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Demonstration of Epstein-Barr Virus Replication in Reed-Sternberg Cells
of Hodgkin's Disease
By Pierre Brousset, Hans Knecht, Bent Rubin, Emmanuel Drouet, Shashikant Chittal, Fabienne Meggetto, Tala1 AI Saati,
Edith Bachmann, GBrard Denoyel, Alain Sergeant, and Georges Delsol
Epstein-Barr virus (EBV) is detectable in approximately
40% of cases of Hodgkin's disease (HD). The viral genomes remain latent but positivestaining with anti-ZEBRA
antibody in a small fraction of Reed-Sternberg (RS) cells of
some cases of HD would suggest possible activation of
EBV replicationwithin these cells. W e report the investigation of 40 cases of EBV-associated HD (including 5 human
immunodeficiency virus [HIVI-positive cases) using antiZEBRA antibodies. Positive staining was found in only
three (HIV-negative) cases. One of these three cases
showed approximately 1 % of ZEBRA-positive tumor cells,
whereas the other two cases showed rare positive cells. In
the case with 1% ZEBRA-positive cells, a strong signal
was obtained with anti-EA-R antibody and BHLFl oligoprobes, which indicatedearly gene expression. EBV replication could be shown in this case by nonisotopic in situ
DNA-DNA hybridization, which showed markedly increased numbers of EBV genomes in a few RS cells. Viral
replication was confirmed using reverse transcriptase and
polymerase chain reaction that detected transcripts from
the BLLFl gene encoding for the membrane antigen
gp350/220. EBV replication in RS cells seems to be an
exceptional event but may provide clues to mechanisms of
control of viral latency and assume clinical implications in
the future.
0 1993 by The American Society of Hematology.
H
cycle. In this study, we reexamined 40 cases of EBV (DNA
and LMP1)-positive H D by using anti-ZEBRA antibodies.
Additionally, anti-EA-R (early antigen restricted), anti-MA
(membrane antigen), and anti-VCA (viral capsid antigen)
antibodies were applied to ZEBRA-positive cases. Nonisotopic in situ hybridization was performed in the same cases
using DNA probes to detect EBV genomes and antisense
oligonucleotides to detect EBER 1/2 gene transcripts (detected during l a t e n ~ y ) ' , ~ . 'and
~ , ' ~BHLFl gene transcripts
(specific of lytic ~ y c l e ) . ' ~Combined
,'~
reverse transcription
and polymerase chain reaction (RT-PCR) were used to
search for BLLFl gene (gp 350/220) transcription" when
material for RNA extraction was available. Last, DNA extraction and Southern analysis with Xho I probe were performed to determine whether EBV genomes are monoclonal or polyclonal and in circular (with fused termini) or in
linear form2'
ODGKlN'S DISEASE (HD), particularly of mixed
cellularity (MC) subtype, is considered to be EBV
associated in about half of the cases. Monoclonal EpsteinBarr virus (EBV) genomes have been found in the neoplastic cells of H D by different molecular methods,lS6and EBV
latent gene transcripts and proteins have been detected by in
situ hybridization and immunohistochemi~try.~-~-'~
The
viral gene expression of EBV-infected Reed-Sternberg (RS)
cells is now better known (EBNAZ-, LMPl+, EBER1/2
transcripts') and confirms that the virus remains latent in
this disease. Pallesen et all' have recently shown that activation of EBV replication could be suspected because of the
detection of ZEBRA (BZLFl) protein in rare cells of a few
cases of EBV (LMP1)-positive HD. This protein, ZEBRA,
originates from the BZLFI (BamHI Z Left Frame 1) sequence and is known to be involved in the switch of EBV
from a latent to lytic (productive) ~ y c l e . ' ~The
- ' ~ expression
of ZEBRA in rare cells would suggest that in these cells,
EBV replication is activated despite the as yet unsuccessful
detection of early (EA), membrane (MA), and viral capsid
(VCA) antigens.'o2'' Thus, it could be considered that this
activation may induce an abortive instead of a full lytic
From the Laboratoire d'Anatomie Pathologique, CHU Purpan.
