JOURNAL OF PATHOLOGY, VOL. 179: 54-59 (1996)

PHENOTYPIC MODULATION OF HODGKIN AND REED-STERNBERG CELLS BY EPSTEIN-BARR VIRUS

HERMANN HERBST, THORSTEN RAFF AND HARALD STEIN

((otisultatit,izs~elrtvum ,fits Lymphknoten- und Hamatopathologie um lnstitut f i r Puthologie, Klii~il~um Benjamin Frunkl~n, Freie Universitat Berlin, 0-12000 Berlin, Gesmuny

SUMMARY

Expression of the Epstein-Barr virus (EBV) gene product L M P l is found in tumour cells in varying proportions of Hodgkin's disease (HD) cases. I t is not clear which cellular genes are influenced by EBV in HD. A total of 387 H D cases were tested for differences among LMP1-positive and -negative cases with respect to age, sex, histotype and immunophenotypic parameters (CD2, CD3, CD4, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD43, CD45RA, CD45R0, CD70, HLA-DR, T-cell receptor &chain, and p53 expression). Comparison of patient age and sex as well as distribution of histotype and tumour cell immunophenotype with published data suggests that the cases in this study are representative of the spectrum of H D in developed countries. L M P l expression was found in 1311387 H D cases (36.4 per cent) with non-homogeneous distribution among H D histotypes, the mixed cellularity type (HDmc) being most frequently EBV-associated (711129 cases, 55 percent). No relationship was found to age and sex. Significant phenotypic differences were restricted to the HDmc histotype, where the tumour cells expressed the activation marker CD30 in a larger proportion, and CD20 in a smaller proportion, when harbouring EBV. These results suggest that EBV may influence the tumour cell phenotype in HD.

KEY WORDS-Epstein-Barr virus; Hodgkin's disease; Hodgkin and Reed-Sternberg cells; activation antigens; B-cell antigens; T-cell antigens; immunophenotype; immunohistochemistry

INTRODUCTION In recent years, a firm association has been established

between Hodgkin's disease (HD) and Epstein-Barr virus (EBV) infection by demonstration of viral genomes and gene products in the neoplastic cell population, Hodgkin and Reed-Sternberg HRS) cells. The proportion of EBV-positive cases shows considerable socio-geographic variation, with a viral association in only 40-60 per cent of Western H D cases in immunocompentent hosts, but in up to 95 per cent of H D in developing countries.'32 Variation was also noled among HD of different histo- logical types, with mixed cellularity HD (HDmc) being most frequently EBV-associated. The monoclonal origin of EBV episomes, as well as the expression of the latent membrane protein LMPl, was considered to support the notion of a athoaetiological role of the virus in this lymphoma.lPLMP1 is the only EBV gene product with oncogenic potential in experimental systems, presum- ably by virtue of inhibiting programmed cell death through interference with hcl-2 gene expre~sion.~

Depending on the pattern of EBV gene expression, latently EBV-infected cells may display phenotypic abnormalities. In Burkitt's lymphoma (BL), EBV seems to down-regulate some antigens such as HLA mol- e c u l e ~ . ~ , ~ In typical BL cells, EBV gene expression is restricted to a minimum of genes; LMPl and all nuclear antigens (EBNAs) are absent, with the exception of EBNAl which is required for the maintenance of the viral episome.6 This situation is different in infectious mononucleosis and lymphoblastoid cell lines, where

Addrcsscc for correspondence: Dr Hermdnn Herbst, lnstitut fur Pathologie, UniversitBtskrankenhaus Eppendorf, 20246 Hamburg, Germany.

CCC 0022-34 17/96/0500!54-06 :<> 1996 by John Wiley &: Sons, Ltd.

expression of LMPl and EBNA2 correlates with expres- sion of activation markers such as CD30 ad CD70 antigen^.^ The contribution of individual EBV genes has been studied in transfection experiments which revealed modulation of CD23, CD21, c-fgr, and lymphocyte function-associated antigens [LFA- 1 a/[] (CDlla , CDlS) and LFA-3 (CDSS)] by EBNA2 in human B-cells." In an epithelial cell environment, LMPl is capable of inducing CD34, CD40, CD58, CD70, and CD80 antigen expression as well as a malignant phenotype, as evidenced by loss of contact inhibition.' ,I2

