Epstein-Barr virus infection and its gene expression in gastric lymphoma of mucosa-associated lymphoid tissue

The role of Epstein-Barr virus (EBV) in the pathogenesis of gastric lymphoma of mucosa-associated lymphoid tissue (MALT) has not been well understood. The aim of the study was to investigate EBV infection and its gene expression in this tumor in order to understand its role in the pathogenesis. EBV infection was screened by in situ hybridization for EBV-encoded nonpolyadenylated RNA (EBER ISH) in 79 cases of gastric MALT lymphoma of nonimmunocompromised patients. The expression of EBV proteins [LMP1 (latent membrane protein 1), EBNA2 (EBV nuclear antigen 2), ZEBRA (switch protein encoded by BZLF1 gene)] was studied by immunohistochemistry in EBER-positive cases. EBV was detected with EBER ISH in 15 (19%) of the 79 cases. EBV was found in virtually all tumor cells in 2 cases of high-grade MALT lymphoma (2.5%) (EBV-associated), and was found only in occasional large or small lymphoid cells in 13 cases (16.5%). False positive EBER signal was detected in the mucinous glandular epithelial cells of gastric antrum with FITC-labeled oligonucleotide probe but not with digoxigenin or ³⁵S-labeled riboprobes. Type II latency (EBER+LMP1+ EBNA2-) was detected in both EBV-associated cases. Type III latency (EBER+LMP1+EBNA2+) was also identified in one EBV-associated case besides latency II. Double labeling showed coexpression of LMP1 and EBNA2 in a small number of tumor cells, indicating the presence of type III latency in single cell level. In cases with only occasional EBER-positive large or small lymphoid cells, LMP1 and EBNA2 were not detected. ZEBRA was negative in all the cases. These findings suggest that EBV may contribute to the pathogenesis of a small proportion of high-grade MALT lymphoma, where virtually all tumor cells harbored EBV and the oncogenic viral protein LMP1 was expressed. Moreover, latency III of EBV infection may exist in nonimmunocompromised patient. J. Med. Virol. 56:342–350, 1998 . © 1998 Wiley-Liss, Inc.     

Presence of Epstein-Barr virus latency type III at the single cell level in post-transplantation lymphoproliferative disorders and AIDS related lymphomas.

AIMS: To investigate the expression pattern of Epstein-Barr virus (EBV) latent genes at the single cell level in post-transplantation lymphoproliferative disorders and acquired immunodefiency syndrome (AIDS) related lymphomas, in relation to cellular morphology. METHODS: Nine post-transplantation lymphoproliferative disorders and three AIDS related lymphomas were subjected to immunohistochemistry using monoclonal antibodies specific for EBV nuclear antigen 1 (EBNA1) (2H4), EBNA2 (PE2 and the new rat anti-EBNA2 monoclonal antibodies 1E6, R3, and 3E9), and LMP1 (CS1-4 and S12). Double staining was performed combining R3 or 3E9 with S12. RESULTS: R3 and 3E9 anti-EBNA2 monoclonal antibodies were more sensitive than PE2, enabling the detection of more EBNA2 positive lymphoma cells. Both in post-transplantation lymphoproliferative disorders and AIDS related lymphomas, different expression patterns were detected at the single cell level. Smaller neoplastic cells were positive for EBNA2 but negative for LMP1. Larger and more blastic neoplastic cells, sometimes resembling Reed-Sternberg cells, were LMP1 positive but EBNA2 negative (EBV latency type II). Morphologically intermediate neoplastic cells coexpressing EBNA2 and LMP1 (EBV latency type III), were detected using R3 and 3E9, and formed a considerable part of the neoplastic population in four of nine post-transplantation lymphoproliferative disorders and two of three AIDS related lymphomas. All samples contained a subpopulation of small tumour cells positive exclusively for Epstein-Barr early RNA and EBNA1. The relation between cellular morphology and EBV expression patterns in this study was less pronounced in AIDS related lymphomas than in post-transplantation lymphoproliferative disorders, because the AIDS related lymphomas were less polymorphic than the post-transplantation lymphoproliferative disorders. CONCLUSIONS: In post-transplantation lymphoproliferative disorders and AIDS related lymphomas, EBV latency type III can be detected by immunohistochemistry in a subpopulation of tumour cells using sensitive monoclonal antibodies R3 and 3E9. Our data suggest that EBV infected tumour cells in these lymphomas undergo gradual changes in the expression of EBV latent genes, and that these changes are associated with changes in cellular morphology.     

