Attempts to demonstrate virus-specific sequences in human tumors. IV. EB viral DNA in European Burkitt lymphoma and immunoblastic lymphadenopathy with excessive plasmacytosis.

Various biopsies from different European malignant lymphomas, two biopsies from nasopharyngeal carcinomas, and material from non-neoplastic lymph nodes were assayed for the presence of Epstein-Barr virus (EBV) DNA by nucleic acid hybridization. Reassociation kinetics of in vitro-labelled EBV DNA were studied in the presence of tumor DNA. The lymphomas tested included among others follicular lymphomas, germinocytomas, immunoblastic lymphomas and lymphoplasmacytoid immunocytomas. Epstein-Barr viral DNA was demonstrated within the two nasopharyngeal carcinoma biopsies as expected. A histologically typical Burkitt lymphoma as well as an immunoblastic lymphadenopathy with excessive plasmacytosis also contained EBV-DNA. The Burkitt biopsy revealed about 15 EBV genome equivalents per cell. Antibodies against EBV-specific antigens were highly elevated in the serum of this patient. The material of the patient with immunoblastic lymphadenopathy contained 2-3 EBV genome equivalents per cell.     

Transformation of lymphocytes by Herpesvirus papio.

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.     

Characterization of the Epstein-Barr virus isolated from a cell line derived from a patient with American Burkitt's lymphoma

Epstein-Barr viral (EBV) DNA is nearly always detectable in African Burkitt's lymphoma (BL) but is infrequently found in the histologically indistinguishable American BL. We have derived a tumor cell line from a patient with American BL which produces EBV, and we have compared this virus isolate [JLP(c)] with African BL EBV. The American JLP(c) virus immortalizes human umbilical cord lymphocytes in vitro, and its DNA is indistinguishable from African BL EBV DNA by nucleic acid hybridization and preliminary restriction endonuclease cleavage analysis.     

Production by EBV infection of an EBNA-positive subline from an EBNA-negative human lymphoma cell line without detectable EBV DNA.

A continuous lymphoma cell line, BJAB, derived from the tumour of an exceptional African case of Burkitt's lymphoma, has previously been described. Unlike 97% of African BL cases studied, neither the original tumour cells nor the cell line contained detectable amounts of EBV (Epstein-Barr virus) DNA, nor did they express the EBV-determined nuclear antigen EBNA. The cells of the established line had the characteristics of B-type lymphocytes and they carried receptors for EBV. EBNA was induced in the majority of BJAB cells after EBV infection. Usually the cells died within 10 days of infection, but it was possible to establish a permanent EBNA-positive variant (GC-BJAB) of BJAB. The patient from whose tumour the original BJAB line was established was seropositive for EBV antigens, indicating previous exposure to and continuing presence of the virus; yet the tumour had not become infected by EBV. This evidence shows that EBV is not readily "picked up" by the lymphoma.     

Heterogeneity of Epstein-Barr virus derived from P3HR-1 cells.

Infection of cells of the EBV-free human B-lymphoma lines BJAB and Ramos resulted in conversion of these cells to EBV-genome carriers expressing EBNA. EBV isolates from P3HR-1 cells induced a heterogeneous EBNA pattern: both a faintly granular pattern and brilliant EBNA-expression were observed. The two types of EBNA-expressing cells could be separated upon cloning. Brilliantly EBNA-expressing cells always segregated varying percentages of EBNA-negative cells. An EBNA-negative subclone derived from these cells was devoid of detectable EBV DNA. Nucleic acid hybridization experiments failed to reveal a correlation between the intensity of EBNA expression and the number of EBV genome equivalents per cell. EBV genome-containing cells had an average of 14-fold more cells showing EA synthesis after superinfection by P3HR-1 virus, when compared with EBNA-negative cells infected under identical conditions. Studies on the kinetics of EA induction in EBNA-positive and EBNA-negative cells indicate that complementation is required for the induction of EA after superinfection.     

