American Journal of Hematology 52:21-28 (1996)

Epstein-Barr Virus Infection and Associated Products

(LMP, EBNA2, vlL-I 0) in Nodal Non-Hodgkin’s Lymphoma
of Human Immunodeficiency Virus-Negative Japanese
Koichi Ohshima, Junji Suzumiya, Kotaro Tasiro, Yasuo Mukai, Tosihiro Tanaka, Akiko Kato,
and Masahiro Kikuchi
Department of Pathology, School of Medicine, Fukuoka University, Fukuoka, Japan

Sixty cases of B-cell nodal nonHodgkin’s malignant lymphoma (B-ML), and 46 cases of
T-cell nodal lymphoma(T-ML) were surveyed for Epstein-Barrvirus (EBV) genomes, RNA,
and associated proteins. We used a Southern blot analysis, polymerase chain reaction
(PCR), and EBV-encoded small RNA-1 (EBER-1) in situ hybridization to investigate the
presenceof EBV. We performed an imrnunohistochernicalstudy on EBV-related oncopro-
teins, such as EBV-determined nuclear antigen9 (EBNA-2), latent membrane protein
(LMP), and viral interleukin-10(vlL-10). In addition, we also analyzed the terminal repetitive
sequence of EBV (EBV-TR) to investigate the EBV-infected cell clonality. Non-Hodgkin’s
lymphomas were grouped into three types by number of EBV-infectedcells: I)almost all
lymphoma cells showed an EBV presence; II) some scattered lymphoma cells showed
an EBV presence; and 111) only a few cells showed such a presence, which was probably
due to a latent EBV infection. In 25 of 60 B-MLs, EBV-infectedcells were found; 7 were
type I,1 was type II, and 17 were type 111. In 27 of 46 T-MLs, EBV-infectedcells were found;
no cases were type I,5 cases were type II, and 22 cases were type 111. Seven B-MLs and
3 T cell lymphomas showed clonal TR bands. Expression of EBNA-2 was found in only
three B-MLs, whereas LMP was seen in four B-MLs and six T-MLs. All EBNA-ZLMP-
positive cases showed an EBV presence. In B-MLs, expression of EBNA-2 and LMP was
detected in almost all lymphoma cells: in T-MLs, however, LMP was found in only a small
portion of the lymphoma cells. Expression of IL-10 was closely associated with LMP. In
summary, it was thus speculated that EBV infection was associated with the various
states of lymphomagenesis. o 1996 ~ i ~ e y - ~ iInc.
ss,

Key words: nonHodgkin’s lymphoma, EBV, LMP, EBNA-2, vlL-10

INTRODUCTION membrane protein (LMP) and nuclear antigen (EBNA).

For example, in HD, LMP was expressed in the H & RS
Epstein-Barr virus (EBV) is a herpes DNA virus that
cells, while EBNA2 was absent [4].In the B-lymphoblas-
is well recognized for its oncogenic properties. Infectious
toid cell lines, both were expressed, while in Burkitt’s
mononucleosis, Burkitt’s lymphoma, nasopharyngeal car-
lymphoma both were absent [6].
cinoma, and secondary B-cell proliferation in immuno-
Sequencing identified the viral BCRF-1 [viral (v) in-
suppressed individuals have all been well demonstrated
terleukin-10 (IL-lo)] gene of EBV, a sequence that is
to be associated with EBV [1,2]. EBV genomes have
highly homologous to the human 1L-10 (hIL-10) gene
been detected in Hodgkin’s disease (HD), and the viral
[7]; it shares most functions with hIL-10, which was
genomes were present as episomes of monoclonal origin
initially purified as a cytokine synthesis inhibitory factor
confined to Hodgkin’s and Reed-Stemberg cells, which
constitute the malignant cell population of HD [3,4].
More recently, EBV genomes have also been found in
the nuclei of adult T-cell leukemiallymphoma (ATLL) Received for publication May 19, 1995; accepted November 8, 1995.
tumor cells [ 5 ] . Address reprint requests to Dr. K. Ohshima, Department of Pathology,
Rowe et al. [6] classified the EBV-associated tumors School of Medicine, Fukuoka University, Nanakuma 7-45- 1, Jonan-
into three types by expression of the EBV-encoded latent ku, Fukuoka 814-01, Japan.
0 1996 Wiley-Liss, Inc.
22 Ohshima et al.
TABLE 1. EBV and Associated Antigens in Malignant Lymphoma

