Epstein-Barr virus latent membrane protein-1 oncogene deletions: correlations with malignancy in Epstein-Barr virus--associated lymphoproliferative disorders and malignant lymphomas

LMP-1, an Epstein-Barr viral (EBV) latency protein, is considered a viral oncogene because of its ability to transform rodent fibroblasts in vivo and render them tumorigenic in nude mice. In human B cells, EBV LMP-1 induces DNA synthesis and abrogates apoptosis. LMP-1 is expressed in EBV-transformed lymphoblastoid cell lines, nasopharyngeal carcinoma (NPC), a subset of Hodgkin's disease (HD), and in EBV-associated lymphoproliferative disorders (EBV-LPDs). Recently, focused deletions near the 3' end of the LMP-1 gene (del-LMP-1, amino acids 346-355), in a region functionally related to the half-life to the LMP-1 protein, have been reported frequently in human immunodeficiency virus (HIV)- associated HD (100%) and EBV+ Malaysian and Danish peripheral T-cell lymphomas (100%, 61% respectively), but less frequently in cases of HD not associated with HIV (28%, 33%) and infectious mononucleosis (33%). To further investigate the potential relationship of del-LMP-1 to EBV- LPDs associated with immunosuppression or immunodeficiency, we studied 39 EBV-associated lymphoproliferations (10 benign, 29 malignant) from four distinct clinical settings: posttransplant (4 malignant, 1 reactive); HIV+ (18 malignant, 2 reactive); nonimmunodeficiency malignant lymphoma (ML) (7 cases); and sporadic EBV infection with lymphoid hyperplasia (7 cases). The presence of EBV within lymphoid cells was confirmed by EBV EBER1 RNA in situ hybridization or by polymerase chain reaction (PCR) analysis. EBV strain type and LMP-1 deletion status were determined by PCR. EBV strain types segregated into two distinct distributions: HIV+ (9 A; 11 B) and non-HIV (19 A, 0 B), consistent with previous reports. Overall, del-LMP-1 were found in 1 of 5 (20%) Burkitt lymphomas (BL); 17 of 24 (71%) aggressive non- Hodgkin's lymphoma (agg-NHL), and 2 of 10 (20%) reactive lymphoid proliferations. Of the agg-NHLs, del-LMP-1 were present in 4 of 4 PT-ML (100%); 10 of 15 HIV+ ML (67%); and 3 of 5 nonimmunodeficiency malignant lymphoma (ML, 60%). A total of 2 of 7 (28%) sporadic EBV- associated lymphoid hyperplasias contained a del-LMP-1. All del-LMP-1 were identical by DNA sequence analysis. No correlation was identified between the presence of del-LMP-1 and the EBV strain type observed. The high incidence of del-LMP-1 observed in agg-NHLs (71%), in contrast to the relatively low incidence observed in reactive lymphoid proliferations (28%), suggests that the deleted form may be preferentially selected in lymphomatous processes. All posttransplant agg-NHLs contained a del-LMP-1, and a similar frequency of del-LMP-1 was observed in both HIV-associated ML (66%) and nonimmunodeficiency ML (60%), suggesting that impairment of immune function alone is not a requirement for the expansion of malignant cells infected by EBV stains containing the deleted LMP-1 gene.     

Apoptosis, proliferative activity and Bcl-2 expression in Epstein-Barr-virus-positive non-Hodgkin's lymphomas

