Burkitt lymphoma and the discovery of Epstein-Barr virus

The chance germinal encounter with the first lecture outside Africa on Burkitt lymphoma is described together with the hypothesis of a viral cause. Repeated virological investigations on lymphoma biopsies proved negative, leading to the idea that a latent virus might be activated if lymphoma cells could be cultured, although no human lymphoid cell had at that time ever been maintained in vitro. A chance event reminding of the need for suspension culture with mouse lymphomas led to success. The cultured cells carried a morphologically unequivocal, strangely inert, herpesvirus shown later to be immunologically, biologically and biochemically unique. How this new agent acquired its name, Epstein-Barr virus, is explained.     

Establishment and characterization of human B-cell lymphoma cell lines using B-cell growth factor

B-cell non-Hodgkin's lymphomas (NHL-B) have been difficult to establish in long-term cell culture using standard techniques. We report the establishment of five representative cell lines from high grade NHL-B using B-cell growth factor (BCGF). The five NHL-B cell lines display the morphologic, immunophenotypic, genotypic, and biologic characteristics of the lymphoma cells present in the original diagnostic specimen. The cell lines showed at least a sevenfold dose-dependent increase in proliferation in vitro over background in the presence of BCGF. Other putative B-cell growth-stimulating cytokines showed no significant proliferative activity or were inhibitory in some cases. NHL-B cell lines secreted growth factor(s) into culture supernatants that mediated at least a fivefold dose-dependent increase in cell proliferation in autochthonous lymphoma cells and a 10-fold or greater stimulation in growth factor-dependent normal B cell lines in vitro. The cell lines show monoclonal rearrangements of IgH genes and nonrandom chromosomal abnormalities characteristic of NHL-B, while the expression of Epstein-Barr virus associated antigen (EBNA-I) is present in two of the five cell lines. The studies show that lineage-specific growth factors may be used to establish neoplastic B cell lines in vitro, which are important experimental systems for cellular and molecular studies in the NHL-B.     

Establishment and characterization of a new Epstein-Barr virus transformed cell line from a human B cell lymphoma

Summary We have established a new cell line from a patient with centrocytic B cell lymphoma. Highly purified peripheral blood B cells from patient DUL (WBC counts 158,000/µl) were infected in vitro with Epstein-Barr virus (EBV), and CD 20⁺ B cells were cloned into 96 well culture plates with the aid of a cell sorter autoclone device. As shown by GTG-banding and Southern blot analysis, outgrowing EBV-positive clones had the same chromosomal abnormalities and identical monoclonal IgH gene rearrangement as the original EBV-genome-negative leukemic B cell clone. Surface marker analysis with a panel of monoclonal antibodies revealed identical patterns on EBV-negative and -positive clones, with the exception of PCA 1 (reactive with plasma cells) which was negative on freshly explanted leukemic B cells but positive on EBV-converted clones.List of abbreviations E sheep erythrocytes - EBV Epstein-Barr virus - FCC-NHL follicular centrocytic non-Hodgkin's Lymphoma - FCS fetal calf serum - LCL lymphoblastoid cell line(s) - mab monoclonal antibody - MNC mononuclear cells - NHL non-Hodgkin's lymphoma This work was supported by the Deutsche Forschungsgemeinschaft (SFB 322/B 2)This work forms part of the Ph. D. thesis of O. Janssen     

T cell leukemia I oncogene expression depends on the presence of Epstein-Barr virus in the virus-carrying Burkitt lymphoma lines

We used a modified subtractive suppression hybridization to identify cellular genes that show altered expression in Burkitt lymphomas (BLs) in the presence of Epstein-Barr virus (EBV). Comparison of the gene expression patterns of an EBV-negative clone of the originally EBV-positive BL line Akata, with its Neo(R)-EBV derivative, revealed a significant difference in the expression of the T cell leukemia 1 oncogene (TCL-1). Subsequent expression studies showed that the original EBV-positive Akata line and the EBV-reconstituted derivative expressed high levels of TCL-1, whereas the EBV-negative variant showed only a low level of expression. Two other independently established EBV-positive BLs (Mutu and OMA) that have also thrown off EBV showed a similar decrease in TCL-1 expression after virus loss. Reinfection with Neo(R)-EBV restored the TCL-1 expression levels in the EBV loss variants to as high a level as the originally EBV-positive lines. High-resolution immunostaining showed that TCL-1 was localized in both the cytoplasm and the nucleus. Our findings suggest that high expression of TCL-1 is necessary for the development of the BL phenotype. In view of the fact that germinal center B cells, regarded as the progenitors of BL, do not express TCL-1, we suggest that constitutive expression of this oncogene occurs by genetic or epigenetic changes in the EBV-negative BLs. In the originally EBV-positive BLs, the ability of the virus to switch on TCL-1 expression would obviate this need.     

Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines.

Two related but differentially expressed potential membrane proteins of Epstein-Barr virus are encoded by the same reading frame in the EcoRI D het region of the viral genome. Potential antigenic sites in the amino acid sequence of these proteins were selected by computer-aided prediction of the secondary structure and two oligopeptides corresponding to regions located in different parts of the proteins were synthesized chemically. Rabbit antisera to these peptides were used for immunoprecipitation of the native viral proteins from Epstein-Barr virus-positive cell lines from various sources. Both predicted membrane proteins could be precipitated from cell lines that had been transformed in vitro with EBV or from cell lines derived from infectious mononucleosis patients. In cell lines established from Burkitt lymphoma, only the smaller polypeptide, which lacks 138 amino acids from the amino terminus, could be identified. Using the synthetic peptides as antigens in ELISA, we detected elevated antibody titers in sera from patients with infectious mononucleosis and nasopharyngeal carcinoma.     

Surface marker characteristics and Epstein-Barr virus studies of two established North American Burkitt's lymphoma cell lines.

Tumor cell lines have been established in continuous culture from two North American Burkitt's lymphomas. The SU-AmB-1 line, derived from a patient with low serum antibody titers to Epstein-Barr virus (EBV), was devoid of EBV genomes by the reaction for EBV-associated nuclear antigen (EBNA), could not be induced to express EBV antigens, and was highly refractory to EBV superinfection. Conversely, the SU-AmB-2 cell line, derived from a patient with "African type" serology, yielded a positive EBNA reaction and was readily inducible and superinfectable. Although both cell lines possessed B (bone-marrow-derived) cell characteristics, they had different surface marker patterns. It is postulated that two different classes of undifferentiated B cell lymphomas exist, one of which is positive for the presence of EBV genomes and occurs endemically in Africa and New Guinea and sporadically in other parts of the world, the other of which is EBV-negative and occurs sporadically throughout the world, including the endemic areas.     

Azidothymidine inhibits NF-kappaB and induces Epstein-Barr virus gene expression in Burkitt lymphoma

The antiviral compound azidothymidine (AZT), alone or in combination with other agents, induces apoptosis in early-passage, Epstein-Barr virus-positive Burkitt lymphoma (EBV+ BL) lines and has clinical activity in EBV+ BL. We report here a mechanism of AZT's antitumor activity. The nuclei of these cells contain activated nuclear factor-kappaB (NF-kappaB) subunits p50, c-Rel, RelB, and p52, but not p65. Treatment of primary EBV+ BL lines with AZT inhibited NF-kappaB within 1 to 2 hours. This was followed by up-regulation of EBV gene expression including viral thymidine kinase (vTK) and apoptosis. Subclones of EBV+ BL cells that demonstrated activated p65 were resistant to AZT. In EBV+ BLs, AZT but not ganciclovir (GCV) was highly phosphorylated to its monophosphate form (AZT-MP). Phosphorylation, as well as apoptosis, was markedly enhanced in the presence of hydroxyurea. AZT inhibits NF-kappaB and up-regulates EBV gene expression in primary EBV+ BLs. AZT with hydroxyurea may represent an inexpensive, targeted regimen for endemic BL.     

