Epstein barr virus IgG and EBER-1 in Burkitt´s lymphoma children at a referral hospital in western Kenya

Epstein barr virus IgG and EBER-1 in Burkitt’s lymphoma children at a referral hospital in western Kenya

Isaac Ndede^1,2,&, Simeon Kipkoech Mining¹, Kirtika Patel¹, Fredrick Muyoma Wanjala², Constance Nalianya Tenge³

¹Department of Immunology, Moi University School of Medicine, Eldoret, Kenya, ²Department of Biological Sciences, University of Eldoret, Eldoret, Kenya, ³Department of Child Health and Paediatrics, Moi University School of Medicine, Eldoret, Kenya

^&Corresponding author
Isaac Ndede, Department of Immunology, Moi University School of Medicine, Eldoret, Kenya

Introduction    

Burkitt's lymphoma (BL) is a tumour of the lymphoid tissues of three different variants; endemic (eBL), sporadic (sBL) and immunodeficiency related. The endemic type is common in equatorial Africa. Burkitt's lymphoma has a non-random clustering and overlap with ecological zone of human pathogens such as Plasmodium falciparum, EBV, Kaposi's sarcoma herpes virus (KSHV), human herpes virus 8 (HHV-8) and human immunodeficiency virus (HIV) in this region [1,2]. Endemic BL commonly involve certain anatomic sites; the jaws tend to predominate in younger children while abdominal locations occur more frequently with increasing age. Sporadic BL occurs worldwide with no geographic or climate association and no apparent infection in its aetiology. This variant usually presents as intra-abdominal swelling and rarely involves the jaw in young adults [3]. Epstein Barr virus is known to preferentially infect resting and memory B lymphocytes. During infection, virus binding is mediated by envelope glycoproteins gp 350 and gp 42 to complement receptors 2 (CR2) and HLA class II proteins on target cells respectively. Majority of EBV infected B cells are recognized and eliminated by host T cell-mediated immune responses, but a fraction of the virus usually remain and enter latency I phase within the germinal centre where they express only three viral genes [4], characterized by expressions of small non coding EBV RNAs (EBERs) and EBNA1.

Latency II is characterized by expression of EBNA-1, latent membrane protein-1 (LMP-1), LMP-2, EBERs and is associated with Hodgkin's lymphomas. While latency III viral products include all EBNAs, EBERs, LMPs, non-translated RNA's-microRNA (miRNA) and is frequently observed in post-transplant lymphoproliferative disorders [5]. Some EBV genome products exhibit homology to a wide variety of cell molecules, such as cytokines and signal transducers important in promoting humans infectivity. Epstein Barr virus proteins such as LMP-1 and LMP-2 have growth transforming ability and may allow unchecked cellular proliferation lymphoma pathogenesis. Another product BHRF-l, a homologue of BCL-2 family, is a known apoptosis suppressor. Growth transformation and uncontrolled proliferation are thought to facilitate oncogenic translocation during pre-B-cell development and immunoglobulin rearrangement [6]. Immune stimulation in response to persistent infections such as EBV in a population probably increases the risk of oncogenic mutations and neoplastic transformation. In addition, climatic and ethnogeographic factors such as, exposure to ultraviolet light, chemical carcinogens, genetic factors and cultural practices among populations have been reported to influence temporal and/or spatial clustering of lymphoma including BL [7]. Our study sought to determine EBV IgG in serum, EBER-1 and MYC in tumours sections in children and adolescent diagnosed with BL at Moi Teaching and Referral Hospital (MTRH) in Eldoret, Kenya.

