title: Next-Generation Genotyping by Digital PCR to Detect and Quantify the BRAF V600E Mutation in Melanoma Biopsies.
abstract: The detection of the BRAF V600E mutation in melanoma samples is used to select patients who should respond to BRAF inhibitors. Different techniques are routinely used to determine BRAF status in clinical samples. However, low tumor cellularity and tumor heterogeneity can affect the sensitivity of somatic mutation detection. Digital PCR (dPCR) is a next-generation genotyping method that clonally amplifies nucleic acids and allows the detection and quantification of rare mutations. Our aim was to evaluate the clinical routine performance of a new dPCR-based test to detect and quantify BRAF mutation load in 47 paraffin-embedded cutaneous melanoma biopsies. We compared the results obtained by dPCR with high-resolution melting curve analysis and pyrosequencing or with one of the allele-specific PCR methods available on the market. dPCR showed the lowest limit of detection. dPCR and allele-specific amplification detected the highest number of mutated samples. For the BRAF mutation load quantification both dPCR and pyrosequencing gave similar results with strong disparities in allele frequencies in the 47 tumor samples under study (from 0.7% to 79% of BRAF V600E mutations/sample). In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples.
authors.affiliation: Department of Biology and Oncogenetics, Institut Régional du Cancer de Montpellier, Montpellier, France; Department of Biobank, Institut Régional du Cancer de Montpellier, Montpellier, France. Electronic address: pierre-jean.lamy@icm.unicancer.fr.
authors.forename: Pierre-Jean
authors.initials: PJ
authors.lastname: Lamy
authors.affiliation: Department of Statistics, Institut Régional du Cancer de Montpellier, Montpellier, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Castan
authors.affiliation: Department of Biology and Oncogenetics, Institut Régional du Cancer de Montpellier, Montpellier, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Lozano
authors.affiliation: Department of Biology and Oncogenetics, Institut Régional du Cancer de Montpellier, Montpellier, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Montélion
authors.affiliation: Department of Biology and Oncogenetics, Institut Régional du Cancer de Montpellier, Montpellier, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Audran
authors.affiliation: Department of Biobank, Institut Régional du Cancer de Montpellier, Montpellier, France; Department of Pathology, Institut Régional du Cancer de Montpellier, Montpellier, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bibeau
authors.affiliation: Department of Biology and Oncogenetics, Institut Régional du Cancer de Montpellier, Montpellier, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Roques
authors.affiliation: Department of Biology and Oncogenetics, Institut Régional du Cancer de Montpellier, Montpellier, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Montels
authors.affiliation: Department of Biology and Oncogenetics, Institut Régional du Cancer de Montpellier, Montpellier, France.
authors.forename: Anne-Claire
authors.initials: AC
authors.lastname: Laberenne
mesh_terms: D001706:Biopsy; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008545:Melanoma / Q000175:diagnosis / Q000235:genetics* / Q000601:surgery; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012878:Skin Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000601:surgery; D000096142:Cutaneous Malignant Melanoma
chemical_list: D004273:DNA, Neoplasm; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf