title: Evaluation of BRAF mutation testing methodologies in formalin-fixed, paraffin-embedded cutaneous melanomas.
abstract: Patients diagnosed with BRAF V600E mutated cutaneous melanoma show response to treatment with the BRAF inhibitor Vemurafenib. Different methods for BRAF mutation detection exist; however, only the Cobas 4800 BRAF V600 Mutation Test has been approved by the US Food and Drug Administration for patient selection. The results from this test depend on the percentage of tumor cells in the samples, which clinically may be estimated with substantial variation. We have evaluated five different methods: the Cobas test, Sanger sequencing, pyrosequencing, TaqMan-based allele-specific PCR, and Competitive Amplification of Differentially Melting Amplicons (CADMA), for detection of BRAF c.1799T>A (V600E) mutations in 28 formalin-fixed paraffin-embedded (FFPE) cutaneous melanoma samples. We show that the frequency of the BRAF V600E mutation is influenced by the analytical sensitivity of the applied method. However, a 100% consensus was observed among all five methods when the tumor tissue fraction was more than 10% of all tissue or more than 50% of cell-dense tissue. When using Sanger sequencing, pyrosequencing, or the Cobas test, it may be advisable to perform macrodissection before mutation testing if the tumor cell fraction is low. CADMA and TaqMan may not require macrodissections for a reliable test. Therefore, the use of more sensitive methods may have a future in testing for BRAF mutations in clinical settings.
authors.affiliation: Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark. johanneromer@hotmail.com
authors.forename: Johanne
authors.initials: J
authors.lastname: Lade-Keller
authors.forename: Kirsten M
authors.initials: KM
authors.lastname: Rømer
authors.forename: Per
authors.initials: P
authors.lastname: Guldberg
authors.forename: Rikke
authors.initials: R
authors.lastname: Riber-Hansen
authors.forename: Lise Lotte
authors.initials: LL
authors.lastname: Hansen
authors.forename: Torben
authors.initials: T
authors.lastname: Steiniche
authors.forename: Henrik
authors.initials: H
authors.lastname: Hager
authors.forename: Lasse S
authors.initials: LS
authors.lastname: Kristensen
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008545:Melanoma / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D009154:Mutation; D016612:Paraffin Embedding; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D013449:Sulfonamides / Q000494:pharmacology; D000077484:Vemurafenib
chemical_list: D007211:Indoles; D013449:Sulfonamides; D005557:Formaldehyde; D000077484:Vemurafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf