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• W2007206568 abstract "Classical Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) infection. Although in developing countries EBV can be demonstrated in Hodgkin-Reed-Sternberg (H-RS) cells in up to 95% of HD cases, in industrialized countries only about 50% of HD cases are associated with EBV. An open question remains whether EBV in the EBV-negative cases has escaped detection by standard screening procedures due to deletions in the viral genome associated with integration of viral fragments into the host cell genome. We, among others, recently described this phenomenon in Burkitt's lymphoma cells. To investigate whether H-RS cells in latent membrane protein-1 (LMP-1)-negative HD cases harbor fragments of the EBV genome, we combined fluorescence in situ hybridization (FISH) using a set of six overlapping DNA probes spanning the whole EBV genome with immunophenotyping of fresh frozen lymphoma sections. Results in the eight cases analyzed were as follows: in three LMP-1-positive cases, FISH analysis yielded specific signals for each EBV DNA probe in H-RS cells, which had been identified by morphology and CD30 staining. In contrast, none of the EBV DNA probes hybridized to the H-RS cells in the five LMP-1-negative cases. Thus, there is no evidence for the presence of fragments of the viral genome integrated into the host cell genome in the LMP-1-negative cases. Furthermore, in the LMP-1-positive cases analyzed, no large deletions in the viral genome were detected. These results show that, in classical HD, LMP-1-negative cases do not harbor EBV DNA within the H-RS cells. Whether, in these cases, a still unknown virus contributes to the transformation and maintenance of the malignant phenotype remains to be established. Classical Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) infection. Although in developing countries EBV can be demonstrated in Hodgkin-Reed-Sternberg (H-RS) cells in up to 95% of HD cases, in industrialized countries only about 50% of HD cases are associated with EBV. An open question remains whether EBV in the EBV-negative cases has escaped detection by standard screening procedures due to deletions in the viral genome associated with integration of viral fragments into the host cell genome. We, among others, recently described this phenomenon in Burkitt's lymphoma cells. To investigate whether H-RS cells in latent membrane protein-1 (LMP-1)-negative HD cases harbor fragments of the EBV genome, we combined fluorescence in situ hybridization (FISH) using a set of six overlapping DNA probes spanning the whole EBV genome with immunophenotyping of fresh frozen lymphoma sections. Results in the eight cases analyzed were as follows: in three LMP-1-positive cases, FISH analysis yielded specific signals for each EBV DNA probe in H-RS cells, which had been identified by morphology and CD30 staining. In contrast, none of the EBV DNA probes hybridized to the H-RS cells in the five LMP-1-negative cases. Thus, there is no evidence for the presence of fragments of the viral genome integrated into the host cell genome in the LMP-1-negative cases. Furthermore, in the LMP-1-positive cases analyzed, no large deletions in the viral genome were detected. These results show that, in classical HD, LMP-1-negative cases do not harbor EBV DNA within the H-RS cells. Whether, in these cases, a still unknown virus contributes to the transformation and maintenance of the malignant phenotype remains to be established. In industrialized countries, Hodgkin-Reed-Sternberg (H-RS) cells in up to 50% of cases of classical Hodgkin's disease (HD) harbor Epstein-Barr virus (EBV) genomes commonly detected by either latent membrane protein-1 (LMP-1. immunostaining or EBV-encoded RNA (EBER) in situ hybridization (ISH)1Herbst H Steinbrecher E Niedobitek G Young LS Brooks L Müller-Lantzsch N Stein H Distribution and phenotype of Epstein-Barr virus habouring cells in Hodgkin's disease.Blood. 1992; 80: 484-491PubMed Google Scholar, 2Weiss LM Chen YY Liu XF Shibata D Epstein-Barr virus, and Hodgkin's disease: a correlative in situ hybridization, and polymerase chain reaction study.Am J Pathol. 