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This study aims to identify DNA methylation biomarkers for multi-cancer detection. Utilizing large tissue datasets, we apply novel search algorithms to discover confined biomarker panels capable of distinguishing tumor from normal and determining the tissue of origin. We explore the applicability to blood-based testing using targeted methylation sequencing followed by machine learning classification. We present an 8-marker panel, which successfully predicts tumors across 14 types with a 91% average sensitivity, maintaining a low false positive rate (&lt; 0.04%). Additionally, a panel of 39 CpG sites exhibits accuracies ranging from 69% to 98% for identifying tissue of origin. When tested on 114 patient plasma samples (colon, liver, pancreatic, prostate, and stomach cancer), the 8-marker panel obtains an AUC of 0.78 with a 78% sensitivity among 32 early-stage patients (stage I-II), and 60% overall. Using the 39-marker panel in a multi-class classification model selecting only the best match, 54% of tumor samples were on average correctly assigned to the tissue of origin, and up to 80% when allowing more inclusive criteria. Using a limited set of biomarkers, our work contributes to advancing non-invasive cancer diagnostics.<\/jats:p>","DOI":"10.1101\/2025.09.16.676542","type":"posted-content","created":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T00:55:16Z","timestamp":1758243316000},"source":"Crossref","is-referenced-by-count":0,"title":["Discovery and performance of DNA methylation panels for cancer detection and classification in blood"],"prefix":"10.64898","author":[{"given":"Gennady","family":"Margolin","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sara R.","family":"Bang-Christensen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sara A.","family":"Grimm","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brian D.","family":"Bennett","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter","family":"Kilfeather","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kevin","family":"Fedkenheuer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sabina","family":"Pathan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nichelle C.","family":"Whitlock","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter A.","family":"Pinto","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leszek J.","family":"Klimczak","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"S.","family":"Katie Farney","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nader","family":"Jameel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yun-Ching","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hanna M.","family":"Petrykowska","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2760-1853","authenticated-orcid":false,"given":"Adam G.","family":"Sowalsky","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6487-081X","authenticated-orcid":false,"given":"Jian-Liang","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1065-019X","authenticated-orcid":false,"given":"Laura","family":"Elnitski","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025092302300580000_2025.09.16.676542v1.1","doi-asserted-by":"publisher","DOI":"10.1200\/JCO.2012.43.1718"},{"key":"2025092302300580000_2025.09.16.676542v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms8686"},{"key":"2025092302300580000_2025.09.16.676542v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/bs.acc.2018.12.005"},{"key":"2025092302300580000_2025.09.16.676542v1.4","doi-asserted-by":"crossref","unstructured":"Gidwani, K. , et al., Nanoparticle-aided glycovariant assays to bridge biomarker performance and ctDNA results. 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