In order to clarify the effects of Epstein-Barr virus (EBV) infection on apoptosis and proliferative activity of non-Hodgkin's lymphoma, 135 Japanese lymphoma cases were investigated for the presence of viral RNA and its correlation withbcl-2 protein (Bcl-2) expression. In addition, the role of EBV in lymphomagenesis was also studied in terms of EBV genotyping and specific deletion in the gene for the latent membrane protein 1(LMP-1). EBER-1 RNA in situ hybridization revealed EBV in 18 cases (13.3%), comprising 12 of 44 T cell (27.3%) and 6 of 91 B cell (6.6%) lymphomas. Type A EBV was found in all 18 cases (100%), and 17 of the 17 (100%) evaluable cases showed a 30-bp deletion within the 3′ end ofLMP-1. Comparison of apoptotic indices (AI), assessed by DNA nick-end labelling, and proliferative activity, estimated in terms of Ki-67 labelling and mitotic indices (KI and MI), demonstrated an overall correlation among AI, KI and MI increases in association with Bcl-2 negativity, indicating a close relation between apoptosis and proliferation. EBV-positive cases showed significantly elevated AI values, independent of Bcl-2 positivity, with no change in KI and MI. These results indicate that EBV in Japanese non-Hodgkin's lymphomas is exclusively of type A with a specific deletion inLMP-1 and that it tends to be present in T cell lymphomas. Moreover, EBV up-regulates apoptosis without any relation to Bcl-2 expression and exerts only minor effects on proliferation.     

Analysis of Epstein-Barr virus gene polymorphisms in normal donors and in virus-associated tumors from different geographic locations

While Epstein-Barr virus (EBV) infection is associated with the development of certain lymphoid and epithelial tumors, the role of the virus in the pathogenesis of these malignancies remains unknown. It has been suggested that EBV strain variation may contribute to tumor development. Two major strains of EBV, type 1 and type 2, have been identified on the basis of genetic polymorphisms and other minor genetic variations give rise to distinct EBV isolates. We analyzed EBV strain variation in healthy individuals and compared them with EBV isolates present in lymphoid and epithelial neoplasms from the same geographic regions. In particular, the incidence of the 30-bp latent membrane protein (LMP1) gene deletion, recently implicated in the development of more aggressive forms of virus-positive lymphomas and Hodgkin's disease [HD], was examined in the normal population. While a preferential association of the LMP1 deletion with the type 2 strain of EBV was observed, there was no increased incidence of virus isolates carrying this deletion in HD, Burkitt's lymphoma, or virus-associated carcinomas compared with the appropriate normal population. A polymorphism in the BamHI F region of the EBV genome, previously identified in Chinese populations, was found at increased incidence in European HD biopsies. Overall, we found that most of the EBV gene polymorphisms detected in EBV isolates from healthy virus carriers occurred with similar frequency in virus-associated tumors from the same geographical region.     

Identification of potential hot spots in the carboxy-terminal part of the Epstein-Barr virus (EBV) BNLF-1 gene in both malignant and benign EBV-associated diseases: high frequency of a 30-bp deletion in Malaysian and Danish peripheral T-cell lymphomas

In this study, we have sequenced the C-terminal part of the Epstein- Barr virus (EBV)-BNLF-1 gene encoding for the latent membrane protein-1 from tissues of EBV-positive Danish Hodgkin's disease (HD) and of Danish and Malaysian peripheral T-cell lymphomas (PTLs) and from tonsils of Danish infectious mononucleosis (IM). Our study showed that some of the 7 single-base mutations and the 30-bp deletion previously detected between codons of amino acid 322 and 366 in the BNLF-1 gene of the nasopharyngeal carcinoma cell line CAO were present in all Malaysian PTLs and in 60% of the Danish PTLs. In HD and the IM cases, the mutations were present in about 30%. The 30-bp deletion and the single base mutations occurred independently, and mutations were detectable in the majority of EBV type B-positive cases. These findings suggest that the 30-bp deletion and the 7 single-base mutations in the C-terminal part of the CAO-BNLF-1 gene do not characterize a new EBV type A substrain. Rather, some of the positions of single base mutations and the 30-bp deletion are hot spots that may have mutated independently through the evolution of EBV strains.     

