The ACC melanoma pilot project: “Real-world” evaluation of an NGS platform for molecular characterization of melanoma in Italy.

Abstract

e14600 Background: Alliance Against Cancer (ACC), the network of Italian Cancer Centers, is involved in designing targeted NGS panels to enable sequencing a significant number of therapeutically actionable genes from FFPE tissue samples at the cost of a routine molecular diagnostic test (RMT). In this retrospective study we used the ACC oncochip v.1 on a cohort of stage III-IV metastatic melanoma patients, with the aim of: a) validating the diagnostic use of this oncochip in comparison with RMTs for BRAF mutation detection; b) evaluating how many additional actionable gene mutations can be identified per patient; c) evaluating possible associations between mutational profiles and outcome. Methods: DNA was extracted from 120 FFPE samples from 9 Italian hospitals previously profiled for BRAF status with a RMT, and matching germline samples. 60% of the patients underwent immunotherapy and 40% targeted therapy, with an overall survival ranging from few days to several years. 10-20 ng of DNA were profiled with the amplicon-based ACC Oncochip v.1, that covers ~700 Kb, including the full coding sequence of 182 genes. Samples were sequenced in a Ion Torrent S5 instrument (ThermoFisher), and mutational profiles were generated with the Ion Reporter software. Results: Adequate coverage of the BRAF codon 600 hotspot was reached in 99% of the samples, and BRAF status was 95% concordant with previous RMTs. In 4/6 discordant cases, BRAF V600 mutations were only detected by NGS (with VAF below 10%); in the remaining two cases NGS did not confirm mutations detected by RMT, possibly due to tumor heterogeneity. In accordance with TCGA data, BRAF was mutated in 55% of patients, NRAS in 23% and NF1 in 13%, with almost complete mutual exclusivity; moreover 86% of triple negative patients showed mutations in other actionable genes. We also observed great variability in mutational load, ranging from ~3 to > 500 somatic mutations per Mb; correlation between mutational profiles and clinical outcome is underway. Conclusions: The ACC Oncochip v.1 NGS panel is accurate and affordable, providing a comprehensive mutational profile for the cost of a RMT and allowing a precision medicine approach to melanoma diagnosis and identification of actionable mutations.