, . 182: 475–479 (1997)

DISAPPEARANCE OF THE EPSTEIN–BARR VIRUS IN ARELAPSE OF HODGKIN’S DISEASE

- , , , , *

Consultation and Reference Center for Lymph Node Pathology and Hematopathology, Institute of Pathology,Klinikum Benjamin Franklin, Free University of Berlin, Berlin, Germany

SUMMARY

Hodgkin’s disease (HD) is associated with the Epstein–Barr virus (EBV) in approximately half of cases. This is a report of a case ofnodular sclerosing HD of the B-cell type that was associated with EBV in the initial manifestation, but was found to be EBV-negativein the relapse of the tumour. Both tumours displayed similar clinical, pathological, and immunohistochemical features. This findingimplies that in a given individual EBV can be lost from malignant tumours and therefore shows that the EBV infection is not requiredto maintain neoplastic growth of HD tumour cells. ? 1997 by John Wiley & Sons, Ltd.

J. Pathol. 182: 475–479, 1997.No. of Figures: 3. No. of Tables: 0. No. of References: 11.

KEY WORDS—Hodgkin’s disease; Epstein–Barr virus infection

INTRODUCTION

Even though its clinical and pathological featureshave been known for a long time, the pathogenesis ofHodgkin’s disease (HD) is still obscure.1 The contribu-tion of the Epstein–Barr virus (EBV) to the neoplastictransformation remains difficult to assess, principallybecause the genome of this herpesvirus is found in theneoplastic cells of HD in only 50 per cent of the cases.2In this study, we report a case of HD that was associatedwith EBV in the initial tumour, whereas the tumour cellswere free of virus at relapse.

CASE REPORT

A 5-year-old boy presented with multiple left cervicaland supraclavicular enlarged lymph nodes as well aswith a pleural effusion. Staging procedures revealeda neoplastic extension to the mediastinum. A supra-clavicular biopsy showed the histological features ofHD with beginning nodular sclerosis. Polychemo-therapy was commenced, followed by local radio-therapy. After 6 months of treatment, the patient wasconsidered to be in complete remission. One year later,the disease relapsed in the right cervical region and inthe mediastinum. A biopsied lymph node showed essen-tially the same features as those in the initial biopsy. The

patient underwent a new chemotherapy regimen associ-ated with radiotherapy and is clinically well 6 monthslater.

MATERIAL AND METHODS

MaterialParaffin-embedded, Giemsa, haematoxylin and eosin

(H & E), and PAS stained sections of the lymph nodespecimens from the initial manifestation and from therelapse were simultaneously reviewed. One formalin-fixed, paraffin-embedded tissue block sample from theinitial lesion and four from the tumour relapse wereused for immunohistological and molecular biologicalexperiments.

Immunohistology

Immunohistochemical studies employed monoclonalantibodies [Ber-H2 (CD30), L26 (CD20), C3D1 (CD15),polyclonal CD3, CS1–4 (LMP-1)] that are reactive inparaffin sections. For antigen retrieval, the sections wereboiled using a pressure cooker prior to immuno-staining.3 Bound antibodies were visualized by thealkaline phosphatase/anti-alkaline phosphatase method(APAAP).4 All antibodies as well as the APAAP com-plex were obtained from Dako (Glostrup, Denmark).

In situ hybridization

In situ hybridization for the detection of EBV-specificRNAs (EBER) was performed as already described.5Briefly, digoxigenin-labelled EBV-specific EBER senseand antisense RNA probes were incubated overnightwith deparaffinized tissue sections. After stringentwashes, hybridization was visualized by incubation of

*Correspondence to: Dr H. Stein, Institute of Pathology,Klinikum Benjamin Franklin, Free University of Berlin, Hinden-burgdamm 30, 12200 Berlin, Germany.

Contract grant sponsor: Deutsche Krebshilfe; Contract grantnumber: M 25/89/St 1.

Contract grant sponsor: European Community; Contract grantnumber: CHRX-CT94-0651.