EPSTEIN–BARR VIRUS (EBV) INFECTION IN INFECTIOUS MONONUCLEOSIS: VIRUS LATENCY,REPLICATION AND PHENOTYPE OF EBV-INFECTED CELLS

Primary Epstein–Barr virus (EBV) infection may manifest itself as a benign lymphoproliferative disorder, infectious mononucleosis (IM). EBV infection has been characterized in lymphoreticular tissues from nine patients with IM using the abundantly expressed EBV-encoded nuclear RNAs (EBERs) as a marker of latent infection. Expression of the virus-encoded nuclear antigen (EBNA) 2 and of the latent membrane protein (LMP) 1 was seen in variable proportions of cells in all cases. Double labelling revealed heterogeneous expression patterns of these proteins. Thus, in addition to cells revealing phenotypes consistent with latencies I (EBNA2⁻/LMP1⁻) and III (EBNA2⁺/LMP1⁺), cells displaying a latency II pattern (EBNA2⁻/LMP1⁺) were observed. Cells expressing EBNA2 but not LMP1 were also detected; whilst this may represent a transitory phenomenon, the exact significance of this observation is at present uncertain. EBER-specific in situ hybridization in conjunction with immunohistochemistry revealed expression of the EBERs mainly in B-lymphocytes, many of which showed features of plasma cell differentiation. By contrast, convincing evidence of latent EBV infection was not found in T-cells, epithelial or endothelial cells. Double-labelling immunohistochemistry revealed expression of the replication-associated BZLF1 protein in small lymphoid cells, often showing plasmacytoid differentiation. There was no unambiguous expression of this protein in other cell types. These results suggest that B-cells are the primary target of EBV infection and that plasma cells may be a source of infectious virus found in the saliva of IM patients. © 1997 John Wiley & Sons, Ltd.     

Humoral immune responses to Epstein-Barr virus encoded tumor associated proteins and their putative extracellular domains in nasopharyngeal carcinoma patients and regional controls

Epstein-Barr virus (EBV) latency proteins EBNA1, LMP1, LMP2, and BARF1 are expressed in tumor cells of nasopharyngeal carcinoma (NPC). IgG and IgA antibody responses to these non-self tumor antigens were analyzed in NPC patients (n=125) and regional controls (n=100) by three approaches, focusing on the putative LMP1, LMP2 extracellular domains. Despite abundant IgG and IgA antibody responses to lytic antigens and EBNA1, patients had low titer (1:25-1:100) IgG to LMP1 (81.2%), LMP2 (95.6%), and BARF1 (84.8%), while immunoblot showed such reactivity in 24.2%, 12.5%, and 12.5% at 1:50 dilution, respectively. Few IgA responses were detected, except for EBNA1. Controls only showed IgG to EBNA1. ELISA using peptides from different domains of LMP1, LMP2, and BARF1 also yielded mostly negative results. When existing, low level IgG to intracellular C-terminus of LMP1 (62.9%) prevailed. Rabbit immunization with peptides representing extracellular (loop) domains yielded loop-specific antibodies serving as positive control. Importantly, these rabbit antibodies stained specifically extracellular domains of LMP1 and LMP2 on viable cells and mediated complement-driven cytolysis. Rabbit anti-LMP1 loop-1 and -3 killed 50.4% and 59.4% of X50/7 and 35.0% and 35.9% of RAJI cells, respectively, and 22% of both lines were lysed by anti-LMP2 loop-2 or -5 antibodies. This demonstrates that (extracellular domains of) EBV-encoded tumor antigens are marginally immunogenic for humoral immune responses. However, peptide-specific immunization may generate such antibodies, which can mediate cell killing via complement activation. This opens options for peptide-based tumor vaccination in patients carrying EBV latency type II tumors such as NPC.     

