116PA targeted genomic analysis uncovered a large spectrum of acquired resistance mechanisms to BRAF inhibitor therapy in metastatic melanoma patients

Background: Several mechanisms have been described to explain the emergence of acquired resistance to MAPK inhibitors. Using tumor DNA sequencing, genetic alterations activating the MAPK and the PI3K/AKT pathways have been associated to emergence of resistance mechanisms. Studies focusing on targeted mRNA analysis have associated gene expression alterations to resistance. Nevertheless, resistance to BRAF inhibitors (BRAFi) remained unexplained for nearly half of melanomas highlighting the need of an approach to exhaustively characterize resistance mechanisms to such therapies. Methods: Tumor samples at baseline and relapse were collected from 20 patients with a BRAFV600 mutated metastatic melanoma. Relapse and best response were assessed using RECIST criteria. After DNA and mRNA extraction, copy number variations (CNV) and mRNA expression were evaluated in a targeted panel of 41 genes with a validated/suggested role in BRAFi resistance using pyrosequencing, Sanger sequencing and quantitative PCR. Differences of mutational gene status, CNVs and mRNA expression were analyzed by comparing relapse to baseline specimens. Results: At least one acquired resistance mechanism was observed for each patient. Alterations of MAPK pathway (BRAF, MAP3K8…), tyrosine kinase receptors (TKR) (KIT, ERBB2…), apoptosis (PTEN, BCL2…) and cell cycle (CDK4, RB1…) related genes were observed in 16, 15, 17 and 20 patients respectively. Five patients had multiple relapse samples (Total= 15) allowing the assessment of tumor heterogeneity. Alterations of MAPK pathway, TKR and apoptosis related genes were thus retrieved in 13/15 samples and cell cycle pathway was altered in all. Conclusions: Using a comprehensive approach combining a panel of genes mutational analysis, CNV and mRNA expression, our study allowed the characterization of at least one resistance mechanism in all relapse tumors. We showed that while there was heterogeneity in the individual genetic alterations, these overlapped when analyzing them within their signaling pathways. This work enlarges the identification of resistance mechanisms to BRAFi and can be combined with those emerging for immunotherapy to improve the clinical management of patients. Legal entity responsible for the study: APHP Funding: Institut National du Cancer Disclosure: M. Battistella: Consulting role for Histalim, Bristol-Myers Squibb, and Innate Pharma. S. Dalle: Principal investigator in studies conducted by Roche-Genentech and Novartis. C. Lebbe: Declares honoraria from Roche, advisory roles at Roche, GSK, Novartis, BMS, MSD, and Amgen and travel accommodation provided by Roche. S. Mourah: Declares a consulting role at Roche, Janssen and Novartis. All other authors have declared no conflicts of interest.