Bile-derived m⁶A as a Proof-of-Concept Liquid Biomarker for Early Pancreatic Cancer

Bile-derived m⁶A as a Proof-of-Concept Liquid Biomarker for Early Pancreatic Cancer | 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 Bile-derived m⁶A as a Proof-of-Concept Liquid Biomarker for Early Pancreatic Cancer Jin-Yi Han¹, Keun Soo Ahn¹, Min Jae Kim¹, Tae-Seok Kim¹, Yong Hoon Kim¹, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7984256/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 Early detection of pancreatic cancer (PCA) remains a major challenge due to the lack of reliable biomarkers. Methods We investigated bile-derived N6-methyladenosine (m 6 A) modification as a diagnostic biomarker. Bile samples were obtained from patients with PCA (n = 7), biliary tract cancer (BTC; n = 35), and common bile duct (CBD) stones (n = 9). Global m6A levels were quantified using an ELISA-based colorimetric assay. Results Global m6A levels were markedly elevated in PCA bile (6.75 ± 1.25%) compared with BTC (1.25 ± 0.15%) and CBD controls (0.5 ± 0.1%) ( p < 0.001; Fig. 1). ROC analysis demonstrated excellent discrimination (AUC = 1.00, optimal cut-off = 1.1%). Consistent results were observed in pancreatic juice samples (AUC = 1.00, cut-off = 3.7%). Conclusions The results demonstrated distinct m⁶A elevation in bile from PCA patients (Fig. 1), supporting its translational potential as a minimally invasive biomarker. Validation in larger cohorts is warranted. These findings provide a proof-of-concept foundation for bile-based RNA methylation profiling in pancreatic cancer. pancreatic cancer m⁶A methylation bile liquid biopsy biomarker epitranscriptomics Figures Figure 1 Figure 2 1. Introduction Pancreatic cancer (PCA) is one of the most lethal malignancies, with a 5-year survival rate below 10%. Its poor prognosis largely reflects delayed diagnosis and the absence of reliable biomarkers. Bile provides a direct and physiologically relevant matrix for detecting tumor-derived nucleic acids [ 1 – 3 ]. N6-methyladenosine (m⁶A) is the most abundant internal mRNA modification and plays a critical role in RNA metabolism and tumor progression [ 4 – 6 ]. Given that bile directly reflects the pancreaticobiliary microenvironment, bile-derived m⁶A profiling may bridge molecular alterations and clinically applicable diagnostics. 2. Methods Bile and pancreatic juice samples were collected via endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic biliary drainage (PTBD), and during operation at Keimyung University Dongsan Medical Center. The cohort included patients with PCA (n = 7), BTC (n = 35), and CBD stones (n = 9). The study was approved by the institutional review board (IRB No. DSMC 2022-07-034). Global m⁶A levels were quantified using an ELISA-based colorimetric assay (EpiQuik m⁶A RNA Methylation Kit, Abcam) according to the manufacturer’s protocol. Statistical comparisons among groups were performed using one-way ANOVA with Tukey’s post hoc test (GraphPad Prism 10.0). ROC analysis determined the diagnostic performance and optimal cut-offs. 3. Results 3.1 Elevated m⁶A levels in PCA bile (Fig. 1 ) Quantitative analysis demonstrated a progressive elevation of global m⁶A modification from benign to malignant states (p < 0.001). The mean m⁶A percentage in PCA bile (6.75 ± 1.25%) was approximately six-fold higher than in BTC (1.1 ± 0.15%) and more than ten-fold higher than in CBD controls (0.5 ± 0.1%). (Fig. 1 ) 3.2. Diagnostic performance ROC analysis demonstrated excellent discrimination between PCA and non-malignant samples (AUC = 1.00, optimal cut-off = 1.1%) (Fig. 2 ). Similar results were observed in pancreatic juice samples (AUC = 1.00, cut-off = 3.7%). The distribution pattern shown in Fig. 1 complements ROC findings (Fig. 2 ), visually confirming the separation between PCA and non-malignant bile. . 4. Discussion This study provides the first evidence that bile-derived m⁶A dysregulation may serve as a biomarker for PCA detection. The strong diagnostic accuracy supports its translational potential for early diagnosis [ 7 ]. Compared to serum-based assays, bile offers a pathophysiologically relevant matrix reflecting pancreatic tumor activity. Our proof-of-concept analysis demonstrates strong diagnostic performance, suggesting that bile m⁶A profiling could complement current cytologic or molecular approaches in ERCP-based evaluation. Although the sample size is small, this work represents a proof-of-concept foundation for larger-scale validation. This proof-of-concept supports the translational feasibility of implementing bile-based m⁶A assays in ERCP-collected samples, enabling real-time, minimally invasive cancer screening during diagnostic procedures. The small sample size and single-center design limit the generalizability of our findings; however, this study provides a strong rationale for multi-institutional validation. Our findings offer a translational bridge between RNA epitranscriptomic profiling and clinical biomarker development, aligning with the mission of BMC Cancer to promote clinically relevant translational discoveries. Translational Relevance