title: 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.
authors.affiliation: EA4340, University of Versailles, Boulogne, France.
authors.forename: Emeline
authors.initials: E
authors.lastname: Colomba
authors.forename: Zofia
authors.initials: Z
authors.lastname: Hélias-Rodzewicz
authors.forename: Andreas
authors.initials: A
authors.lastname: Von Deimling
authors.forename: Cristi
authors.initials: C
authors.lastname: Marin
authors.forename: Nathalie
authors.initials: N
authors.lastname: Terrones
authors.forename: Dominique
authors.initials: D
authors.lastname: Pechaud
authors.forename: Sylvie
authors.initials: S
authors.lastname: Surel
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Côté
authors.forename: Frédérique
authors.initials: F
authors.lastname: Peschaud
authors.forename: David
authors.initials: D
authors.lastname: Capper
authors.forename: Hélène
authors.initials: H
authors.lastname: Blons
authors.forename: Ute
authors.initials: U
authors.lastname: Zimmermann
authors.forename: Thierry
authors.initials: T
authors.lastname: Clerici
authors.forename: Philippe
authors.initials: P
authors.lastname: Saiag
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Emile
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007211:Indoles / Q000627:therapeutic use; D008545:Melanoma / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D013449:Sulfonamides / Q000627:therapeutic use; D000077484:Vemurafenib
chemical_list: D007211:Indoles; D048493:Proto-Oncogene Proteins B-raf; D018719:Erb-b2 Receptor Tyrosine Kinases; D013449:Sulfonamides; D000077484:Vemurafenib; C508053:ERBB2 protein, human; C482119:BRAF protein, human