Thus, the question arises as to whether EBV may modulate the expression of differentiation and/or acti- vation molecules in HRS cells of EBV-associated HD as well. We have previously observed a reduced reactivity of latently EBV-infected small lymphoid cells with a CD20-specific antibody as compared to neighbouring small B lymphocytes, in double staining experiments employing in situ hybridization for the demonstration of EBER molecules combined with immunohistology. '' Recently, Bai et al. reported on the observation of an apparent down-regulation of CD20, CD3, and epithelial membrane antigen (EMA) expression in LMP1-positive compared with LMPl-negative HD cases,I4 in a series largely comprising HDns cases. These differences seemed to be supported by statistical analysis and it was concluded that EBV may be responsible for the frequent absence of leucocyte differentiation markers from a large proportion of HD cases.I4 This attractive hypothesis prompted us to evaluate the immunophenotypic data of HD cases stained with LMPl-specific antibodies and characterized for the expression of up to 18 different immunophenotypic markers, as well as patient age and

Received 111 July 1995 Acceplcd 23 November 1995

EPSTEIN-BARR VIRUS AND HODGKIN’S DISEASE 55

sex, in correlation with the presence or absence of LMP1.

MATERIALS AND METHODS Tissues

HD cases were submitted for diagnosis or as reference material to the Consultation Centre. Tissue was fixed in buffered formol and paraffin-embedded according to standard procedures. Frozen tissue was additionally available in 79 cases. Diagnosis and histotype assign- ment followed the Rye classification criteria. The diag- nosis of lymphocyte-depleted HD (HDld) was restricted to cases with typical HRS cells and an incoherent growth pattern; otherwise, cases with prevalence of mononuclear atypical cells and cohesive growth were considered CD30-positive anaplastic large cell (ALC) lymphomas of Hodgkin-like type, according to the Revised European-American Lymphoma (REAL) ~1assification.l~ In 46 cases, no definitive histotype assignment was possible, most frequently because of the size of the biopsy sample. In these cases, morphological and phenotypic properties suggested a differential diag- nosis of ‘classical’ HD forms (HDmc, HDns, and HDld).

Immunohistology

Four-micrometre sections of routinely formol-fixed/ paraffin-embedded tissue blocks or cryostat sections mounted on 3-aminopropylethoxysilane-treated slides were stained by the immunoalkaline phosphatase (APAAP) method.16 Monoclonal antibodies MT910

L26 (CD20), IF8 (CD21), ACT-1 (CD25), Ber-H2

(CD45RA), Ki-24 (CD70), DO-7 (anti-p53), and CS1-4 (LMPl), as well as a polyclonal anti-CD3 rabbit anti- body, were obtained from Dako, Glostrup, Denmark. Antibody @Fl, directed against the T-cell receptor (TCR) @-chain, was from T-Cell-Sciences, Cambridge, MA, U.S.A., and S-HCL-1 (CD22) was from Becton- Dickinson, Sunnyvale, CA, U.S.A. Tul (CD23) and Tii35 (HLA-DR) were a kind gift from Dr A. Ziegler, Berlin, Germany. Antibodies against CD2, CD4, CD 19, CD22, CD23, CD25, CD70, and HLA-DR were used on cryostat sections. Antigen retrieval by microwave irra- diation prior to immunostaining of paraffin sections was performed for staining with antibodies, L26, Ber-H2, 4KB5, DF-T1, CSl-4, and DO-7. Formol-fixed sections required a proteolytic treatment with 1 mg/ml Strepto- myces griseus protease (Sigma, Munich, Germany) for 10 min prior to incubation with the monoclonal anti- bodies Ber-H2, lF8, and @F1. Immunophenotypic markers were considered present on HRS cells when a minimum of 1 out of 20 tumour cells scored positive compared with adjacent CD30-stained sections.

(CD2), CD4 (MT310), C3D-1 (CD15), HD37 (CD19),

(CD30), DF-TI (CD43), UCHLl (CD45RO), 4KB5

Statistical analysis Homogeneity of LMPl expression across HD histo-

types was tested using Pearson’s chi-square test of

observed vs. expected values. Correlations between expression of differentiation antigens and LMPl within the groups of different HD histotype were analysed using Pearson’s chi-square test corrected for continuity as devised by Yates. All P values were two-tailed and considered statistically significant if PX0.05.

