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Frequent Latent Epstein-Barr Virus Infection of Neoplastic T Cells and
Bystander B Cells in Human Immunodeficiency Virus-Negative European
Peripheral Pleomorphic T - c e l l Lymphomas
By Petra Korbjuhn, loannis Anagnostopoulos, Michael Hummel, Markus Tiemann, Friederike Dallenbach,
Mohammed R. Parwaresch, and Harald Stein
W e investigated 8 1 cases of peripheral pleomorphic T-cell
lymphoma (PMTCL) occurring in human immunodeficiency
virus-negative Europeans for the presence of Epstein-Barr
virus (EBV)-DNA through polymerase chain reaction (PCR)
for the presence of EBV-encoded small nuclear RNAs
(EBER) and immediate early mRNAs (Bam H-fragment,
lower strand frame [BHLF]) by in situ hybridization (ISH)
and for EBV-encoded latent membrane protein (LMP) and
nuclear antigen 2 (EBNAZ) by immunohistology (IH).
EBER-ISH, which could be applied on all cases, showed an
overall incidence of EBV-infected cells in 38 of 8 1 cases
(47%)of PMTCL. These data could be confirmed by PCR,
which produced congruent results in the cases with amplifiable DNA. By EBER-ISH, the virus was located in the tu-
mor cells in 30 of the 38 EBV-positive cases, with the proportion of the infected cells ranging from 1 % to 100%. In
1 8 of these cases and in the 8 cases without EBV-infected
tumor cells, the virus was, respectively, either additionally
or exclusively detectable in occasional nonmalignant lymphoid bystander cells. An LMP expression was observed in
several of the EBER-expressing tumor cells in 1 8 cases,
whereas EBNA2 was detectable only in one case, which
also displayed signs of viral replication. Some nonmalignant EBV-infected B immunoblasts also expressed LMP in
several cases. Primary cutaneous and enteropathy-associated PMTCL displayed less frequent EBV infection when
compared with other extranodal or nodal manifestations.
0 7993 by The American Society of Hematology.
T
was diagnosed according to the criteria ofthe Updated Kiel Classification.” These lymphomas consisted of sheets of T cells with medium-sized to large and irregular nuclei, small to moderately prominent nucleoli, and a varying amount of usually pale cytoplasm.
Mitotic figures were frequent but varied from area to area. The
architecture of the infiltrated lymph nodes was completely effaced,
with the exception that, in some instances, B-cell zones were partially spared. The pleomorphic T cells in the skin frequently showed
a marked epidermotropism with infiltration of the basal cell layer
often accompanied by ulceration. The manifestations of these lymphomas in nasopharyngeal and oral areas exhibited prominent vasoinvasion leading to extensive necrosis, corresponding to the clinical diagnosis of lethal midline granuloma. The collection of
PMTCL also included 10 neoplasms from the jejunum showing
villous atrophy and dense infiltration of the mucosa by the neoplastic intraepithelial T cells. Thus, these lymphomas fulfilled the criteria of enteropathy-associated T-cell lymphoma.21 As a control
group, multiple, small-sized axillary lymph nodes removed from 12
patients during the course of mastectomy were used. Histologically,
they did not display any signs of activation and are therefore referred to as normal lymph nodes.
