Identification of methylation biomarkers in colorectal carcinoma and their detection in circulating tumour cells

Identification of methylation biomarkers in colorectal carcinoma and their detection in circulating tumour cells | 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 Identification of methylation biomarkers in colorectal carcinoma and their detection in circulating tumour cells Zhuo Chen, Jun yu Woon, Neda Moetamedirad, Md Sajedul Islam, Gihani Vidanapathirana, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8234790/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 Circulating tumour cells (CTCs) provide a minimally invasive approach for cancer detection and disease monitoring in metastatic carcinoma. This study aimed to identify methylation biomarkers and assess their translational potential for CTC-based liquid biopsy in colorectal carcinoma (CRC). Predictive methylation sites were screened using the TCGA-Colon Adenocarcinoma dataset (430 cancer and 39 non-cancer colon mucosae) through differential methylation analysis, Lasso regression, and Cox modelling. Three representative sites— GPR125 , GDNF , and ADCY9 —were validated in 62 patients with colorectal adenocarcinoma using quantitative methylation-specific PCR, and comparative analyses between paired CTC and tissue samples were performed in 32 cases. The prediction model yielded a hazard ratio of 4.23 ( p < 0.001) with 1-, 3-, and 5-year area under the curve (AUC) values of 0.718, 0.765, and 0.787, respectively. GDNF showed the strongest CTC detection performance, with 81.2% sensitivity, 93.8% specificity, and an AUC of 0.887. Clinical association analyses revealed that GDNF methylation was significantly correlated with age ( p = 0.040), primary tumour site ( p = 0.004), tumour grade ( p = 0.017), T stage ( p = 0.027), and lymphovascular invasion (LVI) ( p = 0.041). GPR125 methylation was associated with tumour grade ( p = 0.017), LVI ( p = 0.041), and presence of other neoplasms ( p = 0.026). ADCY9 methylation showed limited clinical associations, correlating only with the presence of other neoplasms ( p = 0.029). Network analysis revealed that GDNF formed a distinct regulatory module with strong clinical relevance. These findings suggest that GDNF methylation holds strong potential as a CTC-based liquid biopsy biomarker in colorectal adenocarcinoma, demonstrating high detection accuracy despite systematic differences from tissue methylation patterns. The limited translational applicability of biomarkers derived from other tissues highlights the challenges of translating bioinformatic discoveries into effective liquid biopsy implementation. Colorectal cancer circulating tumour cells DNA methylation GDNF liquid biopsy Full Text Additional Declarations No competing interests reported. Supplementary Files SFigure1.tif SupplementaryTable1PrimerandprobesequencesusedforquantitativemethylationspecificPCR.xlsx SupplementaryTable2ClinicalassociationsofmethylationlevelsatprognosticCpGsitesincirculatingtumorcellsandtumortissues.xlsx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-8234790","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":558295969,"identity":"83efd666-a543-48c4-b75b-c68b5226a409","order_by":0,"name":"Zhuo Chen","email":"","orcid":"","institution":"Griffith University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Chen","suffix":""},{"id":558295970,"identity":"00bd5ecc-4e3c-4135-924c-fe3119cb59c4","order_by":1,"name":"Jun yu Woon","email":"","orcid":"","institution":"Griffith 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cells","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":"Colorectal cancer, circulating tumour cells, DNA methylation, GDNF, liquid biopsy","lastPublishedDoi":"10.21203/rs.3.rs-8234790/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8234790/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCirculating tumour cells (CTCs) provide a minimally invasive approach for cancer detection and disease monitoring in metastatic carcinoma. This study aimed to identify methylation biomarkers and assess their translational potential for CTC-based liquid biopsy in colorectal carcinoma (CRC). Predictive methylation sites were screened using the TCGA-Colon Adenocarcinoma dataset (430 cancer and 39 non-cancer colon mucosae) through differential methylation analysis, Lasso regression, and Cox modelling. Three representative sites—\u003cem\u003eGPR125\u003c/em\u003e, \u003cem\u003eGDNF\u003c/em\u003e, and \u003cem\u003eADCY9\u003c/em\u003e—were validated in 62 patients with colorectal adenocarcinoma using quantitative methylation-specific PCR, and comparative analyses between paired CTC and tissue samples were performed in 32 cases. The prediction model yielded a hazard ratio of 4.23 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) with 1-, 3-, and 5-year area under the curve (AUC) values of 0.718, 0.765, and 0.787, respectively. \u003cem\u003eGDNF\u003c/em\u003eshowed the strongest CTC detection performance, with 81.2% sensitivity, 93.8% specificity, and an AUC of 0.887. Clinical association analyses revealed that \u003cem\u003eGDNF\u003c/em\u003emethylation was significantly correlated with age (\u003cem\u003ep\u003c/em\u003e = 0.040), primary tumour site (\u003cem\u003ep\u003c/em\u003e = 0.004), tumour grade (\u003cem\u003ep\u003c/em\u003e = 0.017), T stage (\u003cem\u003ep\u003c/em\u003e= 0.027), and lymphovascular invasion (LVI) (\u003cem\u003ep\u003c/em\u003e = 0.041). \u003cem\u003eGPR125\u003c/em\u003emethylation was associated with tumour grade (\u003cem\u003ep\u003c/em\u003e = 0.017), LVI (\u003cem\u003ep\u003c/em\u003e= 0.041), and presence of other neoplasms (\u003cem\u003ep\u003c/em\u003e = 0.026). \u003cem\u003eADCY9\u003c/em\u003emethylation showed limited clinical associations, correlating only with the presence of other neoplasms (\u003cem\u003ep\u003c/em\u003e = 0.029). Network analysis revealed that \u003cem\u003eGDNF\u003c/em\u003eformed a distinct regulatory module with strong clinical relevance. These findings suggest that \u003cem\u003eGDNF\u003c/em\u003e methylation holds strong potential as a CTC-based liquid biopsy biomarker in colorectal adenocarcinoma, demonstrating high detection accuracy despite systematic differences from tissue methylation patterns. The limited translational applicability of biomarkers derived from other tissues highlights the challenges of translating bioinformatic discoveries into effective liquid biopsy implementation.\u003c/p\u003e","manuscriptTitle":"Identification of methylation biomarkers in colorectal carcinoma and their detection in circulating tumour cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 08:35:29","doi":"10.21203/rs.3.rs-8234790/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":"954ccab9-2fde-4631-a976-46de8a0ac0b2","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-30T09:25:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 08:35:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8234790","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8234790","identity":"rs-8234790","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}