Toulouse, France; Division d'Himatologie du Dbpartement de
Midecine, Lausanne, Switzerland; CNRS/CRPG. CHU Purpan,
Toulouse, France; Dbpartement de Biologie Clinique, Institut Pasteur, Lyon, France; and ENS-CNRS UMR49, Ecole Normale
Supirieure de Lyon, Lyon, France.
Submitted February 1. 1993; accepted March 15, 1993.
Supported by grantsfvom INSERM (CRE), Fidiration Nationale
des Centres de Lutte Contre le Cancer and Fondation pour la Recherche Midicale.
Address reprint requests to Pierre Brousset, MD, Laboratoire
dgnatomie Pathologique, Chu Purpan, Place du Dr Baylac, 31059,
Toulouse Cedex, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8203-0033$3.00/0
872
MATERIALS AND METHODS
Cases selection. We selected 40 cases of H D (5 acquired immunodeficiency disease (AIDS)-related cases) in which EBV DNA and
EBV latent membrane protein 1 (LMPl) were detected by in situ
hybridization and immunohistochemistry, respectively," in tissues
processed by the (ModAMeX) method." Details of the ModAMeX
method have been already de~cribed.'.'~Immunohistochemistry
was performed by the alkaline phosphatase and monoclonal antialkaline phosphatase (APAAP) method." Dual labeling was also
performed by combined in situ hybridization with BamHI W probe
and immunohistochemistry with anti-CD30/HSR4, anti-CDI 5 /
ION1 (Immunotech, Marseille, France), anti-LMP1 (CS 1-4, Dako,
Copenhagen, Denmark)," or anti-ZEBRA antibodies (see below).
Immunohistochemistry. All 40 cases were investigated on ModAMeX sections by APAAP technique with four different anti-ZEBRA antibodies: clone BZI (Dako) and clones (AZ94, AZ125,
AZI 30). These three AZ-antibodies were produced with recombinant ZEBRA protein obtained after transfecting b12 1 bacteria with
the expression factor pRT3CZ25 as described el~ewhere.'~
The ZEBRA protein obtained was used to immunized BALB/c mice. The
three MoAbs isolated consisted of clone AZ 125 (IgG2a) against the
region located between amino acids 59 and 93 (transcriptional activation domain) and the clones AZ94 and A21 30 (IgC1 and IgC2b)
against ZEBRA basic region located between amino acids 145 and
200. ZEBRA-positive cases were immunostained with monoclonal
Blood, VOI 82,NO 3 (August l ) , 1993:pp 872-876
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a73
EBV REPLICATION IN HODGKIN’S DISEASE
anti-EA-R (DuPont, Billerica, MA), anti-MA (DuPont), and antiVCA (DuPont) antibodies.
In situ hybridization and Southern analysis. ZEBRA-positive
cases were also investigated by in situ hybridization using fluorescein isothiocyanate (FlTC)-labeled antisense EBER 1/2 and
BHLFl oligonucleotides on routinely processed sections. In situ
hybridization was performed with the Dako hybridization kit as
described previou~ly.~~
Southern blotting was performed in ZEBRA-positivecases when frozen tissues were available. The details
of this procedure have been described elsewhere?6 The total DNA
was digested with BumHI (Bethesda Research Laboratories [BRL],
Rockville, MD). After transfer and immobilization onto nylon
membrane, digested DNA was hybridized with BamHI W (3.1 kb)
and Xho I (1.9 kb) EBV probes labeled by random priming with
dCTP-alpha-”P.