DNA analysis
Southern ISHb
No. of blot (EBER-1) Immunological staining
Diagnosis” cases W TR PCR ++ + +I- LMP EBNA-2 1L-I0

B-cell lymphoma (total) 60 8 7 10 7 1 17 4 3 2‘

Diffuse large (largehmmunoblastic) 36 6 5 6 5 1 9 3 2 2‘
Diffuse medium (small cleaved) 13 0 0 2 0 0 6 0 0 0
Follicular medium (small cleaved) 8 0 0 0 0 0 2 0 0 0
Burkitt (small noncleaved) I 0 0 0 0 0 0 0 0 0
Anaplastic large 2 2 2 2 2 0 0 1 1 I
T-cell lymphoma (total) 46 5 3 14 0 5 22 6d 0 4d
ATLV(-) (subtotal) 29 4 2 11 0 4 11 5d 0 0
Diffuse large (largefimmtinoblastic) 5 0 0 0 0 0 2 0 0 0
Diffuse medium (small cleaved) 7 0 0 3 0 0 4 2d 0 2d
Diffuse pleomorphic (immunoblastic) 4 I I 1 0 1 1 0 0 0
AILD-T 13 3 1‘ 7 0 3 4 3d 0 2’
ATLV( +) (subtotal) 17 1 1 3 0 1 II Id 0 0
Diffuse large (largeiiminunoblastic) 5 1 1 1 0 1 4 Id 0 0
Diffuse medium (small cleaved) 7 0 0 1 0 0 5 0 0 0
Diffuse pleomorphic (immunoblastic) 5 0 0 1 0 0 3 0 0 0
Non-sDecitic lvmohadenitis 13 0 0 6 0 0 6 0 0 0

aDefinitions in parentheses are according to the Working Formulation (WF); diffuse large (LSC) includes all diffuse large (WF) and some diffuse
iinmunoblastic lymphoma (WF), which shows mild nuclear pleomorphism.
h++, > 50%; +, 2-50%; +I-, < 2%.
‘One of two cases shows a few positive cells.
“A few cells are positive.
‘Two bands.

[8,9]. In addition, vIL-I0 has also been established as a was kept at - 80°C for immunological analysis using
new latency gene with a direct transformation-prerequi- monoclonal antibodies. We used 13 cases of lymph nodes
site function [lo]. Study of the terminal repeat (TR) se- with non-specific lymphadenitis (showing no history of
quences of the EBV genome is considered to be useful infectious mononucleosis) as a control study.
as a means of assessing the clonality of the cellular popu-
lation harboring the virus [ 111. We thus investigated EBV lmmunohistochemistry
infection in nodal non-Hodgkin’s lymphoma using poly- Monoclonal antibodies for EBV-encoded LMPl (Da-
merase chain reaction (PCR), Southern blot analysis, and kopatts, Copenhagen, Denmark) and EBNA2 (Novocas-
in situ hybridization. In addition, expression of the EBV- tra, UK) were also employed. In addition, monoclonal
associated oncoproteins LMP, EBNA-2, and IL-10 were antibodies for human and viral IL-10 (anti-h- and -vIL-
also analyzed. lo), which have a cross-reaction with both human and
viral IL-10, and viral IL-10 (anti-vIL-10) (Pharmingen,
San Diego, CA, USA) were also used. Antibodies to L26
MATERIALS AND METHODS
(CD20), UCHL-1 (CD4SRO) (Dako), LeuM1 (CDlS)
From this tissue specimens filed in the Department of (Becton-Dickinson, Mountain View, CA, USA), and
Pathology, Fukuoka University, we selected lymph node BerH2 (CD30) (Dakopatts) were also studied in the paraf-
specimens from 106 cases histologically diagnosed as fin-embedded samples. A portion of each lymph node,
non-Hodgkin’s lymphoma. The lymphomas were divided kept at -80°C, was examined using monoclonal antibod-
into T- or B-cell type by both phenotype and genotype ies for T cells (CDl, CD2, CD3, CD4, and CD8), B cells
and then histologically subdivided by the lymphoma (CD19 and CD20), and Kil (CD30) (Becton-Diclunson,
study group (LSG) classification. Almost all cases used Dakopatts, Pharmingen, or Novocastra).
have been previously reported [12]. The LSG classifica-
tion is almost the same as the Working Formulation (WF) In Situ Hybridization
(Table I). We fixed the lymph nodes in either buffered EBV RNA in situ hybridization (ISH) was performed,
formalin or B5 solution, embedded them in paraffin, and with a 30-base oligonucleotide of the EBER-1 gene por-
stained them with hematoxylin-eosin, Giemsa, and Go- tion, using a previously described method [13]. In brief,
mori’s silver impregnation. A portion of each lymph node 3 p m paraffin sections were deparaffinized in xylene,
 EBN in Non-Hodgkin’s Lymphoma 23