1991; 139: 1259-1265PubMed Google Scholar, 3Wu TC Mann RB Charache P Hayward SD Staal S Lambe BC Ambinder RF Detection of EBV gene expression in Reed-Sternberg cells of Hodgkin's disease.Int J Cancer. 1990; 46: 801-804Crossref PubMed Scopus (212) Google Scholar in these cells. By combining Southern blot analysis and ISH, it has been shown that, in EBV-positive HD, the H-RS cells harbor clonal EBV copies.4Anagnostopoulos I Herbst H Niedobitek G Stein H Demonstration of monoclonal EBV genomes in Hodgkin's disease and Ki-1-positive anaplastic large cell lymphoma by combined Southern blot and in situ hybridization.Blood. 1989; 74: 810-816PubMed Google Scholar, 5Gulley ML Eagan PA Quintanilla-Martinez L Picado AL Smir BN Childs C Dunn CD Craig FE Williams Jr, JW Banks PM Epstein-Barr virus DNA is abundant, and monoclonal in the Reed-Sternberg cells of Hodgkin's disease: association with mixed cellularity subtype and Hispanic American ethnicity.Blood. 1994; 83: 1595-1602PubMed Google Scholar Furthermore, the same clonal EBV population can be detected in different tissues affected by HD during the course of the disease.6Brousset P Schlaifer D Meggetto F Bachmann E Rothenberger S Pris J Delsol G Knecht H Persistence of the same viral strain in early and late relapse of Epstein-Barr virus associated Hodgkin's disease.Blood. 1994; 82: 2447-2451Google Scholar In these cases, the expression of latent viral genes may be needed to maintain the malignant phenotype. In this context, the expression of LMP-2A in EBV-positive H-RS cells may contribute to the survival of these cells by mimicking B-cell receptor-derived signals.7Caldwell RG Wilson JB Anderson SJ Longecker R Epstein-Barr virus LPM2A drives B cell development, and survival in the absence of normal B cell receptor signals.Immunity. 1998; 9: 405-411Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar Furthermore, EBV-positive H-RS cells might be rescued from apoptotic death by activation of the nuclear factor κB through the LMP-1-mediated induction of A20 expression.8Laherty CD Hu HM Opipari AW Wang F Dixit VM The Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B.J Biol Chem. 1992; 267: 24157-24160Abstract Full Text PDF PubMed Google Scholar LMP-1 itself is known to exhibit an oncogenic potential in B cells, because LMP-1-transgenic mice develop B-cell lymphomas.9Kulwichit W Edwards RH Davenport EM Baskar JF Godfrey V Raab-Traub N Expression of the Epstein-Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice.Proc Natl Acad Sci USA. 1998; 95: 11963-11968Crossref PubMed Scopus (328) Google Scholar H-RS cells, which represent the tumor cells in classical Hodgkin's disease, are of clonal germinal center B-cell origin.10Küppers R Rajewsky K The origin of Hodgkin and Reed Sternberg cells in Hodgkin's disease.Annu Rev Immunol. 1998; 16: 471-493Crossref PubMed Scopus (270) Google Scholar It was speculated that H-RS cells as a rule do not express a B-cell receptor with high affinity to the respective antigen and that, under physiological conditions, H-RS cells would be committed to apoptosis within the germinal center.11Kanzler H Küppers R Hansmann ML Rajewsky K Hodgkin and Reed Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.J Exp Med. 1996; 184: 1495-1505Crossref PubMed Scopus (604) Google Scholar Thus, EBV infection indeed might be a crucial early step in the rescue of these cells from apoptosis and in the development of the malignant phenotype.However, because EBV-negative HD cases amount to 50% of HD cases in the Western world, the occurrence of these cases must be further elucidated. In this context, three possible explanations must be considered. First, one can speculate that EBV in these cases has never infected the H-RS cells, which accordingly must have been transformed by another mechanism. Second, EBV may have infected the H-RS precursor cells but might have been lost at later stages of lymphoma development when the H-RS cells acquired additional transforming events. If the EBV genome is frequently lost from H-RS cells during subclinical stages of the disease, the initial loss of EBV would never be perceptible during clinical manifestation of HD and, thus, could not be further