Alterations of 5-hydroxymethylation in circulating cell-free DNA reflect molecular distinctions of subtypes of non-Hodgkin lymphoma

The 5-methylcytosines (5mC) have been implicated in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the role of 5-hydroxymethylcytosines (5hmC) that are generated from 5mC through active demethylation, in lymphomagenesis is unknown. We profiled genome-wide 5hmC in circulating cell-free DNA (cfDNA) from 73 newly diagnosed patients with DLBCL and FL. We identified 294 differentially modified genes between DLBCL and FL. The differential 5hmC in the DLBCL/FL-differentiating genes co-localized with enhancer marks H3K4me1 and H3K27ac. A four-gene panel (CNN2, HMG20B, ACRBP, IZUMO1) robustly represented the overall 5hmC modification pattern that distinguished FL from DLBCL with an area under curve of 88.5% in the testing set. The median 5hmC modification levels in signature genes showed potential for separating patients for risk of all-cause mortality. This study provides evidence that genome-wide 5hmC profiles in cfDNA differ between DLBCL and FL and could be exploited as a non-invasive approach.