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

doi:10.21203/rs.3.rs-7984256/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-7984256/v1

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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 (m⁶A) 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

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.

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.

3.1 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.

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.

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

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No competing interests reported.

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You are reading this latest preprint version

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

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

1. Introduction

2. Methods

3. Results

3.1 Elevated m⁶A levels in PCA bile (Fig. 1)

3.2. Diagnostic performance

4. Discussion

Declarations

Conflict of Interest

Ethics statements

Funding

Author Contribution

Acknowledgement

Availability of data and materials

References

Additional Declarations

Supplementary Files

Status:

Version 1