investigated. Third, EBV might have infected the H-RS cells persistently, but might escape detection by standard screening methods such as immunohistochemistry or oligonucleotide ISH due to deletions in the viral genome or absence of latent antigen expression. In this scenario, fragments of the viral genome may be retained in the H-RS cells, because this retention has been demonstrated for rare cases of sporadic Burkitt's lymphoma classified as EBV-negative, based on missing Epstein-Barr nuclear antigen-1 (EBNA-1. expression.12Razzouk BI Srinivas S Sample CE Singh V Sixbey JW Epstein-Barr virus recombination, and loss in sporadic Burkitt's lymphoma.J Infect Dis. 1996; 173: 529-535Crossref PubMed Scopus (95) Google ScholarTo analyze whether such truncated EBV fragments can be detected in H-RS cells classified as EBV-negative due to the absence of the LMP-1-protein, eight cases of HD were investigated for EBV latent gene expression and detection of the viral genome. Because H-RS cells represent only up to 1% of the heterogeneous lymphoma cell population, an ISH method was established to detect EBV DNA fragments on frozen lymph node sections and to simultaneously identify the H-RS cells by fluorescence immunophenotyping. Using this method, we wanted to clarify whether, in H-RS cells that do not express EBV-derived genes, the virus persists in a truncated form.Materials and MethodsLymphoma TissueLymph node specimens of eight cases of classical HD were chosen from the files of the lymph node registry at the Institute for Pathology, University of Würzburg. Six biopsies were taken out for primary diagnosis of HD. Two biopsies were taken out at first relapse of HD. Clinical features of the HD patients are summarized in Table 1.Table 1Clinical Features of the HD PatientsCase no.Age (years)/ sexDisease subtypeDisease courseLymph node localizationH-RS cells LMP-1+ (%)H-RS cells cMSal-A (%)H-RS cells cMB-14 (%)H-RS cells cM302-23 (%)H-RS cells cM301-00 (%)H-RS cells cM302-21 (%)H-RS cells pM966-20 (%)163 /FNS1st relapseAxillar98 /100 (98)67 /100 (67)20 /23 (86.96)60 /100 (60)22 /27 (81.48)20 /30 (66.67)25 /40 (62.5)223 /FNS1st diagnosisSupraclavicular100 /100 (100)27 /33 (81.82)18 /30 (60)32 /45 (71.11)32 /44 (72.73)22 /28 (78.57)120 /200 (60)325 /MNS1st diagnosisCervical65 /100 (65)37 /47 (78.72)28 /34 (82.35)29 /30 (96.67)34 /38 (89.47)32 /35 (91.43)87 /100 (87)421 /MNS1st diagnosisSupraclavicular0 /1000 /310 /450 /300 /380 /400 /99532 /MNS1st diagnosisCervical0 /1000 /800 /800 /800 /800 /800 /100617 /FNS1st diagnosisSupraclavicular0 /1000 /400 /400 /500 /500 /500 /100740 /MNS1st relapseSupraclavicular0 /1000 /1000 /1000 /1000 /1000 /1000 /100831 /FNS1st diagnosisSupraclavicular0 /1000 /800 /800 /800 /800 /800 /60NS, nodular sclerosis. Open table in a new tab Control Cell Lines and Control HD TissueCytospins and tumor tissue sections of the Burkitt's lymphoma cell line BL60-P7,13Wolf J Pawlita M Bullerdiek J zur Hausen H Suppression of the malignant phenotype in somatic cell hybrids between Burkitt's lymphoma cells and Epstein-Barr virus immortalized lymphoblastoid cells despite deregulated c-myc expression.Cancer Res. 1990; 50: 3095-3100PubMed Google Scholar established after subcutaneous inoculation of cells into nude mice, as well as tissue sections of a known LMP-1-positive HD case of nodular sclerosis subtype obtained from the lymph node registry in Cologne, were used for the establishment of the ISH methods to detect EBV DNA. Both sources subsequently served as positive controls. The myeloid cell line U937 served as negative control.EBV DNA ProbesThe cosmid clones cMSal-A, cMB-14, cM302-23, cM301-00, cM302-21 as well as the plasmid probe pM966-20 were kindly provided by A. Polack.14Polack A Hartl G Zimber U Freese UK Laux G Takaki K Hohn B Gissmann L Bornkamm GW A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to the Epstein-Barr virus isolates.Gene. 1984; 27: 279-288Crossref PubMed Scopus (113) Google Scholar Together, these DNA probes span the whole EBV genome in an overlapping way. The cosmid clone cMSal-1 contains about 37 kb of the viral genome hybridizing to the 5′ end of the viral genome. The 3′ end of the cMSal-1-derived