Hodgkin's disease and peripheral T-cell lymphoma: composite lymphoma with evidence of Epstein-Barr virus infection

This paper reports the case of a patient with a composite lymphoma consisting of nodular sclerosing Hodgkin's disease and peripheral T-cell lymphoma. The Hodgkin and Reed-Sternberg (HRS) cells harboured the Epstein-Barr virus (EBV) and displayed a type II EBV latency (LMP1(+)/EBNA2(-)), whereas the neoplastic T-cells were EBV-negative. Four years later, the patient presented with a relapse of the peripheral T-cell lymphoma. In situ hybridization revealed numerous EBV-carrying lymphocytes, which were shown to be polyclonal B-cells with a latency III pattern of EBV gene expression (LMP1(+)/EBNA2(+)). This observation suggests that impairment of EBV-specific immunity in the micro-environment of T-cell lymphomas may facilitate the outgrowth of EBV-carrying B-lymphocytes and emphasizes the importance of determining the phenotype of EBV-infected cells, particularly when studying T-cell lymphomas. The results further suggest that the HRS cells and neoplastic T-cells were of different clonal origins. The detection of EBV-carrying cell populations admixed with the neoplastic T-cells at primary presentation and at relapse raises the possibility that the growth of the T-cell lymphoma was dependent on the presence of such cells.     

Distinct Patterns of Mitogen-Activated Protein Kinase Phosphorylation and Epstein–Barr Virus Gene Expression in Burkitt's Lymphoma Cell Lines versus B Lymphoblastoid Cell Lines

In order to understand mechanistic relationships between signaling pathways regulating mitogen-activated protein kinase (MAPK) phosphorylation and Epstein–Barr virus (EBV) reactivation, we compared MAPK phosphorylation, and EBV reactivation and latency in Burkitt's lymphoma cell lines (BLCLs) versus B lymphoblastoid cell lines (LCLs). EBV was reactivated in the BLCLs Akata and Raji, and in a LCL OB-R33 cells after cross-linking surface immunoglobulin (sIg) with anti-Ig. After stimulation with anti-Ig, MAPK phosphorylation was strongly induced in all BLCLs and in a few LCLs, but not in other LCLs. MAPK was constitutively phosphorylated in most LCLs but not in BLCLs. Expression of EBNA2 and LMP1, and LMP2A was analyzed with both immunoblotting and RT-PCR. EBNA2 and LMP1 were expressed in most LCLs and in some BLCLs. LMP2A was expressed in all BLCLs and LCLs except Namalwa cells. To test the hypothesis that LMP1 induces constitutive MAPK phosphorylation, the LMP1 expression vector was transfected into Akata cells. MAPK phosphorylation was not induced in such transfected cells. Our results indicate that BLCLs and LCLs respectively have distinct MAPK phosphorylation patterns, and that induction of MAPK phosphorylation correlates with EBV reactivation in a few cell lines but not in most of the tested cell lines.     

Correlation of the expression of Epstein-Barr virus latent membrane protein and in situ hybridization with biotinylated BamHI-W probes in Hodgkin''s disease.

The detection of Epstein-Barr virus (EBV) nucleic acids by in situ hybridization (ISH) with biotinylated BamHI-W probes was correlated with the expressions of EBV latent membrane protein (LMP) and EB nuclear antigen 2 (EBNA2), in 107 cases of Hodgkin's disease (HD) of different immunomorphologic subtypes. Epstein-Barr virus nucleic acids were present and restricted to the pathogenic cells in 4 of 40 (10%) cases of nodular sclerosis (NS) and 33 of 55 (60%) cases of mixed cellularity (MC), but were undetectable in other subtypes. Of the 37 cases positive for EBV nucleic acids, 35 (95%) showed the expression of LMP. Epstein-Barr virus nucleic acids and LMP were restricted to Reed-Sternberg cells and variants. Only 1 case (MC) showed LMP expression in the absence of EBV detection. The correlation was strengthened by the finding of LMP expression at first diagnosis in 6/7 EBV positive cases at relapse (14-126 months) (5/5 EBV negative cases at relapse were LMP negative at first diagnosis). EBNA2 was absent in all 13 (NS, 2; MC, 11) EBV+ and LMP+ cases tested. Both LMP and EBNA2 were expressed in control EBV-positive tissues and cell lines. EBV serology in MC HD was indicative of latent EBV infection, but neither serology nor clinical parameters correlated with the presence or the absence of EBV, over a short-term follow-up (median, 20 months). The findings, although not proving EBV as the etiologic agent of HD, suggest that: 1) LMP expression alone may be adequate for identifying EBV-associated HD, 2) the MC subtype has a stronger relation with EBV presence, and 3) the regulation of EBV genes in HD is different from other EBV-associated disorders. The clinical implications still remain to be discovered.     