Diagnostic challenges in a case of B cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma

Unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) is a recently recognized category of mature B-cell lymphoma. This represents a heterogeneous group of diseases which often pose diagnostic problems in clinical practice, yet its distinction from DLBCL to BL is important for its therapeutic and prognostic implications. We report the challenging diagnostic process of a 60-year-old man. He had a CD10 and BCL2-positive, BCL6-negative B-cell malignancy with loss of multiple B-cell markers including surface immunoglobulin. The karyotype was complex and unusual, including t(2;18)(p12;q21), t(8;14)(q24;q32) and a derivative chromosome 22 mimicking a Philadelphia chromosome that led to initial diagnostic confusion. A triple-hit gray zone B-cell lymphoma with rearrangements of MYC, BCL2, BCL6, both alleles of IGH and likely IGK and IGL was finally diagnosed upon additional fluorescence in situ hybridization (FISH) studies. His disease was nonresponsive to intensive combination chemotherapy and he died 4 months after presentation. This case illustrates the diagnostic difficulty encountered in such group of B-cell lymphomas and emphasizes the need to integrate morphological, immunophenotypic and genetic data in making a diagnosis.     

Unusual skin reactions after mosquito bites and Epstein-Barr virus reactivation in a patient with mantle cell lymphoma

We detected Epstein-Barr virus (EBV) reactivation in a patient with mantle cell lymphoma (MCL). The patient, a 53-year-old Japanese man, had been referred to our hospital because of generalized lymphadenopathy, hepatosplenomegaly and lymphocytosis and gave a history of intense skin reactions to mosquito bites. The biopsied lymph node contained a monotonous proliferation of medium-sized lymphocytes with scant cytoplasm and slightly irregular nuclei that were CD5+, CD20+ and CD23-. Antibody titers of IgG against EBV viral capsid antigen and early antigen were increased, and EBV was detected in the lymphoma cells. This case may suggest a relationship between EBV and MCL.     

Recurrent B-cell non-Hodgkin's lymphoma in two brothers with X-linked lymphoproliferative disease without evidence for Epstein-Barr virus infection

We present two male siblings suffering from recurrent manifestations of B-cell non-Hodgkin's lymphoma (NHL) and recurrent infections of the lower respiratory tract associated with bronchiectasis. Immunodeficiency could not be demonstrated by any laboratory investigation. In both patients, lymphomas developed without evidence for Epstein-Barr virus (EBV) infection, i.e. no antibody response to EBV-specific antigens, negative EBV-PCR (polymerase chain reaction) in peripheral blood cells, and absence of latent membrane protein (LMP) and EBV-encoded RNA (EBER) in lymphoma cells. Molecular analysis of the SH2D1A, the gene for X-linked lymphoproliferative disease (XLP) led to the identification of a deletion in the first exon in both patients. Therefore, we postulate that the genetic defect and the following dysregulation of the B-/T-cell interaction rendered these patients susceptible to the early onset of B-cell NHL and that EBV infection is not an obligate prerequisite.     

Pathogenesis of B cell lymphoma in a patient with AIDS

Lymphoma occurs at increased frequency in patients with the acquired immunodeficiency syndrome (AIDS). We studied, using serologic and molecular techniques, one such lymphoma for (a) evidence of infection with human T lymphotropic virus, type III (HTLV-III), and Epstein-Barr virus (EBV), (b) monoclonal rearrangement of immunoglobulin and T cell receptor genes, and (c) rearrangement of the c-myc oncogene. Immunoglobulin and T cell receptor gene studies demonstrated that the tumor was of monoclonal B cell origin. Similar to cases of Burkitt's lymphoma unrelated to AIDS, there were DNA sequences in the lymphoma that hybridized to EBV-specific probes and demonstrated evidence of c- myc rearrangement. HTLV-III sequences were not detected in the malignant B cells. The pathogenesis of some B cell neoplasms in patients with the syndrome may involve transformation by EBV and deregulation of oncogene expression without direct infection of the malignant B cells by HTLV-III.     

Hyperphosphatemia and hypocalcemia accompanying rapid cell lysis in a patient with Burkitt's lymphoma and Burkitt cell leukemia

Hyperphosphatemia, hypocalcemia and acute oliguric renal failure resulting from uric acid nephropathy developed in a patient with Burkitt's lymphoma and Burkitt cell leukemia after effective chemotherapy. A review of other reported cases in which the patients had similar metabolic abnormalities is presented, and the pathophysiology is discussed. The clinical setting in which these metabolic developments are most likely to occur is defined, and an approach for their prevention and management is presented.