Application of the High-Throughput TAB-Array for the Discovery of Novel 5-Hydroxymethylcytosine Biomarkers in Pancreatic Ductal Adenocarcinoma

The clinical outcomes of pancreatic ductal adenocarcinoma (PDAC) remain dismal, with an estimated five-year survival rate of less than 5%. Early detection and prognostic approaches, including robust biomarkers for PDAC, are critical for improving patient survival. Our goal was to explore the biomarker potential of 5-hydroxymethylcytosines (5hmC), an emerging epigenetic marker with a distinct role in cancer pathobiology, yet under-investigated, due largely to technical constraints relating to PDAC. The TET-assisted bisulfite (TAB)-Array assay represents state-of-the-art technology and was used to directly profile 5hmC at single-base resolution with the Illumina EPIC array (~850,000 cytosine modification sites) in 17 pairs of tumor/adjacent tissue samples from US patients collected at the University of Chicago Medical Center. The TAB-Array data were analyzed to explore the genomic distribution of 5hmC and evaluate whether 5hmC markers were differentially modified between tumors and adjacent tissues. We demonstrated distinctive distribution patterns of 5hmC in tissue samples from PDAC patients relative to cis-regulatory elements (e.g., histone modification marks for enhancers), indicating their potential gene regulatory relevance. Substantial differences in 5hmC-modified CpG sites were detected between tumors and adjacent tissues in genes related to cancer pathobiology. The detected 5hmC-contaning marker genes also showed prognostic value for overall survival in the US patients with PDAC from the Cancer Genome Atlas Project. This study demonstrated the technical feasibility of the TAB-Array approach in cancer biomarker discovery and the biomarker potential of 5hmC for PDAC. Future studies using tissues and/or liquid biopsies may include 5hmC as a potential epigenetic biomarker target for PDAC.