P-162: Cell-free DNA Whole Genome Sequencing for Non-Invasive MRD Detection in Multiple Myeloma

Introduction: Accurate detection of minimal residual disease (MRD) is crucial for evaluating treatment efficacy in multiple myeloma (MM), yet current methods are invasive and often limited by bone marrow (BM) sample quality. We therefore compared standard MRD methods with whole-genome sequencing (WGS) of cell-free DNA (cfDNA) for less invasive monitoring.

Methods: The MM Molecular Monitoring (M4) prospective cohort study included 45 newly diagnosed transplant-eligible MM patients (pts) uniformly treated with standard-of-care frontline therapy at 8 Canadian sites. MRD testing was performed at 100 days post-autologous stem cell transplant (ASCT) (n=39) and/or after one year of lenalidomide (len) maintenance (n=33). We analyzed 43 pts by multiparameter flow cytometry (MFC) (71 samples, CytoQuest Technologies), 39 by EasyM (57 samples, Rapid Novor), 28 by clonoSEQ (Adaptive Technologies), and by 18 PET/CT imaging. We performed 30-40X WGS on CD138+ selected BM cells pre-treatment initiation to inform of somatic mutations (n=11) and tracked them by 30-40X WGS in longitudinal peripheral blood cfDNA samples (cfWGS, n=12).

Results: MRD-negative rates at 100 days post-ACST were 0% for EasyM (n=30), 20% for cfWGS (n=5), 45% for clonoSEQ (n=11), and 49% for MFC (n=39). After 1-year of len maintenance, MRD-negative rates were 22% for EasyM (n=27), 29% for cfWGS (n=7), 41% for clonoSEQ (n=17), 59% for MFC (n=32), and 83% for PET (n=18). Among EasyM-positive samples at 100 days post-transplant, only 21/27 remained positive after one year of maintenance therapy, likely due to delayed M-protein clearance.



By May 2024, 12/45 pts had relapsed, with an average time to relapse of 714 days (SD=375) after initiating len. At post-ASCT, all relapsed pts were positive by EasyM (n=6) and clonoSEQ (n=2), 67% (n=6/9) by MFC (mean proportion aberrant cells 0.006%, limit of detection (LOD) range 0.00038%-3.4%) and 50% (n=1/2) by cfWGS. One year post maintenance, all relapse samples were positive by EasyM (n=8), 67% by clonoSEQ (n=4/6), 78% by MFC (n=7/9, LOD 0.00035%-0.38%), 100% by cfWGS (n=3), and 17% by PET (n=1/6). All negative MFC and cfWGS relapse cases were positive below LOD. No EasyM-negative pts relapsed within 2 years (n=6). Relapse within 2 years occurred in 17% of clonoSEQ-negative (n=2/12), 5% of MFC-negative (n=2/38), 33% of cfWGS-negative (n=1/3), and 33% of PET-negative (n=5/15) samples.



cfWGS showed 82% concordance to EasyM (n=11), 67% to PET (n=6), 50% to MFC (n=12), and 25% to clonoSEQ (n=8). The lower concordance to MFC and clonoSEQ was mainly due to cfWGS positives missed by these methods but identified by EasyM. However, 2 clonoSEQ-positive pts were below LOD by cfWGS.

Conclusions: cfWGS is a promising MRD alternative which is less invasive than MFC and clonoSEQ. It benefits non-secretory and some light chain only patients where EasyM is not currently feasible and uniquely informs of clonal dynamics at progression. Future work will aim to improve sensitivity and validate findings in a larger cohort.