Low incidence of minor BRAF V600 mutation-positive subclones in primary and metastatic melanoma determined by sensitive and quantitative real-time PCR.

BRAF V600 mutation is an important biological marker for therapeutic guidance in melanoma, where mutation-positive cases are candidates for therapy targeting mutant B-Raf. Recent studies showing intratumor variation in BRAF mutation status have caused concern that sensitive mutation analysis can lead to mutation-positive results in patients with melanomas with small subsets of mutation-positive cells who may not benefit from therapy targeting mutant B-Raf. Mutation analysis with high analytical sensitivity is generally preferred, to reduce the risk of false-negative results. In this study, sensitive and quantitative BRAF V600E and V600K mutation-specific real-time quantitative PCR was used to study the occurrence of small subsets of mutation-positive cells in primary melanomas and melanoma metastases. The BRAF V600E mutation was detected in 39 of 82 melanoma patients. We observed a highly dichotomous pattern, with most samples either testing mutation positive in a high fraction of alleles (median, 51%) or negative with a high sensitivity (median, 0.06%). This finding demonstrates that the occurrence of small subsets of mutation-positive cells was rare in our study population and indicates that sensitive mutation analysis can generally be expected to produce clinically relevant results in melanoma patients.