CD21非依赖性EB病毒对人胃印戒细胞癌细胞系的感染

目的　探讨CD2 1非依赖性EB病毒 (EBV)对人胃印戒细胞癌细胞系 (HSC 39)的感染作用。方法　用Akata和P3HR 1EBV毒株感染HSC 39,有限稀释法对感染细胞进行克隆。结果　两种EBV毒株感染细胞中均可检测到EBV编码的小RNA(EBER)的表达 ,两种EBV毒株感染的亲代细胞及大多数细胞克隆表达EBV核抗原 (EBNA1) ,但不表达EBNA2、潜伏期膜蛋白 (LMP1)和LMP2A。表现为潜伏Ⅰ型感染。未感染的HSC 39细胞及P3HR 1感染的细胞克隆CD2 1表达阴性 ,而AkataEBV感染的部分细胞克隆CD2 1mRNA阳性。结论　EBV可能通过不依赖CD2 1受体的途径感染HSC 39,印戒细胞癌细胞系可用作EBV感染的靶细胞。     

Long-term carriers generate Epstein-Barr virus (EBV)-specific CD4(+) and CD8(+) polyfunctional T-cell responses which show immunodominance hierarchies of EBV proteins

T cells simultaneously producing multiple cytokines and possessing cytotoxic capacity termed polyfunctional cells (PFCs) are increasingly recognized as the immune correlate of protection against pathogenic viruses. We investigated co‐expression of four cytokines (interferon‐γ, macrophage inflammatory protein 1‐α, tumour necrosis factor‐α and interleukin‐2) and degranulation capacity (CD107a surface expression) of Epstein–Barr virus (EBV) ‐specific CD4⁺ and CD8⁺ T cells upon stimulation by overlapping peptides of EBV lytic (BZLF1) and latent (EBNA1, EBNA3 and LMP2) proteins, in 20 healthy Chinese long‐term carriers. Two patients with post‐transplant lymphoproliferative disorder (PTLD), who had impaired T‐cell immunity, were studied for comparison. Both EBV‐specific CD4⁺ and CD8⁺ PFCs were readily generated in long‐term carriers and showed immunodominance hierarchies of latent proteins (EBNA1 > EBNA3/LMP2 and EBNA3 > LMP2 > EBNA1 for CD4⁺ and CD8⁺ T cells, respectively), as evidenced by a higher proportion of PFCs generated by immunodominant EBV proteins than by subdominant viral proteins. In contrast, the proportion of EBV‐specific PFCs was markedly decreased in patients with PTLD. The EBV‐specific PFCs produced more cytokine per cell than single‐functional T cells and comprised different subsets. Five‐functional CD4⁺ and CD8⁺ T cells were detected and four‐functional CD4⁺ T cells were mainly CD107a negative and expressed all four cytokines whereas four‐functional CD8⁺ T cells were mainly CD107a positive and expressed three of the four cytokines (interleukin‐2‐negative). We conclude that EBV‐specific PFCs are generated in much higher proportions in the long‐term carriers than in the patients with PTLD and maintain the immunodominant characteristics of the virus.     

