Epstein-Barr Virus In Burkitt's Lymphoma Research Articles

Clinicopathologic Insights and Molecular Oncogenesis: Understanding Epstein-Barr Virus-Induced B-cell Lymphoproliferations.

Epstein-Barr virus (EBV) is a highly prevalent virus among adults worldwide. In an immunocompetent individual, EBV infection generally results in lifelong latency of the virus and no sequelae. However, in the setting of immune dysfunction, EBV can induce the development of autoimmune disorders, hyperplastic proliferations, and cancers, including lymphoma. Here, we explore the pathogenic and oncogenic role of EBV in Burkitt lymphoma, diffuse large B-cell lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorders and lymphoproliferative disorders associated with immune deficiency and dysregulation. In addition to describing general mechanisms of EBV-associated oncogenesis, we also discuss EBV-associated oncogenesis in the context of each disorder, as well as their microscopic, phenotypic, and clinical presentations.

Epstein-Barr Virus in Burkitt Lymphoma in Africa Reveals a Limited Set of Whole Genome and LMP-1 Sequence Patterns: Analysis of Archival Datasets and Field Samples From Uganda, Tanzania, and Kenya.

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.

Study of Epstein-Barr virus expression in Burkitt's lymphoma by polymerase chain reaction and in situ hybridization: A study in Iran.

Background:The association of Epstein-Barr virus (EBV) with Burkitt's lymphoma (BL) is variable in different geographic regions. In developing countries, the association of EBV with BL is regarded to be of an endemic-type in equatorial Africa (> 95%) and sporadic-type in the developed countries (15-30%). The purpose of this study is to assess the frequency of EBV infection in BL, in Iran. The study also aims to compare Ribonucleic acid (RNA) in situ hybridization (RISH), the standard diagnostic method, with the polymerase chain reaction (PCR)-based method for diagnosing BL.Materials and Methods:In this epidemiological study, the paraffinized specimens of 18 cases of BL were selected. Next, the ISH of EBV-encoded RNA (EBER-RISH) and PCR assays that were based on Epstein Barr Nuclear Antigen 2 (EBNA2) amplification were used. The EBV strain was determined by PCR. The data were analyzed using the SPSS10 software and by performing Pearson correlation coefficient formula at a significant level of 0.05.Results:EBV RNA was detected in 50% of the BL specimens. Type 1 and 2 accounted for 70 and 30% of the cases, respectively. Regarding RISH as the standard method for EBV diagnosis, the PCR assays showed a sensitivity and specificity of 100 and 88.9%, respectively.Conclusion:According to the obtained findings, the frequency of EBV in BL was 50% and PCR and RISH showed high concordance and sensitivity in EBV detection. Therefore, PCR can be used as a faster method for EBV detection in high-risk geographical regions.

