JOURNAL OF PATHOLOGY, VOL.

168: 365-369 (1 992)

RAPID COMMUNICATION

EPSTEIN-BARR VIRUS (EBV) AND HODGKIN’S DISEASE IN CHILDREN: INCIDENCE OF EBV LATENT MEMBRANE PROTEIN IN MALIGNANT CELLS M. WEINREB*, P. J. R. DAY’, P. G. MURRAY$, F. RAAFAT?, J. CROCKERg, S. E. PARKESII, N. A. G . COADT, J. T. JONES** AND J. R. MA”*

‘Oncology and ?Histopathology Departments, Children’s Hospital, Birmingham, U.K.; Biomedical Research Laboratories, Wolverhampton, U.K.; §Department of Histopathology, East Birmingham Hospital, Birmingham, U.K.; West Midlands Regional Children’s Tumour Research Group, U.K.; TDepartment of Paediatrics, Walsgrave Hospital, Coventry, U.K.; **Department of Histopathology, Princess Royal Hospital, Telford, U.K. Received 15 July I992 Accepted I October I992

SUMMARY Previous studies have detected EBV DNA by Southern blotting or in situ hybridization in biopsy material from up to 30 per cent of adult cases of Hodgkin’s disease. Here we have used monoclonal antibodies specific for the EBV latent membrane protein LMPl to examine archival material from children with Hodgkin’s disease. Material from 74 cases (54 males and 20 females) was examined and 37 (30 males and 7 females) were classified as LMP1-positive in the malignant cells. LMPl positivity was present in 4/13 (31 per cent) of lymphocyte predominant, 14/36 (39 per cent) of nodular sclerosis, 17/20(85 percent) of mixed cellularity, 1/2(50 per cent) of lymphocyte depletion, and 1/3 (33 per cent) of unclassified subtypes. The positive cases by clinical stage were 19/22 (41 per cent), I1 9/20 (45 per cent), I11 11/24(46 per cent), and IV 8/8 (100 per cent). LMPl positivity was present in 2/5 (40 per cent) children aged less than 5 years, 12/ 27 (44 per cent) aged 5-10 years, and 23/42 (48 per cent) aged between 10 and 15 years. The association between EBV and Hodgkin’s disease in children thus appeared to be more frequent in patients with mixed cellularity and advanced disease, but examples of EBV-positive tumours were found in all histological subtypes, stages, and ages. Stepwise discriminant function analysis showed that clinical stage IV and mixed cellularity histology are independently associated with LMPl positivity. These observations indicate that Hodgkin’s disease in children is at least as strongly linked to EBV as is the disease in adults. KEY WORDS-Hodgkin’s

disease, children, EBV, LMPl .

INTRODUCTION Epidemiological’ and s e r ~ l o g i c a studies l ~ ~ ~ have suggested that infective agents, especially EpsteinBarr virus (EBV), may play a role in the aetiology of Hodgkin’s disease (HD). In recent work, EBV DNA has been detected in up to 30 per cent of adult cases Addressee for correspondence: Dr M. Weinreb, Oncology Department, Children’s Hospital, Birmingham B 16 SET, U.K. 0022-341 7/92/12036545 $07.50 0 1992 by John Wiley & Sons, Ltd.

using Southern blotting4 and/or in situ hybridizati01-1;~~ this latter technique revealed viral sequences present selectively in malignant cells. Furthermore, the resident EBV genome was monoclonal, as shown by analysis of terminal repeat region clonality, suggesting that the virus was present before clonal expansion of the Reed-Sternberg (RS) and Hodgkin’s (H) Further evidence for an active role for EBV in the pathogenesis of Hodgkin’s disease came with the demonstration

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that EBV-positive malignant cells express the virus latent membrane protein LMPI,''-'3 one of the key effectors of EBV-induced cell transformation in vitro.14Indeed, there appears to be almost complete concordance between EBV genome status and LMPl status of RS/H cells when H D biopsies have been screened by the two parameters. Interestingly, the EBV genome-positive, LMP1-positive cells in H D do not appear to express one of the other viruscoded latent proteins, the EB nuclear antigen EBNA 2." This pattern of virus gene expression resembles that seen in an EBV-associated e ithelial malignancy, nasopharyngeal carcinoma," and is quite different from that seen in in vitro transformed B cell lines. Most previous work in this field has concentrated on H D in adults and has found a tendency for increased EBV positivity amongst certain histological subtypes, in particular the mixed cellularity subtype. In England and Wales, Hodgkin's disease has an incidence of 4.1 cases per million per annum amongst children, most commonly in the 10-1 5 year age group.I6In this study we have examined archival biopsy material from a range of childhood H D and have screened the malignant cells for LMPl expression. MATERIALS AND METHODS

