The analysis of cancer genetic alterations is used for diagnostic, prognostic and treatment purposes, and recently also for monitoring the Tumor Mutational Burden (TMB) in response to immunotherapy. These tests are usually performed using the DNA and/or RNA obtained from a fragment of the primary tumor or from a metastasis. However, tissue biopsies may not always be easily performed because of their invasive nature. Liquid biopsy based on circulating free DNA (cfDNA) is a non-invasive technique for the detection of mutations in tumor DNA circulating in plasma or serum (ctDNA, circulating tumor DNA).
This project aims to develop a liquid biopsy approach for application in clinical practice, by the Next Generation Sequencing (NGS) of ctDNA. The first Task will refer to the validation of the SiRe NGS panel as a non-invasive predictive biomarker of response to molecularly targeted therapies. Patients with advanced CRC, NSCLC, melanoma and GIST will be enrolled and mutations in six clinically relevant genes (EGFR, KRAS, NRAS, BRAF, cKIT, PDGFRa) will be analysed by SiRe on plasma and serum.
The second Task will concern the development and validation of a TMB NGS protocol of liquid biopsy for the management of NSCLC patients candidate to immunotherapy. The TMB-panel will investigate if the mutational status at multiple loci, as a surrogate for high TMB, might be a biomarker of response to immunotherapies.
The main objective of this project is to establish the applicability of ctDNA liquid biopsy by NGS in a clinical context. The final aim is the successful treatment and management of oncologic patients by non-invasive blood-based markers.
This project concerns a NGS approach aimed to establish the clinical applicability of ctDNA-based genomic profiling of cancer-related alterations. To date, molecular information about the genetic background of tumor tissues is essential to guide the treatment decision. However, despite advances in modern biotechnologies, including NGS platforms, challenges do exist, such as the dynamic clonal evolution of cancer, which results in intra-tumor spatial and temporal heterogeneity. To address such challenges, liquid biopsy detecting ctDNA might enable a real-time assessment of the molecular profile of cancer and identify the emergence of resistant clones at earlier time-points.
NGS biomarker testing on ctDNA, which enables the sequencing of large genomic regions or several exons on ctDNA, through the longitudinal assessment of dominant cancer clones, might fasten the process towards precision medicine in cancer treatment.
Two different NGS panel will be applied in this multi-institutional study. The first one, the narrow SiRe panel, will be studied for routine testing of relevant tumor mutations in ctDNA of patients most commonly tested for molecular alterations (i.e. NSCLC, CRC and metastatic melanoma). Unlike other wider NGS approaches, the SiRe panel allows the ultra-deep sequencing of currently used disease-specific biomarkers (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRa) both for prospective tests in molecular predictive pathology practice, and for monitoring the disease course.
The second NGS panel (TMB-panel) employed in this study, is aimed to monitor the response to anti-PD-1 immunotherapy. Checkpoint inhibitor immunotherapies have unquestionably changed the treatment paradigm for a variety of cancers, including lung cancer, with 20¿40% of patients who receive these therapies showing remarkable responses. To date, the controversial results on the predictive value of PD-L1 expression in NSCLC and response to PD-1/PD-L1 inhibitors do not allow for any conclusive consideration regarding the static assessment of PD-L1 as a biomarker. There is an urgency to identify patients most likely to respond to checkpoint inhibitor therapies with easily accessible and repeatable analyses. High TMB represent a genomic landscape which facilitates the occurrence of neo-antigen-specific effector T cell responses following PD-1 blocked immunotherapy, thus making the treatment effective[20]. Adapting the TMB-immune panel to ctDNA analysis, we might identify a new candidate biomarker of response to immune checkpoint inhibitors and introduce a new tool that has the potential of guiding clinical practice.