RNA extraction and RT-PCR. RNA extraction was performed
in 10 EBV-positive cases as previously described.z6After RT with
an oligo dT primer, PCR amplification of the cDNA was performed
as follows: precycle at 95OC for 2 minutes, and cycles 1 to 25 and 26
to 50 at 93°C for 1 minute (strand separation),60°C for 30 seconds
(annealing), and 72°C for 1 minute (primer extension). Between
cycles 25 and 26 and after cycle 50, the reaction was held at 72°C
for 5 minutes. The primers for the BLLFl gene were, 5‘-TATCCACTGCAGGTGATGTT-3 (downstream) and 5’-AGCCTGGAATCTGTAATGT-3’(upstream). They delineate a 335-bp sequence
of the BLLFl fragment (late gene) of the EBV genome encoding for
membrane antigen (gp 350/220). The internal control probe consisted of 5’-TTAAGTTTGCCAACGTCAGC-3’and was 5’ end labeled in gamma with 32P.The details of the protocol with specific
controls have been described e1~ewhere.l~
To confirm that a specific
BLLFl amplification product was obtained from cDNA and not
from contaminating genomic DNA, a part of the LMPl (latent
membrane protein 1) gene including the first 87-bp long intron was
amplified simultaneouslyusing the same cDNA as a template. The
primers used were 5’-GACTGGACTGGAGGAGCCCTC-3’ ( u p
stream) and 5’-TGCCTGTCCGTGCAAATTCCA-3’ (downstream). The control of specificity was performed with the internal
oligoprobe 5’-AGACCTTCTCTGTCCACTTG-3’. Amplification
products from cDNA templates were 177 bp long, whereas amplification products from genomic DNA were 255 bp long. Last, expression of human recombination activating gene 1 (RAGl)” was
searched for with the following primers: HK3 1, S-GCCATGAAGAGCAGTGAATTA-3 (upstream), HK32, 5-AGGAATTAACTCACAAACTGC-3’ (downstream), and the internal oligoprobe 5’GAGACAGTCCCTTCCATAGAT-3’.
RESULTS
All results are summarized in Table 1. Among the 40
cases of HD tested, three cases ofthe MC subtype, occurring
in nonimmunocompromised patients, showed ZEBRA exTable 1. Results Obtained in the 40 EBV-Positive Cases of HD
With the Different Methods Used to Detect EBV Replication
Cases
Anti-ZEBRA
Anti-EA-R
6HLFl
EBV DNA+++
BLLF1
S6
1/10=
2/27
Same Case H1
PIT
3/40
113
113
113
Abbreviations: PIT,positive/tested; EBV DNA+++, strong signal obtained by in situ DNA-DNA hybridization. SB, Southern blotting.
10 EBV-positive cases have been tested.
t Absence of linear component of EBV genome including the case
with BLLFl transcripts (HI).
pression on ModAMeX sections. The staining was nuclear,
strong, and exclusively localized to large cells (Fig 1A). The
signals obtained were comparable with the four antibodies
used. Approximately 1% of tumor cells were labeled in one
case (case Hl), and rare scattered cells were labeled in the
other two. It was not possible to detect MA or VCA antigens
in these three cases, but the results were difficult to interpret
because of the background yielded by these antibodies on
tissues sections, particularly by cross-reactivity with macrophages. However, a strong labeling was noted in few cells
only in the case H 1 containing numerous ZEBRA-positive
cells, with anti-EA-R antibody (cytoplasmic) and BHLFl
oligoprobes (nuclear) (Fig 1B). The number of EA-R/
BHLFl-positive cells was slightly lower than those with
anti-ZEBRA antibodies. Surrounding small lymphocytes
were distinctly negative with anti-ZEBRA and anti-EA-R
antibodies and by in situ hybridization using BHLFl oligoprobe in all investigated cases. The results of the in situ
hybridization with BamHI W probe indicated weak but
clear nuclear signals in a high percentage of cells in these 3
cases as well as in the 37 other cases (Fig 2, arrows). But the
in situ hybridization signals with BamHI W probe were exceptionally strong in few tumor cells of the case H 1 containing numerous ZEBRA+/EA-R’/BHLFl+ cells (Fig 2) instead of the usual scant granular hybridization product in
the nuclei of most infected cells (Fig 2, arrows). This was
indicative of greater quantity of the hybridization targets.
