Claire Franczak1, Julia Salleron2,3, Cindy Dubois1, Pierre Filhine-Trésarrieu1, Agnès Leroux2,4,1, Jean-Louis Merlin2,4,1, Alexandre Harlé5,6,7. 1. Institut de Cancérologie de Lorraine, Service de Biopathologie, 6 Avenue de Bourgogne, CS 30519, 54 519, Vandoeuvre-lès-Nancy Cedex, France. 2. Université de Lorraine, Nancy, France. 3. Institut de Cancérologie de Lorraine, Cellule data management et biostatistique, Vandoeuvre-lès-Nancy, France. 4. CNRS UMR 7039 CRAN, Nancy, France. 5. Université de Lorraine, Nancy, France. a.harle@nancy.unicancer.fr. 6. CNRS UMR 7039 CRAN, Nancy, France. a.harle@nancy.unicancer.fr. 7. 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.
Abstract
BACKGROUND: 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. METHODS: 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). RESULTS: 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.
BACKGROUND: 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. METHODS: 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). RESULTS: 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.
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