title: A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma.
abstract: Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3-4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.
authors.affiliation: School of Surgery, University of Western Australia, Nedlands, Australia.
authors.forename: Anna
authors.initials: A
authors.lastname: Richter
authors.forename: Fabienne
authors.initials: F
authors.lastname: Grieu
authors.forename: Amerigo
authors.initials: A
authors.lastname: Carrello
authors.forename: Benhur
authors.initials: B
authors.lastname: Amanuel
authors.forename: Kateh
authors.initials: K
authors.lastname: Namdarian
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Rynska
authors.forename: Amanda
authors.initials: A
authors.lastname: Lucas
authors.forename: Victoria
authors.initials: V
authors.lastname: Michael
authors.forename: Anthony
authors.initials: A
authors.lastname: Bell
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Fox
authors.forename: Chelsee A
authors.initials: CA
authors.lastname: Hewitt
authors.forename: Hongdo
authors.initials: H
authors.lastname: Do
authors.forename: Grant A
authors.initials: GA
authors.lastname: McArthur
authors.forename: Stephen Q
authors.initials: SQ
authors.lastname: Wong
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
authors.forename: Barry
authors.initials: B
authors.lastname: Iacopetta
mesh_terms: D001315:Australia; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D008297:Male; D008545:Melanoma / Q000235:genetics*; D016612:Paraffin Embedding / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D016037:Single-Blind Method; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf