CONTEXT: A polymerase chain reaction-based companion diagnostic (cobas 4800 BRAF 
V600 Mutation Test) was recently approved by the US Food and Drug Administration 
to select patients with BRAF-mutant metastatic melanoma for treatment with the 
BRAF inhibitor vemurafenib.
OBJECTIVES: (1) To compare the analytic performance of the cobas test to Sanger 
sequencing by using screening specimens from phase II and phase III trials of 
vemurafenib, and (2) to assess the reproducibility of the cobas test at 
different testing sites.
DESIGN: Specimens from 477 patients were used to determine positive and negative 
percent agreements between the cobas test and Sanger sequencing for detecting 
V600E (1799T>A) mutations. Specimens were evaluated with a massively parallel 
pyrosequencing method (454) to resolve discordances between polymerase chain 
reaction and Sanger results. Reproducibility of the cobas test was assessed at 3 
sites by using 3 reagent lots and an 8-member panel of melanoma samples.
RESULTS: A valid cobas result was obtained for all eligible patients. Sanger 
sequencing had a failure rate of 9.2% (44 of 477). For the remaining 433 
specimens, positive percent agreement was 96.4% (215 of 223) and negative 
percent agreement, 80% (168 of 210). Among 42 cobas mutation-positive/Sanger 
V600E-negative specimens, 17 were V600E positive and 24 were V600K positive by 
454. The cobas test detected 70% of V600K mutations. In the reproducibility 
study, a correct interpretation was made for 100% of wild-type specimens and 
specimens with greater than 5% mutant alleles; V600E mutations were detected in 
90% of specimens with less than 5% mutant alleles.
CONCLUSIONS: The cobas test (1) had a lower assay failure rate than that of 
Sanger, (2) was more sensitive in detecting V600E mutations, (3) detected most 
V600K mutations, and (4) was highly reproducible.