dehydrated in ethanol, air dried, predigested with pronase, the ISH target consisted of up to lo7 copies of EBER-1
prehybridized, and hybridized overnight with a digoxi- RNA [13].
genin-labeled oligo-antisense probe (EBER- l), which
was synthesized on the basis of a published report [13]. EBV-Infected Cells: Distribution by ISH
After washing, hybridization was detected with an antidi- EBV-infected cells could be found in 52 of 106 MLs
goxigenin antibody-alkaline phosphatase conjugate. We (49%) by ISH. In non-specific lymphadenitis, EBV was
used a sense probe as a negative control. found in 6 of 13 cases (46%) (Fig. 2 and Table I). Morpho-
Southern Blotting Analysis logically, the EBV-infected cells were confined to
lymphoid cells. No other cell types (fibroblasts, endothe-
Part of the frozen material was used for DNA isolation lial cells, or histiocytes) showed any EBV infection.
and gene analysis. Details of the examination methods Based on the number of EBV-infected cells, MLs were
have been reported previously [12]. The T-cell receptor groups into three types: I) almost all lymphoma cells
(TCR) gene Cp, Jy, the immunoglobulin heavy chain showed an EBV presence; 11) some scattered lymphoma
(JH) gene, proviral DNA of human T-cell lymphotropic cells showed an EBV presence; and 111) only a few cells
virus type 1 (HTLV-1) (full length; gag, pol, env, pX, showed EBV. Infected cells made up 50-100% of the
LTR), the EBV gene (BamHI W region; Enzo, Hudson, entire lymphoma cell population in type I, 2-50% in type
NY, USA), and EBV-TR repeat (kindly provided by Dr. K. 11, and under 2% in type 111.
Hirai, Department of Virology and Immunology, Tokyo In 25 of 60 B-MLs, EBV-infected cells were found; 7
Medical and Dental University, Tokyo, Japan) were used were type I, 1 was type 11, and 17 were type 111. In 27
as probes. We digested DNA with restriction enzymes of 46 T-MLs, EBV-infected cells were found; 5 cases
EcoRI, HindIII, or BamHI. were type 11, and 22 were type 111. In 6 of 13 lymph nodes
Polymerase Chain Reaction with non-specific lymphadenitis, EBV-infected cells were
found, and all were type 111. Two of 60 B-MLs were
Isolated DNAs were used for PCR. We synthesized metastases of pyothorax-associated lymphoma (PAL),
the specific primers to examine the presence of EBV on and the two cases were type I.
the basis of the published DNA sequence, which corres-
ponded to the EBV W region [14]. After PCR amplifica- EBV-Associated Products (LMP, EBNA-2,
