PROJECT SUMMARY Multiple myeloma (MM), the second most common hematologic malignancy in the United States, is an incurable plasma cell malignancy with standardized incidence rates that are typically 2- to 3-times higher among African Americans (AA) compared to European Americans (EA). Reasons for this apparent racial/ethnic disparity remain largely unclear. Genetic susceptibility, socioeconomic factors, and obesity are important risk factors for MM, but they do not fully explain the excess risk of MM in AA. Epigenetic modifications, particularly cytosine modifications, play a critical role in the development and progression of MM. However, unlike solid tumors (e.g., breast, prostate, colon, etc.) where distinct epigenetic changes in racial/ethnic groups have been shown to account for the differences in tumor initiation, progression, and aggressiveness, the epigenetic contributions to the excess risk of MM in AA are not well characterized. Differences in epigenetic modifications are an intrinsic feature between human populations and associated with complex traits and diseases. The majority of previous epigenetic studies have used technologies that cannot distinguish 5- hydroxymethylcytosines (5hmC), a biochemically stable epigenetic mark showed distinct genome-wide distributions and regulatory roles from the well-studied modified cytosines, 5-methylcytosines (5mC). In addition, epigenetic epidemiology studies have predominantly used DNA from peripheral blood lymphocytes as surrogate specimens because obtaining CD138+ tumor cells from the bone marrow aspirates in healthy individuals is not feasible. Therefore, we propose to elucidate the influence of novel DNA modifications, specifically the 5hmC in circulating cell-free DNA (cfDNA) on racial/ethnic disparities in MM. Circulating cfDNA fragments are released into the bloodstream by circulating dead or proliferating cancerous cells. Thus, cfDNA produced by tumor cells hiding in the bone marrow, bone marrow microenvironment, or extramedullary disease can be detected in plasma. We have demonstrated the relevance of cfDNA-derived 5hmC in MM and other hematological malignancies, including that specific 5hmC modifications in cfDNA were associated with overall survival of MM; distinct 5hmC signatures reflected molecular differences between subtypes of lymphoma; and population-specific pathways involving 5hmC were identified between MM and its precursors. Our central hypotheses are that specific 5hmC signatures associated with MM in cfDNA reflect primary tumor cells, and specific 5hmC modifications contribute to the excess risk in AA. We will identify genome-wide 5hmC signatures for MM in cfDNA (Aim 1) and investigate MM-associated 5hmC in cfDNA-paired bone marrow tumor cells and microenvironment (Aim 2). We will elucidate population-specific 5hmC signatures and pathways between EA and AA patients with MM (Aim 3). This project is significant because it offers a timely and comprehensive strategy to identify...