Open AccessDetection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma
Author(s) -
Alexandre Harlé,
Julia Salleron,
Claire Franczak,
Cindy Dubois,
Pierre Filhine-Tressarieu,
Agnès Leroux,
JeanLouis Merlin
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0153576
Subject(s) - genotyping , immunohistochemistry , high resolution melt , mutation , metastatic melanoma , medicine , melanoma , polymerase chain reaction , molecular diagnostics , microbiology and biotechnology , cancer research , pathology , genotype , biology , bioinformatics , gene , genetics
Background Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies. Methods Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM) PCR, Real-time allele-specific amplification (RT-ASA) PCR, Next generation sequencing (NGS), immunohistochemistry (IHC) and the fully-automated molecular diagnostics platform Idylla TM . Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays. Results BRAF mutations were found in 28(47.5%), 29(49.2%), 31(52.5%), 29(49.2%) and 27(45.8%) samples with HRM, RT-ASA, NGS, Idylla TM and IHC respectively. Twenty-six (81.2%) samples were found bearing a c.1799T>A (p.Val600Glu) mutation, three (9.4%) with a c.1798_1799delinsAA (p.Val600Lys) mutation and one with c.1789_1790delinsTC (p.Leu597Ser) mutation. Two samples were found bearing complex mutations. Conclusions HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, Idylla TM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. Idylla TM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays.