Variations of BRAF mutant allele percentage in melanomas

Abstract

BackgroundBRAF mutations are present in 40 % of human skin melanomas. Mutated tumors with an increased percentage of BRAF mutant alleles (BRAF-M%) may have a better response to RAF/MEK inhibitors. We evaluated the BRAF-M% in melanomas, and the genetic causes of its variation.MethodsBRAF-M% was quantified by pyrosequencing, real-time PCR (rtPCR) and/or picoliter-droplet PCR (dPCR). BRAF mutant expression was detected by immunohistochemistry. Chromosomal alterations were analyzed with fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) arrays.ResultsBRAF-M% quantification obtained with pyrosequencing was highly correlated (R = 0.94) with rtPCR, and with dPCR. BRAF-M% quantified from DNA and RNA were also highly correlated (R = 0.98). Among 368 samples with >80 % tumor cells, 38.6 % had a BRAFV600E mutation. Only 66.2 % cases were heterozygous (BRAF-M% 30 to 60 %). Increased BRAF-M% (>60 %) was observed in 19 % of cases. FISH showed a polysomy of chromosome 7 in 13.6 %, 35.3 % and 54.5 % of BRAF wild-type, heterozygous and non-heterozygous BRAF-mutated samples, respectively (P < 0.005). Amplification (5.6 %) and loss (3.2 %) of BRAF locus were rare. By contrast, chromosome 7 was disomic in 27/27 BRAF-mutated nevi.ConclusionsBRAF-M% is heterogeneous and frequently increased in BRAF-mutant melanomas. Aneuploidy of chromosome 7 is more frequent in BRAF mutant melanomas, specifically in those with high BRAF-M%.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1515-3) contains supplementary material, which is available to authorized users.

Clinical and therapeutic implications of BRAF mutation heterogeneity in metastatic melanoma

“…Similar to us, Hélias‐Rodzewicz et al. () found a wide range of AF (10–90%) in 142 samples of BRAF ‐mutated melanomas containing more than 80% tumour cells, as analysed by pyrosequencing. Lamy et al.…”

Increase in NRAS mutant allele percentage during metastatic melanoma progression

“…In our previous study on variations in BRAF mutant allele percentage , FISH analysis found more frequent polysomy of chromosome 7 in homozygous BRAF V600E mutant melanomas than in heterozygous BRAF V600E mutant melanomas (as defined by 30 to 60% of BRAF mutant alleles), suggesting, as in the present report, that high mutant allele percentage is often due to a CNG through polysomy of the chromosome harbouring the mutant allele.…”

Reply to “Clinical and therapeutic implications of BRAF mutation heterogeneity in metastatic melanoma” by Mesbah Ardakani et al.

“…A high BRAF AF (>60%) is not a rare event, representing in our series 24.4% of our samples. This frequency was similar (19%) in our previous study reporting variations of BRAF AF in melanoma (Hélias-Rodzewicz et al, 2015). However, using a higher cutoff, we decreased the number of samples in the high-BRAF AF group, which diminished the statistical power of our study.…”

“…Patients were statistically comparable in both cohorts; no difference in age at diagnosis, gender, initial histological characteristics, or AJCC stage prior to treatment was found (Table 1) The use of the median of BRAF AF does not necessarily reflect the concept of mutant allele-specific imbalance as BRAF mutation in melanoma is a somatic mutation with a heterozygous pattern (Soh et al, 2009). As it has been previously proposed by Hélias-Rodzewicz et al (Hélias-Rodzewicz et al, 2015), with a purity of tumor cells around 90%, BRAF-mutated samples can be considered heterozygous when they harbor a BRAF AF of 45% ± 15%. To better correlate BRAF AF with mutant allelic imbalance, we therefore chose in our study a cutoff of 60% to consider that a sample had a high BRAF AF.…”