Burkitt Lymphoma.

Epstein-Barr virus (EBV) is associated with endemic Burkitt lymphoma (eBL), but the contribution of EBV variants is ill-defined. Studies of EBV whole genome sequences (WGS) have identified phylogroups that appear to be distinct for Asian versus non-Asian EBV, but samples from BL or Africa, where EBV was first discovered, are under-represented. We conducted a phylogenetic analysis of EBV WGS and LMP-1 sequences obtained primarily from BL patients in Africa and representative non-African EBV from other conditions or regions using data from GenBank, Sequence Read Archive, or Genomic Data Commons for the Burkitt Lymphoma Genome Sequencing Project (BLGSP) to generate data to support the use of a simpler biomarker of geographic or phenotypic associations. We also investigated LMP-1 patterns in 414 eBL cases and 414 geographically matched controls in the Epidemiology of Burkitt Lymphoma in East African children and minors (EMBLEM) study using LMP-1 PCR and Sanger sequencing. Phylogenetic analysis revealed distinct genetic patterns of African versus Asian EBV sequences. We identified 281 single nucleotide variations (SNVs) in LMP-1 promoter and coding region, which formed 12 unique patterns (A to L). Nine patterns (A, AB, C, D, F, I, J, K and L) predominated in African EBV, of which four were found in 92% of BL samples (A, AB, D, and H). Predominant patterns were B and G in Asia and H in Europe. EBV positivity in peripheral blood was detected in 95.6% of EMBLEM eBL cases versus 79.2% of the healthy controls (odds ratio [OR] =3.83; 95% confidence interval 2.06-7.14). LMP-1 was successfully sequenced in 66.7% of the EBV DNA positive cases but in 29.6% of the controls (ORs ranging 5-11 for different patterns). Four LMP-1 patterns (A, AB, D, and K) were detected in 63.1% of the cases versus 27.1% controls (ORs ranges: 5.58-11.4). Dual strain EBV infections were identified in WGS and PCR-Sanger data. In conclusion, EBV from Africa is phylogenetically separate from EBV in Asia. Genetic diversity in LMP-1 formed 12 patterns, which showed promising geographic and phenotypic associations. Presence of multiple strain infection should be considered in efforts to refine or improve EBV markers of ancestry or phenotype.Lay SummaryEpstein-Barr virus (EBV) infection, a ubiquitous infection, contributes to the etiology of both Burkitt Lymphoma (BL) and nasopharyngeal carcinoma, yet their global distributions vary geographically with no overlap. Genomic variation in EBV is suspected to play a role in the geographical patterns of these EBV-associated cancers, but relatively few EBV samples from BL have been comprehensively studied. We sought to compare phylogenetic patterns of EBV genomes obtained from BL samples in Africa and from tumor and non-tumor samples from elsewhere. We concluded that EBV obtained from BL in Africa is genetically separate from EBV in Asia. Through comprehensive analysis of nucleotide variations in EBV’s LMP-1 gene, we describe 12 LMP-1 patterns, two of which (B and G) were found mostly in Asia. Four LMP-1 patterns (A, AB, D, and F) accounted for 92% of EBVs sequenced from BL in Africa. Our results identified extensive diversity of EBV, but BL in Africa was associated with a limited number of variants identified, which were different from those identified in Asia. Further research is needed to optimize the use of PCR and sequencing to study LMP-1 diversity for classification of EBV variants and for use in epidemiologic studies to characterize geographic and/or phenotypic associations of EBV variants with EBV-associated malignancies, including eBL.

A prospective epidemiological study was carried out in the West Nile District of Uganda from 1972 to 1979 in order to investigate the aetiological role of the Epstein-Barr virus (EBV) in Burkitt's lymphoma (BL). By 1976, fourteen BL cases had been detected among the 42,000 children originally bled in the study area. Testing of sera from BL candidates and neighbourhood controls showed that children who develop BL later have EBV/VCA titres several dilutions higher than their age- and sex-matched neighbours. This appearance of a strong EBV activity long before BL development was taken as evidence of a causal role of EBV in BL. In order to add to the unique material of pre-bled BL cases, BL detection was continued up to March 1979 when field work became impossible in Uganda. Two additional pre-bled BL cases were found during this extension of the study. The serological and virological evaluation of these additional cases showed that the EBV/VCA titres, but not the EA and EBNA titres, were about two dilutions higher in the BL candidates than in the controls. Hybridization assays showed that both lymphomas contained EBV/DNA in the tumour cells. These additional results thus confirm the findings in the first 14 cases and strengthen the epidemiological evidence for a causal role of the EBV in endemic BL.

