Wong, Aimée A.

Abstract Introduction: Early relapse limits durable remissions in multiple myeloma (MM). Cell-free DNA (cfDNA) fragment patterns capture tumor burden and nucleosome positioning. We tested whether a fragmentomics signature trained at MRD time points stratifies progression-free survival (PFS) when applied to pretreatment plasma. Methods: We performed 30-40× whole-genome sequencing of plasma cfDNA from 43 newly diagnosed MM patients, with 65 additional MRD timepoint samples from 41 of those patients (post-ASCT n=30; 1-year maintenance n=30; other maintenance n=5) across eight Canadian sites (TFRIM4 and IMMAGINE studies) and one U.S. site (Mayo SPORE). MRD testing used multiparameter flow cytometry for all follow-up samples (n=65) and clonoSEQ in a subset (n=31). Logistic and elastic-net models were trained at MRD timepoints on fragmentomics features (short-fragment burden; a combined model integrating fragment-length distributions with inferred nucleosome positioning at MM-specific chromatin accessibility regions [Ordoñez et al., Genome Res. 2020]). A decision threshold was fixed by the Youden index during training and applied unchanged to diagnosis samples. PFS was analyzed by Kaplan-Meier, log-rank, and Cox models. Results: After a median follow-up of 49.1 months, 16 of 43 patients (37%) progressed. Using the prespecified locked threshold, patients classified as MRD+ by the proportion-of-short-fragments model had significantly shorter PFS than MRD- patients (HR = 5.12; 95% CI 1.16-22.57; log-rank p = 0.016), with median PFS of 49.2 months for MRD+ cases versus not reached for the MRD- group. Fixed-horizon PFS favored MRD- samples at 24/36/48 months (100/94/87% vs 85/81/53% for MRD+). A combined model integrating weighted fragment lengths with inferred nucleosome positioning also separated outcomes (log-rank p=0.054); the Cox HR was not estimable because no MRD- patients relapsed. At 24, 36, and 48 months, baseline MRD positivity by the short-fragment model identified 100%, 83%, and 86% of patients who relapsed within those intervals (specificity 44%, 43%, and 52%), while the combined model detected all early relapses across the same horizons (specificity 15-21%), indicating consistently high sensitivity but modest specificity for predicting early progression. Conclusions: A cfDNA fragmentomics signature trained at MRD time points stratified PFS when applied to diagnostic plasma, identifying patients at risk of early relapse. Short-fragment burden was most discriminative, and a combined model with nucleosome positioning recapitulated it with higher sensitivity, albeit lower specificity. These data support cfDNA fragmentomics as a practical liquid-biopsy risk biomarker in MM and suggest higher signals may proxy aggressive disease with greater tumor shedding and chromatin disorganization. Future work will validate findings in larger cohorts and integrate cytogenetic and immune features. Citation Format: Dor David Abelman, Jenna Eagles, Aimee Wong, Saumil Shah, Jeffrey Bruce, Stephanie Pedersen, David S. Scott, Cecilia Bonolo de Campos, Signy Chow, Darrell White, Irwindeed Sandhu, Kevin Song, Esteban Braggio, Shaji Kunnathu Kumar, Alli Murugesan, Tony Reiman, A. Keith Stewart, Suzanne Trudel, Trevor J. Pugh. Baseline cfDNA fragmentomics identifies multiple myeloma patients at risk of early progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2597.

Abstract Introduction: Monitoring minimal residual disease (MRD) is critical in multiple myeloma (MM) to predict outcomes and guide therapy. Traditional bone marrow (BM) aspirates for MRD detection are invasive and limited by sample quality. We therefore explored cell-free DNA (cfDNA) fragmentation as a less invasive alternative for MRD detection. Methods: We performed 30-40X whole-genome sequencing (WGS) on peripheral blood cfDNA from 45 MM patients, collecting baseline (n = 45) and follow-up samples (n = 98) from eight Canadian sites (M4 and IMMAGINE studies) and one U.S. site (SPORE study), plus 25 healthy controls. Samples were collected post-induction therapy (n = 13), 100 days post-autologous stem cell transplantation (ACST, n = 36), and during maintenance therapy (n = 49). Multiparameter flow cytometry (MFC) MRD testing was conducted on 77 samples. We evaluated insert size metrics and MM-specific chromatin accessibility (from Ordoñez et al., 2020) using Griffin (described in Doebley et al., 2022). Results: Baseline samples had a higher proportion of short fragments (20-150 bp) than follow-up samples (p = 0.0037) and healthy controls (p = 0.019). A fragment score (FS) based on weighted fragment distribution (per Vessies et al., 2022) was highest at baseline (mean = -0.384), followed by MRD-positive (mean = -0.495) and MRD-negative (mean = -0.550) samples. MRD-negative FS was lower than baseline samples (p < 0.001) but higher than healthy controls (mean = -0.639; p = 0.016). Baseline samples had lower coverage at MM-specific chromatin regions than MRD timepoints (p < 0.05; baseline mean = 0.982; MRD-positive = 0.989; MRD-negative = 0.994; healthy = 0.993), suggesting higher transcriptional activity pre-treatment. MRD-negative samples had higher coverage than MRD-positive cases (p < 0.01). Using logistic regression, we assessed the predictive performance of FS and coverage at MM-specific sites. FS alone achieved an AUC of 0.632 (sensitivity [SN] 39.4%, specificity [SP] 92.1%, accuracy [AC] 67.6%). Coverage yielded an AUC of 0.681 (SN 75.8%, SP 60.5%, AC 67.6%). Combining FS and coverage at MM sites improved performance (AUC = 0.734; SN 48.5%, SP 92.1%, AC 71.8%). We next evaluated whether adjusting the BM tumor cell percentage cutoff by MFC could improve performance. At 0.017% (1 in 5, 789 cells) for MRD positivity, the combined model’s accuracy reached 83% with an AUC of 0.757 (SN 17%, SP 97%). Conclusions: cfDNA fragmentomic analysis achieved high specificity (92-97%) in detecting MRD, providing a less invasive alternative to facilitate serial monitoring. While sensitivity remains an area for improvement, the high specificity of cfDNA makes it valuable for confirming MRD negativity and reducing invasive procedures. Future efforts aim to enhance sensitivity by integrating additional fragmentomic features. Citation Format: Dor D. Abelman, Jenna Eagles, Aimee Wong, Saumil Shah, Stephanie Pedersen, Stephenie Prokopec, David S. Scott, Sarah Bridges, Darrell White, Irwindeep Sandhu, Kevin Song, Esteban Braggio, Alli Murugesan, Anthony Reiman, Suzanne Trudel, Trevor J. Pugh. Tumor-independent monitoring of minimal residual disease in multiple myeloma using cfDNA fragmentomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3250.