Paediatric Hodgkin's disease in Spain: association with Epstein-Barr virus strains carrying latent membrane protein-1 oncogene deletions and high frequency of dual infections

The present report analyses the distribution of 30-base pair (bp) latent membrane protein-1 (LMP-1) oncogene deletions in 24 cases of Epstein-Barr virus (EBV)-positive paediatric Hodgkin's disease (HD) and 39 normal controls.
The 30 bp deletion was identified in 19/24 paediatric HD cases (79.2%), of which seven (29.2%) showed the deleted fragment alone, whereas in the remaining 12 (50%) it was accompanied by the nondeleted fragment. Conversely, the deletion was found in 8/22 (36.4%) EBV-positive healthy children, in two (9.1%) of whom the deleted fragment was alone, and was coinfecting with the nondeleted fragment in the other six (27.3%). The LMP-1 deletion was significantly associated with paediatric HD, both including dual infections ( P  = 0.006) or excluding them ( P  = 0.01).
Type 2 EBV was carried by 25% of HD children, whereas all controls harboured type 1 EBV. The 30 bp deletion was present in all the paediatric HD specimens that contained type 2 EBV, suggesting that a deleted type 2 EBV strain may be more tumourigenic than a nondeleted type 2 EBV strain.
These findings indicate that EBV strains carrying a 30 bp deletion in the third exon of the LMP-1 oncogene may have a more important role in the pathogenesis of paediatric HD than full-length EBV strains. Dual infection by LMP-1 deleted and nondeleted EBV strains is a frequent event both in healthy children and in the paediatric HD population.     

The molecular epidemiology and evolution of Epstein-Barr virus: sequence variation and genetic recombination in the latent membrane protein-1 gene

The phylogeny and evolution of Epstein-Barr virus (EBV) genetic variation are poorly understood. EBV latent membrane protein-1 (LMP-1) gene sequences are especially heterogeneous and may be useful as a tool for EBV genotype identification. Therefore, LMP-1 sequences obtained directly from EBV-infected human tissues were examined by PCR amplification and cloning. EBV genotypes were defined as "strains" from among 22 identified LMP-1 sequence patterns. Three molecular mechanisms were identified by which genetic diversity arises in the LMP-1 gene: point mutation, sequence deletion or duplication, and homologous recombination. The rate of LMP-1 gene evolution was found to be accelerated by coinfection with multiple EBV strains. The results of this study refine our understanding of LMP-1 sequence variation and enable accurate discrimination between independent EBV infection events and the consequence of intrahost EBV evolution. Thus, this LMP-1 sequence-based approach to EBV molecular epidemiology will facilitate the study of intrahost EBV infection, coinfection, and persistence.     

Presence of Epstein-Barr virus in extranodal T-cell lymphomas: differences in relation to site

T-cell lymphomas with similar morphology but with different sites of origin have a different clinical behavior. The theoretical explanation for this finding originates from the hypothesis that non-Hodgkin's lymphomas (NHLs) are neoplastic equivalents of immunological reactions involving tissue-restricted lymphocytes. This hypothesis also implies that T-NHLs originating from different sites differ in their genesis, and thus may differ in oncogen expression, expression of adhesion molecules, or presence of certain DNA/RNA viral sequences. Therefore, we have investigated in T-cell lymphomas with similar morphology originating from different sites, ie, nose (n = 5; all pleomorphic small- or medium- and large-cell T-cell lymphomas [PTL]), skin (PTL, n = 6; anaplastic large-cell [ALCL], n = 11), gut (PTL, n = 8; ALCL, n = 4), and lung (PTL, n = 6), the presence of Epstein-Barr virus (EBV) at the DNA, RNA (EBER 1 and EBER 2), and protein level (LMP-1). A double- staining technique was used to detect EBER 1/2, LMP-1, and differentiation markers at the single-cell level. High numbers of EBER 1/2-positive tumor cells (> 100 per medium power field [mpf]) were found in five of five nasal T-cell lymphomas, none of 17 primary cutaneous T-cell lymphomas, one of 12 gastrointestinal T-cell lymphomas (ALCL), and two of six pulmonary T-cell lymphomas. These lymphomas are therefore called EBV-associated lymphomas. In contrast to our earlier findings in lymph nodes, no extranodal lymphomas were found, with only a few EBV-positive tumor cells. Five gastrointestinal cases positive for EBV by polymerase chain reaction (PCR) showed that EBER 1/2 was only found in sporadic nonneoplastic, ie, reactive lymphocytes. Angiocentricity was present in 18 PTL and one ALCL, but not associated with the presence of EBV. These results indicate that the presence of EBV in extranodal T-cell lymphomas is site-restricted and argues for a different pathogenesis of T-cell lymphomas with similar morphology but originating from different sites. The presence of EBV in most tumor cells in these EBV-associated lymphomas suggests that when present, EBV might be important in the pathogenesis of these lymphomas.     