As expected, the greatest proportion of tumor cells and few
scattered small lymphocytes were stained with EBER 1/2
oligoprobes in all cases. Double labeling obtained with biotinylated BamHI W probe and anti-ZEBRA antibodies
clearly indicated that all cells with strong in situ hybridization signal were ZEBRA positive, but not all ZEBRA-positive cells contained abundant EBV DNA. Double labeling
with anti-CD30/HSR4 or anti-CD1 5/ION 1 antibodies and
BumHI W probe confirmed that hybridization signal was
restricted to CD 15+/CD30+cells. Last, a careful examination of the routine sections of the case H 1 showed the presence of big inclusions with irregular outline (possibly related
to high amount of viral genomes), different from the usual
large nucleolus of RS cells, and frequent mitotic features
(Fig IC). Southern analysis confirmed the presence of EBV
genomes in the case H1 and in another ZEBRA-positive
case. The Southern analysis also demonstrated that the viral
genomes were monoclonal and in a circular episomal configuration (with fused terminal repeats) with Xho I probe,
which hybridized a unique band between 9 and 23 kb in
size, but a linear component was not detectable. Nevertheless, EBV replication could be finally confirmed in case H 1
by RT-PCR, which detected transcripts of the BLLFl gene
with a specific band 335 bases long positively hybridized
with internal oligoprobe (Fig 3). No BLLFl gene transcription was detectable in the other cases, including one case
containing rare ZEBRA-positive cells. No RAG-1 gene expression (Fig 3) and no Ig and T-cell receptor gene rearrangements were found for these two cases (not shown).
DISCUSSION
This study confirms the activation of replication of EBV
in few cells of a minority of EBV-associated HD as shown
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074
BROUSSET ET AL
Fig 1. (A) Positive nuclear staining with antiZEBRA antibody in a RS cell in case H1. ModAMeX
sections X 800, APAAP method, and new fuscin.
Hematoxylincounterstaining. (B)Same case showing strong nuclear labeling of rare tumor cells by in
situ hybridizationwith FITC-labeled BHLFl oligonucleotides. Routinely processed sections X 250,
new fuschin. Hematoxylin counterstaining. (C)
Same case showing the presence of RS cells with
big nuclear inclusions (arrow). Routinely processed
section X 310. Giemsa counterstaining.
by Pallesen et al." This could be demonstrated by immunohistochemistry with anti-ZEBRA antibodies in 3 of 40
cases, with a higher proportion of cells in 1 of 3 cases (case
H 1). However, it was not possible to detect the expression of
MA or VCA by the mean of immunohistochemistry. It
would seem therefore that either EBV is involved in an
abortive lytic cycle or that these different antigens are expressed at a low level in EBV-infected RS cells and thus are
not detectable by our immunohistochemical methods with
Fig 2. In situ hybridization findings with
biotinylated BamHl W probe in the case H1
containing numerous ZEBRA-positive cells.
Exceptionally strong nuclear signal in the
form of sizable dots in 1%of cells (bottom
left and insets) instead of the usual scant
granular hybridization product in the nuclei
of most infected cells (arrows). ModAMeX
sections, X 310, BCIP-NBT. Methyl green
or no counterstaining.
the antibodies tested. Using both anti-EA-R antibody and
antisense oligonucleotides specific for BHLFl transcripts
(hallmark of the lytic cycle),'"'* it was possible to detect
positive RS cells in one case only (case H 1). In this case,
numerous tumor cells were also positive with anti-ZEBRA
antibodies. Thus, the pathways of activation of EBV replication involve an immediate early gene, BZLFl, and at least
two of the early gene expression. This finding prompted us
to analyze the results of the in situ hybridization with DNA
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EBV REPLICATION IN HODGKIN'S DISEASE
Fig 3. Detection of the BLLFl mRNA in HD using RT-PCR. cDNAfrom HD case H1 (ZEBRA+)and
H2 (ZEBRA-) were assessed for BLLFl and LMPl
gene expression by RT-PCR. Genomic DNA from
Jijoye (Ji)cell line sewed as positive control. (a) Gel
electrophoresis profile of the amplification products. With the BLLFl set of primers, specific bands
(335 bp) are seen in the positive control Ji and also
in H1 lanes. No amplification product is detectable
for case H2. Amplification with the LMPl set of
primers results in a 255-bp long band of genomic
DNA-specific amplification product in lane Ji and
in two 177-bp long bands of cDNA amplification
products in lanes H1 and H2. Absence of additional
bands at 255 bp indicates high purity of input
cDNA in H1 and H2 and simultaneously provesthat
the BLLFl amplification product detected in H1
originates from cDNA (mRNA). No RAG-1 gene expressionwas detected in both cases H1 and H2. (b)
Southern blot of gel shown in (a). Internal oligoprobe hybridization proves specificity of the BLLFl
amplification products. (c) Southern blot shown in
(b) was washed and rehybridized with an
LMPl -specific intemal oligonucleotide. Specific
signals are seen in the genomic DNA lane (Ji) and in
both HD cases H1 and H2. Absence of additional
band at 255 bp confirms the purity of cDNA in H1
and H2.
a75
BLLFl
RAG1
Ji H1 H 2 Ji H1 H2 Ji
LMP
H1
H2
I
i
!