Histology and immunohistology. Four-micron sections were
cut and stained with hematoxylin and eosin (H &E), Giemsa, peri-
HE EPSTEIN-BARR virus (EBV) is a DNA gamma
herpes virus capable of transforming B cells in vitro
and has been found to be the causative agent of infectious
mononucleosis.’ Since its first isolation from cell lines established from African Burkitt’s lymphoma,2EBV has been
linked with a variety of neoplasms, including nasopharyngeal ~ a r c i n o m a , Hodgkin’s
~,~
d i ~ e a s e polymorphic
,~
B-cell
lymphomas and atypical B-cell lymphoproliferations in immunocompromised individuals,6-’’ and some nasopharyngeal T-cell lymphomas occurring most frequently in Asian
patient^.'^,'^ To determine the incidence of EBV infection
in non-Hodgkin’s lymphomas (NHL) from Northern European patients without congenital or acquired immunodeficiency we screened more than 600 NHLs collected from
former West and East Germany for the presence of EBVDNA using the polymerase chain reaction (PCR). We noticed, to our surprise, that, apart from T-cell lymphoma of
angioimmunoblastic-lymphadenopathytype (AILD-TCL),I4
EBV-DNA was more frequently detected in peripheral pleomorphic T-cell lymphomas (PMTCL) than in any other European NHL type. This finding prompted us to purposely
collect a large series of 8 1 PMTCL cases from two German
lymphoma reference centers (Berlin and Kiel) and to further analyze them in addition to the presence of EBV-DNA
for cellular localization of the EBV-encoded transcripts
EBER-1, EBER-2,’5,’6and BHLF,17 and the expression of
latent infection gene products, such as the latent membrane
protein (LMP)I7-I9and the nuclear antigen 2 (EBNA2).I7
MATERIALS AND METHODS
Bioptic material. Paraffin-embedded specimens with PMTCL
from lymph nodes (44), skin (15), nasopharynx/oral cavity (6),gastrointestinal tract (15), and spleen ( I ) obtained from 81 patients
were selected from the files of the Institute of Pathology, Klinikum
Steglitz, Free University, Berlin (Berlin, Germany) and from the
Institute of Hematopathology, University of Kiel (Kiel, Germany).
None of the patients was of Asian descent or evidenced a history or
other signs of acquired or congenital immunodeficiency. PMTCL
Blood, VOI 82, NO 1 (July 1). 1993: pp 217-223
From the Konsultations und Referenzzentrum fur Lymphknotenund Hamatopathologie am Institut fur Pathologie, Klinikum Steglitz, Freie Universitat Berlin, Berlin: and the Institut fur
Hamatopathologie, Universitat Kid, Kiel, Germany.
Submitted August 14, 1992; accepted February 12, 1993.
Supported by the Deutsche Forschungsgemeinschaft. Projekt HU
557/1-1, and the Deutsche KrebshilJe Projekt M 25/89/St 1.
Address reprint requests to Harald Stein, MD, Institute of Pathology, Klinikum Steglitz, Free University Berlin, Hindenburgdamm
30, 1000 Berlin 45, Germany.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-49 71/93/8201-0010$3.00/0
217
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218
KORBJUHN ET AL
odic acid (PAS), and Gomori silver impregnation. Immunohistologic analysis involved the use of monoclonal antibodies (MoAbs)
and polyclonal antibodies for the detection of lineage-specific or
lineage-characteristic antigens in paraffin sections. The anti-T-cell
antibodies CD3 polyclonal, CD4 (MT310), CD8 (DK25), CD43
(DF-T l), and CD45RO (UCHLI); the anti-B-cell antibodies CD20
(L26) and CD45RA (4KB5); the antimacrophage antibody CD68
(PG-Ml); the anti-CD30 antibody Ber-H2; the a n t X 3 d receptor/
antifollicular dendritic cell antibody CD2 1 (1F8); the anti-EBVLMP antibody CS1-4; and the anti-EBNA2 antibody PE2 were obtained from Dako (Glostrup, Denmark). The anti-T-cell receptor @
chain antibody OF1 was purchased from T Cell Sciences (Cambridge, MA). The antibodies CD45RB (G-B3), Ki-M4p (FDC),
and CD68 (Ki-M Ip) were provided by the laboratory of Dr Panvaresch (Gel, Germany). The bound antibodies were made visible by
using the alkaline phosphatase antialkaline phosphatase (APAAP)
method in association with the New Fuchsin development according to Cordell et a]*’ or with the streptavidin-biotin complex
method (ABC) described el~ewhere.’~
PCR. PCR for the detection of EBV- and @-globin-specificsequences in paraffin-embedded tissue sections was performed as described previously.’4
In situ hybridization (ISH). EBER-ISH was performed with in
vitro-transcribed digoxigenin (DIG)-labeled sense (negative control) and antisense RNA probes on paraffin sections (3 to 5 pm) for
the detection of EBER-positive cells. After dewaxing, rehydration,
and proteinase K digestion, the sections were hybridized overnight
at 42°C in a solution of 50% formamide containing 5 ng DIG-labeled probes. After washing in Tris buffered saline (TBS) buffer and
RNAse treatment, detection of the bound labeled probes was
achieved by incubation with a monoclonal DIG-specific antibody
conjugated with alkaline phosphatase. The EBV-positive cells became visible after development involving Naphtol-As-Biphosphate
and New Fuchsin (Merck, Darmstadt, Germany). The sensitivity of
our approach and the suitability of the tissue sections used were
assessed by a former parallel hybridization of Hodgkin’s disease24
and AILD-TCLI4 tissue sections with radioactively and DIG-labeled in vitro-transcribed probes and by hybridization with a @-actin probe, respectively.
