Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: Application of clinical Next Generation Sequencing and liquid biopsy to the management of Colorectal Cancer patients

Abstract Colorectal cancer (CRC) is among the most lethal and prevalent malignancies in the world. Due to the existence of heterogeneity in biology, therapy response and prognosis, extensive disease stratification is required. Therefore, parameters which include TNM stage, microsatellite status, tumor grade and lymphovascular invasion, and others are assessed in the pathology report to indicate the extent and prognosis of the disease. CRC characterization has largely benefited of Next-generation sequencing (NGS) during the last few years, and the advancement in sequencing technology has improved the molecular diagnosis, treatment and prediction of prognosis of these tumors. Liquid biopsy is also a revolutionary technique that continues to provide an unexpected perspective. It consists of the detection and isolation of circulating tumor cells (CTC), circulating tumor DNA (ctDNA), circulating exosomes (and other analytes) in body fluids, such as serum, plasma, urine, etc. which is becoming more and more relevant in cancer diagnosis, prognosis and prediction of response or resistance to given treatments. In this doctoral project we used both approaches, NGS and liquid biopsy, for a better clinicopathological characterization of CRC patients. In a first study, we analyzed 639 mCRC samples by multigene panel sequencing. We revealed pathogenic mutations in 523 out of 639 mCRC samples. In order to study mutation associations, a pairwise association analysis was initially performed for those genes with a mutation’s frequency >1.5%, which revealed a positive association between EGFR, KRAS and SMAD4 mutations, while BRAF was significantly associated with PTEN. At divergence from KRAS, NRAS mutation is significantly associated with TP53. Furthermore, we identified four different mutation association patterns (MAPs) grouping samples with potentially similar biology based on a KRAS and TP53 mutation status, which might provide greater opportunities in therapeutic decisions for the majority of mCRC cases. To validate this classification, we acquired the TCGA public mutational datasets and examined the correlation of this classification with gender and tumor site. In a second study, we performed ctDNA analysis on 11 mCRC patients with a primary diagnosis of RAS mutant. They were characterized by disease progression (PD) and failure of anti-VEGF based treatments. RAS mutational status assessed at progression in plasma samples by the Idylla™ system resulted in a ratio of 55% (6/11) versus 45% (5/11) for RAS mutation and wild type, respectively. Plasma DNA samples from four out of five patients who tested negative for RAS mutational status by Idylla were also subjected to IT-PGM sequencing. Neither the RAS mutations originally identified in the primary tumor nor de novo RAS mutations were detected in these samples. Therefore, these 4 patients underwent anti-EGFR treatment and achieving significant clinical benefit. Based on this, we proposed a hypothetical algorithm that accounts for the transient disappearance of RAS mutant clones over time, which might extend the continuum of care for mutant RAS colorectal cancer patients with the delivery of a further line of therapy.