Demonstration of Epstein-Barr virus in malignant non-Hodgkin's lymphomas

Lymph nodes or tumor biopsies of 60 persons suspected of having a malignant lymphoma were examined for the presence of Epstein-Barr virus nuclear antigen (EBNA) by anticomplement immunofluorescence. In 8 cases the tissue specimens were also assayed for Epstein-Barr virus (EBV) DNA by nucleic acid hybridization. Serum samples of patients and controls were tested for EBV-related antibodies. The histological tests in 37 cases showed a malignant non-Hodgkin lymphoma, and in 23 cases a reactive lymphadenopathy. A Burkitt lymphoma of a European boy and a polymorphic centroblastoma contained EBNA and approximately 27 or 30 genome equivalents EBV DNA per cell, respectively. EBNA was also demonstrated in about 20% of the cells of a lymph node from a patient with recurrent reactive lymphadenopathy.     

Relationship between Epstein-Barr virus (EBV) DNA and the EBV-determined nuclear antigen (EBNA) in Burkitt lymphoma biopsies and other lymphoproliferative malignancies

Twenty-six of 27 African Burkitt lymphomas with histologically confirmed diagnosis contained relatively large amounts of EBV DNA (10–101 viral genomes per cell), as determined by nucleic acid hybridization. Twenty-five of the 26 EBV DNA-positive lymphomas contained the EBV-determined nuclear antigen, EBNA, in the majority of the nuclei. Technical reasons may have accounted for the apparent EBNA-negativity of one EBV DNA-positive biopsy. Four African lymphoma biopsies, one with a definite diagnosis of Burkitt's lymphoma and three with a questionable diagnosis of the same disease, were all EBV DNA- and EBNA-negative. The same was true for a collection of Swedish cases of Hodgkin's disease, lymphocytic lymphoma, chronic lymphatic leukemia and some other lymphoproliferative malignancies. Thus, there is excellent agreement between the presence of EBV DNA and of EBNA in tumor biopsies. The EBNA antigen test therefore appears a relatively simple way of testing for the presence of the virus genome, provided it is carried out with appropriate controls. Several of the EBV-genome and EBNA-negative cases came from patients with high serum titers of EBV antibodies. It is concluded that the virus does not really travel along with malignant lymphomas as a passenger in the seropositive patients. In comparison with other lymphomas, African Burkitt's lymphoma of the high endemic areas is unique in that the tumors (with rare exceptions) represent the proliferation of an EBV-genome carrying clone. These findings stress the necessity to distinguish between EBV-seropositive status and evidence for EBV-genome-carrying neoplastic cells.     

Restrictions upon Epstein-Barr virus infection of the leukemic cell are demonstrated in patients with hairy cell leukemia

We tested the hypothesis that Epstein-Barr virus (EBV) might actually infect leukemic hairy cells in vivo by examining those cells for the EBV-receptor, EBV nuclear antigen (EBNA) and membrane antigen (MA), for spontaneous transformation and rescue of infectious virus and for presence of EBV genome. EBV-receptors were found on subpopulations of leukemic cells from each of 7 patients with hairy cell leukemia (HCL) tested. MA was present on low numbers (1-5 per cent) of fresh leukemic cells of 7 patients and in some instances occurred with a greater frequency after 3 to 5 days in culture, with or without 12-O-tetradecanoylphorbol-13-acetate. In 11 fresh leukemic cell preparations from 8 HCL patients, no EBNA was demonstrated. However, 2 samples after 4 days in culture expressed low frequencies of EBNA-positive cells. Spontaneous, EBV-positive cell lines were established with a high transformation efficiency from 3 HCL blood samples but not from 8 other specimens. Infectious EBV could be rescued from some hairy leukemic cell preparations by co-cultivation with cord blood lymphocytes. These results demonstrated that leukemic cell populations harbored infectious EBV, that the leukemic cells expressed virus receptors and suggested that a small subpopulation of leukemic cells might become infected in vivo at least transiently and possibly transformed in vitro by EBV. To test for the extent of occult in vivo infection of leukemic cells with EBV, Southern type hybridization studies were performed with a probe for EBV genome (Bam HI W). At a sensitivity level of 0.1 genome per cell, EBV genome was not detected in the leukemic cell populations of 7 patients. We conclude that host defence mechanisms protecting these individuals from EBV also prevent infections of the leukemic cell and/or most hairy leukemic cells are not suitable targets for both infection and transformation.     

Epstein-Barr virus in an American patient with Burkitt's lymphoma: detection of viral genome in tumor tissue and establishment of a tumor-derived cell line (NAB).

