Plasma methylation markers for early colorectal cancer detection

doi:10.5306/wjco.125345

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Publication Name

World Journal of Clinical Oncology

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2218-4333

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

Translational Research

Copyright: ©Author(s) 2026. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial (CC BY-NC 4.0) license. No commercial re-use. See permissions. Published by Baishideng Publishing Group Inc.

World J Clin Oncol. Oct 24, 2026; 17(10): 125345
Published online Oct 24, 2026. doi: 10.5306/wjco.125345

Plasma methylation markers for early colorectal cancer detection

Ze-Feng Liu, Jin-Hua Jiang, Wen-Jie Huang, Yue Wang, Gao-Wei Fan, Bang-Guo Chen, Zhi-En Lan, Lin-Hao Xu, Chun-Hui Cui

Ze-Feng Liu, Jin-Hua Jiang, Wen-Jie Huang, Yue Wang, Gao-Wei Fan, Bang-Guo Chen, Zhi-En Lan, Chun-Hui Cui, Department of Gastrointestinal Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong Province, China

Lin-Hao Xu, Department of R&D, Guangzhou Biotron Technology Co., Ltd, Guangzhou 510530, Guangdong Province, China

Co-first authors: Ze-Feng Liu and Jin-Hua Jiang.

Co-corresponding authors: Lin-Hao Xu and Chun-Hui Cui.

Author contributions: Liu ZF and Jiang JH participated in manuscript drafting, revision and response to reviewers, and share equal contributions to the acquisition, analysis and interpretation of the research data presented in this article as co-first authors; Liu ZF conceived the research framework, performed statistical analyses of all plasma and tissue data; Jiang JH enrolled clinical subjects, collected paired tissue and plasma specimens, and sorted clinicopathological information; Huang WJ conducted multiplex probe amplification marker screening and multiplex methylation quantitative real-time polymerase chain reaction experiments, interpreted raw molecular testing data; Wang Y assisted with cell-free DNA extraction and bisulfite conversion procedures; Fan GW organized Gene Expression Omnibus public methylation data and completed in silico receiver operating characteristic validation; Chen BG sorted the follow-up data of gastrointestinal tumor patients; Lan ZE performed pathological review of all tissue samples; Cui CH acted as the primary corresponding author responsible for all journal communications; Xu LH and Cui CH co-supervised the whole project, made substantial and equal contributions to study conception and design, experimental supervision, data interpretation, manuscript revision, and final approval of the submitted version as co-corresponding authors.

AI contribution statement: ChatGPT-4 was used solely for English-language polishing and proofreading during manuscript revision. No AI tools were applied for study design, data analysis, result interpretation, conclusion development, original manuscript drafting, table-figure organization or reference sorting. All scientific content was independently completed by the authors, who take full responsibility for the originality, integrity and scientific accuracy of this manuscript.

Supported by National Natural Science Foundation of China, No. 81974069; the Guangdong Province Natural Science Foundation, No. 2022A1515012587; and the Key Clinical Research Project of Southern Medical University, No. LC2019ZD011.

Conflict-of-interest statement: All authors declare no conflict of interest in publishing the manuscript.

Corresponding author: Chun-Hui Cui, Chief Physician, Professor, Department of Gastrointestinal Surgery, Zhujiang Hospital, Southern Medical University, No. 253 Industrial Road, Guangzhou 510282, Guangdong Province, China. drcuich@163.com

Received: July 6, 2026
Revised: August 15, 2026
Accepted: October 8, 2026
Published online: October 24, 2026
Processing time: 109 Days and 13.3 Hours

Abstract

BACKGROUND

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. Early detection significantly improves patient outcomes, but current screening methods have limitations in terms of sensitivity, specificity, and patient compliance. Circulating cell-free DNA in the bloodstream, including methylated DNA fragments, offers a promising noninvasive alternative for cancer detection.

AIM

To identify a high-specificity plasma methylation panel for early noninvasive CRC detection.

METHODS

We conducted a pilot recruiting CRC patients, benign gastrointestinal lesion cases and healthy volunteers. Genomic DNA from paired tumor-paracancerous tissues and white blood cells, plus plasma cell-free DNA, was extracted. We used multiplex probe melting assay to screen markers with minimal leukocyte methylation background, followed by quantitative real-time polymerase chain reaction verification. Independent in silico sequencing data validated the panel’s discriminatory power. We further analyzed associations between panel positivity and clinicopathological features, as well as its diagnostic specificity across other gastrointestinal malignancies.

RESULTS

The analysis revealed a three-marker methylation panel comprising IKZF1, ITGA4, and C9orf50, with a sensitivity of 81.03% and a specificity of 100% for healthy individuals, 85.71% for benign diseases, and an overall specificity of 95.83% in distinguishing CRC cases from controls in plasma samples. The area under the receiver operating characteristic curve for this panel was 0.8962 (95%CI: 0.8339-0.9585; P < 0.0001). The positivity of the panel was associated with the size and advancement of the tumor and the clinical specificity, especially in asymptomatic gastrointestinal patients who showed good potential for CRC diagnosis.

CONCLUSION

The three-marker methylation panel offers high diagnostic accuracy and could be integrated into routine clinical practice, such as screening and auxiliary diagnosis of CRC.

Keywords: Colorectal cancer; Cell-free DNA; DNA methylation; Multiplex probe melting assay; Plasma biomarker; Early detection

Core Tip: This study developed a three-marker plasma DNA methylation panel – comprising IKZF1, ITGA4, and C9orf50 – specifically designed for early detection of colorectal cancer. Leveraging multiplex probe melting assay screening, the panel effectively suppresses confounding methylation signals derived from white blood cells. It demonstrates superior diagnostic performance for colorectal cancer compared to the clinically established SEPT9 methylation blood test, achieving higher sensitivity at matched specificity and exhibiting strong potential as a noninvasive complementary diagnostic tool.