insert overlaps with the 5′ end of the insert of CMB–14, containing about 40 kb of the EBV genome. The 3′ end of the insert of cMB-14 overlaps with the 5′ end of the insert of the clone cM302-23 containing 29 kb of the viral genome. The 3′ end of the insert of cM302-23 overlaps with the 5′ end of the insert of cM301-00 containing about 35 kb of the viral genome. The 3′ end of the insert of cM301-00 borders on the 5′ end of the insert of cM302-21 containing about 40 kb of the viral genome. The plasmid pM966-20 contains about 17 kb of the viral genome hybridizing to the terminal repeats, as well as to the LMP-1-gene. The 5′ end of the insert ligated into pM966-20 overlaps with the 3′ end of the insert ligated into cM302-21. The 3′ end of the pM966-20-derived insert overlaps with the 5′ end of the insert ligated into cMSal-A.Fluorescence Immunophenotyping and Subsequent Fluorescence in Situ HybridizationThe fluorescence immunophenotype analysis in combination with fluorescence in situ hybridization (FISH) was performed by the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms method15Weber-Matthiesen K Pressl S Schlegelberger B Grote W Combined immunophenotyping and interphase cytogenetics on cryostat sections by the new FICTION method.Leukemia. 1993; 7: 646-649PubMed Google Scholar with minor modifications. For immunostaining, cryostat sections (10 μm) of HD-affected lymph nodes were fixed in an ice-cold mixture of methanol and glacial acetic acid (ratio, 1:1). The slides were washed twice in phosphate-buffered saline (PBS) and once in PBS supplemented with 0.1% (w/v) bovine serum albumin (BSA). The slides were then incubated with the first antibody (for CD30 staining, Ber H2; Dako, Hamburg, Germany. dilution 1:25 in PBS/0.1% BSA). After three washes with PBS/0.1% BSA, the antibody staining was detected by using a second antibody (rabbit anti-mouse Cy3; Jackson Laboratory; dilution 1:800 in PBS/01% BSA) conjugated with Cy3. This detection was enhanced using a third antibody (goat anti-rabbit Cy3; Dianova, Hamburg, Germany. dilution 1:800 in PBS/01% BSA) conjugated with Cy3. The slides were fixed in methanol/glacial acetic acid (3:1) followed by an incubation in 1% paraformaldehyde in 2× standard saline citrate (SSC). The slides were then incubated for 10 minutes in 0.1 mmol/L HCl at 37°C and washed in PBS. For subsequent FISH, the EBV DNA probes were labeled with biotin-11-dUTP (Sigma Chemical Co., St. Louis, MO) by using a nick translation kit (Life Technologies, Inc., Gaithersburg, MD). Unincorporated nucleotides were removed by chromatography (Sephadex G50; Pharmacia, Uppsala, Sweden). DNA (125 ng) was ethanol-precipitated and vacuum-dried. Salmon sperm DNA (50 μg; Sigma) was added, and the DNA was resuspended in 25 ml of hybridization mixture, containing 50. formamide/1× SSC/1× standard saline phosphate-ethylenediaminetetraacetic acid/20% dextran sulfate/0.5. Tween 20. Probe denaturation, prehybridization, hybridization, and posthybridization washes were done as previously described.16Kievits T Dauwerse JG Wiegant J Devilee P Breuning MH Cornelisse CJ van Ommen GJ Pearson PL Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization.Cytogenet Cell Genet. 1990; 53: 134-136Crossref PubMed Scopus (161) Google Scholar Hybridized probes were detected by fluorescein-isothiocyanate conjugated to avidin (Vector Laboratories, Burlingame, CA). FISH analysis was performed in a blinded fashion. Thus, the LMP-1 status of H-RS cells in each case was unknown. Specific FISH signals can be distinguished from the possible background by their circular shape, their small size, and their bright fluorescence. These signals can be mainly attributed to the nucleus of a cell and were, in general, not detected in the tissue background. In addition, large fluorescent plaques or dots of irregular shape that differ in brightness and color from FISH-derived signals were occasionally detected in the tissue sections. These plaques and dots are most likely derived from autofluorescent tissue structures because they can also be observed in unstained tissue sections.Bright-Field ImmunohistochemistryThe morphological features of all cases were assessed on hematoxylin