Epstein-Barr virus infection in colorectal neoplasms associated with inflammatory bowel disease: detection of the virus in lymphomas but not in adenocarcinomas

Epstein-Barr virus (EBV) is associated with several lymphoid and epithelial human malignancies. The latter include gastric adenocarcinomas, while sporadic colorectal adenocarcinomas (CRCs) have been reported to be EBV-negative. Recently, increased numbers of EBV-infected B lymphocytes have been detected in intestinal mucosal samples affected by ulcerative colitis (UC) and, to a lesser extent, Crohn's disease (CD). Both CRC and colorectal non-Hodgkin's lymphoma (NHL) are recognized complications of inflammatory bowel disease (IBD), but it is unclear to what extent EBV contributes to the development of these neoplasms. Seventeen cases of IBD-associated CRC and nine cases of IBD-associated colorectal NHL were therefore studied for the presence of EBV by in situ hybridization. EBV-positive cases were further studied for the expression of the EBV-encoded nuclear antigen (EBNA) 2 and the latent membrane protein (LMP) 1 of EBV by immunohistochemistry. Four out of seven cases of colorectal NHL associated with UC were shown to be EBV-positive. In addition, two of two colorectal NHLs developing in patients with CD were EBV-positive. Of the EBV-positive lymphomas, three displayed a pattern of EBV latent gene expression consistent with type I latency (EBNA2(-)/LMP1(-)), two a type II pattern (EBNA2(-)/LMP1(+)), and one a type III pattern (EBNA2(+)/LMP1(+)). These findings suggest that EBV infection is involved in the pathogenesis of a proportion of colorectal NHLs developing in IBD. Iatrogenic immunosuppression may contribute to the development of these lymphomas. By contrast, all 17 IBD-associated CRCs were EBV-negative, including a case of CRC occurring synchronously with an EBV-positive NHL. In conjunction with previous reports on sporadic CRCs, this suggests that EBV is not involved in the pathogenesis of CRC.     

Syndromes mononucléosiques et pathologies hématologiques liés au virus d'Epstein-Barr

The Epstein-Barr virus (EBV), which preferentially infects B cells, persists in the infected subject as a latent asymptomatic infection. In adolescents, infectious mononucleosis is the symptomatic manifestation of primary EBV infection. The viral latency in the memory B-cells, the reservoir cells in peripheral blood in individuals is controlled by CD4 and CD8 positive T-cells. Immunodeficient patients are at high risk of developing EBV driven B-cell lymphomas as the consequence of the expression of oncogenic latency proteins LMP1 and EBNA2. These proteins expressed in infected B cells identify latency III or proliferation program in virus transformed B-cell, leading to lymphoid proliferation. In addition to immunodeficiency-related lymphomas, the most frequent lymphoid malignancies associated with EBV are the endemic Burkitt lymphoma, Hodgkin lymphoma and nasal type T-cell lymphoma.     

Epstein-Barr virus in B-cell non-Hodgkin's lymphomas: Unexpected infection patterns and different infection incidence in low- and high-grade types

Two hundred and eight cases of B-cell non-Hodgkin's lymphoma (B-NHL) occurring in Europeans without any signs of HIV infection were investigated for their association with an Epstein-Barr virus (EBV) infection. The polymerase chain reaction (PCR) was applied for EBV-DNA detection, in situ hybridization (ISH) for the cellular localization of EBV-encoded small nuclear RNAs (EBER) and immediate-early RNAs (BHLF), and immunohistology (IH) for the detection of EBV-encoded latent membrane protein (LMP) and EBV nuclear antigen 2 (EBNA2) expression. PCR and EBER-ISH produced congruent results in those cases with amplifiable DNA. EBV was present overall in 26 per cent (54/208) of the B-NHL cases. Through EBER-ISH, the virus could be localized merely in rare non-neoplastic bystander lymphocytes in 27 and additionally in tumour cells of 27 cases. Unexpectedly, the proportion of EBV-infected tumour cells present in the different cases varied between 1 and 100 per cent. All but three of the cases with infected tumour cells were of high-grade malignancy. Correlation with the morphological and immunological tumour phenotype revealed that all cases with more than 80 per cent EBER-positive tumour cells were either B-anaplastic large cell lymphomas (B-ALCL), sporadic Burkitt's lymphomas, or B-NHLs with partial or full plasmacellular differentiation. LMP was consistently absent from Burkitt's lymphomas and constantly expressed in B-ALCLs with EBER-positive tumour cells, while in all other instances it varied greatly and was rarer than EBER expression. The above findings suggest that in cases where EBV is present in 80–100 per cent of tumour cells, EBV infection takes place before malignant transformation and thus may have contributed to the malignant phenotype, whereas in the other cases with only a smaller number of infected tumour and single bystander cells, EBV infection may have occurred following malignant transformation. In these cases, infection would appear to be of little or no significance in tumourigenesis.     