Epstein-Barr virus (EBV) DNA (17.7 genome equivalents/cell) was found in tumor tissue from an American patient with Burkitt's lymphoma who had never traveled outside the United States. A lymphoid cell line (NAB) containing the EBV genome was established from tumor tissue from this patient; characteristics of this cell line were described. Previous Burkitt's tumors found in Americans and examined by molecular hybridization were negative for EBV DNA. Our results suggested that EBV is associated with at least some American Burkitt's tumors.     

Association of BK virus with human brain tumors and tumors of pancreatic islets

BK virus (BKV) DNA was detected by Southern blot hybridization in 19 out of 74 (25.6%) human brain tumors and in 4 out of 9 (44.4%) human tumors of pancreatic islets. BKV DNA was free, in an episomal state and generally in a low copy number (0.2 to 2 genome equivalents per cell). Only occasional tumors contained 10 to 20 genome copies per cell. Viral DNA sequences integrated into cellular DNA were not detected. A number of tumors expressed BKV-specific RNA and T antigen. By transfection of total tumor DNA into human embryonic fibroblasts, viruses with the biological and antigenic properties of BKV were rescued from 6 brain tumors and from 2 tumors of pancreatic islets. Restriction endonuclease mapping of the genomes of the rescued viruses showed that they differ from wild-type BKV. They are all similar to each other and to BKV-IR, a virus previously rescued from a human tumor of pancreatic islets, suggesting the possible association of a BKV variant with specific types of human neoplasms. The significance of the relationship of these BKV variants to human tumors and their possible etiologic role in human oncogenesis are discussed.     

Segregation of the EBV-determined nuclear antigen (EBNA) in somatic cell hybrids derived from the fusion of a mouse fibroblast and a human Burkitt lymphoma line

Four independently derived hybrids between the mouse fibroblast line A9 and the human, Burkitt-lymphoma-derived lymphoblastoid cell line Daudi were studied for the presence of the Epstein-Barr virus (EBV) genome, the EBV-determined nuclear antigen (EBNA), other EBV-associated antigens, human surface immunoglobulin and the presence of human chromosomes. The four lines differed in the number of their EBV genomes. There was a parallelism between this number, as detected by c/RNA/DNA hybridization, and the frequency of EBNA-positive nuclei. None of the other EBV-antigens, EA, VCA or MA, was expressed at any time, either in the untreated hybrid cells or after IUDR-treatment. The hybrids did not carry detectable surface-associated immunoglobulin or EBV-receptors. The presence of the EBV genome was coincident with the maintenance of human chromosomes, but the hybrids that have lost detectable viral genomes and EBNA still contained a considerable number of human chromosomes, suggesting that the viral genome may be associated with a few chromosomes only.     

Association of Epstein - Barr virus and breast cancer in Eritrea

Background

The oncogenic potential of Epstein-Barr virus (EBV) in breast cancer is being increasingly recognized. Despite some controversies regarding such role, new evidence is suggesting a culpability of EBV in breast cancer, particularly in Africa where the virus has been originally associated with causation of several solid and hematological malignancies. One example is a report from Sudan implicating EBV as a prime etiologic agent for an aggressive type of breast cancer, where nearly 100% of tumor tissues were shown to carry viral signatures. To get a broader view on such association, other nearby countries should be investigated. The present study aims to determine the prevalence and possible associations of the virus in Eritrean breast cancer patients.

Methods

Detection of EBV genome using primers that target Epstein Barr Encoded RNA (EBER) gene and Latent Membrane Protein-1 (LMP-1) gene sequences was performed by polymerase chain reaction (PCR) on DNA samples extracted from 144 formalin fixed paraffin embedded breast cancer tissues and 63 non-cancerous breast tissue as control group. A subset of PCR positive samples was evaluated for EBER gene expression by in situ hybridization (ISH). Expression of Latent Membrane Protein-2a (LMP2a) was also assessed by immunohistochemistry in a subset of 45 samples.

Results

Based on PCR results, EBV genome signals were detected in a total of 40 samples (27.77%) as compared to controls (p-value?=?0. 0031) with a higher sensitivity when using the EBER primers. Five out of the 14 samples stained by EBER-ISH 35.71% were positive for the virus indicating the presence of the viral genome within the tumor cells. Of those stained for IHC 7 (15.55%) were positive for LMP2a showing low viral protein frequency.

