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(American Journal of Pathology. 2000;156:209-216.)
© 2000 American Society for Investigative Pathology

Regular Articles

Detection of Epstein-Barr Virus in Hodgkin-Reed-Sternberg Cells

No Evidence for the Persistence of Integrated Viral Fragments inLatent Membrane Protein-1 (LMP-1)-Negative Classical Hodgkin’s Disease

Andrea Staratschek-Jox^*, Sascha Kotkowski^*, Gazanfer Belge, Thomas Rüdiger, Jörn Bullerdiek, Volker Diehl^* and Jürgen Wolf^*

From the Department of Internal Medicine I,^*
University of Cologne, Cologne, Germany; the Center for Human Genetics and Genetic Counseling,
University of Bremen, Bremen, Germany; and the Institute for Pathology,
University of Würzburg, Würzburg, Germany

	Abstract

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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.

	Introduction

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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.¹²

To 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 Methods

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Lymphoma Tissue

Lymph 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 .

Cytospins and tumor tissue sections of the Burkitt’s lymphoma cell line BL60-P7,¹³ 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 Probes

The 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.¹⁴ 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 Hybridization

The 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 method¹⁵ 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 2x 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/1x SSC/1x standard saline phosphate-ethylenediaminetetraacetic acid/20% dextran sulfate/0.5% Tween 20. Probe denaturation, prehybridization, hybridization, and posthybridization washes were done as previously described.¹⁶ 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 Immunohistochemistry

The 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.

	Results

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To establish the FICTION method, cytospins were used of the Burkitt’s lymphoma cell line BL60-P7 and tumor tissue sections¹³ 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.¹⁷ 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.

View larger version (97K): [in this window] [in a new window]   Figure 1. Immunophenotyping and fluorescence in situ hybridization on fresh-frozen HD tissue sections. H-RS cells were stained using the anti-CD30 antibody (red staining, Ber-H2). Subsequently, multiple EBV copies were visualized by hybridizing various EBV DNA probes (bright green signals marked by arrows). An example for each probe is given (a–f). Note that the signals obtained after hybridization of the pM966–20 probe are dim, but clearly visible. Large fluorescent plaques of irregular shape are due to autofluorescent tissue structures.

View larger version (87K): [in this window] [in a new window]   Figure 2. Combined 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 larger version (124K): [in this window] [in a new window]   Figure 3. Detection 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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In Burkitt’s lymphoma cells not expressing the EBNA-1 gene, the presence of integrated EBV genomes harboring deletions was demonstrated by Southern blot analysis.¹² 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 -2.²⁰ Absence of this region due to viral integration was also found in primary Burkitt’s lymphoma tissues¹² and after the infection of a Burkitt’s lymphoma cell line with the B95–8 virus.²¹ 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.²² Moreover, integration of part of the viral genomes has been observed in vitro for B cells that carry multiple episomal copies of EBV.^23-25 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.²⁶ 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.^23,25

To detect EBV fragments in H-RS cells classified as EBV-negative due to the absence of LMP-1 expression, FISH analysis was performed on five LMP-1-negative HD cases, using the whole set of EBV DNA probes. None of the EBV probes gave rise to a hybridization signal in the H-RS cells of these five cases. Thus, there is no indication for the former infection of these cells by EBV. Moreover, in these cases, the malignant phenotype of the tumor cells is not maintained by a truncated EBV genome that escapes detection by standard screening methods.

	Footnotes

 
Address reprint requests to Andrea Staratschek-Jox, M.D., Department of Internal Medicine I, University of Cologne, D-50924 Cologne, Germany.

Supported by a grant from the Frauke Weiskam Stiftung and by a grant from the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 502.

Accepted for publication September 16, 1999.

	References

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Staratschek-Jox, A. Articles by Wolf, J. Search for Related Content PubMed PubMed Citation Articles by Staratschek-Jox, A. Articles by Wolf, J.