For thedetection ofBHLF-mRNA, a mixture ofthree oligonucleotides complementary to two abundant immediate early mRNAs
encoding proteins belonging to the subgroup of EA-D of EBV-encoded antigens”,” were used for ISH. The procedure was performed according to the manufactor’s protocol (Dako; code no. Y
018) with the exception that we used Naphtol-As-Biphosphate/
New Fuchsin instead of nitro-blue-tetrazolium/brom-chlorindoxylphosphate (NBT/BCIP) for the detection of BHLF-expressing
cells.
Double lubeling. To determine the B- or T-cell nature of EBVinfected cells, we performed a double-labeling procedure using immunohistology (IH) and EBER-ISH. The immunohistologic demonstration of CD20 and CD45RO antigens involved the ABC
method (DAKO) that was applied before EBER-ISH. EBER-ISH
was performed as described above using a development either involving Naphtol-As-Biphosphate and New Fuchsin or alternatively
NBT/BClP (BioRad, Munich, Germany). All steps were performed
under RNAse-free conditions. After development, the slides were
washed in phosphate-buffered saline (PBS) buffer and submitted to
EBER-ISH as described above. As a result of this procedure, EBVinfected B or T cells became visible by their purple-red or deep blue,
respectively, nuclei and brown membrane staining.
RESULTS
EBV-DNA detection by PCR. In 30 of the 8 I PMTCL
cases and 2 of the 12 normal lymphoid tissue samples, the
extracted DNA was not amplifiable, as shown by the nonappearance of the P-globin-specific band after two-step nested
primer PCR with the appropriate primers. In an additional
19 PMTCL cases, the scarcity of embedded tissue did not
allow the application of PCR. PCR analysis of the remaining 32 PMTCL cases detected EBV-specific sequences in 20
cases. EBV-specific DNA was only present in 2 of the remaining 10 normal lymphoid tissue samples with amplifiable DNA.
Correlation of PCR and EBER-ISH results. The comparison of the EBER-ISH data with the PCR results obtained from the 32 cases in which extracted DNA was suitable for amplification showed a complete congruence of the
findings, except for one case. In that instance, EBV-specific
sequences were shown by PCR, whereas no positive cells
could be identified after EBER-ISH. This difference implies
that the positive PCR result was due to the presence of one
single EBV-infected cell in the section used for DNA extraction and that the section used for EBER-ISH was devoid of
such a cell.
Detection of EBER and BHLF by ISH. By application
of DIG-labeled antisense probes specific for EBER-1 and
EBER-2, EBV-infected cells could be shown in 38 of the 8 1
PMTCL cases as well as in 3 of the 12 normal lymphoid
tissue samples. Only one case was found to be positive when
a mixture of three BHLF-specific oligonucleotides was used
for ISH.
The preservation of morphology achieved by application
of DIG-labeled probes for ISH allowed precise morphologic
evaluation of the EBV-infected cells. It could be clearly
shown in each case that only lymphoid cells were EBV-positive. All other cell types, including macrophages, granulocytes, endothelial cells, fibrocytes, plasma cells, and epithelial cells, were EBV-negative. For lineage allocation of the
EBV-infected cells, double labeling was performed on all
cases containing EBER-positive tumor cells. This procedure
led to satisfactory results in 20 of 30 cases. The lineage of the
EBV-infected cells in the remaining cases was established
after comparing the EBER-ISH with immunohistologic
staining reactions performed on serial sections. The following groups of EBV presence could be distinguished (see also
Table 1).