RESULTS

A total of 387 HD biopsies were evaluated. Patient data as to sex and age were available from 370 and 376 patients, respectively. The age distribution in general and categorized by sex, as well as the histological types categorized by sex, are in good agreement with data of other studies on HD in Western c o u n t r i e ~ . ’ ~ - ~ ~ A total of 387 cases were studied for the expression of LMP1, 131 of which proved to display this antigen in a pro- portion of cells ranging between approximately 5 and 90 per cent, but usually between 10 and 50 per cent, of the HRS cell number as determined by CD30 staining of adjacent sections. There were no significant differences in LMPl expression between male and female HD patients (Fig. 2A). The expression of LMPl (P<O.Ol) was non-homogeneously distributed among the four different histotypes recognized by the Rye classification (Fig. 2B), thus precluding the consideration of HD as a homogeneous entity with respect to LMP 1 expression. In agreement with previous studies, LMPl expression was most frequently found in tumour cells of HDmc (71 of 129 cases, 55 per cent) and was only occasionally present in tumour cells of HDlp cases (5 of 34, 14.7 per cent), whereas the proportions were intermediate for HDns and HDld with 31 out of 155 (20 per cent) cases and 6 out of 23 (26 per cent) cases, respectively (Table I). The age distribution of our LMPl-positive and -negative cases categorized by histological type did not disclose a predilection of a specific age group for LMPI-positive HD (Figs 2C-2F). The number of paediatric patients, however, was very small in our series.

CD30 was detectable in the vast majority of HD cases. In most cases analysed on cryostat sections, at least one of the additionally studied activation markers, CD25, CD70, and HLA-DR, was expressed. Although still detectable in the majority of cases, activation markers were less frequently expressed in tumour cells of HDlp. The comparison between LMP1-positive and -negative cases indicated a tendency of LMP1-positive cases to be more frequently associated with detectable expression of activation markers. This notion is supported by statisti- cal significance, however, only for CD30 staining in HDmc cases.

Expression of B-cell differentiation markers was dis- played by almost all cases of HDlp. The proportions of HDns, HDmc, and HDld cases expressing B-cell mark- ers were considerably smaller; approximately 20 per cent of HDmc cases, for example, presented with a CD20- positive phenotype. CD21 and CD23 expression was noted in only a small proportion of cases. Expression of T-cell markers was most frequently found in HDns, with 20 per cent of these cases scoring positive for TCR jl-chain and/or CD3 expression by HRS cells. The CD 15

43

40

! 35

G3

0

2 23

2m

5 1

5 z

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10

20

30

40

50

60

70

80

90

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Bs

o

50

L 40

B b =

20

; 30

s z 10

100,

i

Fig.

I-(A

) A

ge d

istri

butio

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s (n=

376)

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play

ing

a bi

mod

al d

istri

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ith p

eaks

in th

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ird a

nd s

ixth

dec

ades

. (B)

Age

dis

tribu

tion

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D c

ases

cat

egor

ized

by

sex

for

mal

e (n

=233

) an

d fe

mal

e (n

=137

) pa

tient

s. (

C)

Dis

tribu

tion

of h

isto

logi

cal

type

in m

ale

(n=2

17)

and

fem

ale

(n=1

24) H

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atie

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A

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07

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00

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Fig.

2-(A

) D

istri

butio

n of

LM

P1-p

ositi

ve a

nd -n

egat

ive

in m

ale

(n=

239)

and

fem

ale (n

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8) p

atie

nts.

(B

) Dis

tribu

tion

of h

isto

logi

cal t

ypes

of H

D w

ith (n=

121)

or

with

out

(n=2

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LM

Pl e

xpre

ssio

n di

spla

ying

pre

pond

eran

ce o

f H

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c hi

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ype

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-pos

itive

HD

. (C

) Age

dis

tribu

tion

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cat

egor

ized

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xpre

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n (p

ositi

ve n

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neg

ativ

e n=

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(D

) A

ge d

istri

butio

n of

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ns c

ateg

oriz

ed b

y L

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exp

ress

ion

(pos

itive

n=3

1, n

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ive n=

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). (E

) Age

dis

tribu

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c ca

tego

rized

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LM

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xpre

ssio

n (p

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ve n

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gativ

e n=51). (

F) A

ge d

istri

butio

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HD

ld c

ateg

oriz

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y L

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exp

ress

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(pos

itive

n=5

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gativ

e n=

18)

Tabl

e I-

Phen

otyp

ic

char

acte

ristic

s of

LM

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tum

our

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umm

ary.