Methods     

A cross sectional study of children and adolescents ≤18 years old with clinical and histology diagnosis of non-Hodgkin's lymphomas, were targeted at MTRH in Eldoret, Kenya, before onset of treatment. Blood and biopsy samples were taken from each patient whose parent or legal guardian provided written informed consent, in accordance with Institutional Research Ethics Committee (IREC). Participants' files and questionnaires were used to obtain clinical and demographic information. To determine EBV IgG, patient sera and controls were incubated in microtitre strip wells coated with EBV-capsid antigen using Enyzme-linked immunosorbent assay (ELISA) kit (Human Gesellschaft, Germany), the plates were then washed and absorbance measured at 450nm within 30 minutes of terminating the reaction in an ELISA microplate reader. Patient's value equal or greater than cut-of-values ± 15% were considered EBV-IgG-antibody positive. Three (3) μm tissue sections from immunohistochemistry confirmed BL tumours (results not shown), were deparaffinised in xylene and rehydrated in a graded series of ethanol before being subjected to epitope retrieval prior to staining with EBER-1 and MYC (DAKO^®) primary antibodies, interposed with washing steps using tris-buffered saline (TBS) at pH 7.6. Endogenous peroxidise was neutralised by peroxidise followed by protein block to reduce non-specific binding of EBER-1 antibody. Followed by 3, 3'-diaminobenzidine tretrahydrochloride dehydrate (DAB) to visualize antibody binding after incubation at room temperature in a Leica Bond III® stainer. Staining relativities were evaluated in at least 10 high-power microscopic fields of the slides. Data were analysed using SAS version 9.1 (SAS Institute, Cary, NC). Unconditional logistic regression was used to estimate odds ratios (ORs), adjusted for child's age and sex.

Ethical approval and consent to participate: institutional Research and Ethics Committee (IREC) of Moi University and Moi Teaching and Referral Hospital approved the study. Written informed consent was obtained from parent or legal guardian of each participant.

Results     

Ninety three (93) percent participants and 86.7% of BL cases were positive for EBV IgG antibodies in serum, and EBER-1 positive respectively, while 13.33% did not carried latent EBV encoded small RNA 1 (EBER-1) (Figure 1). All (100%) jaw BL tumours were positive for EBER-1 and 90.1% expressed MYC protein (Table 1). Bivariate associations using a log-linear model, adjusted for age and gender, showed that BL occurrence was 1.39 times greater in participants who tested positive for EBER-1 than those who tested negative, adjusted odds ratio (AOR) 1.39, 95% CI: 0.16-12.19) regardless of age and gender.

Discussion     

Nearly every participant in the study setting had been exposure to EBV as measured by EBV IgG. The association of EBER-1 and BL tumours in this current study suggest a role of EBV in BL pathogenesis in support of some past studies [8,9]. Sustained immune stimulation in response to persistent infection such as EBV is thought to trigger oncogenic mutations during VDJH and VJL immunoglobulin genes recombination phase during B cell activation in response to a pathogen [10]. This step probably involves Ig/c-myc gene translocation as indicated by many MYC+ protein positive BL tumours in this study. The fact that not every participant was positive for EBV IgG, EBER-1 and MYC negative developed BL may be interpreted to mean that persistence of EBV is usually benign and appears to cause disease when the host-virus balance is upset [11].

Conclusion     

BL is associated with EBV in the study setting. The existence of BL tumours negative for EBER-1 and MYC Burkitt's lymphoma may be suggestive of alternative pathogenesis mechanisms or variant(s) and needs further studies.

Competing interests     

The authors declare no competing interests.

Authors’ contributions     

IN, SKM and KP conceptualized and designed the study, carried out analysis and interpretation of data, drafted the initial manuscript, reviewed, revised the draft manuscript and approved the final manuscript. FMW and CT carried out data collection, reviewed, revised the draft manuscript, and approved the final manuscript.

Acknowledgments     

We are grateful to staff at the Department of Human Anatomy and Pathology University of Siena Italy for confirming BL cases and the families of study patients for their participation in this study.

Table and figure     

Table 1: tumour site and EBER-1/MYC status in BL cases

Figure 1: EBER-1in BL tumour and EBV IgG in participants’ sera

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