Group I displayed a diffuse and focal distribution of
many EBV-infected tumor cell nuclei in 8 of 81 cases (Fig
1A). The labeled cells corresponded in size and cytology to
the neoplastic blasts expressing T-cell antigens. The amount
of the labeled cells made up 20% to 100% of the whole
tumor cell population (Fig 1B). Additional EBER-positive
small B lymphocytes and B immunoblasts could be identified in 5 cases (Fig IC), whereas coexistent infected small T
cells occurred only in 1 case. In addition to the cases with
latent EBV infection, only I case displayed signs of a lytic
infection in single neoplastic T cells as proved by BHLFISH (Fig 1E).
Group I1 showed an EBV infection of scattered tumor
cells, often with focal accumulation, in 22 of 8 1 cases (Fig
2A). The amount of EBV-infected tumor cells accounted
for 1% to 20% of the tumor cell population (Fig 2B). In 13
cases of this group, a coexistent labeling of several B im-
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219
EBV IN PLEOMORPHIC T-CELL LYMPHOMAS
Table 1. Cellular Localization and Frequency of EBER and L M P Expression in PMTCL
EBER Expression in
6-16 and/or
Small 8
Total
No. of
Tumor Cells
Cases'
8
22
8
43
Group I
Group II
Group Ill
Group IV
Lymphocytes
8 (20-1OO%)t
22 (1-20%)t
0
0
LMP Expression in
Small T
Lymphocytes
5
13
8
0
Tumor Cells
6-16
6
12
0
2
4*
0
0
0
The figures in the columns indicate number of cases
Abbreviation: E-IB, E-immunoblasts.
* Of the various groups of cases examined, a number showed labeling of not only EBV-positive tumor cells, but also nonmalignant bystander cells.
t Percentage of EBER-positive tumor cells per case.
$ Two of these cases exclusively contained LMP-positive E-IB
munoblasts and small-sized B cells was observed, lying in
the remnants of B-cell zones, whereas a labeling of small T
lymphocytes was observed in one specimen.
Group 111 presented with an occasional occurrence of
EBER-positive cells in 8 of 8 1 TCL. These cells possessed a
round or slightly irregular nucleus that resembled that of the
rare EBER-positive lymphoid cells encountered in 3 of I2
normal lymphoid tissue samples.
Group IV consisted of 43 cases without any EBER-labeled cells.
Detection of EBV-encoded LMP and EBNAZ. The
cocktail of four MoAbs against different epitopes on the
LMP led to the staining of lymphoid cells in 20 of the 81
T-cell lymphomas (Table 1). The number of LMP-positive
cells was in all instances much less than the number of
EBER-positive cells. The intensity of the LMP staining varied considerably within the same case and between the
cases. The comparison with the infection patterns seen with
EBER-ISH led to the following findings: Of the eight cases
of infection pattern group I displaying a high proportion of
EBV-infected tumor cells, six contained LMP-expressing
tumor cells (Fig 1D). In addition to LMP expression, one of
these cases displayed EBNA2 expression in single tumor
cells, as shown by application of the MoAb PE2. This case
also showed an expression of BHLF-mRNA. Furthermore,
two of these cases also contained LMP-positive B immunoblasts. The remaining 14 cases with LMP expression belonged to group 11. Twelve of these cases showed LMP staining in neoplastic T blasts with additional occurrence of
LMP-positive B immunoblasts in 2 cases. In 2 further cases,
the LMP expression among the B cells was restricted to B
immunoblasts. No LMP expression could be detected in the
third group with occasional EBER-positive small lymphoid
cells, similar to the EBER-positive normal lymphoid tissue
samples.
Correlations of EBV infection patterns with patients' age
and tumor localization. The male/female ratio of the lymphoma patients was 2.2: 1. The groups with EBV-positive
tumor cells (I and 11) and the EBV-negative group (IV) displayed similar age distribution, with an increased incidence
in those of greater age (61 to 90 years), whereas an even
distribution was observed in the group with EBV-harboring
bystander cells (111). The correlation with the tumor localiza-
tion showed preferential EBV infection of PMTCL that primarily occurred in the nasopharynx, lymph nodes, and gastrointestinal tract, with a trend of higher incidence in the
nasopharynx. In contrast, EBV infection in primary cutaneous and enteropathy-associated PMTCL was significantly
lower (Table 2).