St

atis

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ly s

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Pear

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Yat

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cor

rect

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P<

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type

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1271

1 3 1

(97%

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/32

(88%

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/34

( 100

%)

2012

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2411

18

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(16%

) 7/

29

(24%

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93

(3%

) 2/

29

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) 1/

86

(1%

) 81

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(1 6%

) 11

11 12

( 1

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29

(21%

) 51

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0132

1/53

6711

06

28/3

3

(1 6%

)

(OYO

)

(2%

)

(63%

)

(85%

)

2281

256

(89'Y

o)

34/4

0 (8

5%)

43/4

5 (9

6%)

25/3

8 (6

6%)

5912

22

(27%

) 8/

47

( 17%

) 9/

45

(20%

) 51

185

7/54

(1

3%)

5114

1 (4

%)

(23%

)

(3%

)

16/7

7

4612

13

1014

3 (2

3%)

11/4

3 (2

6%)

5/49

( 1

OYO)

8/

93

(9%

) 14

0120

6 (6

8%)

77/9

0 (8

6%)

(22%

)

518

(63%

) o/ 1

(O

YO)

111

(1 00

%)

111

(1 00

%)

718

(88%

) o/ 1

(0

%)

o/ 1

(OYO

) 01

7 (0%

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I (0%

) 1 I6

(1

7%)

o/ 1

(0%

) 01

8 (0%

) 01

1 (0

%)

o/ 1

(OY"

) 01

3 (0

%)

015 (0%

) 11

6

212

( 100

~0)

( 17%

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14/2

6 (5

4%)

213

(67%

) 31

3 ( 1

00%

) 31

3 (1

00%

) 22

/23

(96%

) 21

3 (6

7%)

213

(67%

) 01

22

(OYO

) 01

3 (O

YO)

3/20

(1

5%)

018 (0%

) 01

22

(0%

) 01

3 (O

YO)

013

(0%

) 01

6 (0%

) 1/

12

(8%

) 4/

20

(20%

) 81

1 1

(73%

)

LMP'

L

MP

- L

MP+

L

MP-

LM

P'

LM

P-

LMP'

L

MP

71/7

1 (1

00%

) 13

11 3

(100

%)

13/1

3 (1

00%

) 61

9 (6

7%)

7/63

(1

1%)

2/12

211 1

(1

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1/

50

(2%

) 1/

20

(5%

) 01

44

(0%

) 2/

22

(9%

) 31

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(5%

) 21

1 1

(1 8%

) 21

1 1

(18%

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16

(OYO

) 01

25

(OYO

) 43

/62

(69%

) 21

/25

(84%

)

(1 7%

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52/5

8 (9

0%)

1011

1

13/1

3 ( 1

00%

) 7/

10

(70%

) 16

/54

(30%

) 21

1 3

(15%

) 11

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(9%

) 3/

43

(7%

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1 7

(OYO

) 1/

36

(3%

) 5/

20

(25%

) 8/

50

(1 6%

) 4/

12

(33%

) 4/

12

(33%

) 21

1 1

(1 W

O)

2/25

(8

%)

3014

8 (6

3%)

14/1

9 (7

4%)

(91?'0)

31/3

1

12/1

3 (9

2%)

14/1

4

9/10

(9

0%)

6/28

3/13

(2

3%)

311 1

(2

7%)

1/24

(4

%)

1 /7

(14%

) 01

22

(0%

) 5/

20

(25%

) 5/

25

3/13

(2

3%)

2/13

(1

5%)

011 1

(O

YO)

1/21

(5

%)

18/2

8 (6

4%)

112

(500

/0)

(100

%)

(1 0

0~

0)

(21%

)

(20%

)

(191

124

(96%

) 20

123

(87%

) 24

/26

(92%

) 13

/23

(57%

) 16

1112

( 1

4%)

3/26

(1

2%)

3/23

(1

3%)