CCC 0022–3417/97/080475–05 $17.50 Received 21 August 1996? 1997 by John Wiley & Sons, Ltd. Accepted 27 February 1997

the slides with digoxigenin-specific Fab fragmentscoupled to alkaline phosphatase and reaction with theNew Fuchsin substrate.

Isolation of single HRS cells from paraffin sections

Paraffin sections were stained for the EBV proteinLMP-1 (initial tumour) or CD30 (relapse) and 20immunostained Hodgkin and Reed–Sternberg (HRS)cells were isolated in each picking session. The isolatedHRS cells from each case were pooled, proteinaseK-digested (10 mg/ml), and the enzyme was inactivatedat 95)C for 10 min.6 For controls, numerous aliquotswere drawn from the buffer covering the sections duringthe isolation procedure.

Polymerase chain reaction (PCR) analysis

DNA was isolated from 20 ìm thick paraffin sectionswith an automatic DNA extractor (Applied Biosystems341A, Weiterstadt, Germany) according to the manufac-turer’s instructions. One paraffin tissue block from theinitial tumour and four from the tumour at relapse wereused for DNA extraction.A nested PCR was carried out for detection of the

EBV genome. In the first round of amplification, theBamHI-W region of the EBV DNA was amplifiedfrom 500 ng of genomic DNA using 100 ng of HPLC-purified oligonucleotides (5*-GCAGTAACAGGTAATCTCTGG-3*, nt 13 980 to 14 000 and 5*-ACCAGAAATAGCTGCAGGACC-3*, nt 14 380 to 14 360) in 100 ìlof reaction mixture 62·5 m KCl, 12·5 m Tris–HCl,1·75 m MgCl2, 0·8 m dNTPs, 2 units of AmpliTaqpolymerase (Perkin Elmer Cetus, Norwalk, CT, U.S.A.).The PCR conditions included 40 cycles (96)C 1 min 15 s;55)C 50 s; 72)C 45 s) in a GeneAmp PCR system 9600A(Perkin Elmer Cetus, Norwalk, CT, U.S.A.). One micro-litre of the amplified product was then reamplifiedusing the 5*-GATTTGGACCCGAAATCTGA-3* (nt14 005 to 14 024) and 5*-CCTCCCTAGAACTGACAATTGG-3* (nt 14 334 to 14 313) oligonucleotides (100 ngper reaction) under the same PCR conditions. Ten to100 pg of DNA extracted from the Burkitt’s lymphoma(BL) cell line Namalwa was used as a positive control,whereas 500 ng of DNA extracted from EBV-negativelymphoma tissue embedded in paraffin provided thenegative control. A nested PCR with primers specific tothe beta-globin gene was run in parallel to ensure thatthe DNA was amplifiable. The following primers wereused for the first amplification, 5*-ATGGTGCACCTGACTCCTGAGG-3* (nt 2205–2226) and 5*-GCCATCACTAAAGGCACCGAGC-3* (nt 2059–2538), andfor the second amplification 5*-TTAGGGTTGCCCATAACAGC-3* (nt 2229–2247) and 5*-ACGTGGATGAAGTTGGTGGT-3* (nt 2501–2483), under the

same conditions as those described for the amplificationof the BamHI-W fragment with the exception of theMgCl2 concentration (3·5 m in the first PCR, 2 m inthe second PCR).The clonality of the HD lesions was analysed using a

