Low Incidence of Minor BRAF V600 Mutation-Positive Subclones in Primary and Metastatic Melanoma Determined by Sensitive and Quantitative Real-Time PCR

Abstract

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.

Detection of BRAF mutations from solid tumors using Tumorplex™ technology

“…BRAF V600E mutation (c.1799T>A, which results in p.Val600Glu amino acid change) [6] is associated with thyroid, melanoma and colorectal cancers. Its detection is vital in diagnosis and treatment modalities [7] , [8] , [9] , [10] , [11] , [12] . The BRAF gene encodes a kinase found in the signal transduction pathway from Ras to MEK 1/2, which plays a key role in the proliferation of melanocytes [13] .…”

Regulation of Reactive Oxygen Species and the Antioxidant Protein DJ-1 in Mastocytosis

“…Cells were placed in puromycin-free culture media for 24 h and then plated in 12 well plates (2 x 10 5 cells/ well; 1 ml) for additional 24 h. Media were collected for measurement of ROS and cells lysates for DJ-1 determinations. Expression of KIT and KIT phosphorylation in the transfected cells was confirmed by western blot and presence of mutation in the obtained cell line was confirmed by allele specific PCR [36]. …”

BRAF Gene Copy Number and Mutant Allele Frequency Correlate with Time to Progression in Metastatic Melanoma Patients Treated with MAPK Inhibitors

“…Both mut-and wtspecific PCRs were performed using the ALB gene as a control to normalize for DNA content. Standard curves with serial dilutions of the commercially available BRAF V600E or V600K 50% allele standards (Horizon Diagnostics) in BRAF wt sample were used to evaluate the reaction efficiency and to calculate the fraction of BRAF V600mut allele as in Kristensen and colleagues (28). The BRAFmut allele % was calculated as a ratio of BRAFmut/BRAF-mutþBRAFwt adjusted by tumor cell percentage.…”

“…The BRAFmut allele % was calculated as a ratio of BRAFmut/BRAF-mutþBRAFwt adjusted by tumor cell percentage. To validate the method, we compared the BRAFmut allele % as measured by allele-specific real-time qPCR with that evaluated by pyrosequencing for five representative samples and for the BRAF V600E HT-29 cell line (22,23,28). The two methods yielded comparable results with an average difference of 10.7%.…”