DISCUSSION
The present study shows in a large series of cases (n = 8 1)
an unexpected high incidence of EBV infection in European
cases of PMTCL. All patients of our study were HIV-negative and did not display any history or signs of congenital or
acquired immunodeficiency. By using two-step PCR, EBVDNA was detected in 20 of 32 cases of PMTCL in which
amplifiable DNA could be extracted from paraffin-embedded biopsies. In contrast, EBV-DNA was detectable in only
2 of 10 normal lymph node samples with amplifiable DNA.
These results could be confirmed by EBER-ISH. The nearly
100%congruence between the results obtained by PCR and
EBER-ISH is in line with findings indicating that EBERISH allows the detection of all latently EBV-infected cells.
The EBER-ISH could also be applied to those cases that
proved to be unsuitable for PCR due to the absence of amplifiable DNA, resulting in the fact that all PMTCL cases
collected could be used for our study. With this approach,
EBV-harboring cells were detected in 38 of 8 1 (47%) of the
PMTCL and in 3 of 12 (25%) of the normal lymphoid tissue
samples. The application of nonisotopic, (DIG)-labeled
EBER probes on routinely processed paraffin sections allowed, thanks to their superb morphologic preservation,
subtle evaluation of both the morphology and the amount
of infected cells. When combined with immunohistologic
analysis of adjacent sections and/or the same sections (double labeling), EBER-ISH could identify most EBV-harboring cells as being neoplastic T cells in 30 of 81 PMTCL
cases, with their percentage ranging from 1% to nearly
100%.Abundant EBV-harboring neoplastic T cells (more
than 20%) occurred in 8 cases. In addition to infected neoplastic T cells, 18 cases displayed EBV-infected small bystander B lymphocytes. In 6 cases, some of the EBV-positive
B cells had the morphology of immunoblasts. Of the remaining 5 1 PMTCL cases without EBV-positive tumor
cells, 8 cases contained occasional EBV-positive small lym-
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220
KORBJUHN ET
AL
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EBV IN PLEOMORPHIC T-CELL LYMPHOMAS
-
~
~
Fig 1. EBV infection pattern I in PMTCL. (A) ISH of a PMTCL
case by using DIG-labeled EBER-1- and EBER-2-specific probes.
Labeling of multiple positive medium- to large-sized pleomorphic
nuclei. (B) Double labeling for CD45RO molecule (MoAb UCHL-1;
ABC reaction) and EBER (DIG-labeledspecific probes) of the same
case as in (A). Note that approximately 90%of the T cells (brown
membrane staining) contain EBER-positive (deep blue) nuclei. (C)
Double labeling for CD20 molecule (MoAb U 6 ;ABC reaction) and
EBER (ISH with DIG-labeledspecific probes) of the same case as in
(A). The staining pattern is complementary to the T-cell labeling
shown in (B). Among the CD20 - EBV-infectedneoplastic T cells,
there are a few CD20+ B cells with EBER-positivepurple-red nuclei
(arrows). (D) LMP immunostaining of the same case as in (A) with
the MoAb CS1-4. The labeling is confined to those cells that resemble neoplastic T cells in cytology and distribution. (E) ISH of another
PMTCL case by using a mixture of three oligonucleotides complementary to immediate early mRNA (BHLF). Labeling of single medium to large-sized pleomorphic nuclei. Original magnification
X 400 is the same for (A) through (E).
phocytes similar to the findings observed in 3 of 12 normal
lymph nodes. It should be stressed that, in all instances,
EBER expression was restricted to cells of lymphoid immunophenotype and/or morphology. As EBER expression
may be downregulated in the case of a lytic infe~tion,'~
the
presence of nuclear immediate early mRNAs was investigated by applying ISH for BHLF detection. BHLF-mRNAs
were present only in one case, which additionally showed
abundant EBER-expressing tumor cells.
