Assessment of Minimal Residual Disease (MRD) in newly diagnosed Multiple Myeloma Patients enrolled in a Clinical Trial: Comparison of Next Generation Flow Cytometry (NGF) and Next Generation Sequencing (NGS).

Multiple Myeloma (MM) is a malignant plasma cell disorder characterized by clonal proliferation within the bone marrow, leading to a wide range of clinical manifestations, including bone lesions, anemia, renal impairment, and hypercalcemia. Despite advancements in treatment, MM remains incurable. Sensitive methods to assess treatment response allowing monitoring of residual disease after treatment are pivotal. The evaluation of Minimal/Measurable Residual Disease (MRD) has become a cornerstone in the management of MM, providing critical insights into treatment efficacy and disease progression. This thesis focuses on the comparison and validation of two sensitive MRD detection techniques— flow-cytometry based (multiparametric flow cytometry (MFC) and Next-Generation Flow Cytometry (NGF)) and sequencing based (Next-Generation Sequencing (NGS))—in the context of MM. NGF, developed by the EuroFlow consortium, offers a standardized approach for detecting residual clonal plasma cells, even in samples with a minimal tumor burden. Similarly, NGS provides unparalleled precision in identifying clonal immunoglobulin gene rearrangements, offering a molecular-level assessment of residual disease. The study longitudinally evaluates MRD in a cohort of MM patients enrolled in the FORTE trial, which compares the efficacy of carfilzomib-based regimens with or without autologous stem cell transplantation (ASCT). MRD assessments were conducted at multiple time points, including post-induction, post-ASCT, and during maintenance therapy. The concordance between flow-based and sequencing-based MRD detection methods was a focal point, with the study aiming to determine whether these technologies can be used interchangeably or if one method holds superiority in specific clinical scenarios. Results demonstrated a high degree of concordance between MFC/NGF and NGS at the 10^-5 sensitivity level, with both methods showing similar predictive capabilities for progression-free survival (PFS) and overall survival (OS). However, discrepancies were noted at the higher 10^-6 sensitivity, with NGS detecting MRD positivity in a subset of patients deemed negative by flow cytometry. This finding suggests that while MFC/NGF are highly effective techniques for MRD detection, NGS may offer a slightly higher sensitivity, particularly in patients with very low levels of residual disease. Moreover, the design of the study allowed us to explores the MRD dynamics over time and in particular the value of sustained MRD negativity, defined as MRD negativity maintained over multiple assessments. Patients achieving sustained MRD negativity exhibited significantly improved PFS and OS, highlighting the importance of continuous MRD monitoring in optimizing long-term outcomes. On the other hand we explored the outcome of the loss of MRD negativity over time (unsustained MRD negativity) and the risk factors associated with the resurgence of a disease clone. The resurgence of residual disease predicted clinical relapse and need for a second line treatment and we identified disease-related (e.g. HRCA) and treatment-related factors predicting unsustained MRD negativity. In conclusion, the research underscores the critical role of MRD evaluation in MM, advocating for the integration of NGF or NGS in routine clinical practice to enhance the precision of disease monitoring. The findings contribute to the ongoing debate regarding the optimal MRD detection strategy and emphasize the need for personalized treatment approaches in MM based on molecular profiling.