semi-nested PCR and oligonucleotides specific to theIgH genes.7 500 ng of genomic DNA was amplifiedusing HPLC-purified consensus oligonucleotides specificto the variable (FR2A, 400 ng per reaction) and joining(LJH, 100 ng per reaction) IgH segments8 in 100 ìl ofreaction mixture (62·5 m KCl, 12·5 m Tris–HCl,2·5 m MgCl2, 0·8 m dNTPs, 2 units of AmpliTaqpolymerase). The PCR conditions included five initialstringent cycles (96)C 15 s; 63)C 30 s; 72)C 30 s) fol-lowed by 35 cycles with a lower annealing temperature(96)C 15 s; 57)C 30 s; 72)C 30 s). One microlitre of theamplified product was then reamplified using theFR2A oligonucleotide (200 ng per reaction) and anoligonucleotide internal to the LJH binding site (VLJH)(200 ng per reaction) under the same conditions with theexception of the MgCl2 concentration (1·5 m). Twenty-five cycles of PCR (96)C 15 s; 63)C 30 s; 72)C 30 s) werecarried out for the reamplification.PCR analysis of IgH rearrangements in single HRS

cells was performed as previously described.6 Briefly, thepooled and proteinase K-digested cells were amplifiedemploying a semi-nested FW2 or FW3 PCR. Both PCRswere performed under the same conditions as describedelsewhere7 with the exception that the MgCl2 concen-tration for the FW3 PCR was higher (2·5 m). Buffercontrols were processed in parallel under identicalconditions.After PCR, the amplified fragments were size-

separated on a 6 per cent polyacrylamide gel andsubsequently stained with ethidium bromide.

RESULTS

Both tumours showed the same typical histologicalfeatures of HD with nodular sclerosis (Figs 1A and 1E).Large mono- or bi-nucleated neoplastic HRS cells witha prominent eosinophilic nucleolus and an abundanteosinophilic cytoplasm were mixed with numerous smallT-lymphocytes, polytypic plasma cells, macrophages,and eosinophils (Figs 1B and 1F). HRS cells showed anintracytoplasmic dot-like positivity for CD30, whereasonly a minority of these cells were weakly positive forCD15. The B-cell-specific antigen CD20 (L26) wasfound to be expressed on numerous neoplastic cells inboth samples (Figs 1C and 1G). An antibody cocktailspecific to the viral latent protein (LMP)-1, CS1–4,stained the majority of neoplastic cells in the initialtumour (Fig. 1D), but the reactivity to this antibody wasabsent in the tumour at relapse. We tested for the

Fig. 1—Morphological and immunohistological features. (A) Initial tumour, low magnification, nodular sclerosis. (B) Initial tumour, highmagnification, numerous mononucleated and binucleated neoplastic cells with one or two prominent eosinophilic nucleoli. (C) Initial tumour,staining with the CD20 specific antibody L26. (D) Initial tumour, staining with the LMP-1-specific antibody CS1–4. (E) Tumour relapse, lowmagnification, nodular sclerosis. (F) Tumour relapse, high magnification, numerous mono- and bi-nucleated neoplastic cells with one or twoprominent eosinophilic nucleoli. (G) Tumour relapse, staining with the CD20-specific antibody L26. (H) Initial tumour, in situ hybridization usingthe EBER probe

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? 1997 by John Wiley & Sons, Ltd. , . 182: 475–479 (1997)

presence of the EBERs in tumoural cells using in situhybridization and a PCR-based method was used inaddition to amplify EBV-specific genomic sequences.With both methods, the virus could be detected in theinitial tumour, but not in the tumour at relapse (Figs 1Hand 2). PCR with primers specific to the beta-globingene gave rise to amplification of all the tested DNAs,indicating amplifiability (Fig. 3).The clonality of both neoplastic lesions was tested by

analysing their IgH rearrangements. Using a semi-nestedFW2 PCR, no clonal B-cell population could be ident-ified in the whole tissue DNA extracted from paraffin-embedded tumour specimens. The investigation ofisolated single CD30-positive or LMP-positive HRScells revealed no amplification products, even afterapplication of a semi-nested FW3 PCR.

