Plasma EBV DNA Methylation: Evidence of EBV Lytic Infection in HIV(+) EBV(+) Plasmablastic Lymphoma

Background: Among people living with HIV, plasmablastic lymphoma (PBL) and classical Hodgkin lymphoma (HL) are almost uniformly associated with Epstein-Barr virus (EBV). Immunohistochemical studies have demonstrated high levels of lytic gene expression in PL,¹ whereas EBV(+) HL shows exclusively latent viral gene expression. EBV virion DNA is never CpG methylated, but tumor-derived plasma EBV is CpG methylated as previously shown by our group in EBV(+) HL and nasopharyngeal carcinoma (NPC).² Others have also presented evidence that patterns of CpG methylation of plasma EBV DNA varies between people with NPC and non-malignant EBV(+) conditions.³ To better characterize the nature of plasma EBV in patients with lymphoma, we investigated EBV DNA copy number and EBV DNA methylation in HIV-associated PBL and HL.

Methods: With appropriate approvals from the Human Research Ethics Committee of the University of the Witwatersrand and the Johns Hopkins Institutional Review Boards, we collected plasma from newly diagnosed untreated patients with HIV and PBL or HL in Johannesburg, South Africa. Diagnostic biopsy specimens were reviewed in Baltimore by a hematopathologist to confirm the diagnosis and Epstein-Barr encoding region (EBER) in situ hybridization was performed to determine EBV association. Plasma EBV copies were measured by standard qPCR targeting the BamW repeats. qPCR using the same primers was also performed after capture with methyl-DNA binding protein 2 (MBD2) magnetic beads, where the captured fraction represents methylated DNA and the flow-through represents unmethylated DNA.

Results: We studied plasma from 7 patients with PBL and 12 patients with HL. Four of five PBL tumors were EBV (+) and eight of 10 HL tumors were EBV (+) by tissue EBER in situ hybridization. Analysis of plasma EBV DNA copy number showed overlapping levels of EBV DNA (median 2.7 Log₁₀ copy/mL in PBL and 3.2 Log₁₀ copy/mL in HL) (Fig. 1). Patients with at least 1.5 Log₁₀ copy/mL of EBV underwent CpG methylation evaluation, which showed very different EBV CpG methylation indices. The EBV methylation index was highly variable in PBL (median of 51%) and was uniformly high in HL (median of 97%).

Conclusions: Although plasma from plasmablastic lymphoma patients show high EBV copy number, the characteristic of that EBV DNA is very different from the plasma EBV detected in Hodgkin lymphoma patients. This difference is hypothesized to be related to evidence of viral lytic gene expression seen in PBL. The degree of plasma EBV DNA methylation may have broad implications for lymphoma diagnosis and subtyping.

References:

1. Ambrosio MR, Mundo L, Gazaneo S, et al. MicroRNAs sequencing unveils distinct molecular subgroups of plasmablastic lymphoma. Oncotarget. 2017;8(64):107356.

2. Shamay M, Kanakry JA, Low JS, et al. CpG methylation in cell-free Epstein-Barr virus DNA in patients with EBV-Hodgkin lymphoma. Blood Advances. 2020;4(8):1624-1627.

3. Lam WJ, Jiang P, Chan KA, et al. Methylation analysis of plasma DNA informs etiologies of Epstein-Barr virus-associated diseases. Nature communications. 2019;10(1):3256.

Xian:Invivoscribe: Honoraria. Martinson:Pfizer: Research Funding.