Abstract
Characterization of tumor epigenetic aberrations is integral to understanding the mechanisms of tumorigenesis and provide diagnostic, prognostic, and predictive information of high clinical relevance. Among the different tumor-associated epigenetic signatures, 5 methyl-cytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two most well-characterized DNA methylation alterations linked to cancer pathogenesis. 5hmC has a tissue-specific distribution and its abundance is subjected to changes in tumor DNA, making it a promising biomarker. Detecting tumor-related DNA methylation alterations in tissues is highly invasive, while the analysis of the cell-free DNA (cfDNA) is poised to supplement, if not replace, surgical biopsies. Despite many studies attempted to identify new epigenetic targets for liquid biopsy assays, little is known about the regulatory roles of 5hmC, its impacts on the molecular phenotypes in tumors. Most importantly, whether the oncogenic-associated 5hmC signatures found in tumor tissues can be recapitulated in patients' cfDNA. In this study, we performed the unbiased and simultaneous detection of 5mC and 5hmC whole-genome DNA modifications at base-resolution from two distinct cancer cohorts, from patients with bladder cancer or B-Cell lymphoma, their corresponding normal tissues, and cfDNAs from plasma. We analyzed tissue-specific methylation patters and searched for signatures in gene coding and regulatory regions linked to cancerous states. We then looked for methylation signatures in patients' cfDNA to determine if they were consistent with the tumor-specific patterns. We determined the functional significance of 5hmC in tissue specific transcription and uncovered hundreds of tumor-associated 5hmC signatures. These tumor-associated 5hmC changes, particularly in genes and enhancers, were functionally significant in tumorigenesis pathways and correlated with tumor specific gene expression. To investigate if cfDNA is a faithful surrogate for tumor-associated 5hmC, we devised a targeted capture strategy to examine the alterations of 5hmC in cfDNA from patients with bladder cancer and lymphoma with sufficient sensitivity and specificity and confirmed that they recapitulated the patterns we observed in tumor tissues. Our results provide analytic validation of 5hmC as a cancer-specific biomarker. The methods described here for systematic characterization of 5hmC at functional elements open new avenues to discover epigenetic markers for non-invasive diagnosis, monitoring, and stratifying cancer.