title: Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients.
abstract: BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients' samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAF(V600E) and BRAF(V600K) mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method.
authors.affiliation: Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Raffaela
authors.initials: R
authors.lastname: Barbano
authors.affiliation: Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Pasculli
authors.affiliation: Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Michelina
authors.initials: M
authors.lastname: Coco
authors.affiliation: Biostatic Unit, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Fontana
authors.affiliation: Biostatic Unit, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Copetti
authors.affiliation: Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.|Department of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Michelina
authors.initials: M
authors.lastname: Rendina
authors.affiliation: Department of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Vanna Maria
authors.initials: VM
authors.lastname: Valori
authors.affiliation: Department of Pathology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG) 71013, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Graziano
authors.affiliation: Department of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Evaristo
authors.initials: E
authors.lastname: Maiello
authors.affiliation: Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.|CIR Laboratory for Molecular Medicine and Biotechnology, University Campus Biomedico, Rome 00128, Italy.
authors.forename: Vito Michele
authors.initials: VM
authors.lastname: Fazio
authors.affiliation: Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotundo (FG) 71013, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Parrella
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis; D005260:Female; D018740:Genetic Heterogeneity; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008543:Melanins / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics*; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000188:drug therapy / Q000235:genetics
chemical_list: D003062:Codon; D008543:Melanins; D047428:Protein Kinase Inhibitors; D048493:Proto-Oncogene Proteins B-raf