Conclusions

Based on these findings it can be concluded that EBV in Eritrea is associated with a smaller subset of tumors, unlike neighboring Sudan, thus pointing to possible differences in population predisposition and diseases epidemiology.

     

Enhancement of replication of Epstein-Barr virus DNA in human lymphoblastoid cell lines by N-methyl-N''-nitro-N-nitrosoguanidine

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a potent carcinogenthat alkyiates nucleic acids. Interaction of MNNG with humanlymphoblastoid cell fines carrying Epstein-Barr virus (EBV)was studied. Treatment of virus-producing cells (P3HR-1) withMNNG resulted in an {small tilde}3-fold increase in EBV genomecopies per cell as determined by cRNA - DNA hybridization. Thiseffect was not observed in a non-virus-producer line (Raji).Dose-response studies indicated that the optimum concentrationwas between 0.5 µg and 2 µg/ml. This same dose rangewas most effective in inhibiting cell proliferation both ofP3HR-1 and Raji cells. Concomitant treatment of P3HR-1 cellswith MNNG and 12-O-tetradecanoyl-phorbol-13-acetate gave anadditive increase to 9-fold of the number of EBV genome copiesper cell. Pretreatment of Raji cells with MNNG followed by superinfectionwith P3HR-1 virus resulted in a 35% enhancement of EBV DNA replicationas analyzed by density centrifugation. In contrast, Raji cellssuperinfected with MNNG-treated EBV showed a marked reductionin EBV DNA replication which indicates that the lesions producedin the viral genome by the drug interfered with the infectiouspotential of the virus.     

Relationship between the Epstein-Barr virus genome and nasopharyngeal carcinoma in Caucasian patients.

In order to explore whether undifferentiated nasopharyngeal carcinoma (NPC) shows a regular association with Epstein-Barr virus (EBV), regardless of the geographical and ethnic origin of the patient, a correlated histopathological and nucleic acid hybridization study was performed on biopsies from Caucasian patients with nasopharyngeal carcinomas and from various controls. Among 12 undifferentiated NPCs, 11 were positive for EBV-DNA, with multiple copies of the viral genome per cell. Serological tests showed elevated anti-VCA and anti-EA(DA) titers. Six NPCs with various degrees of squamous differentiation, four malignant lymphomas of the nasopharynx and seven carcinomas located outside the nasopharynx were EBV-DNA negative. These findings further stress the uniqueness and regularity of the association between EBV-DNA and undifferentiated NPC. Clearly, the association extends over geographical barriers and holds true not only in the previously studied, moderate-incidence African ethnic group, but also in the low-incidence Western patients.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

Geographic variation in Epstein-Barr virus-associated Burkitt's lymphoma in children from Brazil

In developing countries, BL has a strong association with EBV infection during childhood. In South America, the data have shown an EBV association intermediate between that reported in the United States (30%) and that in equatorial Africa (95%). Early age at EBV infection and lower socioeconomic status have been related to increased EBV-associated BL in developing countries. In Brazil, there are not enough data on childhood BL related to EBV infection. Our aim was to evaluate the clinicopathologic features and EBV association of 44 children with NHL from the state of Rio de Janeiro, situated in the southeast of Brazil. EBV was detected using RNA in situ hybridization in 36 biopsy specimens. DNA from fresh tumor samples and from paraffin-embedded tissues of patients were analyzed by PCR, in which the first reaction included primers for an EBNA-2 common region while the nested reaction amplified the region discriminating between EBV types 1 and 2 in separate reactions. EBV was detected in 21 of 29 BLs (72%), and type 1 virus infected the majority of EBV-positive BLs (18/21). There was a trend for younger age in children with EBV-positive BL compared to EBV-negative BL (median age 4 compared to 6 years, respectively; p = 0.056). Our study confirmed that in the southeast of Brazil BL had an intermediate association with EBV. A higher rate of EBV-associated BL was described in the northeast of Brazil. These differences are probably related to regional socioeconomic status. In conclusion, our study suggests that early infection with EBV in the background of a low socioeconomic condition associated with other environmental factors could contribute to BL in Brazil.     