5
3
335-
- 255 gDNA
-177
a
cDNA
335 -
b
- 255 g DNA
- 177 cDNA
C
BarnHl W probe to see whether the signal was accentuated
in a few cells, as it would be expected after viral replication.'2*28The use of nonisotopic in situ hybridization was
helpful because ofthe high resolution ofthe labeling and the
absence of background.6*28.Moreover, this method allows
semiquantification ofthe numberofEBV genomes. Interestingly, in the case containing 1% of ZEBRA-positive cells
(HI), it was possible to show a signal of unusual strength in
few cells, similar to that commonly observed in EBV-infected epithelial cells of oral hairy leukoplakia. This strong
signal seems to indicate that, in rare cells (ZEBRA', EA-R',
BHLFI'), EBV replication does occur and that, in these
cells, EBV genomes are probably in high copy number, similar to the demonstration of Tagaki et aI2' in EBV-infected
cell lines.
The expression of BLLFl gene (expressed after viral replication and encoding for membrane antigen gp 350/220)
confirmed the replication of EBV in H D tissues as previously shown in one case by Joske et aI.l9 Together with the
labeling with anti-ZEBRA and anti-EA-R antibodies and
with BHLFI oligoprobe, the demonstration of BLLFl expression clearly indicates that EBV replication occurs
within RS cells. The negative staining of non-neoplastic
surrounding lymphocytes with anti-ZEBRA and anti-EAR antibodies and with BHLFI oligoprobe makes it unlikely
that this replication could take place in these non-neoplastic
cells. One would expect that viral replication yields linear
EBV genomes.l6V2' This finding could not be confirmed by
Southern blot analysis and only the circular form of EBV
genome was detected in this case as well as in the lymphoblastoid cell line used as control. The most probableexplanation is that only few cells are involved by EBV replication,
and thus linear EBV DNA cannot be detected by Southern
blotting because of the extreme dilution in total cellular
DNA. The activation of EBV replication in H D occurs in
sporadic cases and seems to involve few cells in each case.
Even though we have confirmed the evidence of EBV replication. it would be difficult to consider the occurrence of a
full lytic cycle in all ZEBRA-positive cells but consideration
of abortive lytic cycles is more appropriate. Instead of being
responsible for the cell death. these abortive lytic cycles
might augment the cell growth and thus the course of the
disease. Indeed, ZEBRA protein is known to be partially
homologous (ie, DNA-binding domains) to c - ~ o s 'and,
~ . ~in~
addition, is capable of transactivating the promoter of cOS.'^ So it is conceivable that during abortive lytic cycles,
ZEBRA and other early gene products such as BMLFI (EAD transa~tivator)'~
might play a role in the proliferation of
EBV-infected RS cells. This was further suggested by the
high number of mitotic figures observed on conventional
examination in the case H I showing RS cells with replicative EBV genomes. EBV replication in RS cells is apparently an unusual event with as yet no clear etiologic and
clinical implications. Spontaneous activation of replication
is constantly observed, although in rare cells (
to
in lymphoblastoid cell lines.30The frequency of this phenomenon compared with its rarity in H D could be explained by the lack of host control in in vitro conditions. An
immunologic mechanism could be therefore envisaged for
the control of viral latency, as suggested by Pallesen et
who found a high incidence of ZEBRA-positive cases in
AIDS-related non-Hodgkin's lymphomas. Surprisingly, no
ZEBRA expression could be detected in our five AIDS-related cases of HD.
ACKNOWLEDGMENT
We thank Daniel Roda. Laboratoire d'Anatomie Pathologique.
Chu Purpan. Toulouse. France. for his expert technical assistance.
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876
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1993 82: 872-876
Demonstration of Epstein-Barr virus replication in Reed-Sternberg
cells of Hodgkin's disease
P Brousset, H Knecht, B Rubin, E Drouet, S Chittal, F Meggetto, TA Saati, E Bachmann, G
Denoyel and A Sergeant
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