and eosin (H&E)-, periodic acid-Schiff (PAS)-, Giemsa-, and Gömöri-stained sections of formalin-fixed, paraffin-embedded tissues. Immunoperoxidase studies were performed on paraffin-embedded lymph nodes from all patients, by a three-stage indirect immunoperoxidase technique (tissueGnost 20053; Merck, Darmstadt, Germany) after antigen retrieval by pressure boiling the slides in citrate buffer for 35 minutes. The mixture of monoclonal mouse anti-LMP-1 antibodies CS1–4 (M0897) was purchased from Dako.ResultsTo establish the FICTION method, cytospins were used of the Burkitt's lymphoma cell line BL60-P7 and tumor tissue sections13Wolf J Pawlita M Bullerdiek J zur Hausen H Suppression of the malignant phenotype in somatic cell hybrids between Burkitt's lymphoma cells and Epstein-Barr virus immortalized lymphoblastoid cells despite deregulated c-myc expression.Cancer Res. 1990; 50: 3095-3100PubMed Google Scholar established after subcutaneous inoculation of BL60-P7 cells into nude mice. In BL60-P7, five copies of EBV are integrated into the host genome. Furthermore, in this cell line the EBV genome contains a large deletion comprising the genes coding for LMP-1, EBER-1, EBER-2, and the origin of viral replication.17Wolf J Jox A Skarbeck H Pukrop T Bartnitzke S Pawlita M Diehl V Bullerdiek J Selective loss of integrated Epstein-Barr virus genome after long term cultivation of Burkitt's lymphoma X B-lymphoblastoid cell hybrids due to chromatin instability at the integration site.Virology. 1995; 212: 179-185Crossref PubMed Scopus (19) Google Scholar As expected, hybridization of the genomic EBV probes cMSal-A, cMB-14, cM302-23, cM301-00, cM302-21 resulted in bright signals on both sources, whereas hybridization of pM966-20 covering the terminal repeats, as well as the LMP-1 gene, did not reveal hybridization signals. To establish the hybridization of this EBV DNA probe on EBV genomes, tissue sections of an LMP-1-positive HD case of nodular sclerosis subtype were used. As expected, hybridization of pM966-20 revealed the detection of EBV genomes in the H-RS cells. Hybridization of none of the EBV DNA probes gave rise to a positive signal in U937 cells known to be of myeloid origin. In the following experiments, the cell line BL60-P7 was used to control the quality of hybridization of cMSal-A, cMB-14, cM302-23, cM301-00, and cM302-21, whereas the hybridization of pM 966-20 was controlled by using the tissue sections of the above mentioned EBV-positive HD case.To analyze LMP-1-positive and LMP-1-negative HD cases for the presence of EBV DNA in the H-RS cells, lymphoma tissues were obtained from eight patients suffering from HD. To detect EBV DNA in H-RS cells, ISH was performed with six different DNA probes covering the whole genome of EBV onto fresh frozen tissue sections in a blinded fashion, ie, without any information concerning the LMP-1 status. Simultaneously, H-RS cells were identified within the sections by fluorescent CD30 immunostaining. For all DNA probes, multiple hybridization signals were seen within the nuclei of the H-RS cells of three cases, indicating the presence of multiple copies of EBV in these cells (Figure 1). The hybridization signals were broadly distributed throughout the nucleus. No substantial deletion of the viral genome was observed in any of the three cases, because FISH analysis of every EBV DNA probe covering at least 17 kb to nearly 40 kb of the entire EBV genome resulted in several hybridization signals. In contrast, none of the EBV probes used in FISH analysis revealed a hybridization signal within the nuclei of H-RS cells of the remaining five cases (Figure 2). When comparing the FISH results with the results obtained from immunostaining to detect LMP-1 expression, it became evident that expression of LMP-1 was detected exclusively in the three cases in which FISH analysis revealed the detection of the viral genome (Figure 3). In the remaining five cases the H-RS cells did not express LMP-1. However, the occasional detection of hybridization signals in very few small CD30-negative cells indicated the presence of EBV-positive bystander cells in the vicinity of the LMP-1 and EBV-negative H-RS cells in lymph