Comparison of BRAF Mutation Screening Strategies in a Large Real-Life Series of Advanced Melanoma Patients

Malignant melanoma (MM) is one of the deadliest skin cancers.BRAFmutation status plays a predominant role in the management of MM patients. The aim of this study was to compareBRAFmutational testing performed by conventional nucleotide sequencing approaches with either real-time polymerase chain reaction (rtPCR) or next-generation sequencing (NGS) assays in a real-life, hospital-based series of advanced MM patients. Consecutive patients with AJCC (American Joint Committee on Cancer) stage IIIC and IV MM from Sardinia, Italy, who were referred for molecular testing, were enrolled into the study. Initial screening was performed to assess the mutational status of theBRAFandNRASgenes, using the conventional methodologies recognized by the nationwide guidelines, at the time of the molecular classification, required by clinicians: at the beginning, Sanger-based sequencing (SS) and, after, pyrosequencing. The present study was then focused onBRAFmutation detecting approaches only.BRAFwild-type cases with available tissue and adequate DNA were further tested with rtPCR (Idylla (TM)) and NGS assays. Globally, 319 patients were included in the study; pathogenicBRAFmutations were found in 144 (45.1%) cases examined with initial screening. The rtPCR detected 11 (16.2%) and 3 (4.8%) additionalBRAFmutations after SS and pyrosequencing, respectively. NGS detected one additionalBRAF-mutated case (2.1%) among 48 wild-type cases previously tested with pyrosequencing and rtPCR. Our study evidenced that rtPCR and NGS were able to detect additionalBRAFmutant cases in comparison with conventional sequencing methods; therefore, we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy ofBRAFdetection.