The m⁶A-based assay could be readily incorporated into existing ERCP workflows, facilitating real-time molecular diagnosis. These findings highlight the potential of bile-based RNA methylation profiling as a minimally invasive diagnostic tool bridging molecular oncology and clinical detection of PCA. Declarations Conflict of Interest The authors declare no competing interests. Ethics statements This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Keimyung University Dongsan Medical Center (IRB No. DSMC 2022-07-034). Because the study used residual clinical bile samples, the requirement for informed consent was waived under Article 16(3) of the Bioethics and Safety Act (Republic of Korea). Funding Supported by the National Research Foundation of Korea (NRF-2022 R1A2C4001769). Author Contribution Jin-Yi Han: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Visualization, Writing – original draft, Writing – review & editing.Keun Soo Ahn: Conceptualization, Supervision, Project administration, Funding acquisition, Writing – review & editing, Corresponding author.Min Jae Kim: Resources, Methodology, Validation, Formal analysis.Tae-Seok Kim: Resources, Data curation, Investigation.Yong Hoon Kim: Resources, Investigation, Validation, Visualization.Kyung In Shin: Resources, Sample acquisition, Data curation.Kwang Bum Cho: Resources, Clinical data collection, Review & editing.All authors read and approved the final manuscript. Acknowledgement Bile collection was assisted by Seo-Yeon Cha, Min-Jung Kim, and So-Jeong Kim. Biospecimens were provided by Keimyung University Dongsan Hospital Biobank, a member of the Korea Biobank Network. Availability of data and materials All data supporting the findings of this study are included in this article. Additional data is available upon reasonable request from the corresponding author References Han JY, Ahn KS, Kim MJ, Kim TS, Kim YH, Cho KB, Kang KJ. Optimization of bile preparation for liquid biopsy in cholangiocarcinoma focusing on circulating tumor DNA and protein stability. Sci Rep. 2025;15(1):12090. Han JY, Ahn KS, Kim TS, Kim YH, Cho KB, Shin DW, Baek WK, Suh SI, Jang BC, Kang KJ. Liquid Biopsy from Bile-Circulating Tumor DNA in Patients with Biliary Tract Cancer. Cancers (Basel) 2021, 13(18). Ahn KS, Kang KJ, Kim YH, Kim TS, Cho KB, Kim HS, Baek WK, Suh SI, Han JY. Diagnostic Role of Bile Pigment Components in Biliary Tract Cancer. Biomol Ther (Seoul). 2023;31(6):674–81. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485(7397):201–6. He Y, Yu X, Liu S, Wang R, Zhao J, Zhang Y, Li X, Sun Y, Yang X, Zhang T, et al. The emerging role of N6-methyladenosine (m6A) modification in gastrointestinal cancers. Nat Rev Gastroenterol Hepatol. 2012;9:211–8. Chen M, Wong CM, Xu M, Li A, Yang J, Xu D, Yu J, Zhang N, Huang J, Huang T. m6A RNA methylation regulates epithelial mesenchymal transition and metastasis of pancreatic cancer. Cancer Lett 2022, 540. Tang R, Zhang Y, Liang C, Xu J, Meng Q, Hua J, Liu J, Zhang B, Yu X, Shi S. m6A-related lncRNAs are potential biomarkers for predicting the overall survival of colorectal cancer. Mol Cancer 2021, 20. Additional Declarations No competing interests reported. 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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-7984256","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":562581507,"identity":"97d70efe-dad3-4f03-8100-b34e77d9add9","order_by":0,"name":"Jin-Yi Han¹","email":"","orcid":"","institution":"Keimyung University Dongsan Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Jin-Yi","middleName":"","lastName":"Han¹","suffix":""},{"id":562581508,"identity":"c937115f-b34e-4c70-be74-db6b934cc1c5","order_by":1,"name":"Keun Soo 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Bars represent mean ± SD. \u0026nbsp;Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7984256/v1/bbffbe1e621b1004170b48fb.png"},{"id":99187697,"identity":"6eeeafd0-4870-4449-9f0f-855ae3dc6994","added_by":"auto","created_at":"2025-12-30 00:11:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23571,"visible":true,"origin":"","legend":"\u003cp\u003eROC curves showing discrimination between PCA and benign biliary diseases. (A) Pancreatic juice (AUC = 1.00, cut-off = 3.7%), (B) Bile (AUC = 1.00, cut-off = 1.1%).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7984256/v1/4f0d22ebce3e655f7d945353.png"},{"id":104779097,"identity":"b1d2b591-31a0-4582-9dc1-fb3253db35b8","added_by":"auto","created_at":"2026-03-17 07:34:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":445079,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7984256/v1/374cbcd6-24b5-4af4-9fbb-e86c21bdc524.pdf"},{"id":99187696,"identity":"7f68cbbf-7b56-46d3-919c-20109cb1c3e8","added_by":"auto","created_at":"2025-12-30 00:11:47","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2062608,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-7984256/v1/55932cc154a39a4ba602f462.