Search

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.

Purpose Oncogenic activation of the BRAF gene has been demonstrated to be involved in the pathogenesis of malignant melanoma (MM). In this study, we investigated the contribution of BRAF to melanoma susceptibility, also making a comparison with frequency of CDKN2A germline mutations in MM patients from different areas in Italy. Patients and Methods Using a combination of denaturing high-performance liquid chromatography analysis and automated sequencing on genomic DNA from peripheral blood or tumor tissue samples, 569 MM patients (211 from northern Italy and 358 from southern Italy) were screened for BRAF mutations. Results Three BRAF germline sequence variants (M116R, V599E, and G608H) were identified in four (0.7%) of 569 MM patients. The most common BRAF mutation, V599E, was detected in one germline DNA sample only; M116R and G608H were newly described mutations. A high frequency (59%) of BRAF mutations was instead observed in tumor samples from patients also undergoing germline DNA analysis; at the somatic level, substitution of valine 599 was found to account for the majority (88%) of BRAF mutations. We then estimated the germline mutation rates in BRAF and CDKN2A among 358 consecutively collected patient samples originating in southern Italy; a low (2.5%) or very low (0.29%) prevalence of CDKN2A and BRAF mutations, respectively, was detected. Conclusion Mutation analysis of either blood DNA from a large collection of MM patients or matched MM tissues from a subset of such patients revealed that BRAF is somatically mutated and does not play a major role in melanoma susceptibility. The present study further suggests that patient origin may account for different mutation rates in candidate genes.

10543 Background: Mutations in the CDKN2A gene underlie melanoma susceptibility in as many as 50% of melanoma kindreds in selected populations, and several CDKN2A founder mutations have been described. Inherited mutations in CDKN2A have been found to be associated with other non-melanoma cancers including pancreatic cancer and neural system tumours. Here we report a novel germline mutation in exon 1 of the CDKN2A gene, E27X, which we first detected in melanoma patients living in or originally from a small geographic area bordering Liguria, in north-western Italy. A subset of melanoma kindreds positive for this mutation displayed pancreatic cancer and neuroblastoma. Methods: We investigated 70 pancreatic cancer patients from a case-control study living in or originally coming from a small geographic area bordering Liguria, in north-western Italy, for CDKN2A mutations and ARF. Results: 2 out of 70 patients displayed the E27X mutation and 1 of the 2 patients had a first degree relative affected by pancreatic cancer. Conclusions: E27X generates a premature stop codon, leading to dramatically reduced protein levels of p16 and leaving p14ARF unaltered. As pancreatic cancer and neural system tumours have been postulated to be preferentially associated with CDKN2A mutations located in exon 2 and/or affecting p14ARF alone, the position of E27X in exon 1a provides interesting insights towards clarifying the mechanisms by which the CDKN2A/ARF locus is involved in cancer predisposition. Its finding in pancreatic cancer patients confirms common susceptibility to melanoma and pancreatic cancer due to this CDKN2A exon 1 mutation. No significant financial relationships to disclose.