A NEXT GENERATION SEQUENCING APPROACH TO GENOMIC LANDSCAPE IN MELANOMA PATIENTS: EVOLUTION OF PATIENT-SPECIFIC RESPONSE TO THERAPY

Until 10 years ago, advanced melanoma was associated with poor survival due to the lack of durable responses to conventional therapy, with a median Overall Survival (OS) of about 6 months in patients with stage IV melanoma. Since 2011, however, the rules for stage IV melanoma treatment have been completely rewritten, with targeted therapies with BRAF and MEK inhibitors (BRAF+MEKi), and immunotherapy with the anti CTLA-4 ipilimumab and the anti-PD-1 nivolumab and pembrolizumab. These new therapeutic approaches improved melanoma prognosis, resulting in a 5-year survival rate of 34–43%. However, mainly because of primary and acquired resistance to treatments, the majority of patients will ultimately relapse, and only patients harbouring a BRAF mutation, observed in about 50% of cutaneous melanoma, can receive a targeted treatment with BRAF+MEKi. To further improve the prognosis of melanoma patients, several preclinical and clinical trials are study new actionable mechanisms and/or molecules to tackle multiple resistance mechanisms simultaneously. The advent of massive parallel sequencing, allowing the simultaneous analysis of several genes, led, in the past two decades, to Whole-Exome Sequencing (WES) and Whole-Genome Sequencing (WGS) studies that allow the identification of several potential therapeutic targets. In light of this, numerous clinical and preclinical trials are ongoing to identify new molecular targets. Since the discovering of the first actionable mutation (BRAF V600), several other genes have been identified as putative drivers of melanomagenesis and/or melanoma progression, and additional candidate drivers are currently being assessed, prompting pharmacogenomics studies on potentially actionable targets. However, melanoma is one of the tumours with the highest mutation burden, and results from different studies were frequently not overlapping, possibly due to dissimilar sample size and cohort characteristics. Although this high mutational burden is one of the reasons behind the success of immunotherapy in this tumour, it makes it hard to clearly identify novel driver genes that could be used for targeted therapies. The landscape of mutated non-BRAF skin melanoma, in light of recent data deriving from WES or WGS studies on melanoma cohorts, established 33 candidate driver genes altered with a frequency greater than 1.5%. Despite the many advances in melanoma therapy and the exciting results achieved, some issues remain unanswered. Among them, the most important is the identification and overcoming of primary and acquired resistance. In this context, the liquid biopsy and the analysis of circulating free DNA (cfDNA) from different body fluids spread in the recent years as a useful biological tool for non-invasive and quantitative characterization of the whole tumour genome, identification of tumour heterogeneity, identification of drug resistance mechanisms, and clonal evolution during treatment and toward disease progression in melanoma patients. In summary, assessing BRAF mutations has become a key diagnostic procedure and a priority in determining the oncologist's choice and course of therapy. In this context, several molecular strategies are available for mutational analysis of the BRAF gene, such as Next-Generation Sequencing (NGS) target techniques. For the employment of this diagnostic approach, several guidelines and recommendations need to be applied to standardize the implementation of NGS-based panels in the clinical setting by prior technical validation to ensure the detection of somatic variants and the high quality of sequencing results. In part II of my dissertation, I discuss a recent collaborative study that we finalized to evaluate the NGS concordance data obtained among two groups using an NGS melanoma panel, aiming to adopt it in clinical as well as in research practice within the Italian Melanoma Intergroup (IMI) (Published; Vanni et al., 2020. doi. 10.1186/s13000-020-01052-5). However, the increasing interest in the molecular characterization of melanoma, aiming to identify additional molecular markers either targetable by new drugs or valuable for predicting response to therapy and prognosis, paves the way to the use of exome/genome level high-throughput sequencing in real-world settings of melanoma patients, with integration of WES and RNA-seq data to provide an extensive melanoma genetic layout, as reported in Part I of my dissertation. This study is preliminary to the analysis of the complex interplay of the tumour cells with the tumour microenvironment and the immune system, including local and systemic factors, which are likely to modulate therapy efficacy. All of these factors are complex and change in time, and their integration in a real-world series of melanoma patients longitudinally followed during therapy is going to yield significant insight in this interplay