Detection of BRAF p.V600E mutations in melanomas: comparison of four methods argues for sequential use of immunohistochemistry and pyrosequencing

Abstract

BRAF p.V600 mutation detection recently became necessary to treat metastatic melanoma patients with vemurafenib. This study compares different methods of detection of BRAF mutations. Melanoma samples from 111 patients were analyzed for BRAF mutations, and for 89 of them, results were obtained with the four following methods: Sanger sequencing, real-time PCR, immunohistochemistry, and pyrosequencing. All samples contained at least 60% of tumor cells. Directional Sanger sequencing of PCR products failed to detect 3 of 40 p.V600E-mutated cases (7.5%) (sensitivity, 92.5%; 95% CI, 78.5% to 98.0%). BRAF p.V600E-specific real-time PCR identified 39 of 40 p.V600E-mutated cases (97.6%) (sensitivity, 97.5%; 95% CI, 87.1% to 99.6%) and all 39 wild-type (WT) cases and surprisingly was also positive for 6/6 p.V600K (specificity, 87.8%; 95% CI, 75.8% to 94.3%). However, other mutations, p.V600R (n = 1), p.K601E (n = 2), and p.600_601delinsE (n = 1), were not detected. Immunohistochemistry with VE1, specific for p.V600E, identified all p.V600E and WT cases (sensitivity, 100%; 95% CI, 91.2% to 100%) but was negative for all other BRAF mutations. Pyrosequencing successfully identified all WT and mutated cases. Immunohistochemistry is highly specific for p.V600E, and could be used as a first-line method, as is currently performed for HER2 amplification detection. Pyrosequencing proved to be the most efficient method to detect BRAF mutations in melanomas and could be performed on VE1-negative or uninterpretable cases.