Evaluation of BRAF Mutation Testing Methodologies in Formalin-Fixed, Paraffin-Embedded Cutaneous Melanomas

Abstract

Patients diagnosed with BRAF V600E mutated cutaneous melanoma show response to treatment with the BRAF inhibitor Vemurafenib. Different methods for BRAF mutation detection exist; however, only the Cobas 4800 BRAF V600 Mutation Test has been approved by the US Food and Drug Administration for patient selection. The results from this test depend on the percentage of tumor cells in the samples, which clinically may be estimated with substantial variation. We have evaluated five different methods: the Cobas test, Sanger sequencing, pyrosequencing, TaqMan-based allele-specific PCR, and Competitive Amplification of Differentially Melting Amplicons (CADMA), for detection of BRAF c.1799T>A (V600E) mutations in 28 formalin-fixed paraffin-embedded (FFPE) cutaneous melanoma samples. We show that the frequency of the BRAF V600E mutation is influenced by the analytical sensitivity of the applied method. However, a 100% consensus was observed among all five methods when the tumor tissue fraction was more than 10% of all tissue or more than 50% of cell-dense tissue. When using Sanger sequencing, pyrosequencing, or the Cobas test, it may be advisable to perform macrodissection before mutation testing if the tumor cell fraction is low. CADMA and TaqMan may not require macrodissections for a reliable test. Therefore, the use of more sensitive methods may have a future in testing for BRAF mutations in clinical settings.

Mutual Exclusivity Analysis of Genetic and Epigenetic Drivers in Melanoma Identifies a Link Between p14ARF and RARβ Signaling

“…Sequence analysis of CDKN2A was conducted as described previously (21). Pyrosequencing of the BRAF c.1799T>A mutation was conducted using the PyroMark Q24 platform (Qiagen) and previously described primers (22). Mutation analysis of BRAF (exons 11 and 15), NRAS (exons 2 and 3), KIT (exons 9, 11, 13, and 17), PIK3CA (exons 9 and 20), PIK3R1 (exon 9, 10, and 12), IDH1 (exon 4), FGFR1 (exon 7), GNAQ (exon 5), TP53 (exons 4-9), CTNNB1 (exon 3), CDK4 (exon 2), RB1 (exons , and PTEN (exons 1-9) was conducted using a combination of PCR and denaturing gradient gel electrophoresis followed by direct sequence analysis.…”

BRAF mutation screening in melanoma

“…Several hypotheses might explain this discordance involving SLNs. First, decreased pyrosequencing sensitivity may be because of low quantities of tumor DNA in the case of contamination by normal tissue [19]. In the current study, the mean DNA concentration was sufficient for pyrosequencing in both SLN (115 ng/μl, range 33-233.8 ng/μl) and other tumor samples (400 ng/μl, range 7-1100 ng/μl).…”

“…Currently, pyrosequencing is the most efficient method used in clinical practice with a higher sensitivity than that of approved commercially available tests (e.g. Cobas 4800 BRAF V600 Mutation Test or Sanger sequencing [12,[19][20][21]). This allows BRAF mutation detection in melanoma tumors, using allele-specific amplification, with a limit of detection of 5% BRAF mutant alleles [22], while the usual threshold is 10% [23].…”

Pathology of Melanoma

“…[32][33][34] The major difference between these 2 categories is that multiple genes can be sequenced simultaneously by next-generation sequencing and to a greater depth. As discussed elsewhere in this article, histologic properties of melanomas are associated with the underlying mutated oncogenes and these could be used to determine an appropriate order of sequencing.…”