Detection 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.
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.



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 IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays.



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, IdyllaTM 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.



HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays.
Université de Lorraine, Faculté de Pharmacie, Nancy, France.|CNRS UMR 7039 CRAN, Nancy, France.|Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandoeuvre-lès-Nancy, France.
Alexandre
A
Harlé
Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandoeuvre-lès-Nancy, France.|Institut de Cancérologie de Lorraine, Cellule biostatistique, Vandoeuvre-lès-Nancy, France.
Julia
J
Salleron
Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandoeuvre-lès-Nancy, France.
Claire
C
Franczak
Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandoeuvre-lès-Nancy, France.
Cindy
C
Dubois
Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandoeuvre-lès-Nancy, France.
Pierre
P
Filhine-Tressarieu
CNRS UMR 7039 CRAN, Nancy, France.|Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandoeuvre-lès-Nancy, France.
Agnès
A
Leroux
Université de Lorraine, Faculté de Pharmacie, Nancy, France.|CNRS UMR 7039 CRAN, Nancy, France.|Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandoeuvre-lès-Nancy, France.
Jean-Louis
JL
Merlin
D000483:Alleles; D001331:Automation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D008545:Melanoma / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D009362:Neoplasm Metastasis; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf