An elevated level of circulating cell-free DNA (cfDNA) has been associated with tumor bulk and poor prognosis in diffuse large B-cell lymphoma (DLBCL), but the tumor-specific molecular alterations in cfDNA with prognostic significance remain unclear. We investigated the association between 5-hydroxymethylcytosines (5hmC), a mark of active demethylation and gene activation, in cfDNA from blood plasma and prognosis in newly diagnosed DLBCL patients. We used 5hmC-Seal, a highly sensitive chemical labeling technique, to profile genome-wide 5hmC in plasma cfDNA from 48 DLBCL patients at the University of Chicago Medical Center between 2010 and 2013. Patients were followed through 31 December 2017. We found a distinct genomic distribution of 5hmC in cfDNA marking tissue-specific enhancers, consistent with their putative roles in gene regulation. The 5hmC profiles in cfDNA differed by cell of origin and were associated with clinical prognostic factors, including stage and the International Prognostic Index. We developed a 29 gene–based weighted prognostic score (wp-score) for predicting event-free survival (EFS) and overall survival (OS) by applying the elastic net regularization on the Cox proportional-hazards model. The wp-scores outperformed (eg, prognostic accuracy, sensitivity, specificity) established prognostic factors in predicting EFS and OS. In multivariate Cox models, patients with high wp-scores had worse EFS (hazard ratio, 9.17; 95% confidence interval, 2.01-41.89; P 5 .004) compared with those in the low-risk group. Our findings suggest that the 5hmC signatures in cfDNA at the time of diagnosis are associated with clinical outcomes and may provide a novel minimally invasive prognostic approach for DLBCL.
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