 mutant melanoma. Therefore, the accuracy of BRAF mutation test is critical.

Methods: BRAF mutations were tested by dual-priming oligonucleotide-polymerase chain reaction (DPO-PCR), direct sequencing and subsequently retested with a real-time PCR assay, cobas 4800 V600 mutation test. In total, 64 tumors including 34 malignant melanomas and 16 papillary thyroid carcinomas were analyzed. DNA was extracted from formalin-fixed paraffin embedded tissue samples and the results of cobas test were directly compared with those of DPO-PCR and direct sequencing.

Results: BRAF mutations were found in 23 of 64 (35.9%) tumors. There was 9.4% discordance among 3 methods. Out of 6 discordant cases, 4 cases were melanomas; 3 cases were BRAF V600E detected only by cobas test, but were not detected by DPO-PCR and direct sequencing. One melanoma patient with BRAF mutation detected only by cobas test has been on vemurafenib treatment for 6 months and showed a dramatic response to vemurafenib. DPO-PCR failed to detect V600K mutation in one case identified by both direct sequencing and cobas test.

Conclusions: In direct comparison of the currently available DPO-PCR, direct sequencing and real-time cobas test for BRAF mutation, real-time PCR assay is the most sensitive method.

Keywords: BRAF mutation; Dual-priming oligonucleotide-PCR; Melanoma; Real-time polymerase chain reaction; Sanger sequencing.

Fig. 1

(A) Results of dual-priming oligonucleotide-polymerase chain reaction (wild) and direct sequencing (wild) in BRAF mutation-positive malignant melanoma case confirmed by cobas 4800 BRAF V600 mutation test. (B) Results of dual-priming oligonucleotide-polymerase chain reaction (failed) and direct sequencing (V600E) in BRAF mutation-positive malignant melanoma case confirmed by cobas 4800 BRAF V600 mutation test. (C) Results of dual-priming oligonucleotide-polymerase chain reaction (wild) and direct sequencing (V600K) in BRAF mutation-positive malignant melanoma case confirmed by cobas 4800 BRAF V600 mutation test.