Epstein-Barr virus (EBV) is an etiologic agent of endemic Burkitt lymphoma (BL), a prevalent pediatric cancer in sub-Saharan Africa. There are two known types of EBV, EBV Type-1 (EBV-1) and EBV Type-2 (EBV-2), both found in eBL patients. To determine the EBV load and type dynamics within the tonsils, saliva, whole blood, and plasma, we enrolled 102 children aged 1-14 undergoing tonsillectomy in a malaria holoendemic region of western Kenya. Additionally, we investigated EBV-2 cell type preference in tonsillar mononuclear cells (TMCs). Saliva, whole blood, and tonsillar tissue were collected at the time of enrollment. Whole blood was centrifuged to separate plasma from red blood cells, and tonsillar mononuclear cells (TMCs) were isolated from tonsils using a ficoll gradient. Using qPCR, we assessed EBV load and type in TMCs, plasma, whole blood, and saliva. Additionally, a subset of samples was subject to cell sorting to determine the infected cell population. We found that EBV loads were significantly higher in TMCs compared to whole blood (P < 0.01) and in saliva compared to plasma (P < 0.01). Children with EBV-2 exhibited higher EBV loads in TMCs (P = 0.016) than those with EBV-1. Both EBV types were detected in T-cells isolated from TMCS and were also observed in different compartments within the same individual. Our findings indicate that EBV-2 infection in tonsils is associated with higher viral loads, consistent with a model where EBV-2 has more lytic replication compared to EBV-1.IMPORTANCEThis study investigates Epstein-Barr virus (EBV) persistence in children residing in a malaria-endemic region of western Kenya. We examined the dynamic interplay of EBV viral load and type distribution across multiple host compartments: tonsils, saliva, whole blood, and plasma. Our key finding reveals significantly higher viral loads in pediatric tonsils infected with EBV-2 compared to EBV-1, providing novel insights into the phenotypic differences between EBV-1 and EBV-2. This suggests a greater propensity for lytic replication in EBV-2. We also observed the presence of both EBV types within the same individual, but in different compartments. Understanding the dynamics of EBV persistence, particularly the association of EBV-2 with increased tonsillar viral loads, is crucial. This knowledge sheds light on the pathophysiology of EBV infections and may indicate more severe or prolonged infections in affected children, ultimately contributing to improved risk assessment and management of EBV-associated diseases.

Epstein-Barr virus (EBV) contributes to ~1.5% of human cancers, including lymphomas, gastric and nasopharyngeal carcinomas. In most of these, nearly 80 viral lytic genes are silenced by incompletely understood epigenetic mechanisms, precluding use of antiviral agents such as ganciclovir to treat the 200,000 EBV-associated cancers/year. To identify host factors critical for EBV latency, we performed a human genome-wide CRISPR-Cas9 screen in Burkitt B-cells. Top hits included the lysine-specific histone demethylase LSD1 and its co-repressors ZNF217 and CoREST. LSD1 removes histone 3 lysine 4 (H3K4) and histone 3 lysine 9 (H3K9) methylation marks to downmodulate chromatin activation. LSD1, ZNF217 or CoREST knockout triggered EBV reactivation, as did a LSD1 small molecule antagonist, whose effects were additive with histone deacetylase inhibition. LSD1 blockade reactivated EBV in Burkitt lymphoma, gastric carcinoma and nasopharyngeal carcinoma models, sensitized cells to ganciclovir cytotoxicity and induced EBV reactivation in murine xenografts. ZNF217 and LSD1 co-occupied the EBV immediate early gene BZLF1 promoter, which drives B-cell lytic cycle, as well as to the oriLyt enhancer regions recently implicated in EBV reactivation. LSD1 depletion increased levels of activating histone 3 lysine 4 (H3K4) methylation but not repressive histone lysine 9 methylation marks at BZLF1 and oriLyt and induced their interaction by long-range DNA looping. An orthogonal CRISPR screen highlighted a key H3K4 methyltransferase KMT2D role in driving EBV reactivation. Our results highlight H3K4 methylation as a major EBV lytic switch regulator and suggest novel therapeutic approaches.

Epstein Barr Virus (EBV) Reactivation After Reduced Intensity Conditioning (RIC) Allogeneic Hematopoietic Stem Cell Transplantation

Chromosomes 2q, 4q and 13q21 Are Preferential Targets of Epstein-Barr Virus (EBV) Integration In Burkitt's Lymphoma Cell Lines

Epstein Barr Virus (EBV) Reactivation After Reduced Intensity Conditioning (RIC) Unrelated Umbilical Cord Blood Transplantation (UCBT)

Prevalence of Epstein-Barr viral sequences and EBV LMP1 oncogene deletions in Burkitt's lymphoma from Pakistan: epidemiological correlations

Natural History of Post-Allogeneic HSCT EBV-Reactivation: A Single Center Retrospective Survey.

