Elsevier

The Journal of Molecular Diagnostics

Volume 15, Issue 1, January–February 2013, Pages 94-100
The Journal of Molecular Diagnostics

Regular article
Detection of BRAF p.V600E Mutations in Melanomas: Comparison of Four Methods Argues for Sequential Use of Immunohistochemistry and Pyrosequencing

https://doi.org/10.1016/j.jmoldx.2012.09.001Get rights and content
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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.

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Supported by grants from the nonprofit associations Ligue contre le Cancer, AREP (Association Pour la Recherche de l’Enseignement en Pathologie), and AROLD (Association Pour la Recherche en Oncologie Digestive).

Disclosures: J.F.E. and P.S. received honoraria for counseling on diagnosis and/or treatment of patients with melanomas from Roche and Glaxo Smith Kline. D.C. and A.V.D. applied for a patent on the diagnostic use of BRAF p.V600E mutant–specific antibody VE1. All terms are being managed by the German Cancer Research Center in accordance with its conflict of interest policies. Funding sources had no involvement in study design, data collection, data analysis, data interpretation, writing of the manuscript, approval of the manuscript, or the decision to submit the manuscript for publication. No one has been paid to write this article by a pharmaceutical company or other agency.