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

AbstractAbstract 4452The Epstein-Barr virus (EBV) was first described in 1964 as a possible cause of Burkitt's lymphoma (BL), the most common childhood cancer in equatorial Africa. EBV is an oncogenic virus found in about 95% of the endemic BL. In latently infected cells, the EBV DNA can be maintained in episomal form, but integrated EBV could also be present. Data on the exact integration loci of EBV in BL are very rare, however, integration of EBV-DNA into the human genome has been pointed as an important mechanism in malignant cellular transformation. We have investigated the EBV integration loci (EBV-IL) in 20 BL cell lines using fluorescence in situ hybridization (FISH).FISH was performed using, as EBV-DNA probe, a biotinylated 3,000 bp fragment amplified by long distance-PCR from DNA of the EBV-positive BL cell line Raji. FISH on metaphase spreads of the BL cell line Namalwa was performed to validate the quality of the EBV-FISH probe. Negative controls were performed on EBV-negative BL cell lines CA-46, CW698, and Tanoue. Integration of EBV was defined by the presence of symmetrical doublet hybridization signals at the same chromosomal loci in both sister chromatids. A minimum of 15 well-conserved and complete metaphases was evaluated in each case.As expected, Namalwa showed symmetrical doublet hybridization signals of our EBV-FISH probe on chromosome 1p35. EBV-negative cell lines did not show hybridization signals of the probe. We detected 632 EBV-IL in the total of metaphases analyzed in 20 BL cell lines. Integration of EBV was seen in all chromosomal arms except Yp, 8p, 14p, 20q, 21p, and 22p. An analysis of the chromosomal distribution of all EBV-IL revealed a pattern of preferential insertion for EBV-IL on chromosomes 13q (14% of the metaphases), 2q and 4q (12%, respectively), 7q (10,5%), 3q (9%), and 17q (6,5%).Regarding the recurrency of EBV-IL, the BL cell lines were divided in 4 groups. The first group consisted of 8 cell lines with high-recurrent EBV-IL at a specific chromosomal band (75%-100% of the metaphases analyzed). This first group showed specific EBV-IL at a precise chromosomal location on homologous chromatids on 2p23 (Seraphine), 2q21~31 (Switzer), 22q12 (PA-682), 7p22 and 13q21 (AG876), 7q11 and 17q25 (Naliaka), and 7q21 and 13q21 (LY-67). Interestingly, in BL60 and Maku, the unique EBV-IL visualized was localized near the translocation breakpoint junction of a der(19)t(17;19) and a der(13)t(3;13), respectively. The second group contained 5 cell lines with medium-recurrent EBV-IL (27%-50%). In this group, we observed metaphases with 1 to 3 recurrent EBV-IL localized on 2p12 (Rael), 15q11~14 (JI), 15q13~15 (JBL2), 4q21 and 4q32 (BL16), and 3q21~26, 4p12, and 13q21 (Akuba). The third and fourth groups comprised 4 cell lines with low-recurrent EBV-IL (13%-20%) and 3 cell lines with absence of recurrent EBV-IL, respectively. Non-recurrently integrated EBV-DNA signals on additional chromosomes were observed in cell lines of all 4 groups, except in 4 of the high-recurrent group. We further investigated the distribution of the EBV-IL of groups 3 and 4, of those in groups 1 and 2 with a recurrency less than 27% of the metaphases, and of those that were non-recurrently integrated. We observed again that the viral integration targeted preferentially chromosomes 2q (21%), 4q (14%), and 3q (11%).Our results identify for the first time the integration loci of EBV in BL cell lines and that this integration succeed preferentially on chromosomes 2q, 3q, and 4q. Moreover, in 12 cell lines, this integration ocurred non-randomly at a specific chromosomal sites, targeting 13q21 recurrently. Whether the integration of EBV in these loci affects the expression of genes important for the pathogenesis of BL or whether the EBV integration contributes to an enhanced chromosomal instability remains to be elucidated. Disclosures:No relevant conflicts of interest to declare.

Three Restricted Forms of Epstein-Barr Virus Latency Counteracting Apoptosis in c-Myc Expressing Burkitt Lymphoma Cells.

Epstein-Barr virus (EBV) is aetiologically linked with Burkitt Lymphoma (BL) but its contribution to lymphomagenesis, versus that of the chromosomal translocation activating c-myc expression, remains unclear. This is in part because the full virus growth transforming programme that is expressed when EBV infects normal resting B cells, is not expressed in BL. Instead EBV in BL normally exhibits a restricted Latency I form of infection characterised by expression of only one latent antigen EBNA1 from the BamHI Q promoter. Here we describe an endemic BL, Awia, which uniquely is heterogeneous at the single cell level for EBV gene expression. Analysis of single cell clones of Awia-BL revealed cells displaying three forms of restricted EBV latency:i.classical Latency I,ii.'Wp-restricted latency' expressing EBNAs 1, 3A, 3B, 3C and -LP, andiii.a novel 'EBNA2+/LMP1- latency' in which all EBNAs including EBNA2 are expressed without the latent membrane proteins LMPs 1 and 2.Comparison with rare EBV-negative clones from the same tumour showed that each form of infection provides the c-myc-expressing BL cells with a specific degree of protection from apoptosis. Microarray analysis was carried out on the isogenic Awia-BL clones to determine the influence of EBV gene expression on cellular transcription. Interestingly it was found that expression of the pro-apoptotic Bcl2 family protein BIM, which has previously been implicated in c-myc induced apoptosis, was downregulated in the apoptosis resistant BL cells. Our work suggests that EBV acts as an anti-apoptotic rather than a growth-promoting agent in BL by downregulating expression of the cellular BIM protein. In addition the microarray analysis on these isogenic Awia-BL clones showed that the EBNA profile influences the differentiation status of the BL cell on the germinal centre to plasmacytoid differentiation pathway. These findings may reflect viral functions that are important not just for BL pathogenesis but also for EBV's normal strategy of persistence in the B cell system.