2110

5

5/30

(1

7%)

0172

9/42

2111

09

5/25

(2

0%)

6/26

(2

3%)

2/28

(7

%)

5/50

(1

0%)

9411

16

(81%

) 45

/47

(2%

)

(OYO

)

(21%

)

(1 9%

)

(960

/0)

616

14/1

7 ( 1

OOYO

) (8

2%)

o/ 1

111

(0%

) (1

00%

) 01

0 11

1 (1

00%

) 01

0 11

1 (1

00%

) 01

5 31

1 6

010

111

(1 OO

YO)

010

111

( 100

~0)

115

0112

(2

0%)

(0%

) o/ 1

1 /3

(O

YO)

(33%

) 01

5 1/

10

(OYO

) (1

0%)

012

212

(0%

) (1

00%

) 01

5 41

1 5

010

111

010

111

012

1 I4

012

015

(OYO

) (0

%)

314

1011

4

(OYO

) (1

9%)

(OYO

) (2

7%)

( 100

~0)

(1 00

%)

(0%

) (2

5%)

(75%

) (7

1%)

212

517

(1 OO

YO)

(71%

)

14/1

5 (9

3%)

314

616

(1 00

%)

415

(80%

) 4/

14

(29%

) 01

6

216

(33%

) 01

7 (O

YO)

010

019

(OYO

) 1 1

6 (1

7%)

311 3

(2

3%)

1 I4

(25%

) 11

6 (1

7Yo)

01

0

010

216

(33%

) 21

2 (1

00~0

)

(75%

)

(OYO

)

2913

1 (9

4%)

112

212

( 100

%)

111

(100

%)

2/17

( 1

2%)

014

(OYO

) 21

7

013

(0%

) 11

1 ( 1

OOYO

) 01

3 (0

%)

015

(OYO

) 13

/17

(76%

) 01

2 (0

%)

o/ 1

(0%

) 01

0

01 1 (0%

) 21

8

516

(50%

)

(290

/,)

(25%

)

(83%

)

TC

R;B

=T-c

ell r

ecep

tor p

-cha

in.

58 n. HERBST ET A L

antigen was detectable in up to 78 per cent of classical HD, such as HDmc, but was less frequently displayed by tumour cells of HDlp (20 per cent). Nuclear staining of tumour cells with the p53-specific antibody was a frequent finding across all histological types of HD.

Statistical evaluatiori of HRS cell antigen expression in LMP1-positive vs. LMPl-negative H D cases showed significant (W0.05) differences for CD30 and CD20 only in HDmc, with CD30 being more frequently, and CD20 being less frequently, expressed in LMPl -positive cases. Differences were also suggested for other antigens, such as CD3 and TCR-8 chain, but without statistical significance (Table I).

DISCUSSION

We have evaluated a total of 387 H D cases for epidemiological differences with respect to LMPl expression and tested for a putative influence of LMPl expression on the phenotypic profile of tumour cells. LMPl expression in the absence of EBNA2-5 expres- sion characterizes latent EBV infection of HRS cells in HD. Analysis of patient age and sex mirrored epidemio- logical features characteristic of H D in Western c o ~ n t r i e s , ' ~ - ~ ~ with predisposition of young female patients for HDns and a bimodal age distribution. In keeping with previous report^,^'-^^ no differences were found with respect to LMP1 expression and patient age. Because of the small number of patients, however, our data are inconclusive as to paediatric HD, which has been suggested to be more frequently related to EBV than in other age groups, also in developed countries." 21 The staining results, with regular expres- sion of activation markers and CD15 in classical HD, expression of B-cell markers in virtually all HDlp cases, " and expression of T- and/or B-cell markers in a pro- portion of HDns, HDmc, and HDlp cases, are in good agreement with the generally accepted immunopheno- typic characteristics of HD.25 2x

As previously noted, EBV infection of HRS cells as reflected by LMPl expression is non-homogeneously distributed among the four histotypes of HD, with a high prevalence of LMP1-positive cases only in HDmc. I. .29.10 Consequently, phenotypic differences considered to be influenced by LMPl expression cannot be statistically analysed across all histotypes. In agree- ment with some previous observations, however, the presence of LMPl did not correlate with CD21, CD23, and p53 expression."." This is well in line with data indicating CD21 and CD23 expression to be influenced by EBNA2, which is not expressed in HD,2"30 rather than by LMPl . l o Diflerences between LMP1-positive and -negative H D were suggested for activation marker expression in cases of classical HD, but proved to be statistically significant only for CD30 expression in HDmc. All cases of LMPI-positive classical H D were uniformly CD30-positive, whereas CD30 was not detect- able in up to 12 per (cent of cases in LMP1-negative classical HD.

