Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: Next Generation Sequencing to identify markers of resistance on cell-free DNA and droplet digital PCR to study measurable residual disease in patients with chronic lymphocytic leukemia treated with venetoclax and rituximab

Chronic lymphocytic leukemia (CLL) represents the most frequent adult leukemia in Western countries. The characteristic clonal expansion of CD5+ mature B cells in the blood and secondary lymphoid organs defines the disease. While CLL remains an incurable condition, the landscape of its management has witnessed a transformative shift with the introduction of groundbreaking inhibitors targeting Bruton's kinase (ibrutinib) and BCL2 (venetoclax) in 2014. The advent of next-generation sequencing (NGS) technologies has unraveled the intricate genetic landscape of CLL, revealing recurrent somatic mutations and shedding light on the genetic heterogeneity between and within patients. Project number 1 aims to explore the potential of NGS on the analysis of cell-free DNA (cfDNA) extracted from plasma of CLL patients before and during first-line treatment with venetoclax + rituximab. The exploration of cfDNA as a source for mutational studies could be relevant due to the multi-compartmental nature of CLL, encompassing bone marrow and lymph nodes beside peripheral blood. The project seeks to identify in cfDNA elusive variants that could serve as resistance markers, moving from the assumption that cfDNA can offer a more informative reservoir for detecting resistance mutations, potentially anticipating a clinical relapse/progression. Project number 2 is focused on measurable residual disease (MRD) monitoring on the same setting of patients. Recognizing the pivotal role that MRD plays in predicting treatment outcomes, the project aims to overcome the limitations of conventional methods of MRD analysis like real-time quantitative polymerase chain reaction (QR-PCR), introducing Digital Droplet PCR (ddPCR), a cutting-edge technology known for its sensitivity and precision. By comparing the performance of these methods to monitor CLL MRD in peripheral and bone marrow samples during and after treatment, the project aspires to provide valuable insights into CLL management, paving the way for a more sensitive and precise technique in the monitoring of this complex hematological malignancy.