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bile-derived m⁶A as a Proof-of-Concept Liquid Biomarker for Early Pancreatic Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePancreatic cancer (PCA) is one of the most lethal malignancies, with a 5-year survival rate below 10%. Its poor prognosis largely reflects delayed diagnosis and the absence of reliable biomarkers. Bile provides a direct and physiologically relevant matrix for detecting tumor-derived nucleic acids [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. N6-methyladenosine (m⁶A) is the most abundant internal mRNA modification and plays a critical role in RNA metabolism and tumor progression [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Given that bile directly reflects the pancreaticobiliary microenvironment, bile-derived m⁶A profiling may bridge molecular alterations and clinically applicable diagnostics.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003eBile and pancreatic juice samples were collected via endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic biliary drainage (PTBD), and during operation at Keimyung University Dongsan Medical Center. The cohort included patients with PCA (n\u0026thinsp;=\u0026thinsp;7), BTC (n\u0026thinsp;=\u0026thinsp;35), and CBD stones (n\u0026thinsp;=\u0026thinsp;9). The study was approved by the institutional review board (IRB No. DSMC 2022-07-034). Global m⁶A levels were quantified using an ELISA-based colorimetric assay (EpiQuik m⁶A RNA Methylation Kit, Abcam) according to the manufacturer\u0026rsquo;s protocol. Statistical comparisons among groups were performed using one-way ANOVA with Tukey\u0026rsquo;s post hoc test (GraphPad Prism 10.0). ROC analysis determined the diagnostic performance and optimal cut-offs.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Elevated m⁶A levels in PCA bile (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eQuantitative analysis demonstrated a progressive elevation of global m⁶A modification from benign to malignant states (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The mean m⁶A percentage in PCA bile (6.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25%) was approximately six-fold higher than in BTC (1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15%) and more than ten-fold higher than in CBD controls (0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Diagnostic performance\u003c/h2\u003e \u003cp\u003eROC analysis demonstrated excellent discrimination between PCA and non-malignant samples (AUC\u0026thinsp;=\u0026thinsp;1.00, optimal cut-off =\u0026thinsp;1.1%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similar results were observed in pancreatic juice samples (AUC\u0026thinsp;=\u0026thinsp;1.00, cut-off =\u0026thinsp;3.7%). The distribution pattern shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e complements ROC findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), visually confirming the separation between PCA and non-malignant bile.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study provides the first evidence that bile-derived m⁶A dysregulation may serve as a biomarker for PCA detection. The strong diagnostic accuracy supports its translational potential for early diagnosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Compared to serum-based assays, bile offers a pathophysiologically relevant matrix reflecting pancreatic tumor activity. Our proof-of-concept analysis demonstrates strong diagnostic performance, suggesting that bile m⁶A profiling could complement current cytologic or molecular approaches in ERCP-based evaluation. Although the sample size is small, this work represents a proof-of-concept foundation for larger-scale validation. This proof-of-concept supports the translational feasibility of implementing bile-based m⁶A assays in ERCP-collected samples, enabling real-time, minimally invasive cancer screening during diagnostic procedures. The small sample size and single-center design limit the generalizability of our findings; however, this study provides a strong rationale for multi-institutional validation.\u003c/p\u003e \u003cp\u003eOur findings offer a translational bridge between RNA epitranscriptomic profiling and clinical biomarker development, aligning with the mission of \u003cem\u003eBMC Cancer\u003c/em\u003e to promote clinically relevant translational discoveries.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTranslational Relevance\u003c/strong\u003e \u003cp\u003eThe m⁶A-based assay could be readily incorporated into existing ERCP workflows, facilitating real-time molecular diagnosis. These findings highlight the potential of bile-based RNA methylation profiling as a minimally invasive diagnostic tool bridging molecular oncology and clinical detection of PCA.\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics statements\u003c/h2\u003e \u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Keimyung University Dongsan Medical Center (IRB No. DSMC 2022-07-034). Because the study used residual clinical bile samples, the requirement for informed consent was waived under Article 16(3) of the Bioethics and Safety Act (Republic of Korea).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eSupported by the National Research Foundation of Korea (NRF-2022 R1A2C4001769).