DISCUSSION

In this paper, we report a case of HD whose tumourcells harboured EBV in the first manifestation but notin the neoplastic lesion that developed 1 year after

a polychemotherapy-induced remission. EBV-specificDNA sequences as well as EBERs and the EBV-encodedLMP-1 could be detected in the first lesion, but not inthe second. This observation is unusual, since the analy-sis of several cases of EBV-positive HD and of theirsubsequent relapses showed that in most cases, the viruspersisted in both manifestations of the disease.9There are two possible explanations for the presence

of EBV in the first tumour and its absence in the second.The first possibility is that the tumour occurring afterthe complete remission represents a re-occurrence of theneoplastic clone of the first manifestation which has lostthe EBV genome. A second explanation is that a newEBV-negative tumour population has developed that isunrelated to the first tumour cell clone. Unfortunately,analysis of VH rearrangements was not able to deter-mine precisely the relationship between the tumour cellsof the first and second lesions, since no clonal amplifi-cates could be obtained from whole tissue DNAextracts. Even the application of single cell PCR to HRScells isolated from the paraffin-embedded sections wasunable to clarify this issue, since no amplification prod-ucts could be generated. However, it is known from

Fig. 2—Association with EBV, detection of viral DNA by PCR. Amplification products afterPCR and PAGE (6 per cent polyacrylamide, staining with ethidium bromide). Tumoursample 1: initial tumour. Tumour samples 2: DNAs extracted from four different paraffinblock samples of the tumour relapse. Namalwa is an EBV-associated cell line that served asa positive control. Negative control: DNA extracted from an EBV-negative lymphoma

Fig. 3—Amplification of the beta-globin gene by PCR. Amplification products after PCR andPAGE (6 per cent polyacrylamide, staining with ethidium bromide). The tested DNAs areidentical to the one tested for EBV association as depicted in Fig. 2

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? 1997 by John Wiley & Sons, Ltd. , . 182: 475–479 (1997)

other single cell studies that HRS cells often carry highloads of somatic mutations as well as deletions orinsertions. These alterations, in combination with deg-radations due to formalin fixation, might be responsiblefor the non-detectability of IgH rearrangements in theHRS cells.It is more likely, however, that the HRS cells in both

lesions belong to the same tumour cell population, basedon the following observations: both tumour lesionspresented were in the same anatomical region, i.e., in thecervical lymph node and in the mediastinum; the histo-type of both tumour lesions was identical, in that theyboth showed the histological features of nodular sclero-sing HD; and the immunophenotype of the tumour cellsof both lesions was the same, i.e., the HRS cellsexpressed CD30 and CD20, with a minority weaklypositive for CD15. The CD20 expression in particular isa strong indicator of a relationship between the tumourcells of the first and second lesions, because CD20 isabsent from HRS cells in the majority of instances.In vitro experiments using the EBV-positive BL cell

line Akata have clearly shown that limiting dilution ofthe cell line allows the isolation of some EBV-negativesubclones that have lost the viral genome.10 This provesthat EBV infection is not required for the growth of BLcell lines, because either genetic abnormalities thatoccurred in vitro substitute for EBV, or the viral infec-tion is not at all necessary for the development of BL.Plasmid constructs harbouring the cis-acting and trans-acting replication elements of EBV transfected in BL celllines in the absence of selectable markers like the neo-mycin resistance gene are eliminated after a few cellulardivisions.11 Therefore it can be hypothesized that inovertly malignant cells, e.g. in BL cell lines or in HRScells, the virus is not required for neoplastic growth andmay be lost.In conclusion, this is the first description of a case of

HD that was initially associated with EBV, but relapsedwith EBV-negative HRS cells. The loss of the EBVgenome modified neither the pathological features of

this case nor its clinical neoplastic behaviour. Thissuggests that EBV infection is not essential for themaintenance of tumour cell growth in HD.

ACKNOWLEDGEMENTS

We thank H. Lammert and H. H. Mueller for experttechnical assistance and J. Yates for her help in editingthe manuscript. This work was supported by a grantfrom the Deutsche Krebshilfe (M 25/89/St 1) and by aEuropean Community contract (CHRX-CT94-0651).

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479EPSTEIN–BARR VIRUS INFECTION AND HODGKIN’S DISEASE

? 1997 by John Wiley & Sons, Ltd. , . 182: 475–479 (1997)