Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a 
small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in 
BRAFV600 mutant melanoma, creating the need for a well-validated companion 
diagnostic to select patients for treatment. We describe analytic performance 
characteristics of the cobas 4800 BRAF V600 Mutation Test, the test used to 
select patients for the pivotal vemurafenib trials. This real-time polymerase 
chain reaction assay was designed to detect the V600E (1799T>A) mutation DNA 
from formalin-fixed paraffin-embedded tissue samples. Sensitivity was assessed 
using blends of cell lines or tumor DNA, and tumor specimens with low levels of 
mutant alleles, as determined by 454 sequencing (a quantitative next-generation 
pyrosequencing method). A >96% hit rate was obtained across all specimen types 
with 5% mutant alleles at a DNA input of 125 ng, an amount readily obtained from 
one 5-μm section. The cobas test showed a higher sensitivity and specificity 
than direct bidirectional sequencing in a panel of 219 melanoma specimens. Cross 
reactivity with V600K and V600D was observed. Repeated testing of 5 specimens by 
2 operators, using different instruments and reagent lots, yielded correct calls 
in 158/160 tests (98.8%). A set of 26 highly pigmented samples were identified 
that gave invalid test results. A simple 1:2 dilution resulted in a valid test 
result of 76% in such cases. The cobas test is a reproducible assay that detects 
some non-V600E mutations and is more accurate than direct sequencing in 
detecting BRAFV600E.