node sections of these five cases (Figure 2). The frequency of those cells varied from 0 to 2 cells per lymph node section. The detection of at least 1 to 2 EBV-positive, CD30-negative small cells in each patient indicated that also the patients suffering from EBV-negative HD were carriers of EBV.Figure 2Combined immunophenotype analysis and FISH. Combined immunophenotype analysis and FISH revealed absence of EBV DNA in CD30-positive H-RS cells obtained from 5 of 8 HD specimens analyzed (a–e). f: FISH-positive CD30-negative non-H-RS cells. Red staining. Detection of CD30 by using the anti-CD30 antibody Ber-H2, FISH signals (green) are marked by arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Detection of LMP-1 in H-RS cells. In three of the eight HD cases analyzed, expression of LMP-1 was detected in the H-RS cells by immunohistochemistry.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionIn a considerable proportion of HD cases, H-RS cells harbor clonal EBV genomes. It has been speculated that EBV contributes to the transformation and maintenance of the malignant phenotype of H-RS cells in these cases. Because the detection of EBV in H-RS cells is mainly based on the detection of the latently expressed gene LMP-1 or on the detection of EBER transcripts, it remained an open question whether, in H-RS cells classified as EBV-negative, the EBV genome might have escaped standard screening methods, due to the absence of latent gene expression. Indeed, the expression level of LMP-1 in EBV-positive H-RS cells varies from cell to cell.18Teramoto N Cao L Kawasaki N Tonoyama Y Sarker AB Yoshino T Takahashi K Akagi T Variable expression of Epstein-Barr virus latent membrane protein I in Reed-Sternberg cells of Hodgkin's disease.Acta Med Okayama. 1996; 50: 267-270PubMed Google Scholar By EBER ISH, the detection level of EBV-positive H-RS cells can be increased. However, the detection of EBV DNA, rather than the detection of viral gene expression, represents the most sensitive method to detect the presence of EBV in the lymph node sections.19Lauritzen AF Hording U Nielsen HW Epstein-Barr virus, and Hodgkin's disease: a comparative immunological, in situ hybridization, and polymerase chain reaction study.APMIS. 1994; 102: 495-500Crossref PubMed Scopus (26) Google ScholarIn Burkitt's lymphoma cells not expressing the EBNA-1 gene, the presence of integrated EBV genomes harboring deletions was demonstrated by Southern blot analysis.12Razzouk BI Srinivas S Sample CE Singh V Sixbey JW Epstein-Barr virus recombination, and loss in sporadic Burkitt's lymphoma.J Infect Dis. 1996; 173: 529-535Crossref PubMed Scopus (95) Google Scholar Thus, the use of DNA detection methods might be more successful in searching for viral fragments that cannot be detected by EBER ISH or immunostaining for LMP-1 due to the deletion of the respective genes. However, in H-RS cells, Southern blot analysis is not useful to detect EBV genomes because H-RS cells represent only a minority of cells within the affected tissue that may contain, in addition, EBV-infected nonmalignant B cells. Thus, from the detection of EBV genomes in HD tissue sections by Southern blot analysis the existence of EBV in H-RS cells cannot be directly deduced.To detect EBV DNA in single H-RS cells, we performed FISH analysis in combination with immunofluorescence. For FISH we used a panel of overlapping EBV DNA clones spanning the whole EBV genome, which also allows detection of viral genomes carrying large deletions. As a control for the detection of an EBV fragment clonally integrated into the host cell genome, the cell line BL-60 p7 was used. In this cell line, the integrated EBV harbors a deletion of about 20 kb covering the left and right termini of EBV as well as the genes for LMP-1 and EBER-1 and -2.20Wolf J Pawlita M Klevenz B Frech B Freese UK Müller-Lantzsch N Diehl V zur Hausen H Down-regulation of integrated Epstein-Barr virus antigen 1 and 2 genes in a Burkitt's lymphoma cell line after somatic cell fusion with autologous EBV-immortalized lymphoblastoid cells.Int J Cancer. 