Azacitidine induces demethylation of the Epstein-Barr virus genome in tumors

Purpose: To determine whether therapy with a DNA methyltransferase inhibitor is effective in achieving demethylation and gene re-expression in tumor DNA in patients. Methods: Biopsy specimens were obtained from patients with Epstein-Barr virus-associated tumors, enrolled on a clinical trial of 5-azacitidine, within 72 hours of the conclusion of the last infusion of the first cycle of therapy, and compared to pretreatment specimens. Methylation-specific polymerase chain reaction, bisulfite genomic sequencing, and immunohistochemistry were used to assess demethylation and gene re-expression. Results: Substantial degrees of demethylation were detected in all latent and lytic Epstein-Barr virus promoters examined. Immunohistochemistry suggested activation of a previously silent viral antigen expression in one instance. Conclusion: Pharmacologic reversal of dense CpG methylation in tumor tissue can be achieved in patients.