Direct analysis of transcription factor protected cfDNA in plasma by ChIP-seq: Measurement of altered CTCF binding in cancer is a novel biomarker for liquid biopsy

Direct analysis of transcription factor protected cfDNA in plasma by ChIP-seq: Measurement of altered CTCF binding in cancer is a novel biomarker for liquid biopsy | 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 Method Article Direct analysis of transcription factor protected cfDNA in plasma by ChIP-seq: Measurement of altered CTCF binding in cancer is a novel biomarker for liquid biopsy Dorian Pamart, Brieuc Cuvelier, Adrien Govaerts, Muriel Chapelier, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8047483/v2 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 2 posted 5 You are reading this latest preprint version Show more versions Abstract Background The aggregate presence of cell free CTCF-DNA (cfCTCF-DNA) nucleoproteins in plasma has been reported. But the occupancy of individual CTCF binding sites in plasma cfCTCF-DNA has not been studied. Results For the first time, we have isolated endogenous plasma cfCTCF-DNA by chromatin immunoprecipitation (ChIP) and sequenced the associated cfDNA (ChIP-Seq) in samples from cancer patients and healthy subjects. The mean observed enrichment of plasma cfCTCF-DNA by ChIP was 180-fold with a mean CTCF recovery of 48%. Background cfDNA, primarily in the form of nucleosomes, was almost completely physically removed (> 99.7%) by ChIP and the remainder was removed bioinformatically. cfCTCF-DNA sequences were partitioned into low or high affinity binding cfCTCF-DNA nucleoproteins in silico , on the basis of binding motif concordance with the consensus CTCF binding motif. We observed that cfCTCF-DNA was quantitatively and qualitatively different in samples from healthy subjects or cancer patients. High affinity cfCTCF-DNA nucleoproteins were present only in the plasma of cancer patients and not in samples from healthy subjects. Testing of small developmental and validation sample cohorts for ChIP-seq peaks for a 14 member panel of high affinity CTCF loci sequences, showed excellent specificity and sensitivity for cancer detection. Conclusions Plasma sequence data sets comprising > 99% pure CTCF protected ctDNA can be prepared using a 2-step process involving the physical removal of most background cfDNA in vitro , followed by removal of all remaining background cfDNA in silico . CTCF plasma ChIP-seq represents a novel liquid biopsy method for a novel class of liquid biopsy biomarkers. Cancer detection cfDNA ctDNA chromatin fragments CTCF ctDNA immunoprecipitation liquid biopsy nucleoproteins NGS ChIP Full Text Additional Declarations Competing interest reported. With the exception of J.-V.T. and A.G., all authors are current employees of Volition. M.C., M.H., A.R., T.B. and J.M. are shareholders of Volition. Volition has a pending patent application covering this technology Supplementary Files SupplementalInformation2.xlsx Cite Share Download PDF Status: Under Revision Version 2 posted Editorial decision: Revision requested 30 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviewers invited by journal 26 Mar, 2026 Submission checks completed at journal 25 Mar, 2026 First submitted to journal 16 Mar, 2026 You are reading this latest preprint version Show more versions 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-8047483","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":614818318,"identity":"bf6b90f9-2c6a-11f1-b2c4-06cc9d20a69f","order_by":0,"name":"Dorian Pamart","email":"","orcid":"","institution":"Belgian Volition SRL","correspondingAuthor":false,"prefix":"","firstName":"Dorian","middleName":"","lastName":"Pamart","suffix":""},{"id":614818407,"identity":"cd88a5ee-2c6a-11f1-b2c4-06cc9d20a69f","order_by":1,"name":"Brieuc Cuvelier","email":"","orcid":"","institution":"Belgian Volition 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But the occupancy of individual CTCF binding sites in plasma cfCTCF-DNA has not been studied.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFor the first time, we have isolated endogenous plasma cfCTCF-DNA by chromatin immunoprecipitation (ChIP) and sequenced the associated cfDNA (ChIP-Seq) in samples from cancer patients and healthy subjects. The mean observed enrichment of plasma cfCTCF-DNA by ChIP was 180-fold with a mean CTCF recovery of 48%. Background cfDNA, primarily in the form of nucleosomes, was almost completely physically removed (\u0026gt;\u0026thinsp;99.7%) by ChIP and the remainder was removed bioinformatically. cfCTCF-DNA sequences were partitioned into low or high affinity binding cfCTCF-DNA nucleoproteins \u003cem\u003ein silico\u003c/em\u003e, on the basis of binding motif concordance with the consensus CTCF binding motif. We observed that cfCTCF-DNA was quantitatively and qualitatively different in samples from healthy subjects or cancer patients. High affinity cfCTCF-DNA nucleoproteins were present only in the plasma of cancer patients and not in samples from healthy subjects. Testing of small developmental and validation sample cohorts for ChIP-seq peaks for a 14 member panel of high affinity CTCF loci sequences, showed excellent specificity and sensitivity for cancer detection.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ePlasma sequence data sets comprising\u0026thinsp;\u0026gt;\u0026thinsp;99% pure CTCF protected ctDNA can be prepared using a 2-step process involving the physical removal of most background cfDNA \u003cem\u003ein vitro\u003c/em\u003e, followed by removal of all remaining background cfDNA \u003cem\u003ein silico\u003c/em\u003e. CTCF plasma ChIP-seq represents a novel liquid biopsy method for a novel class of liquid biopsy biomarkers.\u003c/p\u003e","manuscriptTitle":"Direct analysis of transcription factor protected cfDNA in plasma by ChIP-seq: Measurement of altered CTCF binding in cancer is a novel biomarker for liquid biopsy","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-03-30 19:12:00","doi":"10.21203/rs.3.rs-8047483/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-30T07:50:11+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"93287693595764039818825525548093318212","date":"2026-03-31T14:35:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-26T07:34:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-26T02:37:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Epigenetics","date":"2026-03-16T19:06:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-epigenetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clep","sideBox":"Learn more about [Clinical Epigenetics](http://clinicalepigeneticsjournal.biomedcentral.com/)","snPcode":"13148","submissionUrl":"https://submission.nature.com/new-submission/13148/3","title":"Clinical Epigenetics","twitterHandle":"@OAgenetics","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2025-12-05 01:55:24","doi":"10.21203/rs.3.rs-8047483/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-03T15:24:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-30T00:07:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-13T19:55:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93287693595764039818825525548093318212","date":"2025-12-05T22:11:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1169152674034032358586456187005958746","date":"2025-12-04T13:50:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276820232076465612144118909842641805994","date":"2025-12-04T11:26:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T11:04:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-12T04:36:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T04:35:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Epigenetics","date":"2025-11-06T11:24:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-epigenetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clep","sideBox":"Learn more about [Clinical Epigenetics](http://clinicalepigeneticsjournal.biomedcentral.com/)","snPcode":"13148","submissionUrl":"https://submission.nature.com/new-submission/13148/3","title":"Clinical Epigenetics","twitterHandle":"@OAgenetics","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"72ad8fb3-a1e2-4135-8ef5-a56d3b39e836","owner":[],"postedDate":"March 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T07:54:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-30 19:12:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-8047483","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8047483","identity":"rs-8047483","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}