A novel Epstein-Barr virus-like virus, HV(MNE), in a Macaca nemestrina with mycosis fungoides.

Epstein-Barr virus (EBV) infection of humans has been associated with the development of lymphoid malignancies mainly of B-cell lineage, although occasionally T-cell lymphomas have been reported. We describe here the characterization of a novel EBV-like virus (HV(MNE)) isolated from a simian T-cell lymphotropic virus type I/II (STLV-I/II) seronegative pigtailed macaque (Macaca nemestrina) with a cutaneous T-cell lymphoma. Immunohistochemistry studies on the skin lesions demonstrated that the infiltrating cells were of the CD3(+)/CD8(+) phenotype. Two primary transformed CD8(+) T-cell lines were obtained from cultures of peripheral blood mononuclear cells (PBMC) and skin, and, with time, both cell lines became interleukin-2-independent and acquired the constitutive activation of STAT proteins. Polymerase chain reaction analysis of the DNA from the cell lines and tissues from the lymphomatous animal demonstrated the presence of a 536-bp DNA fragment that was 90% identical to EBV polymerase gene sequences, whereas the same DNA was consistently negative for STLV-I/II sequences. Electron microscopy performed on both cell lines, after sodium butyrate treatment, showed the presence of a herpes-like virus that was designated HV(MNE) according to the existing nomenclature. In situ hybridization studies using EBV Epstein-Barr viral-encoded RNA probes showed viral RNA expression in both CD8(+) T-cell lines as well as in the infiltrating CD8(+) T cells of skin-tissue biopsies. Phylogenetic analysis of a 465-bp fragment from the polymerase gene of HV(MNE) placed this virus within the Lymphocryptovirus genus and demonstrated that HV(MNE) is a distinct virus, clearly related to human EBV and other EBV-like herpesviruses found in nonhuman primates.     

Association of Epstein-Barr virus with human immunodeficiency virus-negative peripheral T-cell lymphomas in Japan

The association of Epstein-Barr virus (EBV) with human immunodeficiency virus-negative T-cell lymphoma was examined in 68 patients using the polymerase chain reaction (PCR) with DNA obtained from formalin-fixed paraffin-embedded tissues and an in situ hybridization technique. EBV-encoded RNA (EBER) was detected in 43 of 68 cases (63%) of peripheral T-cell lymphoma: in 100% (11 of 11 cases) of NK/T-cell lymphomas, 70% (14 of 20 cases) of angioimmunoblastic T-cell lymphomas (AILT) and 49% (18 of 37 cases) of other types of peripheral T-cell lymphoma. A positive band was also detected at high incidence (36 of 65 cases; 55%) in a PCR analysis using primers to detect the Bam HI-W fragment of EBV. In the immunohistochemical analysis using a monoclonal antibody to latent membrane protein 1 (LMP-1) of EBV, one of the EBV-encoded latent gene products, LMP-1, was found to be expressed in 13 of 64 cases (20%), but EBNA-2 was not expressed in all the cases examined (0 of 59 cases; 0%). The 5-yr survival rate was 28% for peripheral T-cell lymphomas overall, 0% for NK/T-cell lymphomas, 38% for AILTs and 28% for other types of peripheral T-cell lymphoma. The difference in the overall survival rate between NK/T-cell lymphoma and non-NK/T-cell lymphoma was significant (P = 0.0498 by Log-rank test). Among peripheral T-cell lymphoma patients overall, the group severely infected with EBV (EBER-ISH ++) had a lower 5-yr survival rate (8%) than the group slightly (EBER-ISH +) or not infected (38%; P = 0.0013).     

