title: Detection of BRAF V600 mutations in metastatic melanoma: comparison of the Cobas 4800 and Sanger sequencing assays.
abstract: Detection of the BRAF V600E mutation is required for use of the BRAF inhibitor, vemurafenib, in patients with metastatic melanoma. Although the Roche Cobas 4800 BRAF V600 Mutation Test is approved, it detects primarily the single-nucleotide V600E mutation and could miss other potentially relevant V600 mutations. To assess the detection rate of the cobas assay for V600 mutations in clinical specimens, we compared the results of this assay with Sanger sequencing in 295 melanoma FFPE samples. Twenty samples were excluded because of invalid results on the cobas (n = 3), sequencing (n = 15), or both (n = 2). V600 mutations were detected by the cobas test in 96 (34.9%) of 275 samples and by Sanger sequencing in 118 (42.9%) of 275 samples. Thus, relative to Sanger sequencing, the cobas test exhibited 80.5% sensitivity (95% CI, 72.4% to 86.6%) and 99.4% specificity (95% CI, 96.5% to 99.9%). Of 23 samples with positive sequencing results but negative cobas results, 21 harbored dinucleotide mutations (V600E in 6, V600K in 10, and V600R in 5); the other two involved single-nucleotide mutations (V600E and V600G). These findings indicate that the cobas assay may miss many V600 mutations in clinical specimens. In our study, the addition of Sanger sequencing for samples with negative cobas results increased the detection rate to 42.9%. This approach could help maximize the number of patients who benefit from BRAF inhibitor treatment.
authors.affiliation: Department of Hematology and Oncology, Quest Diagnostics Nichols Institute, San Juan Capistrano, California. Electronic address: kevin.z.qu@questdiagnostics.com.
authors.forename: Kevin
authors.initials: K
authors.lastname: Qu
authors.forename: Qiulu
authors.initials: Q
authors.lastname: Pan
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.forename: Luis
authors.initials: L
authors.lastname: Rodriguez
authors.forename: Ke
authors.initials: K
authors.lastname: Zhang
authors.forename: Hairong
authors.initials: H
authors.lastname: Li
authors.forename: Albert
authors.initials: A
authors.lastname: Ho
authors.forename: Heather
authors.initials: H
authors.lastname: Sanders
authors.forename: Anthony
authors.initials: A
authors.lastname: Sferruzza
authors.forename: Shih-Min
authors.initials: SM
authors.lastname: Cheng
authors.forename: Diedre
authors.initials: D
authors.lastname: Nguyen
authors.forename: Dan
authors.initials: D
authors.lastname: Jones
authors.forename: Frederic
authors.initials: F
authors.lastname: Waldman
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D004252:DNA Mutational Analysis; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000556:secondary; D008875:Middle Aged; D020125:Mutation, Missense; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf