Monitoring EBV-associated lymphoma tumor progression based on a novel herpes virus methylation assay

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and has been associated with several human illnesses. Over 90% of adults are latently infected with EBV worldwide. In the majority of infected individuals, EBV infection is asymptomatic, but in certain cases it can lead to the development of several malignancies. EBV is associated with 30-50% of Hodgkin lymphoma (HL) cases, and over 90% of nasopharyngeal carcinoma (NPC) cases. Viral copy number in the blood as a diagnostic tool for the detection and monitoring of EBV-associated NPC is already in practice. The major challenge of viral copy number in the blood is to draw the threshold line to distinguish between healthy EBV-carriers and patients with EBV-associated malignancies, since it varies significantly between individuals. In latently infected cells, including tumor cells, the viral episomal genomes are CpG methylated, as opposed to the un-methylated virion associated viral DNA. We hypothesized that CpG methylated EBV genomes will be present preferentially in the blood of patients with EBV-associated HL and that this methylation will decrease upon successful treatment. The use of paramagnetic beads linked to methyl CpG binding domain (MBD) protein allowed separation of virion from cell-derived EBV DNA. We followed CpG methylation of EBV sequences in cell-free (cf) DNA isolated from the plasma of patients with EBV-positive HL before and after chemotherapy. Our results show dramatic decrease in EBV methylation rates after chemotherapy treatment. This study offers a new method for monitoring EBV-associated HL tumor progression by evaluating methylation rates of the virus.