Fulminant EBV(+) T-cell lymphoproliferative disorder following acute/chronic EBV infection: a distinct clinicopathologic syndrome

This study describes the clinicopathologic features of 5 patients who developed a fulminant Epstein-Barr virus (EBV)-positive clonal T-cell lymphoproliferative disorder (LPD) after acute EBV infection. One additional patient developed a similar disorder in the setting of long-standing chronic active EBV infection. Detailed immunophenotyping, in situ hybridization for EBV early RNA-1 (EBER1) and polymerase chain reaction (PCR) analyses for immunoglobulin (Ig) heavy chain and T-cell receptor (TCR)-gamma gene rearrangements were performed on paraffin-embedded tissue from all patients. In addition, EBV strain typing and detection of the characteristic 30-bp deletion of the latent membrane protein-1 (LMP-1) gene were performed by PCR. Controls included 8 cases of uncomplicated infectious mononucleosis (IM). Patients included 4 males and 2 females with a median age of 18 years (2-37 years). Three patients were Mexican, 2 were white, and 1 was of Asian descent. All presented with fever, hepatosplenomegaly, and pancytopenia; 5 were previously healthy, but had a clinical history of a recent viral-like upper respiratory illness (1 week to 2 months), and 1 patient had documented chronic active EBV infection for 7 years. Serologic data for EBV were incomplete but titers were either negative or only modestly elevated in 3 cases. In 1 case serology was consistent with severe chronic active EBV infection. In the remaining 2 cases serologic studies were not performed. All patients died within 7 days to 8 months of presentation with T-cell LPD. On histologic examination, the liver and spleen showed prominent sinusoidal and portal lymphoid infiltrates of CD3(+), beta F1(+), EBER1(+) T cells lacking significant cytologic atypia. Two cases were CD4(+), 2 cases were CD8(+), and 2 cases had admixed CD4(+) and CD8(+) cells without clear subset predominance. All were TIA-1(+), CD56(-). Only rare B cells were noted. Marked erythrophagocytosis was present. Molecular analysis revealed identical T-cell clones in 2 or more sites (liver, spleen, lymph node) in 5 cases. All patients carried type A EBV; 4 cases had wild-type EBV-LMP, and 2 showed the 30-bp deletion. This fulminant T-cell LPD after acute/chronic EBV infection is characterized by hepatosplenomegaly, often without significant lymphadenopathy, fever, liver failure, pancytopenia, and erythrophagocytosis indicative of a hemophagocytic syndrome. EBV serology may be misleading, with lack of elevated titers. The presence of an EBER1(+) T-cell infiltrate with scant B cells should alert one to this diagnosis. Although cytologic atypia is minimal, studies for T-cell clonality confirm the diagnosis. (Blood. 2000;96:443-451)     

Biological aspects of Epstein-Barr virus (EBV)-infected lymphocytes in chronic active EBV infection and associated malignancies

Most primary Epstein-Barr virus (EBV) infections are clinically inapparent, but occasionally EBV infection can cause acute infectious mononucleosis. EBV has been linked to a variety of hematologic and non-hematologic malignancies. Chronic active EBV (CAEBV) infection designates a recently identified EBV-associated syndrome characterized by a variety of serious hematological disorders, including malignant lymphoma. EBV was found to infect circulating T- and/or NK-cells in patients with CAEBV infection. These EBV-infected T- and/or NK-cells express EBNA-1, LMP-1, and LMP-2A, a type II form of EBV latency, which is also observed in nasopharyngeal carcinoma (NPC), Hodgkin's disease (HD), and peripheral T-cell lymphoma. CAEBV infections may thus represent a subset of EBV-associated T- and/or NK-cell lymphoproliferative disorders.     