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJin-Yi Han: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.Keun Soo Ahn: Conceptualization, Supervision, Project administration, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing, Corresponding author.Min Jae Kim: Resources, Methodology, Validation, Formal analysis.Tae-Seok Kim: Resources, Data curation, Investigation.Yong Hoon Kim: Resources, Investigation, Validation, Visualization.Kyung In Shin: Resources, Sample acquisition, Data curation.Kwang Bum Cho: Resources, Clinical data collection, Review \u0026amp; editing.All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eBile collection was assisted by Seo-Yeon Cha, Min-Jung Kim, and So-Jeong Kim. Biospecimens were provided by Keimyung University Dongsan Hospital Biobank, a member of the Korea Biobank Network.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eAll data supporting the findings of this study are included in this article. Additional data is available upon reasonable request from the corresponding author\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHan JY, Ahn KS, Kim MJ, Kim TS, Kim YH, Cho KB, Kang KJ. Optimization of bile preparation for liquid biopsy in cholangiocarcinoma focusing on circulating tumor DNA and protein stability. Sci Rep. 2025;15(1):12090.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan JY, Ahn KS, Kim TS, Kim YH, Cho KB, Shin DW, Baek WK, Suh SI, Jang BC, Kang KJ. Liquid Biopsy from Bile-Circulating Tumor DNA in Patients with Biliary Tract Cancer. \u003cem\u003eCancers (Basel)\u003c/em\u003e 2021, 13(18).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn KS, Kang KJ, Kim YH, Kim TS, Cho KB, Kim HS, Baek WK, Suh SI, Han JY. Diagnostic Role of Bile Pigment Components in Biliary Tract Cancer. Biomol Ther (Seoul). 2023;31(6):674\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485(7397):201\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Y, Yu X, Liu S, Wang R, Zhao J, Zhang Y, Li X, Sun Y, Yang X, Zhang T, et al. The emerging role of N6-methyladenosine (m6A) modification in gastrointestinal cancers. Nat Rev Gastroenterol Hepatol. 2012;9:211\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen M, Wong CM, Xu M, Li A, Yang J, Xu D, Yu J, Zhang N, Huang J, Huang T. m6A RNA methylation regulates epithelial mesenchymal transition and metastasis of pancreatic cancer. Cancer Lett 2022, 540.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang R, Zhang Y, Liang C, Xu J, Meng Q, Hua J, Liu J, Zhang B, Yu X, Shi S. m6A-related lncRNAs are potential biomarkers for predicting the overall survival of colorectal cancer. Mol Cancer 2021, 20.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"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":"pancreatic cancer, m⁶A methylation, bile, liquid biopsy, biomarker, epitranscriptomics","lastPublishedDoi":"10.21203/rs.3.rs-7984256/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7984256/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEarly detection of pancreatic cancer (PCA) remains a major challenge due to the lack of reliable biomarkers.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe investigated bile-derived N6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) modification as a diagnostic biomarker. Bile samples were obtained from patients with PCA (n\u0026thinsp;=\u0026thinsp;7), biliary tract cancer (BTC; n\u0026thinsp;=\u0026thinsp;35), and common bile duct (CBD) stones (n\u0026thinsp;=\u0026thinsp;9). Global m6A levels were quantified using an ELISA-based colorimetric assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGlobal m6A levels were markedly elevated in PCA bile (6.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25%) compared with BTC (1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15%) and CBD controls (0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;1). ROC analysis demonstrated excellent discrimination (AUC\u0026thinsp;=\u0026thinsp;1.00, optimal cut-off =\u0026thinsp;1.1%). Consistent results were observed in pancreatic juice samples (AUC\u0026thinsp;=\u0026thinsp;1.00, cut-off =\u0026thinsp;3.7%).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe results demonstrated distinct m⁶A elevation in bile from PCA patients (Fig.\u0026nbsp;1), supporting its translational potential as a minimally invasive biomarker. Validation in larger cohorts is warranted. These findings provide a proof-of-concept foundation for bile-based RNA methylation profiling in pancreatic cancer.\u003c/p\u003e","manuscriptTitle":"Bile-derived m⁶A as a Proof-of-Concept Liquid Biomarker for Early Pancreatic Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 00:11:42","doi":"10.21203/rs.3.rs-7984256/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":"fd1cbf69-40be-498d-87d6-65dfbfff5325","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-12T12:39:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 00:11:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7984256","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7984256","identity":"rs-7984256","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}