title: Comparison of Five Different Assays for the Detection of BRAF Mutations in Formalin-Fixed Paraffin Embedded Tissues of Patients with Metastatic Melanoma.
abstract: Metastatic or unresectable melanoma is a serious and deadly disease. Anti-BRAF and immunotherapy improved overall survival in patients with metastatic disease. Thus, BRAF genotyping is important to choose the right therapy.
  
  
  
  In our study, we assessed and compared BRAF mutations in 59 formalin-fixed and paraffin-embedded tumor samples of patients with metastatic melanoma with next-generation sequencing (NGS), Cobas®4800 BRAF V600 mutation test CE-IVD commercial kit, high-resolution melting PCR (HRM), multiplex real-time allele specific amplification (multiplexed RT-ASA) and immunohistochemistry (IHC).
  
  
  
  Thirty-one samples were found bearing a BRAF mutation with NGS (52.5%), 28 with Cobas®test (47.5%), 28 with HRM (47.5%), 29 with multiplexed RT-ASA (49.2%) and 27 with IHC (45.8%). Based on NGS data, 26 (81.2%) were c.1799 T>A (p.Val600Glu), 3 (9.4%) were c. 1798-1799 GT>AA (p.Val600Lys), 1 was c.1789_1790 CT>TC (p.Leu597Ser) and 2 were complex mutations. Sensitivity was 90.3% for Cobas®test, 93.1% for multiplexed RT-ASA and 87.1% for IHC and HRM. Specificity was 100% for Cobas®test, IHC and multiplexed RT-ASA and 96.4% for HRM. The reference assay was NGS. Rare mutations were detected with NGS and HRM: c.1789_1790 CT>TC (p.Leu597Ser) mutation and the complex mutation c.1796 A>T; c.1797_1798 insACT (p.Thr599Thr; p.Thr599_Val600insThr). Our data suggest that multiplexed RT-ASA is the most sensitive assay but specific primers for each mutation are needed. HRM can detect all exon 15 mutations but has a lower sensitivity. Because of its specificity for Val600Glu mutation, IHC may be considered only as a screening tool and testing should be completed by a method able to detect other V600 mutations. BRAF Cobas®assay is Val600Glu-specific and has poor sensitivity for the other V600 mutations; thus, it looks important to use multiplex assays able to detect all V600 mutations because a false-negative result will deprive the patient of an important treatment option.
authors.affiliation: Institut de Cancérologie de Lorraine, Service de Biopathologie, 6 Avenue de Bourgogne, CS 30519, 54 519, Vandoeuvre-lès-Nancy Cedex, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Franczak
authors.affiliation: Université de Lorraine, Nancy, France.|Institut de Cancérologie de Lorraine, Cellule data management et biostatistique, Vandoeuvre-lès-Nancy, France.
authors.forename: Julia
authors.initials: J
authors.lastname: Salleron
authors.affiliation: Institut de Cancérologie de Lorraine, Service de Biopathologie, 6 Avenue de Bourgogne, CS 30519, 54 519, Vandoeuvre-lès-Nancy Cedex, France.
authors.forename: Cindy
authors.initials: C
authors.lastname: Dubois
authors.affiliation: Institut de Cancérologie de Lorraine, Service de Biopathologie, 6 Avenue de Bourgogne, CS 30519, 54 519, Vandoeuvre-lès-Nancy Cedex, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Filhine-Trésarrieu
authors.affiliation: Université de Lorraine, Nancy, France.|CNRS UMR 7039 CRAN, Nancy, France.|Institut de Cancérologie de Lorraine, Service de Biopathologie, 6 Avenue de Bourgogne, CS 30519, 54 519, Vandoeuvre-lès-Nancy Cedex, France.
authors.forename: Agnès
authors.initials: A
authors.lastname: Leroux
authors.affiliation: Université de Lorraine, Nancy, France.|CNRS UMR 7039 CRAN, Nancy, France.|Institut de Cancérologie de Lorraine, Service de Biopathologie, 6 Avenue de Bourgogne, CS 30519, 54 519, Vandoeuvre-lès-Nancy Cedex, France.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Merlin
authors.affiliation: Université de Lorraine, Nancy, France. a.harle@nancy.unicancer.fr.|CNRS UMR 7039 CRAN, Nancy, France. a.harle@nancy.unicancer.fr.|Institut de Cancérologie de Lorraine, Service de Biopathologie, 6 Avenue de Bourgogne, CS 30519, 54 519, Vandoeuvre-lès-Nancy Cedex, France. a.harle@nancy.unicancer.fr.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Harlé
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D009154:Mutation; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D048493:Proto-Oncogene Proteins B-raf