tion, one-tenth of the reaction mixture was then analyzed and vlL-10)
by Southern blot.
Expression of LMP was demonstrated on the cell mem-
brane and cytoplasm (Fig. 3 and Table I). In addition,
RESULTS LMP was found in four B-MLs and six T-MLs. In four B-
A total of 119 cases were examined: 106 with non- MLs, LMP was expressed in almost all EBER-l-positive
Hodgkin’s malignant lymphoma (ML), and 13 with non- lymphoma cells; however, LMP of T-MLs was found in
specific lymphadenitis. ML was categorized into T-cell some scattered lymphoma cells in T-MLs. The number
lymphoma by the genotype of TCR and the phenotype of LMP-positive cells was less than the number of EBER-
of UCHL-1, CD2, CD3, CD4, and/or CD8 or B-cell l-positive cells by ISH. Expression of vIL-10 was con-
lymphoma by JH, L26, CD19, and/or CD20. Anaplastic fined to the LMP-positive cases, and the number of vIL-
large cell lymphoma was identified by morphology and 10-positive cells was less than the number of LMP-posi-
CD30 (Table I). HTLV-1 proviral DNA was found in 17 tive cells. In the serial sections, vIL- 10-positive cells were
cases of T-ML. positive for LMP. Expression of EBNA-2 was found in
only three B-MLs, and EBNA-2 was expressed in almost
EBV Presence: Correlation of Southern Blot, all the lymphoma cells. One of the three EBNA-2-positive
PCR, and ISH cases was an aged patient with anaplastic large cell
EBV genomes and EBV-infected cells were found in lymphoma; the other two had pyothorax-associated
13 of 106 MLs (12%) by Southern blot analysis, in 24 lymphoma (PAL).
of 106 MLs (23%) by PCR, and in 52 of 106 MLs (49%)
by ISH (Fig. 1 and Table I). In non-specific lymphadenitis, Clonality of EBV-Infected Cells
EBV was found in 6 of 13 cases (46%) by PCR and ISH. Seven B-MLs and three T-cell lymphomas showed
The density of the DNA bands and amplified DNA and clonal TR bands. One case of T-cell lymphoma with
the number of ISH-positive cells varied, considered to angioimmunoblastic lymphadenopathy features (AILD-
be due to the varying numbers of EBV-infected cells. The T) displayed two clonal bands, and the other showed a
sensitivity of ISH was the best, since the Southern blot monoclonal band. The density of the bands varied, but
and PCR targets consisted of only a few copies of the it was related to the number of EBER-l-positive cells.
EBV-W region DNA in the EBV-infected cell, whereas In type I1 T-MLs, the band showed a very weak density,
24 Ohshima et al.

