Fiche publication
Date publication
juillet 2015
Journal
The Journal of molecular diagnostics : JMD
Auteurs
Membres identifiés du Cancéropôle Est :
Pr BIBEAU Frédéric
Tous les auteurs :
Lamy PJ, Castan F, Lozano N, Montélion C, Audran P, Bibeau F, Roques S, Montels F, Laberenne AC
Lien Pubmed
Résumé
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.
Mots clés
Biopsy, DNA Mutational Analysis, methods, DNA, Neoplasm, analysis, High-Throughput Nucleotide Sequencing, methods, Humans, Melanoma, diagnosis, Mutation, genetics, Polymerase Chain Reaction, methods, Proto-Oncogene Proteins B-raf, genetics, Skin Neoplasms, diagnosis
Référence
J Mol Diagn. 2015 Jul;17(4):366-73
