Global DNA methylomes reveal oncogenic-associated 5-hydroxylmethylated cytosine (5hmC) signatures in the cell-free DNA of cancer patients

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. Competing Interest Statement The authors have declared no competing interest. Funding Statement Research reported in this manuscript was supported by grants from the US National Institutes of Health (NCI Cancer Center Support Grant 5 P30 CA034196-36), the Integrated Translational Cancer Science Center (ITSC), The Hope Foundation and The Jackson Laboratory Scientific Service Process Improvement Funds. 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 conducted in accordance with the principles of the Helsinki Declaration and were approved by the appropriate Institutional Review Boards: Oregon Health Science University (OHSU) IRB#4422 and IRB#4918, The Jackson Laboratory IRB Number 2018-025. All patients provided written informed consent. 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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data described in this study can be found at the NCBI Short Read Archive, BioProject: PRJNA1184365. Computational methods and tools used in this study are listed in the Methods and can be downloaded from https://github.com/gabyrech/tumorMethylome. Further information and requests for resources and reagents should be directed to and will be fulfilled by lead contact Chia-Lin Wei (weicl{at}uw.edu).