IL-10 can induce the expression of EBV-encoded latent membrane protein-1 (LMP-1) in the absence of EBNA-2 in B lymphocytes and in Burkitt lymphoma- and NK lymphoma-derived cell lines

EBV-positive nasopharyngeal carcinoma and Hodgkin, T, and natural killer (NK) lymphomas express EBNA-1 and the latent membrane proteins (LMP1-2; type II latency). In contrast to type III EBV-transformed lymphoblastoid cell lines, in these cells the LMPs are expressed in the absence of EBNA-2. We have previously reported that exposure to CD40 ligand and IL-4 could induce LMP-1 in an in vitro EBV-infected Hodgkin lymphoma-derived cell line, which expressed only EBNA-1. We show now that both human and EBV-encoded IL-10 can induce LMP-1 in the absence of EBNA-2 in the Daudi, P3HR1, and other BL cell lines. Interestingly, induction of LMP-1 was not accompanied by the downregulation of BCL-6. IL-10 could also induce LMP-1 in the conditional lymphoblastoid cell line ER/EB2-5 where EBNA-2 was downregulated in the absence of estrogen. Moreover, IL-10 could induce the expression of LMP-1 in tonsillar B cells infected with the nontransforming, EBNA-2-deficient EBV strain P3HR1 and enhance LMP-1 expression in 2 EBV-positive NK lymphoma lines. The demonstration that IL-10 can induce the expression of LMP-1 in an EBNA-2-independent manner shows that the major transforming EBV gene LMP-1 can be induced by extracellular signals in lymphoid cells, and IL-10 might contribute to the establishment of type II EBV latency.     

Nasopharyngeal carcinoma in Indonesia has a low prevalence of the 30-base pair deletion of Epstein-Barr virus latent membrane protein 1

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC), one of the highest incidence of tumors in Indonesia. EBV infection is ubiquitous around the world, but NPC occurs with a remarkable geographic distribution. This phenomenon suggests that there are subtypes of EBV, some of which may have greater tumorigenic potential. The latent membrane protein 1 (LMP 1) gene encoded by EBV is tumorigenic due to its ability to transform rodent fibroblast. It was originally shown that the LMP 1 gene from NPC of Chinese patients harbors a deletion of 30-bp in the carboxyl terminal of the gene. However, the deletion is also present in healthy control and in other EBV-positive tumors. We examined the polymorphism of LMP 1 in 56 tumor biopsies of Indonesian patients with NPC and identified low prevalence of the 30-bp deletion of LMP 1. Sequence analysis showed unique mutations of LMP 1 which suggests that strain-specific variations of EBV are found in Indonesia. The low frequency of 30-bp deletion in the country with high prevalence of NPC indicates that the deletion may represent a geographic polymorphism rather than a predisposing factor in the development of NPC.     

Characterization of Epstein-Barr virus genotype in AIDS-related non-Hodgkin's lymphoma.

In the present study sequence variations at the C terminus of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), EBV-encoded latent membrane protein 1 (LMP-1), and EBNA-2 and EBNA-3C genes were investigated in 64 cases of EBV-positive AIDS-related diffuse large cell lymphoma (AIDS-DLCL), both systemic (12) and localized primarily to the central nervous system (52), and in 12 cases of EBV-positive AIDS-related Burkitt's lymphoma (AIDS-BL). Sequence analysis of the EBNA-1 C-terminal region led to the distinction of two major unrelated EBV strains, termed strain P (prototype) and strain V (variant), and their related subtypes, namely P-ala, P-thr, V-leu, V-val, and V-pro. Analysis of the LMP-1 gene was performed to assess the frequency of the C-terminus deletion variant, whereas analysis of EBNA-2 and EBNA-3C genes led to the identification of the distribution of the EBV type 1 and type 2 strains. The frequency of EBNA-1 subtypes was assessed in 49 cases of AIDS-NHL, including 37 cases of AIDS-DLCL and 12 cases of AIDS-BL. The P strain was detected in 45 of 49 cases (91.8%) whereas the V strain was found in 4 of 49 samples (8.1%). A significant difference in the distribution of the P and V strains was found between AIDS-DLCL and AIDS-BL (p < 0.01), because of the exclusive infection by the P strain of the AIDS-DLCL samples analyzed. The frequency of LMP-1 deletion variants and of EBV type 1 and type 2 strains in AIDS-DLCL overlapped with that of the general population, and no correlation was found with the evaluated clinicoepidemiological data of patients, that is, disease site, tumor histology, CD4(+) cell counts, and HIV transmission route. In conclusion, we found that the distribution of the EBV genotype in all of the AIDS-NHL samples analyzed is similar to the viral representation found in control individuals of both immunocompetent and immunocompromised populations.     