To clarify whether the functional state of EBV genomes
differs between the latently infected tumor and bystander
cells, the expression of EBV-encoded LMP and of EBNA2
was studied by IH. Small EBV-infected bystander cells
proved to be constantly LMP-negative, in contrast to the
neoplastic T cells, which were LMP-positive in 18 of the 30
cases of PMTCL containing EBER-positive tumor cells. Interestingly, LMP was also detectable in some EBER-positive B immunoblasts. In contrast to LMP, EBNA2 was detectable only in one case that also contained EBV in its
replicative form, as described above. In all instances, the
number of LMP- and EBNAZpositive cells was considerably lower than the number of EBER-positive cells. Previous studies on EBV gene expression during latency have
found concordant expression of LMP and EBNA2 in most
cases of posttransplant lymphomas" and nasal T-cell lymphomaZ6similar to the EBV phenotype observed in lymphoblastoid cell lines. The lack of EBNA2 expression in the vast
majority of our LMP-positive cases is similar to the discordant patterns of expression described in Hodgkin's disease," in most EBV-positive acquired immunodeficiency
syndrome (AIDS)-related lymphomas,28and in nasopharyngeal ~arcinoma.'~
Because LMP and EBNA2 detection
by the MoAbs used is slightly reduced in paraffin sections, it
is likely that the expression of these proteins may be more
221
Table 2. Site of Primary Manifestation of EBV-Positive
and EBV-Negative PMTCL
Average
Manifestation
Frequency
Site of Primary
Manifestation
(Group I
Group I
Group II
Group 111
Group IV
+ II)
(Group 'I1+ 1")
Lymph node
Skin
Nasopharynx/
mouth
Gastrointestinal
Spleen
Jejunum'
3
2
18
0
5
1
18
12
0.97
0.17
3
0
0
0
0
2
0
2
0
0
0
2
3
3
1
6
1.o
0.67
Total no. of
cases
8
22
8
43
-
0.25
The figures in the first four columns indicate number of cases. For the
definition of the groups I to IV, see Table 1.
Associated with celiac disease.
extensive than what we were able to document in this study.
Our findings show that EBV is transcriptionally active in
the infected neoplastic T cells. Because LMP has transforming p~tential~',~'
and can prolong cell survival,32it might
well be that EBV is involved as a cofactor in the genesis of
EBV-positive PMTCL. Our results are principally in accordance with observations made in two recent s t ~ d i e sthat
~~,~~
report on a relatively frequent presence of EBV-DNA and
its gene products in PMTCL. However, due to their inherent insensitivity, the techniques applied in these studies (ie,
Southern blot and/or ISH for EBV-DNA detection; IH for
LMP and EBNA2 expression) could not detect the entire
amount of EBV-harboring cases nor were they capable of
providing precise information regarding the amount, morphology, and lineage allocation of the infected cells.
However, the role of EBV in PMTCL cannot be clearly
understood unless the reason for the presence of EBV in
only a proportion of and not in all neoplastic cells in the
majority of PMTCL studied is clarified. It is also necessary
to explain the frequent coinfection of B cells in addition to
the EBV infection of the tumor cells. EBER-ISH has shown
that in other EBV-associated neoplasms, such as Burkitt's
lymphoma (from our own unpublished data) and Hodgkin's disease,24all tumor cells harbor the virus, implicating
that such neoplasms are derived from a single EBV-infected
lymphocyte. Four assumptions appear to be conceivable for
explaining EBV detection in only a proportion of tumor
cells (Fig 3): (1) some of the tumor cells either eliminate or
lose (through unequal distribution of the episomes during
cell division) the virus, or the viral genes encoding EBER- 1
and EBER-2 become inactive or defective; (2) the tumor is
oligoclonal and derives from EBV-positive and EBV-nega-
4
Fig 2. EBV infection pattern II in PMTCL. (A) ISH of a PMTCL case using DIG-labeledEBER-1 and EBER-2 probes. Labelingof occasional
pleomorphic nuclei. (B) Double labeling of the same case (A) for CD45RO molecule (MoAb UCHL-1; ABC reaction) and EBER (DIG-labeled
specific probes). Only a few of the neoplastic T cells show a colocalizationof both labels. Original magnification X 400 for both (A) and (B).
