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2137
Nasal T-cell Lymphoma Causally Associated with
Epstein-Barr Virus
Clinicopathologic, Phenoiypic, and Genotypic Studies
Yasuaki Harabuchi, M.D., Ph.D.'
Shosuke Imai, M.D., Ph.D.2
Junichi Wakashima, M.D.~
Motoyasu Hirao, M.D., Ph.D.1
Akikatsu Kataura, M.D., Ph.D.'
Toyoro OSatO, M.D., Ph.D.2
Shinichiro Kon, M.D.,Ph.D.3
' Department
of Otolaryngology, School of
Medicine, Sapporo Medical University, Sapporo, Japan.
Department of Virology, Cancer Institute,
School of Medicine, Hokkaido University, Sapporo, Japan.
Department of Pathology, School of Medicine,
Sapporo Medical University, Sapporo, Japan.
This work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Science, and Culture of Japan.
Presented in part at the 4th International Academic Conference on lmmunobiology in Otology, Rhinology and Laryngology, Oita, Japan,
April 4 to 7, 1994, and at the 13th International
Symposium on lnfectiori and Allergy of the
Nose, Copenhagen, Denmark, June 19 to 23,
1994.
The authors thank Prof. K. Kikuchi of Sapporo
Medical University for providing the L26 monoclonal antibody; Dr. T. Miyashita of National
Children's Medical Research Center, Tokyo, for
genotypic study on a few cases; and Mr. D. Day
of Sapporo Medical University for his helpful
comments.
Address for reprints: Yasuaki Harabuchi, Department of Otolaryngology, School of Medicine, Sapporo Medical University, SlW16,
Chuo-Ku, Sapporo, 060, Japan.
Received July 14, 1995; revisions received November 15, 1995, and February 5, 1996; accepted February 5, 1996.
0 1996 American Cancer Society
BACKGROUND. The authors have previously demonstrated nasal T-cell lymphoma
(NTL) associated with Epstein-Barr virus (EBV). The detailed clinical, phenotypic,
and genotypic features and the role of EBV in lymphomagenesis remain to be
clarified.
METHODS. The study group consisted of 18 patients with NTL. The phenotype was
determined by immunoperoxidase staining with various monoclonal antibodies.
Genotypic study was done using Southern blot hybridization. The presence of EBVencoded small nuclear early region (EBER) RNA and EBV DNA were determined by
in situ hybridization. The expression of EBV-encoded nuclear antigen (EBNA) and
latent membrane protein (LMP1) were identified by immunohistologic methods.
Clonotypic analysis of EBV genomes was performed by Southern blot hybridization
with EBV termini fragment probe.
RESULTS. The clinical features of NTL were characterized as prolonged fever (16
patients), widespread dissemination into distant sites (13 patients), and poor prognosis with a median survival of only 6 months. EBER transcripts were identified
in 16 of 18 patients. Monoclonal EBVgenomes EBNAl and LMPl were also detected
in all EBER-positive cases tested. All 18 patients expressed pan-T antigens such as
MT1, CD45R0, andlor CD2. The rearrangements of T-cell receptor (TCRI-P, =y,
andlor -6 genes were shown in all 11 patients tested. The natural killer (NK) cell
phenotype CD56 was expressed in all EBV-positive cases tested, and was not detected in EBV-negative cases. Seven EBV-positive cases expressed a TCR-b chain
with rearranged TCR-y or -6 genes whereas both EBV-negative cases corresponded
to aPT-cell lymphoma, which expressed a TCR-P chain with a rearranged TCR-~3
gene.
CONCLUSIONS. These data suggest that EBV-positive NTL may be derived from thle
lineage of NK-like T-cells or yJT-cells, and that EBV may play a role in lymphomagenesis. Therefore, we propose that NTL which has peculiar clinical and histologic
features, could be classified as a new lymphoma entity. Cancer 1996; 77213749. 0 1996 American Cancer Society.
KEYWORDS: Epstein-Barr virus, lethal midline granuloma, nasal T-cell lymphomai,
polymorphic reticulosis, ysT-cells, natural killer cells.
L
ethal midline granuloma (LMG)' is a clinical illness characterized b y
progressive unrelenting ulceration and necrosis of the nasal cavity and
midline facial tissues. Histologic features of LMG, displayed by angiocentric and polymorphous lymphoreticular infiltrates, have been called polymorphic reticulosis.' Several investigators, including us3-' on the basis of
phenotypic analysis, have characterized LMG (polymorphic reticulosis)
as T-cell lymphoma, and the term nasal T-cell lymphoma (NTL) has since
been used widely. Recently, the coexpression of the natural killer (NK)
cell phenotype CD56 was seen in some
Molecular studies as-
2138
CANCER May 15,1996 / Volume 77 / Number 10
sessing the clonality of NTL cells have been performed
on only a limited number of cases and their results have
not been uniform."-16 With regard to etiologic factors,
we previously showed Epstein-Barr virus (EBV) genetic
DNA and EBV-oncogenic proteins in five cases of NTL.17
In addition to this report, the presence of EBV genomes
in several cases of NTL from Chinese and Western populations has also been
Recently, we demonstrated clonotypic EBV genome in six cases of the disease?' However, many questions remain. Are the tumor
cells indeed clonal neoplastic T-cells? What subpopulation of T-cell lineage develop to tumor cells? How frequently is EBV detected in a larger series of NTL? Is EBV
only a "silent passenger" or is it closely related to lymphomagenesis?What are the clinical characteristics of EBVpositive NTL?
