Integrated ctDNA Methylation Profiling of P2RX1, CBFA2T3, and CERS4 as Pancreatic Ductal Adenocarcinoma Biomarkers: Identification and Mechanistic Insights

Background: Pancreatic ductal adenocarcinoma (PDAC) stands as one of the most formidable cancers globally, yet its diagnosis continues to be a pressing challenge, largely owing to the inherent limitations of current diagnostic methodologies. Methods: Utilizing whole-genome bisulfite-sequencing (WGBS), differentially methylated genes in the promoter region were identified from the plasma of PDAC patients. Subsequently, screening of candidate methylation genes, methylation-specific primers and probes for P2RX1 , CBFA2T3 and CERS4 were designed, and a stable multiple quantitative methylation-specific polymerase chain reaction (M-QMSP) system was established to detect the circulating tumor DNA (ctDNA) methylation level of these genes in PDAC. Finally, the disease-driving mechanism of the biomarker CERS4 was explored separately in vitro and in vivo. Results: A total of 112,294 differential methylation regions (DMRs) were precisely identified by WGBS, encompassing 88,210 hypermethylation DMRs and 24,084 hypomethylation DMRs. Notably, three differentially methylated genes, P2RX1 , CBFA2T3 and CERS4 (nominated as P2CC model), were astutely pinpointed as potential plasma methylation biomarkers. The M-QMSP system was established through the prioritization of these genes, which demonstrated that the combined diagnostic prowess of P2CC modelnotably outperformed carbohydrate antigen 19-9 (CA19-9). In PDAC, overexpression of CERS4 has been observed to suppress tumor growth in nude mice xenografts and inhibit cell proliferation and migration. Additionally, CERS4 played a pivotal role in promoting lipid metabolism. Conclusion: Based on WGBS screening and M-QMSP validation, we have determined that the P2CC model can serve as the combined diagnostic biomarkers for PDAC, superior to CA19-9. Moreover, as a tumor suppressor gene, CERS4 regulates the metabolism of sphingolipids.