Identification of multi-omic biomarkers from Fecal DNA for improved Detection of Colorectal Cancer and precancerous lesions

Background Timely diagnosis and intervention of colorectal cancer (CRC) at curable stages is essential for improving patient survival. Stool samples carry exfoliation of intestinal epithelium, therefore providing excellent opportunity for non-invasive diagnosis of CRC as well as precancerous lesions. In this study, we aimed to conduct multi-dimensional analysis of fecal DNA and investigate the utility of different types of biomarkers for CRC detection. Method In this case-control study, we performed comprehensive analyses of the genomic, epigenomic, and metagenomic features of fecal DNA from CRC patients, individuals with advanced precancerous lesions (APLs) and controls. DNA methylation markers were identified by whole genome bisulfite sequencing of paired colorectal cancer and normal tissues. A multi-gene fecal DNA methylation test was then developed based on three marker genes ( SDC2, ADHFE1 and PPP2R5C ) using quantitative methylation-specific PCR (qMSP), and validated on fecal DNA samples. Genomic mutation profiles as well as microbiome signatures of fecal DNA were analyzed using high-throughput sequencing. Results The methylation-based fecal DNA test demonstrated an overall sensitivity of 88% for CRC and 46.2% for APL respectively, and a specificity of 91.8% for controls. On the other hand, the mutation-based diagnostic model yielded limited sensitivity, and combined detection of methylation markers and mutation in fecal DNA did not improve the assay performance. Meanwhile, a diagnostic model based on the relative abundance of bacterial species showed inferior performance than the methylation-based model. Finally, integrated diagnostic model combining both methylation and microbial markers showed an enhanced performance (AUC= 0.95) compared to methylation markers alone. Conclusions The multi-gene fecal DNA methylation test provided remarkable diagnostic performance for CRCs and APLs. Furthermore, multi-target assay integrating both methylation and microbial markers may further improve the diagnostic performance. Our findings may aid in the development of novel diagnostic tools for CRC. ### Competing Interest Statement JP, ZL, RJ, YS, JS, DZ, YW are employees of Envelope Health Biotechnology Co. Ltd., BGI-Shenzhen. SZ is an employee of BGI Genomics, BGI-Shenzhen. ### Funding Statement This study was supported by Guangzhou Science and Technology Plan Projects (Health Medical Collaborative Innovation Program of Guangzhou, 201803040019), Guangzhou Key Research and Development Project (No. 202206080008) and Shenzhen Engineering Laboratory for Innovative Molecular Diagnostics (DRC-SZ[2016]884) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Institutional Review Board at Sun Yat-sen University Cancer Center (B2019-068-X01) and BGI (BGI-IRB 20039) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors