Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

Raffaela Barbano,Paolo Graziano,Andrea Fontana,Michelina Rendina,Michelina Coco,Evaristo Maiello,Barbara Pasculli,Paola Parrella,Vanna Maria Valori,Massimiliano Copetti,Vito Michele Fazio

doi:10.1038/srep18592

Abstract

BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method.

Highlights

Melanomas are malignant tumours arising from the pigment producing cells melanocytes which are derived from the neural crest and are widely distributed throughout all cutaneous, ocular and most of the mucosal surfaces[1,2,3,4]
Comparison between Cast-PCR and Sanger sequencing for the detection of BRAFV600E and BRAFV600K in clinical samples. κ − statistic showed a significant agreement between the two techniques (κ = 0.85, CI: 0.710–0.990; p < 0.001)
We found that the mutant alleles were well detectable up to the 1:100 Mut/not mutated (NMut) ratio for both case 2 (BRAFV600E), and case 23 (BRAFV600K) by Cast-PCR

Summary

Introduction

Melanomas are malignant tumours arising from the pigment producing cells melanocytes which are derived from the neural crest and are widely distributed throughout all cutaneous, ocular and most of the mucosal surfaces[1,2,3,4]. It has long been known that activation of the RAS–RAF–MEK–ERK–MAPK (mitogen activated protein kinase, MAPK) pathway plays an important role in melanoma development[5]. Somatic mutations of the BRAF gene encoding for a serine/threonine kinase that transduces regulatory signals from RAS through MEK to MAPK are detected in 40–60% of melanoma cases[6,7,8,9,10]. The most common mutations of the BRAF gene in melanoma occur within exon 15 codon 600 are the BRAFV600E(c.1799. Cutaneous melanoma accounts for only 4% of all dermatologic cancers, it is responsible for 80% of the deaths[2]. The prognosis of metastatic melanoma has recently changed substantially thanks to the approval of the kinase inhibitors Vemurafenib[8,9,10,11,12] and Dabrafenib[13,14,15], and the immune checkpoint inhibitor Ipilimumab[16,17]

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Results

Conclusion