cfDNA-NGS Liquid Biopsy as a new strategy to face advanced cancers: the usefulness of Liquid Biopsy in Cancer molecular characterization, detection of targetable mutations and insights into treatment resistance.

Despite advances in oncology, many patients face limited options after exhausting standard treatments and effective treatments for advanced solid tumours remain a challenge. Liquid Biopsy, a minimally invasive technique, offers a promising alternative by detecting key mutations and monitoring cancer progression, overcoming the limitations of traditional tissue biopsies. Circulating tumour cell-free DNA (cfDNA) serves as a highly sensitive biomarker to identify targetable and therapy-escaping mutations. This thesis applied cfDNA-NGS Liquid Biopsy to molecularly characterise and monitor clonal evolution in 291 advanced cancer patients across several solid tumour types, including breast, lung, colorectal, pancreatic, and prostate cancers. Using the Avenio Expanded genes panel (Roche), 1,478 variants were detected, of which 15% were classified as pathogenic or likely pathogenic. Key mutations associated with treatment resistance were identified, such as ESR1, PIK3CA, and TP53 in breast cancer, KRAS, EGFR, BRAF and TP53 in lung cancer, and APC, KRAS, BRAF, MAPK1, and TP53 in colorectal cancer. In pancreatic and prostate cancers, coupled KRAS/TP53 mutations and BRCA1, BRCA2 mutations, along with TP53 and AR mutations, respectively, were associated with worse clinical outcomes and could explain treatment resistance. Furthermore, targeted therapies may benefit patients with some of the mutations identified. During my research at the Institute of Cancer Research in Sutton and University College of London, I explored treatment resistance in metastatic castration-resistant prostate cancer (mCRPC), trying to detect epigenetic signatures on cfDNA and tissues of patients unresponsive to Olaparib (PARP inhibitor) in the TOPARP-B clinical trial, as well as analysing cfDNA and post-mortem metastatic tissues of CASCADE patients from the LuPARP clinical trial. These studies further highlighted the potential of cfDNA-NGS in monitoring metastatic processes and treatment escapism. In conclusion, cfDNA-NGS Liquid Biopsy proved to be a powerful, non-invasive tool for profiling tumours, offering insights into cancer progression and treatment resistance. This work underlines the importance of a personalised approach in cancer treatment, as different genetic signatures can significantly influence tumour aggressiveness and response patterns, even within the same histological cancer type.