EBV status of lymphoma specimens from Ethiopia: Initial categorization of pathology and epigenetic state of EBV

Abstract Background: Epstein-Barr virus (EBV) is an oncogenic herpesvirus that infects nearly all individuals by adulthood, with over 90% of the global population harboring it. The Epstein- Barr encoding region (EBER) in situ hybridization is a widely utilized technique for detecting EBV-infected cells in tissue specimens, essential for diagnosing EBV-related diseases and understanding the virus's pathophysiology. However, no studies have been conducted in Ethiopia using this method to detect the virus in lymphoma specimens. This research aims to be the first to present EBV detection through EBER-ISH and comprehensive epigenetic status of lymphoma specimens collected at a large tertiary medical referral center in Ethiopia. Methods: The study was conducted at Tikur Anbessa Specialized Hospital in Addis Ababa, Ethiopia. A total of 305 participants were enrolled, both retrospectively and prospectively after obtaining informed consent. Samples of formalin-fixed paraffin-embedded (FFPE) tissues, blood, and lymph node biopsies were collected. Serum plasma, polymorphonuclear, and blood mononuclear cells (PBMCs) were isolated from whole blood samples. EBER- ISH technique and immunohistochemistry for CD3, CD20, and CD56 were performed. Additionally, the viral epigenome methylation profile was assessed using a multiplex PCR-mass-spectrometry assay (iPLEX) quantifying methylation at n=24 EBV gene loci. Results: Out of the total 305 study participants, 288 individuals were diagnosed with lymphoma based on confirmed pathology reports. Among these, 32% (n=91) were diagnosed with Hodgkin lymphoma, while 68% (n=197) had non-Hodgkin lymphoma (NHL). Within the NHL subgroup, small lymphocytic lymphoma accounted for the highest percentage (18%), followed by diffuse large B-cell lymphoma (13%). EBER analysis was conducted on 78 FFPE samples, revealing an EBER positivity rate of 26.9% (n=21). Of these, six cases (28.6%) were linked to EBV-associated lymphomas and the rest of the samples (n=15), had scattered EBV positive cells, which was concluded to be due to latent EBV infection. EBV was detected in 25.4% of non-Hodgkin lymphoma samples, while 42.9% of Hodgkin lymphoma samples tested positive for the virus using EBER-ISH. CD56 expression was observed in 27.4% of lymphomas, predominantly in B cell lymphoma specimens (64%). EBV DNA methylation patterns across lymphoma samples revealed intermediate to high average methylation (46.9±7.4%), with heterogeneous methylation at the Cp- (66.3±34.6%) and LMP1 (14.5±27.2%) promoters indicative of heterogeneous (type 2/3) EBV latency states. Conclusion: EBER-ISH on biopsy samples serves as a reliable indicator of EBV status in lymphoma patient samples from Ethiopia. The nature of EBV infection is consistent with latent state. EBV status serves as an independent prognostic variable in lymphoma, thus, establishing laboratory services for routine EBER-ISH in EBV diagnosis is crucial for improving disease evaluation and prognosis. Keywords: EBV, EBER-ISH, Lymphoma, Methylation, Ethiopia