Detection of Epstein-Barr Virus DNA in Reed-Sternberg Cells of Hodgkin's Disease Using the Polymerase Chain Reaction and in situ Hybridization

Thirty-one cases of Hodgkin's disease were examined for the occurrence of Epstein-Barr virus (EBV) genome by using the polymerase chain reaction (PCR) of DNA in formalin-fixed paraffin-embedded tissues and the in situ hybridization technique. The cases were subdivided into 17 cases of nodular sclerosis (NS), nine cases of mixed cellularity (MC), four cases of lymphocyte predominance (LP), and one case of lymphocyte depletion (LD). EBV DNA was detected in eight cases including four cases of NS, three cases of MC and one case of LP. The sensitivity of PCR was higher than that of Southern blot hybridization of DNA from fresh frozen tissue, because Southern blot hybridization using the Bam HI-W fragment of EBV detected virus DNA only in two of three cases which were positive by PCR. The results of in situ hybridization studies confirmed that EBV genome was localized within the nuclei of Reed-Sternberg (RS) cells and their mononuclear variants. Furthermore, double-labeling studies combining in situ hybridization and immunocytochemistry using CD30 (BerH2) and CD15 (LeuM1) as markers of RS cells, as well as pan B-marker (L26) and pan T-marker, CD45RO (UCHL1), were performed to demonstrate the phenotype of EBV DNA-positive cells, confirming that EBV DNA was present in RS cells but not in lymphocytes. The results of this study indicate a significant association between EBV and some cases of Hodgkin's disease.     

Detection of Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin's disease using the polymerase chain reaction and in situ hybridization

Thirty-one cases of Hodgkin's disease were examined for the occurrence of Epstein-Barr virus (EBV) genome by using the polymerase chain reaction (PCR) of DNA in formalin-fixed paraffin-embedded tissues and in situ hybridization technique. The cases were subdivided into 17 cases of nodular sclerosis (NS), nine cases of mixed cellularity (MC), four cases of lymphocyte predominance (LP), and one case of lymphocyte depletion (LD). EBV DNA was detected in eight cases including four cases of NS, three cases of MC and one case of LP. The sensitivity of PCR was higher than that of Southern blot hybridization of DNA from fresh frozen tissue, because Southern blot hybridization using the BamHI-W fragment of EBV detected virus DNA only in two of three cases which were positive by PCR. The results of in situ hybridization studies confirmed that EBV genome was localized within the nuclei of Reed-Sternberg (RS) cells and their mononuclear variants. Furthermore, double-labeling studies combining in situ hybridization and immunocytochemistry using CD30 (BerH2) and CD15 (LeuM1) as markers of RS cells, as well as pan B-marker (L26) and pan T-marker, CD45RO (UCHL1), were performed to demonstrate the phenotype of EBV DNA-positive cells, confirming that EBV DNA was present in RS cells but not in lymphocytes. The results of this study indicate a significant association between EBV and some cases of Hodgkin's disease.     

Sporadic Activation of Epstein-Barr Virus in Thyroid Lymphoma

The causal role of Epstein-Barr virus (EBV) in the development of B-cell lymphoma, especially in immunocompromised individuals, has been suggested. The purpose of the present study was to evaluate an association of EBV with thyroid lymphoma (TL) and chronic lymphocytic thyroiditis (CLTH) which is known to play an important role in the development of TL. Thirty cases with TL and 28 with CLTH were studied for presence or absence of EBV genome in the lesions using the polymerase chain reaction (PCR) and the in situ hybridization method. EBV genomes were detected by PCR in one and two cases with CLTH and TL. respectively. Subtyping of EBV genome was possible in one TL case showing B-type in EBNA-2 coding region. In situ hybridization revealed positive signals in the nucleus of lymphoma cells, which also expressed latent membrane protein-I. The present findings indicate that activation of EBV in TL is not common.     

Blastogenic response and EBNA induction in human lymphocytes by Epstein-Barr virus only requires B cells but not macrophages.

The postulated role of macrophages in the primary infection of human lymphocytes by Epstein-Barr virus (EBV) was examined. Macrophage removal had no effect on the blastogenic response but slightly reduced the percentage of EBNA-positive cells induced by the B 95-8 virus strain. Both the rate of DNA synthesis and the appearance of EBNA-positive cells could rather be related to the number of B cells in the infected populations. Macrophages are thus not required for the initiation of EBV-induced lymphocyte transformation.