1993; 53: 621-627Crossref PubMed Scopus (8) Google Scholar Absence of this region due to viral integration was also found in primary Burkitt's lymphoma tissues12Razzouk BI Srinivas S Sample CE Singh V Sixbey JW Epstein-Barr virus recombination, and loss in sporadic Burkitt's lymphoma.J Infect Dis. 1996; 173: 529-535Crossref PubMed Scopus (95) Google Scholar and after the infection of a Burkitt's lymphoma cell line with the B95–8 virus.21Trivedi P Cuomo L de Campos-Lima PO Imreh MP Kvarnung K Klein G Masucci MG Integration of a short Epstein-Barr virus DNA fragment in a B95–8 virus converted Burkitt lymphoma line expressing Epstein-Barr nuclear antigens EBNA2 and EBNA5.J Gen Virol. 1993; 74: 1393-1398Crossref PubMed Scopus (5) Google Scholar Among the EBV DNA clones used in our analysis, the plasmid pM966-20 covers this region including the gene for LMP-1. As expected, in BL60-P7 cells this probe did not result in a hybridization signal, whereas the hybridization of all other cosmid clones resulted in bright hybridization signals. However, hybridization of pM 966-20 and of each of the other EBV DNA probes on tissue sections of three LMP-1-positive HD cases revealed hybridization signals in H-RS cells. Owing to its smaller size, the hybridization signals of pM966-20 on single EBV copies were dim but clearly visible. Thus, no large deletion of the EBV genome, affecting fragments containing the sequence recognized by at least one EBV DNA probe, was detected within the cases in which the H-RS cells express LMP-1. These results suggest the presence of the whole EBV genome in the H-RS cells of these cases.By FISH on interphase nuclei, the EBV genomes can be detected in H-RS cells. The pattern of hybridization signals that are distributed throughout the nucleus of H-RS cells would be compatible with the presence of multiple episomal copies of EBV. Indeed, when EBV latently infects B cells, the majority of copies persist in an episomal form.22Dambaugh T Hennessy K Fennewald S Kieff E The virus genome and its expression in latent infection.in: Epstein MA Achong GG The Epstein-Barr Virus: Recent Advances. William Heinemann, London1986: 13-45Google Scholar Moreover, integration of part of the viral genomes has been observed in vitro for B cells that carry multiple episomal copies of EBV.23Delecluse HJ Bartnizke S Hammerschmidt W Bullerdiek J Bornkamm GW Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines.J Virol. 1993; 67: 1292-1299PubMed Google Scholar, 24Anvret M Karlsson A Bjursell G Evidence for integrated EBV genomes in Raji cellular DNA.Nucleic Acids Res. 1984; 12: 1149-1161Crossref PubMed Scopus (17) Google Scholar, 25Srinivas SK Sample JT Sixbey JW Spontaneous loss of viral episomes accompanying Epstein-Barr virus reactivation in a Burkitt's lymphoma cell line.J Infect Dis. 1998; 177: 1705-1709Crossref PubMed Scopus (43) Google Scholar However, by using FISH on interphase nuclei, whether EBV persists exclusively in H-RS cells in an episomal status cannot be distinguished, nor, in addition, whether integration into the host cell genome took place.It was speculated that H-RS cells classified as EBV-negative might have been initially infected by EBV, but that EBV was lost during lymphoma progression.26Jox A Rohen C Belge G Bartnitzke S Pawlita M Diehl V Bullerdiek J Wolf J Integration of Epstein-Barr virus in Burkitt's lymphoma cells leads to a region of enhanced chromosome instability.Ann Oncol. 1997; 8: 131-135Abstract Full Text PDF PubMed Scopus (31) Google Scholar If integration of a fragment of the viral episomes into the host cell genome is a frequent event during EBV infection, one would expect detection of these integrated fragments in EBV-negative H-RS cells as vestiges of a former infection. Such integrated viral fragments resulting from a recombination event were detected in some cases of Burkitt's lymphoma (see above). Furthermore, loss of the episomal copies of EBV was observed in several Burkitt's lymphoma cell lines so that, in these cell lines, integrated EBV genomes were exclusively retained.23Delecluse HJ Bartni" @default.
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• W2007206568 title "Detection of Epstein-Barr Virus in Hodgkin-Reed-Sternberg Cells" @default.
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