Epstein-Barr viral DNA in acute large granular lymphocyte (natural killer) leukemic cells.

Serologic studies in a male Caucasian presenting with an acute hepatitis-like illness, associated with an increase in peripheral blood large granular lymphocytes (LGLs), suggested a chronic or reactived Epstein-Barr virus (EBV) infection. The LGL were shown to have a natural killer (NK) cell, CD3- CD16- CD56+ CD57- phenotype and mediated strong nonspecific major histocompatability complex-unrestricted (NK) cytotoxic activity. A progressive increase in the peripheral blood LGL count was associated with a rapid deterioration, hepatic necrosis, and death. Widespread organ infiltration with LGLs suggested a malignant lymphoproliferative condition, but no lymphoid (T-cell receptor or IgH) gene rearrangement or cytogenetic marker was detected. However, molecular analysis identified EBV genomic DNA present in a single episomal form within the LGL, establishing the clonal nature of the LGL proliferation. Confirmation that the EBV had infected the leukemic LGL was obtained by in situ hybridization studies that showed EBV RNA within the LGLs. Immunoblotting of LGL protein extracts established that, of the EBV gene products, EBV nuclear antigen-1 (EBNA-1) was expressed but EBNA-2 and the latent membrane protein (LMP-1) were not detectable in the leukemic cells. These results suggest that EBV may be involved directly in LGL cell transformation, in a manner similar to EBV-associated B-cell lymphomas, although other molecular changes probably contribute to the evolution of a fully malignant leukemic clone.     

Antisense oligodeoxynucleotides to latent membrane protein 1 induce growth inhibition, apoptosis and Bcl-2 suppression in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells, but not in EBV-positive natural killer cell lymphoma cells

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) is essential for immortalization of B cells by EBV, protects the infected cells from apoptotic cell death and induces Bcl-2 expression. Suppression of LMP-1 expression by antisense oligodeoxynucleotides (AS-oligo) to LMP-1 inhibits proliferation, promotes apoptosis and suppresses Bcl-2 expression in EBV-transformed B cells. However, the function of LMP-1 expression in EBV-positive natural killer (NK) cell lymphoma cells has not been reported previously. We examined the function of LMP-1 in two EBV-positive NK cell lymphoma cell lines (NK-YS and YT) through suppressing LMP-1 expression by AS-oligo to LMP-1. The AS-oligo to LMP-1 suppressed LMP-1 mRNA and protein expression in two EBV-positive NK cell lymphoma cell lines, as well as in an EBV-transformed B-cell line (CMG-1). Proliferation was inhibited, apoptosis was induced and Bcl-2 expression was suppressed in CMG-1 cells, but none of these events were observed in NK-YS or YT cells. These results suggest that proliferation, inhibition of apoptosis and Bcl-2 expression in EBV-positive NK cell lymphoma cells are not directly regulated by LMP-1 as in EBV-transformed B-cell lines, but are probably mediated through other signal transducing systems.     

