PROJECT SUMMARY In the United States, it is estimated there are ~730,000 new cases of prostate cancer, lung cancer, colorectal cancer (CRC), and ovarian cancer annually. Advances in therapies during the past decades have significantly improved clinical outcomes. For many common cancers, however, treatment options are limited when the cancer is diagnosed with distant metastasis at later stages. Although early detection may reduce cancer mortality, systematic screening programs are available only for a limited number of cancers (e.g., CRC), which also faces challenges such as patient compliance and potential complications. Therefore, minimally-invasive blood-based tools that could detect these common cancers at earlier stages are likely to be paradigm-shifting in cancer control and care. The Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial offers a unique opportunity and resource to investigate the possibility of applying our established 5hmC-Seal assay to: 1) explore cancer-specific early epigenetic marks, specifically 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA), when the cancer is not “overt”; and 2) develop a multi-cancer detection (MCD) tool that is minimally-invasive and possesses high sensitivity and specificity. Current MCD tests, e.g., Grail’s blood test, are not suitable to exploit the PLCO samples (e.g., that contain very limited amounts of DNA). In contrast, the 5hmC-Seal assay, only requiring nanograms amounts of DNA, together with the next-generation sequencing (NGS) holds the promise to address the challenge as a highly sensitive approach for PLCO biospecimens. Of note, our compelling preliminary results demonstrated: 1) the feasibility of applying the 5hmC-Seal assay to PLCO biospecimens (~200 µL plasma/sample); 2) analysis of genome-wide 5hmC from PLCO samples successfully identified a 5hmC-based model that detected CRC cases months or even years prior to overt disease; 3) altered 5hmC signatures associated