Biopsy material Cases of H D diagnosed in children aged below 15 years in the West Midlands Health Authority Region between 1957 and 1992 were identified through the records of the West Midlands Regional Children's Tumour Research Group. Archival biopsy material was reviewed by a panel of three histo athologists and subclassified according to the Rye" convention. Clinical staging in all such cases was determined according to the Ann Arbor scheme." Archival lymph node biopsies from children with benign lymphoid hyperplasia were used as controls in parallel with the archival H D material; these gave uniformly negative results in staining assays. Immunohistological staining Sections of each biopsy were stained with a pool of four mouse monoclonal antibodies (CS 1, CS 2, CS 3, CS 4) specific for LMP1;19 in addition, antibodies CS 1 and CS 2 (known to be reactive against different epitopes of LMPI, M. Rowe, personal

communication) were used individually on sections of each biopsy. Parallel staining was carried out in each case with non-immune serum of the same IgG subclass. Dewaxed paraffin sections were treated with normal rabbit serum at a dilution of 1/10 in Tris-buffered saline (TBS) for 5 min. Mouse monoclonal antibodies CS 1 and CS 2 were then used as hybridoma supernatants at a dilution of 1/10, and CS I 4 as pooled hybridoma supernatants at a dilution of 1/50 in TBS for 60 min. The slides were washed for 5 min in TBS before incubation with rabbit anti-mouse IgG at a 1/20 dilution in TBS for 5 min, and staining was developed by double-layer alkaline phosphatase/anti-alkaline phosphatase (APAAP) using the method as originally developed by Murray et al.I3 As reference material, we used formalin-fixed tissue of EBV-positive lymphoblastoid tumours from SCID mice2' and of known EBV-associated cases of adult HD." For those cases of childhood H D where frozen biopsy was also available, frozen sections of the tumour were stained with CS 1-4 and with the EBNA 2specific mouse monoclonal antibody P E 2 as described;2' frozen sections from appropriate SCID mouse tumours and adult H D were used as controls in these cases. The relationship between LMPl status and sex, age group, histological subtype, and clinical stage was investigated using univariate (chi-square test and test for trend) and multivariate (stepwise discriminant function analysis) methods.22 RESULTS Archival material was available from 74 childhood cases of H D (54 males and 20 females). In some cases, biopsies had been taken from more than one site and thus the total number of tissue biopsies which could be analysed was 89. In each case, the biopsy could be classified unequivocally by monoclonal antibody staining as LMPl -positive in the majority of the malignant cell population (Fig. la) or as uniformly LMP1-negative (Fig. Ib). Concordant results were obtained using the different LMPI-specific antibody preparations, i.e., CS 1 alone, CS 2 alone, or the CS 1 4 pool. In certain biopsies, we observed some non-specific staining of plasma cells and smooth muscle cells with the CS 1 4 pool, but not with CS 1 or CS 2 alone. Of the 74 cases studied, 37 (30 males and 7 females) were classified as LMPl -positive in the malignant cells; when multiple biopsies were available from the

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EBV IN CHILDHOOD HODGKIN’S DISEASE

Fig. 1-a) A paraffin section from a specimen of mixed cellularityHodgkin’s disease. Malignant cells are positive for LMPI. (b) A paraffin section from a specimen of nodular sclerosis Hodgkin’s disease. Malignant cells are negative for LMPl

same patient, these always gave concordant results. Table I presents a summary of the results for the 74 patients classified in terms of histological subtype, clinical stage at presentation, and age at presentation. All but four of the 74 cases could be divided into histological subtypes according to the Rye classification. The incidence of LMPl positivity was significantlyhigher (85 per cent) in mixed cellularity cases compared with the other subtypes (P = 0.001, chi-square test). However, there were some cases of LMPl-positive HD in each of the four subtypes. In terms of clinical staging, we noted using a test for trend that the incidence of LMPl positivity was significantly higher ( P = 0.037) in tumours which were more widespread at presentation. In particular, all eight cases presenting with stage IV disease were LMPl -positive. This cannot be explained by a skewing of the histopathological subtype distribution in stage IV disease, since three of these cases were nodular sclerosing, three mixed cellularity, one was lymphocyte depleted, and one unclassified. The stepwise discriminant function analysis showed that clinical stage IV disease and mixed cellularity histology are independently associated with LMPl positivity.

Table I-Results of LMPl stains and relation to histological subtype, disease status, and age LMPI-positive Total Histological subtype Lymphocyte predominant Nodular sclerosis Mixed cellularity Lymphocyte depletion Unclassified Total

37 (500/,)

13 36 20 2 3 74

Clinical stage I I1 111 IV Total

9 (41%) 9 (45%) 11 (46%) 8 (100%) 37 (50%)

22 20 24 8 14

Age 0 < 5 years 5 < 10 years l o < 15 years

2 (40%) 12 (44%) 23 (48%)

5 21 42

4 (31%) 14 (39%) 17 (85Yo) 1 (50%) 1 (33%)

Figure 2 presents a histogram of the incidence of LMPl -positive cases versus age of presentation.

