BRAFV600E is a unique molecular marker for metastatic
melanoma, being the most frequent somatic point mutation in this
malignancy. Detection of BRAFV600E in blood could have prognostic and predictive value and could be useful for monitoring response to BRAF-targeted
therapy. We developed a rapid, sensitive method for the detection and quantification of BRAFV600E in circulating free
DNA (
cfDNA) isolated from plasma and serum on the basis of a quantitative 5'-nuclease PCR (Taqman) in the presence of a
peptide-nucleic acid. We validated the assay in 92 lung, colon, and
melanoma archival serum and plasma samples with paired
tumor tissue (40 wild-type and 52 BRAFV600E). The correlation of
cfDNA BRAFV600E with clinical parameters was further explored in 22 metastatic
melanoma patients treated with BRAF inhibitors. Our assay could detect and quantify BRAFV600E in mixed samples with as little as 0.005% mutant
DNA (copy number ratio 1 : 20 000), with a specificity of 100% and a sensitivity of 57.7% in archival serum and plasma samples. In 22
melanoma patients treated with BRAF inhibitors, the median progression-free survival was 3.6 months for those showing BRAFV600E in pretreatment
cfDNA compared with 13.4 months for those in whom the mutation was not detected (P=0.021). Moreover, the median overall survival for positive versus negative BRAFV600E tests in pretreatment
cfDNA differed significantly (7 vs. 21.8 months, P=0.017). This finding indicates that the sensitive detection and accurate quantification of low-abundance BRAFV600E alleles in
cfDNA using our assay can be useful for predicting treatment outcome.