Fig. 1. A Southern bot and PCR analysis of the EBV ge- lane 4 is T-ML (AILD-T), and lane 5 is T-ML (ATLL). The BamHl
nomes. In the Southern blot of EBV-W and TR, lane l is a restriction enzyme is used. In the PCR analysis, lane 1 is a
negative control (placental DNA), lane 2 is a positive control negative control, lane 2 is a positive control, lane 3 is B-ML,
(EBV+ lymphoepithelioma),lane 3 is B-ML (diffuse large), lane 4 is T-ML, and lane 5 is non-specific lymphadenitis.

and in type I B-MLs, the band showed a strong density malignant transformation, whereas in the other cases, with
(Fig. 1 and Table I). a smaller number of infected cells, EBV infection may
have occurred following malignant transformation [ 151.
Our Japanese nodal B-MLs displayed more frequent EBV
DISCUSSION
infection than European cases, but the difference does
The present study shows an unexpectedly high inci- not seem to be significant. Our seven cases (12%, 7/
dence of EBV infection in nodal non-Hodgkin’s 60) with over 50% EBV-infected tumor cells were also
lymphoma (ML). By EBER-ISH, EBV infection was anaplastic large cell lymphomas or large cell lymphomas,
found in 52 of 106 cases of ML, but the number of which included PAL, or large cell lymphoma with plasma-
the EBV-infected cells varied and the proportion of the cellular differentiation.
infected cells ranged from 1% to 100%. From the number EBNA-2 is a kind of oncoprotein that, in association
of EBV-infected cells, non-Hodgkin’s lymphomas were with LMPs, plays an important role in B-lymphocyte
grouped into three types: I) almost all lymphoma cells transformation [16]. The cytotoxic T cells of the host
showed an EBV presence; 11) some scattered lymphoma recognize the LMP molecules and induce cell-mediated
cells showed an EBV presence; and 111) only a few cells cytolysis. In the absence of LMP, the EBV-induced trans-
showed EBV, probably due to latent EBV infection. In formants cannot be lysed by cytotoxic T cells [15]. Rowe
25 of 60 B-MLs, EBV-infected cells were found; 7 were et al. [6] classified EBV-associated tumors into three types
type I, 1 was type 11, and 17 were type 111. In 27 of 46 based on expression of LMP and EBNA. For example,
T-MLs, EBV-infected cells were found; no cases were in Hodgkin’s disease, LMP was expressed, whereas
type I, 5 were type 11, and 22 were type 111. EBNA-2 was absent (latency 11) [4]. In the B-lymphoblas-
Previously 208 cases of B-MLs without human immu- toid cell lines, both were expressed (latency III), and in
nodeficiency virus (HIV) infection were investigated for Burkitt’s lymphoma both were absent (latency I) [6]. In
an association with EBV infection in Europeans using this study, LMP was found in four B-MLs and six T-
PCR and ISH [15]. EBV was present overall in 26% (54/ MLs. In four B-MLs, LMP was expressed in almost all
208). Through EBER-ISH, EBV could be localized in EBER- 1-positive lymphoma cells, but LMP was found
tumor cells of 27 cases (13%, 27/208), but the proportion in only a few scattered lymphoma cells in T-MLs. Expres-
of EBV-infected cells in the different cases varied between sion of EBNA-2 was found in only three B-MLs, in which
1 and 100%. Morphologically, the 17 cases (8%) with EBNA-2 was expressed in almost all lymphoma cells.
over 80% EBV-infected tumor cells were either anaplastic Three cases of EBNA-2-positive B-MLs, in which LMP
large cell lymphomas, sporadic Burkitt’s lymphomas, or was positive, were classified as latency I11 type, and
high-grade lymphoma with plasmacellular differentiation. the other seven LMP-positive B-MLs and T-MLs were
In these cases, EBV infection probably takes place before latency I1 type. In addition, small EBV-infected bystander
 EBN in Non-Hodgkin’s Lymphoma 25

Fig. 2. Light microscopical appearance (a, c, e) and in situ cells show an EBV infection (b). A diffuse proliferation of
hybridization (EBER-1) (b, d, f). a and b, c and d, and e and lymphoma cells (c; ATLL) and some lymphoma cells show
f are the same cases. A diffuse proliferation of atypical large EBV (d). A diffuse proliferation of lymphoma cells (e; AILD-
lymphoid cells is found (a; B-ML), and almost all lymphoma T); only one cell shows an EBV infection (f).

cells proved to be constantly LMP negative; they were berculous pyothorax [17]. Most of these lymphomas are
thus probably latency I type. In our nodal lymphomas, B-cell lymphomas and are closely associated with EBV.
no specific pattern of LMP and EBNA-2 expression Both EBNA-2 and LMP were demonstrated in PAL, as
was found. in immune-deficient EBV-infected lymphoma [ 171.
PAL is a rare tumor associated with long-standing tu- Southern blot analysis showed a single terminal fragment
26 Ohshima et al.