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count

1

1

2

3

4

5

6

7

8

9 1 0 1 1 1 2 1 3 1 4

Age

Fig. 2-Age distribution of Hodgkin's disease in children by LMPl positivity. Ratio male:female= 54:20

In eight of the 74 H D cases (five LMPI-positive, three LMPl-negative), frozen biopsy material was available. Frozen sections were stained using CS 14 and also the EBNA 2-specific monoclonal antibody PE 2. These tests confirmed LMPl positivity in the malignant cells of the five cases found to be LMP1-positive in paraffin sections, but no ENBA 2 staining was detected in any case. DISCUSSION

In fact, our results show that this is not the case, even amongst those tumours presenting in children within the first 10 years of life. This supports the hypothesis that EBV plays an aetiological role in the development of HD, particularly in tumours of the mixed cellularity subtype. Precisely what this role is remains to be determined, but the fact that LMPl is expressed at high levels in tumour cells may be of significance; this protein has multiple dosedependent effects on cell rowth and cell survival in experimental systems2G2Fandmay contribute to a multistep oncogenic process in vivo. EBV is oncogenic for B cells.29Immunoblastic lymphomas in immunosuppressed individuals express the full spectrum of virus latent genes (including EBNA 2 and LMPl), exactly like virus~ ~ seems likely, transformed B cells in v i t r 0 . 2 ' ~ It therefore, that EBV is the primary driving force in such tumours.20The situation in HD is different in that the malignant cells display a more restricted pattern of viral gene expression (EBNA 2-negative, LMPl -positive). This probably reflects the fact that in H D the malignant cells are of different origin from those of other lymphomas and EBVinfection almost certainly represents one of several cooperating factors bringing about malignant changes. In the 74 cases of childhood H D studied here, we noted a possible link between EBV genome positive status and the stage of the disease at presentation. This was not apparently an effect of subtype distribution per se; hence, further studies are required to examine the possible influence of EBV positivity on disease progression.3o Multivariate analysis of age, sex, histological subtype, and clinical stage showed that histological subtype and clinical stage were both independently associated with LMPl positivity. Although not described here in detail, 15 of the 37 EBV-positive cases in this study subsequently relapsed (7/7 relapse biopsies were again found to be LMP1-positive), whereas only 7 of the 37 EBV-negative cases showed relapse. Thus, even though a recently published preliminary study has not revealed any clear influence of EBV status as a prognostic indicator in HD,30proper prospective studies will be needed to address this issue thoroughly.

Whilst the association between EBV and adult H D has recently been the subject of several studies using archival material, there are as yet only very limited data with respect to HD in childhood ( < 15 In the present work we have screened a large group of childhood HD cases by immunohistochemical staining for the EBV-coded LMPl protein. The results show that the relationship between EBV and childhood HD is at least as strong as that recently observed with adult disease. Thus, up to 50 per cent of cases are EBV-positive as reflected by LMPl expression in malignant cells. Furthermore, the tendency of mixed cellularity subtype tumours to show a higher incidence of EBV positivity, first noted in adult series," is again seen with the childhood tumours. In our study, 4/13 cases of lymphocyte-predominant disease were LMPlpositive, a higher proportion than is seen in adults. Further studies will be required to determine the significance of this finding. The EBV status of childhood HD, especially in relation to the age of first presentation, is of interest ACKNOWLEDGEMENTS since in the U.K. a significant proportion ofchildren We thank Professor A. B. Rickinson for his remain uninfected by the virus up to age 15 years.25 If the virus were an innocuous passenger in HD, we advice and assistance in undertaking these studies, would expect the proportion of EBV-positive HD and the Leukaemia Research Fund and the Special cases to be lower in childhood than in adult disease. Trustees of the Research Endowment Fund of the

EBV IN CHILDHOOD HODGKIN’S DISEASE

former United Birmingham Hospitals for financial support. We also thank Mr A. Brownhill for technical support in the laboratory, Dr M. Rowe for supplying the antibodies, the panel of reviewers of lymphomas for the West Midlands Regional Children’s Tumour Research Group (Professor D. H. Wright, Professor E. L. Jones, and Dr A. H. Cameron), Dr J. Powell for the statistical analyses, and many consultant and medical records staff in the West Midlands Region for access to records and pathology material from their patients. REFERENCES I. Meuller N. An epidemiologist’s view of the new molecular biology findings in Hodgkin’s disease. Ann Oncol1991; 2 (Suppl): 23-28.

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