Global Hypomethylation in Cell-free DNA Enables Non-invasive Colorectal Cancer Screening: Results from a Retrospective Validation Study

Global Hypomethylation in Cell-free DNA Enables Non-invasive Colorectal Cancer Screening: Results from a Retrospective Validation Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Global Hypomethylation in Cell-free DNA Enables Non-invasive Colorectal Cancer Screening: Results from a Retrospective Validation Study Shahdeep Kaur, Shefali Lathwal, Smita Agrawal, Tina Mahmoudi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8682249/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Global DNA hypomethylation is a fundamental epigenetic hallmark of colorectal cancer, yet it remains difficult to measure in circulating cell-free DNA using existing technologies that focus on a limited number of genomic loci. As a result, global hypomethylation has not been effectively translated into non-invasive diagnostics. We evaluated a label-free electrical impedance assay that measures cell-free DNA aggregation, a biophysical property linked to genome-wide methylation state, to determine whether global hypomethylation can be detected and clinically leveraged for colorectal cancer screening and monitoring. Results Plasma samples from 46 treatment-naïve colorectal cancer patients and 33 healthy individuals were analyzed. The assay distinguished cancer from healthy samples with 95.65% sensitivity and 93.94% specificity across all disease stages, including early-stage cancers. Longitudinal analysis of six patients showed concordance between assay output and clinical course, including response to treatment and disease recurrence. Transmission electron microscopy confirmed methylation-dependent differences in cell-free DNA aggregation underlying the electrical signal. To contextualize these findings biologically, we analyzed 11 public DNA methylation datasets. Conventional whole-array analyses underestimated global hypomethylation due to overrepresentation of CpG island probes. Restricting analysis to OpenSea regions, which better represent genome-wide methylation and are enriched in cell-free DNA, revealed a significant 5.8% reduction in global methylation in colorectal cancer tissue compared to adjacent normal tissue, consistent across molecular subtypes. In contrast, blood-derived immune cell genomic DNA showed no comparable hypomethylation, supporting a predominantly tumor-derived contribution to the observed circulating signal. Conclusions These results demonstrate that global DNA hypomethylation is detectable in circulating cell-free DNA and can be captured using a rapid, amplification-free electrical assay. By measuring structural consequences of pan-genomic epigenetic alterations rather than locus-specific changes, this approach enables accurate, non-invasive detection and monitoring of colorectal cancer. The findings support global hypomethylation as a clinically actionable epigenetic biomarker and establish a new framework for cell-free DNA–based cancer diagnostics. Full Text Additional Declarations Competing interest reported. SG (Founder & CEO), SK, and TM are employees of Asima Health Inc. and may financially benefit from this role, including through equity or stock options. Supplementary Files CRCManuscriptSI.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8682249","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596252782,"identity":"4d6b2283-c2cd-4074-b415-c6842319f625","order_by":0,"name":"Shahdeep Kaur","email":"","orcid":"","institution":"Asima Health Inc.","correspondingAuthor":false,"prefix":"","firstName":"Shahdeep","middleName":"","lastName":"Kaur","suffix":""},{"id":596252787,"identity":"cc878c81-79ed-43c6-b80c-91e3505e016d","order_by":1,"name":"Shefali Lathwal","email":"","orcid":"","institution":"101 GenAI Inc.","correspondingAuthor":false,"prefix":"","firstName":"Shefali","middleName":"","lastName":"Lathwal","suffix":""},{"id":596252790,"identity":"b797232e-9caa-4029-9894-bd33ed3ac6d2","order_by":2,"name":"Smita Agrawal","email":"","orcid":"","institution":"101 GenAI Inc.","correspondingAuthor":false,"prefix":"","firstName":"Smita","middleName":"","lastName":"Agrawal","suffix":""},{"id":596252792,"identity":"237f3753-bbd9-4ec3-90bc-1a6d9bb07477","order_by":3,"name":"Tina Mahmoudi","email":"","orcid":"","institution":"Asima Health Inc.","correspondingAuthor":false,"prefix":"","firstName":"Tina","middleName":"","lastName":"Mahmoudi","suffix":""},{"id":596252798,"identity":"066ec26e-47ed-4697-8087-1e9bc43b984a","order_by":4,"name":"Shalini Gupta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDACCQbGAwkMEnIMDIwNIMTAx8BDUAsDSIsxXAsbGzFagFRiA4hDlBb52c0PDjzcYZHe33+4dcPPHTbybPK9Bxh+VGzDqcXgzjGDA4lnJHJn3Ehsu9l7Js2wjY0vgbHnzG3cWiQSgFraJHIbbjC23eBtO8zYxsZjwMzYhluL/Iz0DyAt6fLnD7bd/Nv2356gFoYbOWBbwHbd5m0DkoS0GNzIKQBpMdwI9Mtt2bbk5Da2HIOD+PwCdNjGhz/b6uTlzh9/dvNtm51tP/MZwwc/KvA4DCs4QKL6UTAKRsEoGAVoAAAj7l3ssTKUSgAAAABJRU5ErkJggg==","orcid":"","institution":"Asima