The impact of newer approaches to haploidentical transplantation on Epstein-Barr virus (EBV) is largely unknown. We prospectively evaluated the incidence of EBV reactivation and its impact on transplantation outcomes in 71 patients undergoing haploidentical transplantation with posttransplantation cyclophosphamide in combination with CTLA4Ig-based T-costimulation blockade. Eight patients developed EBV reactivation at a median of 96 days with no incidence of lymphoproliferative disorder. There was no impact of EBV reactivation on acute graft-versus-host disease (GVHD), nonrelapse mortality, progression-free, or overall survival. Despite an overall incidence of 19%, there was a significant increase in chronic GVHD following EBV reactivation (62.5% versus 8%; P = 0.01). NKG2A subset of CD56 natural killer cells increased substantially and persisted following EBV reactivation and chronic GVHD, with a reciprocal decrease in NKG2C subset, whereas the reverse was witnessed in those without chronic GVHD (P < 0.01). Increase in NKG2C subset and a decrease in the NKG2A subset were witnessed within 3 months of subsidence of chronic GVHD. Thus, CTLA4Ig-based haploidentical transplantation was associated with a low incidence of EBV reactivation without EBV-lymphoproliferative disorder. However, EBV reactivation was associated with a sustained alteration in NKG2A and NKG2C subsets of CD56 natural killer cells which might have a pathogenic role in chronic GVHD.

Comparative Analysis of Endemic and Sporadic Burkitt Lymphoma By RNA Sequencing

Epstein-Barr virus (EBV) is associated with many human neoplasms, including Burkitt's lymphoma (BL). Endemic BL in central Africa is more often EBV-associated than BL in the United States, where seroconversion for EBV occurs somewhat later than in Africa. Therefore, the EBV association rate in BL may correlate more with the socioeconomic status of the population studied, which influences the age of EBV seroconversion, than with such factors as malaria, which may relate to the overall higher incidence rate in endemic regions. Forty-one patients with BL in Egypt, which differs both climatically and racially from central African countries (i.e., Kenya, Uganda) where BL is endemic, were analyzed. All biopsies were evaluated for EBV-encoded RNAs (EBER1) by RNA in situ hybridization, analyzed for p53 protein expression using the monoclonal antibody D07, and immunophenotyped using a panel of monoclonal antibodies that included L26 (CD20), Leu 22 (CD43), and A6 (CD45RO). Twelve cases were evaluable for EBV subtype by polymerase chain reaction with EBV-specific primers. The median age at diagnosis was 9 years (range, 2-22 years). The biopsy site was extranodal in 29 patients and nodal in 12 patients. All 41 cases were documented as B-cell neoplasms. A hybridization signal for EBER1 RNA was identified in greater than 95% of the neoplastic cells in 30 of 41 cases (73%), whereas no signal was observed in 11 cases (27%). Epstein-Barr virus subtype 1 was found in 10 patients, subtype 2 in two patients. Immunostaining for p53 was observed in greater than 5% of the neoplastic cells in 9 of 37 cases (24%). No significant correlation was observed between EBV positivity and sex, biopsy site, or p53 immunostaining. The prevalence of EBV in BL from Egypt is slightly lower than in BL in endemic regions, but significantly higher than in sporadic BL. Epstein-Barr virus positivity probably reflects the socioeconomic status of the patient population, and age at seroconversion. The prevalence of EBV subtype 1 suggests that immunodeficiency does not play a role in Egyptian Burkitt's lymphoma, in contrast to endemic Burkitt's lymphoma, in which holoendemic malaria is thought to contribute to immunodeficiency, a higher incidence rate, and the observed prevalence of subtype 2.

Burkitt lymphoma (BL) is one of the most common childhood cancers in sub-Saharan Africa and is etiologically linked to malaria. However, evidence for an effect of malaria interventions on BL is limited. To investigate the potential population-level association between large-scale rollout of insecticide-treated bed nets (ITNs) in sub-Saharan Africa in the 2000s and BL incidence. In this systematic review and meta-analysis, a search was conducted in the Embase, Global Health, and Medline databases and in cancer registry publications between January 1, 1990, and February 27, 2023. All epidemiologic studies on BL incidence rates in children and adolescents aged 0 to 15 years in sub-Saharan African countries where malaria is endemic were identified by 2 reviewers blinded to each other's decision. The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses reporting guideline. Data were extracted independently by 2 reviewers, and quality was scored based on 3 predefined criteria: data collection, case ascertainment, and calculation of person-time at risk. Incidence rates of BL during childhood and mean ITN use in the population. Data were analyzed using a random-effects negative binomial regression model. Of 2333 studies meeting selection criteria, 23 comprising 66 data points on BL incidence were included based on 5226 BL cases from locations with large-scale ITN use in 17 countries. Rates of BL were 44% (95% CI, 12%-64%) lower in the period after ITN introduction compared with before. The adjusted pooled incidence rates of BL were 1.36 (95% CI, 0.88-2.10) and 0.76 (95% CI, 0.50-1.16) per 100 000 person-years before and after introduction of ITNs, respectively. After adjusting for potential confounders, a 1-percentage point increase in mean ITN use in the population in the 10 years before BL data collection was associated with a 2% (95% CI, 1%-4%) reduction in BL incidence. In this systematic review and meta-analysis, large-scale rollout of ITNs in the 2000s was associated with a reduction in BL burden among children in sub-Saharan Africa. Although published data may not be representative of all incidence rates across sub-Saharan Africa, this study highlights a potential additional benefit of malaria control programs.

Incidence and Risk Factors of Epstein-Barr Virus Viremia in Patients Undergoing Haploidential Transplantation with Post Translant Cyclophosphamide

Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.