Characterizing The CpG Methylation Of Epstein-Barr Virus DNA In The Plasma Of Patients With Hodgkin Lymphoma and HIV-Associated Burkitt Lymphoma

Epstein-Barr virus (EBV) is harbored as latent virus within the tumor cells of a subset of classical Hodgkin lymphoma (HL) and HIV-associated Burkitt lymphoma (BL). In HL, we and others have previously shown that elevated EBV DNA copies in plasma accurately distinguish EBV(+) tumors from EBV(-) tumors. However, EBV DNA may be present in plasma due to apoptosis of latently infected EBV(+) tumor cells (or latent EBV(+) benign lymphocytes), as a result of replicative lytic infection (particularly in immunocompromised hosts), or both. Thus, detecting elevated EBV DNA copies in plasma by does not always signify the presence of an EBV(+) tumor. However, a distinction is that the EBV DNA derived from latently infected cells is replicated by cellular DNA polymerase and is usually CpG methylated, whereas EBV DNA in lytic infection is replicated by the viral DNA polymerase and is never CpG methylated.

To improve upon plasma EBV DNA as a tumor-specific marker in EBV(+) lymphomas, we assessed the CpG methylation of EBV DNA in a subset of plasma specimens from immunocompetent patients with HL (enrolled in the Eastern Cooperative Oncology Group E2496 trial) and a subset from patients with HIV-associated BL (enrolled in the AIDS Malignancy Consortium 048 trial). The EBV tumor status was determined by in situ hybridization (ISH) on tissue for EBV RNAs (EBER). For the CpG methylation assay, methyl binding domain paramagnetic beads were used to first fractionate CpG methylated DNA from CpG unmethylated DNA in plasma. EBV DNA in each fraction were quantified by real time polymerase chain reaction (qPCR) to determine the percentage of methylated EBV DNA. Specimens with > 100 EBV DNA copies/mL plasma were analyzed.

Among patients with EBER(+) HL and elevated EBV DNA in plasma prior to therapy (n=8), EBV DNA was largely CpG methylated (median 95%, range 54%-99%). By contrast, in the rare cases where patients with EBER(-) HL tumors had high EBV DNA copy number in plasma prior to therapy (4 of 92, 4%), only small percentages of EBV DNA were methylated in 3 patients (5%, 9%, and 12%). In the fourth patient, the plasma EBV DNA was 99% CpG methylated. We suspect that patient 4 had an EBV-associated HL with either a technical failure of EBER-ISH or an instance of a tumor that harbors viral DNA but does not express EBERs. In patients with HIV-associated BL, plasma EBV DNA copies were elevated in 100% of patients with EBER(+) BL (n=4), but also in 4 of 9 (56%) patients with EBER(-) BL. This propensity to detect EBV DNA in the plasma of patients with HIV but without EBV(+) BL likely reflects replicative lytic infection in immunocompromised hosts, making plasma EBV DNA less reliable as a marker of EBV(+) lymphoma in this setting. Thus, we applied the methylation assay to specimens from patients with HIV and BL. Patients with HIV and EBER(+) BL tumors (n=4), EBV DNA was 28%, 46%, 47%, and 60% methylated, whereas a patient with HIV and EBER(-) BL had lower methylation at 8%. To better elucidate the meaning of plasma EBV DNA elevations during lymphoma treatment, we applied the methylation assay to specimens from patients with EBV(+) HL and elevated EBV DNA in plasma at month 6 of therapy (n=4). Dense CpG methylation (96%) of plasma EBV DNA was detected in 1 patient who went on to relapse at Month 15, while the plasma EBV DNA was largely unmethylated in 3 patients who remain in remission each with over 5 years of follow-up.

In settings where EBV DNA is detected in plasma, the CpG methylation assay helps differentiate patients with EBV(+) HL or BL from those with EBV(-) tumors. In patients with HIV where lytic EBV replication can contribute greatly to elevations of EBV DNA in plasma, the fractionated quantification of plasma EBV DNA by the methylation assay aids in distinguishing EBV DNA that is tumor-derived from that which is the result of lytic replication. With growing interest in plasma EBV DNA as a serial marker of disease activity in EBV(+) lymphomas, the methylation assay is promising in its ability to differentiate patients with elevated EBV DNA in plasma but ongoing remission from those in whom plasma EBV DNA is tumor-derived and an early harbinger of relapse.

No relevant conflicts of interest to declare.