PROJECT SUMMARY Multiple myeloma (MM) affects ~35,000 adult patients in the United States and causes ~12,000 deaths each year. Novel immunomodulatory drugs and effective multidrug combinations have improved the prognosis for patients, but the vast majority of patients eventually relapse. Even among patients achieve a complete response, ~25% progress within 2 years. Most MM relapses can be attributed to the persistence of measurable (minimal) residual disease (MRD). MRD status has emerged as one of the most important prognostic markers, has therapeutic implications, and is incorporated in the International Myeloma Working Group response criteria for therapeutic response assessment. The gold standard of MRD assessment includes multiparameter flow cytometry and next-generation sequencing (NGS) based on bone marrow aspirates. However, evaluation of MRD using only bone marrow aspirates is prone to false-negatives due to patchy disease involvement, hemodilution of bone aspirate, and extramedullary disease. In addition, bone marrow biopsy is an invasive procedure, hence cannot be performed frequently to monitor MRD. Positron emission tomography/computed tomography (PET/CT) is complementary to bone marrow assessment although gaps remain. We have shown that the 5-hydroxymethylcytosine (5hmC), a stable epigenetic marks generated from active DNA demethylation, in plasma cell-free DNA (cfDNA) could be complementary to PET/CT and was associated with overall survival of patients with MM. Here we propose to apply the highly sensitive mapping of genome-wide 5hmC in cfDNA to identify the optimal combination of serial cfDNA-based 5hmC markers with PET/CT for detecting emergence of MRD. Our central hypothesis is that altered 5hmC signatures in cfDNA are associated with clinically detectable disease by PET/CT and/or NGS, thus offering opportunities for accurate yet less invasive approaches to complement bone marrow-based MRD assessment by NGS. Specifically, we propose to identify 5hmC in cfDNA associated with MRD-positivity (Aim 1), determine cfDNA-based temporal dynamics of 5hmC and fluorodeoxyglucose (FDG)-PET/CT changes associated with MRD (Aim 2), and evaluate performance of different MRD modalities in real-world patients (Aim 3). To address these aims, we have assembled an interdisciplinary team with extensive and complementary expertise. The study leverages the established resources of the UChicago Myeloma Epidemiology Study and our leadership in three clinical trials with banked serial blood samples, bone marrow-paired peripheral blood, and known MRD status by NGS. The knowledge gained from this application may improve clinical utilization of MRD in clinical decision making by proper combination of serial MRD assessments (i.e., cfDNA, PET/CT, and bone marrow) for sensitive tracking of MRD as well as provide novel insights into the epigenetic contribution to MM progression. Identifying patients at high risk of progression after successful treatment will allow ...