Distribution and phenotype of Epstein-Barr virus-infected cells in human pharyngeal tonsils.

Although Epstein-Barr virus (EBV) is often found in human tonsils, it remains to be precisely determined in what cells and microenvironment the virus is present. Although generally regarded as a B lymphotropic virus, EBV is associated with non-B-cell tumors, for example, NK/T-cell lymphoma, carcinoma, and leiomyosarcoma. To provide a basis for understanding the origin and biology of EBV-infected non-B cells, the immunophenotype of all EBV-infected cells in reactive human tonsils was determined by subjecting tonsil sections to dual/triple EBER in situ hybridization and immunohistochemistry with monoclonal antibodies to T cells (CD3, CD4, CD8, CCR3), B cells (CD20), plasma cells (CD138), natural killer (NK) cells (PEN5), and epithelial cells (cytokeratin), as well as frozen section immunostaining with antibodies to EBV latent proteins EBNA1, EBNA2, LMP1, and EBV early protein BZLF1. Most tonsils contained nearly equal numbers of EBNA1- and LMP1-positive cells (latency program) while only a few contained EBNA2-positive cells (growth program). More than 1000 EBER-positive cells from six tonsils were detected in the interfollicular zone (59%), tonsillar crypts (26%), and follicles (15%). Most (82%) EBER-positive cells are CD20-positive B cells, 7% are CD3-positive T cells, and 11% are cells of indeterminate lineage, often with plasmacytoid morphology. However, no EBER-positive plasma cells were identified. Rare EBER-positive NK cells and EBER/BZLF1-positive epithelial cells were identified. The direct demonstration of EBV within rare T cells, NK cells, and epithelial cells in reactive human tonsils provide a basis for further understanding of the origin of EBV-associated tumors of non-B-cell type.     

A naturally derived gastric cancer cell line shows latency I Epstein-Barr virus infection closely resembling EBV-associated gastric cancer

In a process seeking out a good model cell line for Epstein-Barr virus (EBV)-associated gastric cancer, we found that one previously established gastric adenocarcinoma cell line is infected with type 1 EBV. This SNU-719 cell line from a Korean patient expressed cytokeratin without CD19 or CD21 expression. In SNU-719, EBNA1 and LMP2A were expressed, while LMP1 and EBNA2 were not. None of the tested lytic EBV proteins were detected in this cell line unless stimulated with phorbol ester. EBV infection was also shown in the original carcinoma tissue of SNU-719 cell line. Our results support the possibility of a CD21-independent EBV infection of gastric epithelial cells in vivo. As the latent EBV gene expression pattern of SNU-719 closely resembles that of the EBV-associated gastric cancer, this naturally derived cell line may serve as a valuable model system to clarify the precise role of EBV in gastric carcinogenesis.     

EBV+ diffuse large B-cell lymphoma arising within atrial myxoma. An example of a distinct primary cardiac EBV+ DLBCL of immunocompetent patients

Cardiac myxoma and diffuse large B-cell lymphoma are uncommon tumors, yet four composite tumors have been reported since 2009. We are reporting on the fifth case providing detailed immunohistochemical and FISH analyses. The lymphoma was present as superficially located nests of large cells with patchy necrosis in the background of a typical atrial myxoma. It displayed features of DLBCL with non-germinal center phenotype, expressed EBER, LMP1, EBNA2 and shared the following features with the previously reported cases: B-cell lineage, high-grade cytology, high proliferation rate, EBV infection in latency type 3 with one tested case and an excellent outcome. The lymphomas arising within myxoma may follow a pathogenic pathway driven by EBV, whose transformation potential is unleashed in the cytokine-rich milieu of a myxoma, presumably accentuating age-related decline of adaptive immunity known as immune senescence. DLBCL arising within atrial myxoma grouped together with EBV+ DLBCL associated with valve prosthesis and with an atrial thrombus differs in the immunocompetent patients from primary cardiac DLBCL, not otherwise specified, in clinical presentation, pathological features and a course of the disease. Distinction between these groups may have important therapeutic consequences.