Health Inc.","correspondingAuthor":true,"prefix":"","firstName":"Shalini","middleName":"","lastName":"Gupta","suffix":""}],"badges":[],"createdAt":"2026-01-23 20:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8682249/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8682249/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104882350,"identity":"86beda8e-2892-4d31-835e-c3b48bd4c855","added_by":"auto","created_at":"2026-03-18 09:29:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1595383,"visible":true,"origin":"","legend":"","description":"","filename":"MainManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8682249/v1_covered_6c2e3db6-e661-4e97-af90-804bfa27347e.pdf"},{"id":103424670,"identity":"b925f191-f892-4591-ab81-22b83a8f112c","added_by":"auto","created_at":"2026-02-25 14:00:16","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7664405,"visible":true,"origin":"","legend":"","description":"","filename":"CRCManuscriptSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-8682249/v1/798e4742190e802ef2d4276c.docx"}],"financialInterests":"Competing interest reported. SG (Founder \u0026 CEO), SK, and TM are employees of Asima Health Inc. and may financially benefit from this role, including through equity or stock options.","formattedTitle":"Global Hypomethylation in Cell-free DNA Enables Non-invasive Colorectal Cancer Screening: Results from a Retrospective Validation Study","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8682249/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8682249/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eGlobal DNA hypomethylation is a fundamental epigenetic hallmark of colorectal cancer, yet it remains difficult to measure in circulating cell-free DNA using existing technologies that focus on a limited number of genomic loci. As a result, global hypomethylation has not been effectively translated into non-invasive diagnostics. We evaluated a label-free electrical impedance assay that measures cell-free DNA aggregation, a biophysical property linked to genome-wide methylation state, to determine whether global hypomethylation can be detected and clinically leveraged for colorectal cancer screening and monitoring.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePlasma samples from 46 treatment-na\u0026iuml;ve colorectal cancer patients and 33 healthy individuals were analyzed. The assay distinguished cancer from healthy samples with 95.65% sensitivity and 93.94% specificity across all disease stages, including early-stage cancers. Longitudinal analysis of six patients showed concordance between assay output and clinical course, including response to treatment and disease recurrence. Transmission electron microscopy confirmed methylation-dependent differences in cell-free DNA aggregation underlying the electrical signal. To contextualize these findings biologically, we analyzed 11 public DNA methylation datasets. Conventional whole-array analyses underestimated global hypomethylation due to overrepresentation of CpG island probes. Restricting analysis to OpenSea regions, which better represent genome-wide methylation and are enriched in cell-free DNA, revealed a significant 5.8% reduction in global methylation in colorectal cancer tissue compared to adjacent normal tissue, consistent across molecular subtypes. In contrast, blood-derived immune cell genomic DNA showed no comparable hypomethylation, supporting a predominantly tumor-derived contribution to the observed circulating signal.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results demonstrate that global DNA hypomethylation is detectable in circulating cell-free DNA and can be captured using a rapid, amplification-free electrical assay. By measuring structural consequences of pan-genomic epigenetic alterations rather than locus-specific changes, this approach enables accurate, non-invasive detection and monitoring of colorectal cancer. The findings support global hypomethylation as a clinically actionable epigenetic biomarker and establish a new framework for cell-free DNA\u0026ndash;based cancer diagnostics.\u003c/p\u003e","manuscriptTitle":"Global Hypomethylation in Cell-free DNA Enables Non-invasive Colorectal Cancer Screening: Results from a Retrospective Validation Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 14:00:11","doi":"10.21203/rs.3.rs-8682249/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af4a6d2b-e44f-457a-bc41-700825f13d5d","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-18T09:29:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 14:00:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8682249","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8682249","identity":"rs-8682249","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}