Search for therapeutic response molecular markers in malignant melanoma through NGS: on the way to an anti-BRAF combination therapy.

Introduction: Recent development of new targeted agents is giving viable treatment options to melanoma therapies. Vemurafenib® specifically targets BRAF-V600E mutation, an highly common alteration found in malignant melanomas (MM). This targeted therapy is very effective on BRAF mutated MM patients but, most of them quickly develops an acquired drug resistance, limiting the therapeutic efficacy. We previously characterized the potential antitumor activity of several compounds on MM cells using a series of cell lines derived from MM patients showing different histopathological features (primary and/or metastatic, different tumor stage and site of onset). Using NGS approaches, we started the molecular characterization of Vemurafenib-resistant BRAF-mutated MM cell lines in comparison with their drug-sensitive counterparts, in order to identify genetic variants associated to the resistance. The evaluation of the antiproliferative activity of some new compounds - like the curcumin-related D6 molecule - on Vemurafenib-resistant MM cell lines is in progress. Materials and Methods: The NGS testing was performed on cell lines using the Ion-Torrent® technology. The validation of new markers will be carried out in vitro (same cell lines) and in vivo (MM paraffinated tissue samples) by Sanger sequencing. BRAF-mutated MM cell lines were treated with Vemurafenib (20???to????? to isolate resistant clones. These were treated with a combination of Vemurafenib and the curcumin-related D6 compound, before performing MTT proliferation assays. Results and discussion: A genetic heterogeneity was observed after characterization of several primitive and metastatic MM cell lines by cancer gene mutation profiling. Among them, four paired BRAF-mutated primitive and metastatic MM cell lines were selected to isolate Vemurafenib-resistant clones. These showed to be sensitive to D6 treatments, thus suggesting a combination therapy using Vemurafenib and D6. NGS results are being evaluated in order to identify alterations acting as markers for predicting the antiproliferative response following this treatment. Moreover, they will allow a better understanding of the molecular mechanisms underlying the development of resistance to Vemurafenib in MM. Conclusion: Our preliminary results suggest the feasibility of Vemurafenib-D6 combination treatment in drug-resistant MM. Our aim is to identify and validate new molecular markers associated to therapeutic response in order to obtain a better classification of the cases to be addressed to the treatment.