Fig. 3. lmmunohistochemical staining. Staining for LMP shows a positive reaction in the
cytoplasm of almost all lymphoma cells (a; B-ML) and a large immunoblast-likecell (b;
AILD-T). EBNAS is positive in the nuclei of almost all lymphoma cells (c; B-ML). IL-10 is
positive inthe cytoplasm of almost all lymphoma cells (d; 6-ML), and IL-10 is positive in
a few cells (e; AILD-T).

in PAL. Our two cases also showed LMP, EBNA-2, and showed clonal bands, but the density of the bands varied.
a clonal terminal fragment in each. The density related to the proportion of EBV-infected
ATLL is a human malignancy associated with HTLV- cells. In those cases in which TR was detected, LMP and
I, which is a retrovirus [ 171. There has also been a report EBNA-2 reaction were positive or negative and showed
in which the C3d receptor expression on the cell mem- no specific patterns. The reason for this is uncertain;
brane of ATLL was detected [ 18,191. Tokunaga et al. [5] possibly clonal pathogenesis is not associated directly
reported that ISH indicated EBV in the nuclei of ATLL with either lymphomagenesis or the immune reactions of
tumor cells in 16 of 96 cases (17%), while the number the hosts.
of EBV-positive cells varied. However, our ATLL study IL-10 was initially purified as a cytokine synthesis
showed a more frequent EBV infection, which may be inhibitory factor, which inhibits many types of cytokine
due to the sensitivity of the methods used. release including interferon-? (IFN-y), which is important
Study of the TR sequences of the EBV genome is in mediating cellular defenses against EBV infection [9].
considered to be useful as a means of assessing the clon- IL-10 also exhibits strong DNA and amino acid sequence
ality of the cellular population harboring the virus [ 111. homology to BCRFl (vIL-lo), an open reading frame in
For example, EBV-associated lymphoepithelioma of the the EBV genome [7]. vIL-10 has apparently conserved
nasopharynx represents a clonal expansion of a single many functions of IL-10. The inhibitory activity would
EBV-infected progenitor cell [ 111. TR analysis revealed also likely benefit the virus, because both T cells and
the oligoclonal or monoclonal origin of B-cell lymphoma natural killer cells will permit an EBV infection and
[20], polyclonal B-cell lymphoproliferation of infectious immortalization of the B cells [8]. Using a wide panel
mononucleosis [21], and monoclonal T-cell lymphoproli- of EBV-positive and EBV-negative cell lines, it has been
feration in chronic active EBV infection [22]. In this shown that EBV-positive B-cell lines derived from pa-
study, all ten cases in which TR bands could be found tients with the acquired immunodeficiency syndrome and
 EBN in Non-Hodgkin’s Lymphoma 27

Burkitt’s lymphoma secrete large quantities of IL-10. By duced polyclonal and monoclonal B cell lymphoproliferative disease
occurring after renal transplantation. Clinical, pathologic, and virologic
contrast, EBV-negative B-cell lines do not express IL-10 findings and implications for therapy. Ann Surg 198:356, 1983.
[23]. LMP induced IL-10 production in EBV-carrying 2. Hamilton-Dutoit SJ, Pallesen G, Franzmann MB, Karkow J, Black
Burkitt’s lymphoma lines, whereas EBNA showed no F, Skinhoj P, Pedersen C: AIDS-related lymphomas. Histopathology,
such induction [24]. In this study, the LMP-positive cases immunophenotype and association with Epstein-Ban virus as demon-
frequently showed expression of IL-10. Thus LMP proba- strated by in situ nucleic acid hybridization. Am J Pathol 138:149,1991.
3. Weiss LM, Movahed LA, Wamke RA, Sklar J: Detection of Epstein-
bly induced IL-10 production in the EBV-infected cells. Ban viral genomes in Reed-Stemberg cells of Hodgkin’s disease. N
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Previously, 81 cases of T-ML occurring in HIV-nega- Lantzsch N, Stein H: Epstein-Barr virus latent membrane protein ex-
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5. Tokunaga M, Imai S, Uemura Y, Tokudome T, Osato T, Sat0 E: Epstein-
cells of 30 cases, with the proportion of the infected cells Barr virus in adult T cell leukemiallymphoma. Am J Pathol
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7. Vieira P, DeWaal-Malefty R, Dang MN, Johnson KE, Kastel-Stein R,
rarely demonstrated in cutaneous and enteropathy-associ- Fiorentino DF, De Vries JE, Roncarolo MG, Monsmann TR, Moore
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