Epstein-Barr virus strain type and latent membrane protein 1 gene deletions in lymphomas in patients with rheumatic diseases

Objective. Recent studies have shown that immunomodulatory therapy for the treatment of rheumatic diseases can be associated with the development of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. The present study was undertaken to determine the strain type of EBV in lymphoproliferative disorders that occur in patients with rheumatic disease and to investigate EBV latent membrane protein 1 (LMP-1) gene deletions that occur in these lymphoproliferative disorders. Methods. Ten EBV-associated lymphoid neoplasms in patients with rheumatoid arthritis or dermatomyositis were analyzed by polymerase chain reaction to determine EBV strain type and to investigate for the presence of a previously characterized 30-basepair deletion in the LMP-1 gene. Results. The results indicated that lymphoproliferative disorders in these patients can harbor EBV strain type A or B, with a predominance of type A infection (80%). It was also shown that both wild-type and mutated LMP-1 genes can be found in these neoplasms, with the deleted form of the LMP-1 gene occurring in one-third of cases in this series. Conclusion. LMP-1 deletions associated with certain aggressive lymphoid neoplasms are not required for the genesis of lymphoproliferative disorders in patients with rheumatic disease. The relative frequencies of type A and type B EBV strains in these lymphoproliferative disorders show similarities to the frequencies in patients with post-solid organ transplantation immunosuppression-associated lymphoproliferative disorders.     

Persistence of the same viral strain in early and late relapses of Epstein-Barr virus-associated Hodgkin's disease

Twelve cases of relapsing Hodgkin's disease were investigated for the presence of Epstein-Barr virus (EBV). Of these, 7 cases contained EBV gene products (LMP1, EBER RNA) in the diagnostic Reed-Sternberg cells and variants at first presentation and at relapse(s), whereas 5 cases were negative at both first diagnosis and relapse. Among the 7 EBV- positive cases, material for DNA extraction was available in 2 cases at both diagnosis and relapse(s). Ig and T-cell receptor gene rearrangements displayed a germline configuration in the 2 cases. However, Southern blot analysis of the terminal repeats (TR) of EBV genome showed that, in 1 of the 2 cases, the fragment was of the same size at diagnosis and in the subsequent two relapses (1 early and 1 late). The second case contained monoclonal EBV genome at diagnosis, but the Southern analysis of the TR was negative at relapse. The latent membrane protein (LMP1) sequence analysis confirmed the persistence of a distinctive viral strain in each of the 2 cases with individual abnormalities within the carboxy terminal region (5 point mutations and a 30-bp deletion for the first case and 6 point mutations for the second case). The persistence of a given strain in early and late relapses is evidence towards the view that in Hodgkin's disease such relapses are related to a single residual tumor cell clone.     

Effect of Epstein-Barr virus infection on response to chemotherapy and survival in Hodgkin's disease.

We have analyzed paraffin sections from 190 patients with histologically confirmed Hodgkin's disease (HD) for the presence of Epstein-Barr virus (EBV) using in situ hybridization to detect the EBV-encoded Epstein-Barr virus early RNAs (EBERs) and immunohistochemistry to identify latent membrane protein-1 (LMP1) expression. EBV was present in the tumor cells in 51 HD cases (27%) and was mainly confined to the mixed cellularity and nodular sclerosis subtypes. There was no difference between EBV-positive and EBV-negative HD patients with regard to age, clinical stage, presentation, and the number of alternating chemotherapy cycles of ChIVPP and PABIOE received. The complete remission rate after study chemotherapy was 80% in EBV-positive patients versus 69% in EBV-negative patients (P =.05). The 2-year failure-free survival rate was significantly better for EBV-positive patients when compared with the EBV-negative HD group (P =.02). Although 2-year and 5-year overall survival rates were better for EBV-positive HD patients, the differences were not statistically significant (P =.18 and P =.40, respectively). In conclusion, the results confirm the favorable prognostic value of EBV in the tumor cells of HD patients and suggest important differences in response to chemotherapy between EBV-positive and EBV-negative patients.