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KORBJUHN ET AL
222
TCL derived from an
EBV-infected T-cell: 100% EBV'
EBV
@
5
7
0
@\
Oliqoclonal TCL derived
?om i n EBV' and EBV- T-cell
EBVi
f
@
0
\ 4"
0-0
formarlon
Secondary EBV-infection
of a monoclonal TCL
Loss of EBV
in a monoclonal T C l
EBV
$,
0
f
@
I,
-0
'.o'
stage represented by the tumor cells in these cases is particularly susceptible to EBV infection. Although EBV receptor
(CD2 1) of low density occurs on up to 40% of circulating T
cells,36the mechanism by which the neoplastic T cells were
infected could not be clarified because the CD2 I molecule
was not detectable on the lymphoid cells in our material.
However, this may be due to the staining of paraffin sections
in which a low expression of these molecules escapes detection.
Correlation of the presence of EBV in tumor cells with the
site of the primary lymphomatous manifestation disclosed
that EBV infection of PMTCL located in the nasopharyngeal region is frequent not only in Asian countries but also
in Northern Europe. However, our report is the first that
describes, based on a large number of cases and the application of two highly sensitive EBV detection systems, a high
frequency of latent EBV infection in nodal and gastrointestinal PMTCL and a much rarer EBV infection of cutaneous
and enteropathy-associated PMTCL (Table 2). The reason
for this difference in the incidence of EBV infection between PMTCL of different localizations requires further
clarification. It may very well be that EBV has less tropism
towards skin and gut mucosa-affine T cells or the tissue-specific homing of the skin or gut mucosa-affine T cells has
been reduced after EBV infection. These issues should be
addressed by a prospective study that includes experimental
investigations and the comparison of the clinical course of
EBV-positive and EBV-negative cases.
ACKNOWLEDGMENT
Fig 3. Possible explanation for the different EBV infection patterns in PMTCL.
tive lymphocytes; ( 3 ) a single EBV-negative lymphocyte
gives rise to malignant cells, of which only some become
secondarily EBV-infected; (4) a combination of ( I ) , (2), and
( 3 ) . There is no published evidence as yet that the first possibility may occur in latently infected cells. The second possibility could not be addressed due to a lack of fresh tissue
from the PMTCL cases studied. However, from previous
studies it is known that the majority of PMTCL disclose a
T-cell receptor gene rearrangement pattern that is consistent with monoclonality of the tumor cells.35In view of
these data, it appears to be more likely that EBV-positive
PMTCL cases of our series are secondarily EBV-infected, ie,
after malignant transformation. The demonstration of latently EBV-infected B immunoblasts and small B lymphocytes in several of the PMCTL cases with EBV-positive tumor cells suggests that the neoplastic T cells might have
become infected from these EBV-cawing B cells. This scenario could explain the different percentages of EBV-infected tumor cells seen in the PMTCL studied by suggesting
that (1) the number of the infected T cells may vary and (2)
the proliferation rate of the infected tumor cells may increase, resulting in an eventual predominance of EBV-positive tumor cells. The high frequency of EBV-infected neoplastic T cells in PMTCL indicates that the differentiation
The authors thank C. Kreschel and H.-H. Muller for their excellent technical assistance and L. Oehring for his photographic assistance. Our special thanks also go out to Yvette F. Maddrey and
J.-A. Sutton for their excellent editorial assistance.
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2. Epstein MA, Achong BG, Barr YM: Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet 1:702, 1964
3. Monoyama M, Huang CH, Pagano JS, Klein G, Singh S:
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4. Zur Hausen H , Schulte-Holthausen H, Klein G, Henle W,
Henle G, Clifford P, Santesson L: EBV DNA in biopsies of Burkitt's
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5. Hummel M, Anagnostopoulos I, Dallenbach F, Korbjuhn P,
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7. Hanto DW, Frizzera G, Purtilo DT: Clinical spectrum oflymphoproliferative disorders in renal transplant recipients and evidence for the role of Epstein-Barr virus. Cancer Res 4 l :4253, 198 l
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EBV IN PLEOMORPHIC T-CELL LYMPHOMAS
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1993 82: 217-223
Frequent latent Epstein-Barr virus infection of neoplastic T cells and
bystander B cells in human immunodeficiency virus-negative
European peripheral pleomorphic T-cell lymphomas
P Korbjuhn, I Anagnostopoulos, M Hummel, M Tiemann, F Dallenbach, MR Parwaresch and H
Stein
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