Epstein-Barr virus nuclear antigen 2 induces FcRH5 expression through CBF1

AbstractFc-receptor homolog 5 (FcRH5) is a recently identified B-cell membrane protein of unknown function. In Burkitt lymphoma cell lines with chromosome 1q21 abnormalities, FcRH5 expression is deregulated, implicating FcRH5 in lymphomagenesis. Epstein-Barr virus infects and immortalizes B cells, and is implicated in the etiology of several tumors of B-cell origin. Overexpression of genes located on 1q21-25 has been proposed as a surrogate for Epstein-Barr virus in Burkitt lymphoma. We now report that Epstein-Barr virus nuclear antigen 2 (EBNA2) markedly induces the expression of the FcRH5 gene, encoded on chromosome 1q21. Induction occurred in the absence of other viral proteins and did not require de novo protein synthesis. EBNA2 lacks a DNA-binding domain and can target responsive genes through the host DNA binding protein CBF1. We show that induction of FcRH5 by EBNA2 is strictly CBF1 dependent, as it was abolished in CBF1-deficient cells. Accordingly, EBNA2 targeted CBF1 binding sites present in the FcRH5 promoter in vivo, as detected by chromatin immunoprecipitation. These results identify FcRH5 as a novel, direct target of EBNA2 that may contribute to the development of Epstein-Barr virus–associated tumors.

Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo.

Epstein-Barr virus (EBV) is a herpesvirus that establishes a lifelong, persistent infection. It was first discovered in the tumor Burkitt's lymphoma (BL). Despite intensive study, the role of EBV in BL remains enigmatic. One striking feature of the tumor is the unique pattern of viral latent protein expression, which is restricted to EBV-encoded nuclear antigen (EBNA) 1. EBNA1 is required to maintain the viral genome but is not recognized by cytotoxic T cells. Consequently, it was proposed that this expression pattern was used by latently infected B cells in vivo. This would be the site of long-term, persistent infection by the virus and, by implication, the progenitor of BL. We now know that EBV persists in memory B cells in the peripheral blood and that BL is a tumor of memory cells. However, a normal B cell expressing EBNA1 alone has been elusive. Here we show that most infected cells in the blood express no detectable latent mRNA or proteins. The exception is that when infected cells divide they express EBNA1 only. This is the first detection of the BL viral phenotype in a normal, infected B cell in vivo. It suggests that BL may be a tumor of a latently infected memory B cell that is stuck proliferating because it is a tumor and, therefore, constitutively expressing only EBNA1.

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

To investigate the potential relationship of socioeconomic status with the prevalence of Epstein-Barr virus (EBV) and to understand the significance of del-LMP-1 within EBV+ cases of Burkitt's lymphoma (BL), we studied 10 cases of BL, 30 cases of diffuse large cell lymphoma (DLCL) arising in nonimmunocompromised patients, and 30 reactive tonsillar biopsy specimens from Pakistan. Each lymphoma was analyzed for EBV by EBER1 RNA in situ hybridization (EBV-RISH). Cases showing hybridization signal within neoplastic cells and all reactive tonsillar tissues were analyzed for EBV strain type by EBNA-2 polymerase chain reaction (PCR) and for the presence of a del-LMP-1 by PCR. Eight of 10 (80%) of BL were EBV+, each containing EBV strain A and a wild-type LMP-1 gene. In contrast, only 4 of 30 DLCL (13%) cases were EBV positive (three strain A, one strain B), each containing a wild-type LMP-1 gene. Fifteen of 30 tonsillar biopsy specimens contained EBV, all of which were strain A and wild-type for LMPI. The prevalence of EBV in BL from Pakistan is slightly lower than in BL in endemic regions, but significantly higher than in BL in North America. EBV positivity probably reflects the socioeconomic status of the patient population and age at seroconversion. The absence of del-LMP-1 within all EBV+ BL cases is consistent with the view that del-LMP-1 is not involved in the pathogenesis of BL, and the presence of del-LMP-1 in EBV+ cases of BL reported in other studies may likely reflect the prevalence of a viral strain containing the 30-bp deletion within the respective population studied.

The investigation of epstein–barr viral sequences in 41 cases of Burkitt's lymphoma from Egypt. Epidemiologic correlations

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.

Final case reporting from the ugandan prospective study of the relationship between ebv and burktit's lymphoma

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.

Detection of Epstein‐Barr virus DNA in American Burkitt's lymphoma

Twenty American patients with Burkitt's lymphoma (BL) were studied serologically and biochemically to detect an association with Epstein-Barr virus (EBV). A wide range of antibody reactivities was encountered. Five patients had serological profiles similar to those of African patients with BL: one and possibly two of these patients harbored EBV-DNA in their tumors. Fifteen patients had low or absent antibody to EBV: tumors from 10 of these patients lacked evidence of EBV-DNA. An unexplained association between elevated antibody (greater than or equal to 1:80) to EBV virus capsid antigen (VCA) and a favorable prognosis was noted in this series; this prognostic observation requires further substantiation. Thus, it appears that EBV-associated BL is inversely proportional to tumor endemicity. An etiological role for EBV in BL remains speculative.