EBV is known to be among the most potent inducers of CD30 expression in peripheral blood lymphocytes.32

Infectious mononucleosis blasts are another example, suggesting an up-regulatory role of LMPl for this activation antigen in lymphoid cells.' Expression of LMPl in AIDS-related lymphomas also appears to correlate with CD30 expression.' Up-regulation of other activation markers such as CD70 has been reported for undifferentiated nasopharyngeal carcinoma, another EBV-associated entity, and LMPl-transfected epithelial cells.12 LMP1-negative latently EBV-infected small lym- phoid cells, characterized by expression of the small nuclear transcripts EBER-1 and -2, lack expression of CD30.12 It is thus possible to assume that the up-regulatory effect of EBV on CD30 expression in HRS cells is related to LMPI. HRS cells express CD30 constitutively, so that LMPl only augments basal expression of CD30 and perhaps of other related acti- vation markers. These markers then become detectable by immunohistochemistry in a larger proportion of EBV-positive than EBV-negative cases.

This raises the question as to the possible mechanism of CD30 up-regulation by EBV. LMPl is known to activate TNF-receptor-dependent intracellular signalling by molecular mimicry of tumor necrosis factor (TNF) receptor cytoplasmic domains.33 Furthermore, tran- scription factors such as NF-KB and A20 zinc-finger protein are activated in this c ~ n t e x t . ~ ~ , ' ~ It will therefore prove interesting to analyse the 5' untranslated region of the CD30 and other activation marker genes for the presence of corresponding binding sites.

CD20, the other molecule found to be associated with LMPl expression, proved to be less frequently detect- able in LMP1-positive HDmc. Contrary to the sug- gested influence of LMPl on CD30 expression, LMPl may not be the EBV gene product regulating CD20. This is supported by the observation of a down-regulatory effect of EBV infection on CD20 expression in small lymphoid cells, which usually do not express LMPI." Similarly, down-regulation of HLA antigen expression is documented for Burkitt's lymphoma type I cell lines. This may represent a common mechanism by which EBV may prevent inadvertent activation of the cells leading to expression of other viral antigens and exposure to cell-mediated imrn~n i ty .~

In conclusion, statistically significant evidence for a modulatory influence of EBV on HD is limited to the predominance of HDmc among LMP1-positive H D and the modulation of CD30 and CD20 antigen expression. It will prove interesting to quantitate the density of these and other surface molecules on fresh H R S cells by cytofluoroinetric analysis. This may ultimately lead to the recognition of other modulatory effects that may not be disclosed by immunohistology. Our data are partially in agreement with a recent report by Bai et on the cumulative statistical analysis of 64 H D cases largely representing the HDns histotype (50 of 64 cases). These authors observed an inverse relationship between expression of CD20, CD3, and epithelial membrane antigen (EMA) on the one hand, and the EBV infection on the other. The finding of EMA expression in a large proportion of HD cases is surprising, because this antigen is generally considered a useful marker for the distinction of ALC lymphomas and HDlp from classical

EPSTEIN-BARR VIRUS AND HODGKIN’S DISEASE 59

HD.366,37 Down-regulation of EMA has been observed in epithelial cells following LMPl transfection.” LMP1- induced regulation of EMA may thus also occur in a lymphoid environment. However, as to CD20 expres- sion, the data presented here, as well as the communi- cation by Bai et al.,I4 seem to indicate that EBV-induced modulation of CD20 may account for the failure to detect this antigen on HRS cells in a proportion of HD cases. Because CD20-specific immunostaining is often used for lineage assignment of HRS cells in paraffin sections, the presence or absence of EBV should be considered when evaluating immunostaining patterns in HD.

ACKNOWLEDGEMENTS

This work was supported by Deutsche Krebshilfe- Mildred-Scheel Stiftung, grant W81/91/Hel.

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