In the present study, we investigated the presence of
EBV-encoded small nuclear early region (EBER) RNA by
in situ hybridization in 18 Japanese patients with NTL. To
clarify the EBV-association,we analyzed the clonotypes of
EBV genomes by Southern blot hybridization and investigated the expression of EBV-oncogenic proteins such as
EBV-encoded nuclear antigen (EBNA) and latent membrane protein type 1 (LMP1) by immunohistology. To determine the cell lineage of NTL, we performed phenotypic
studies using a wide variety of monoclonal antibodies
and investigated rearrangements of the T-cell receptor
(TCR) genes by Southern blot hybridization. Finally, clinical characteristics of the EBV-positive NTL were discussed.
PATIENTS AND METHODS
Patients
The study group consisted of 18 Japanese patients (9 males
and 9 females with a median age of 46 years; range, 28 to
72 years) who were treated in our clinic between 1974 and
1994. They were clinically diagnosed with LMG based on a
relentless, destructive ulceration and necrosis involving the
nasal cavity and/or midline facial structures. Individuals
with Wegener's granulomatosis, chronic inflammation due
to infections, and any kind of carcinoma were excluded
from this study. Clinical data and part of the immunohistologic reviews of 4 patients (Patients2,3,5, and 18)have been
reported el~ewhere.5"~
Six patients (Patients 8- 13)were also
previously demonstrated to have clonotypic EBV genome
and EBNA in tumor
Clinical Evaluation
Pertinent clinical information and follow-up data were
obtained from hospital charts for all patients. The clinical
records of each patient were reviewed for types and durations of symptoms, initial sites, and involvement of distant sites found during the clinical course. The clinical
stage according to the Ann Arbor systemz4was assessed
by physical examination, gallium 67 (67Ga)and techne-
tium 99m ("Tc) scintiscans, computerized tomography
scan, lymphangiogram, echogram, roentgenographic and
fiberscopic examination of the gastrointestinal tracts, and
bone marrow aspiration.
Histopathologic Study
Biopsy specimens from midline tumor tissues were fixed
in 10% buffered formalin, embedded in paraffin, and
stained with hematoxylin and eosin (H&E)for histologic
studies. Lymphomas were classified according to the
Working Formulation System of the National Cancer In~titute.~~
EBV Studies
The identification of EBER RNAs, which are actively transcribed in latently EBV infected cells,26was performed
by in situ hybridization using fluorescein isothiocyanate
(F1TC)-conjugatedEBV (EBER1 and 2) oligonucleotides
complementary to nuclear RNA portions of the EBERl
and 2 genes (Dako AIS, Glostrup, Denmark). Formalin
fixed paraffin embedded tissue sections were deparaffinized, rehydrated, and treated with 0.2 N hydrochloride
and proteinase K (20 PglmL). The proteolysed sections
were treated with 0.1 M triethanolamine (pH 8) containing 0.25% acetic anhydrate, dehydrated, and airdried. The sections were then hybridized overnight at 42
"C with 30 pL of FITC-conjugated EBER oligonucleotides.
After washing with Tris-buffered saline containing 0.1%
Triton X-100 (TBS-T),the sections were incubated with
1:20 diluted mouse anti-FITC monoclonal antibody
(DakoA/S), 1:lOO diluted biotinylated goat antimouse immunoglobulin (Ig) G (Vector Laboratories, Burlingame,
CA),and 1:lOO diluted streptavidin-biotinylated alkaline
phosphatase complex (Dako A/S), for 45 minutes each at
room temperature. After each incubation, the sections
were washed three times with TBS-T. Colorimetric detection was then performed by incubation with 5-bromo-4chloro-3-indoxyl phosphate and nitro blue tetrazolium
chloride solution (BCIPINBT; Dako AIS) for 10 minutes.
In situ hybridization for EBV genomic DNA was performed as described previously." Briefly, the fresh-frozen
biopsy sections were fixed in Cornoy's B solution, washed
with 2xSSC (sodium chloride-sodium citrate), and
treated with RNase (200 ,ug/mL).After passing through a
series of ascending ethanol, the sections were incubated
with prehybridization solution containing 50% deionized
formamide, 10%dextran sulfate, 2xSSC, 0.12 M ethylenediamine-tetraacetic acid (EDTA),and 0.33 mg/mL salmon
sperm DNA, at 42 "C for 1 hour. The sections were then
heated with undiluted biotinylated EBV Barn HI-V region
probe (Enzo Biochem. Inc., New York, Ny) at 92 "C for 5
minutes for denaturation and incubated at 42 "C overnight. The slides were washed with 2xSSC and incubated
with peroxidase-labeled avidin-biotin complex (Vector
Laboratories)for 45 minutes at room temperature. Color-
Nasal T-cell Lyrnphorna/Harabuchi et al.
imetric detection was then performed by 3-3' diaminobenzidine. EBV-positive Raji cells, lymphoblastoid cells,
and the sections of nasopharyngeal carcinoma were used
as positive controls for EBER and EBV DNA detection.
EBV-negative BJAB cells, palatine tonsillar sections from
an EBV-seronegative donor, and the hybridization mixture without probe were used as negative controls. Controls were run parallel to each other in each batch of
experiments. The EBER signal was abolished after RNase
treatment, confirming the specificity of the reaction.
The detection of intracellular EBV-encoded nuclear
antigen type 1 (EBNA1) was performed by anticomplement immunofluorescence.'7~27The acetone-methanol
(1:l) fixed-frozen tissue sections were incubated with 1:5
diluted EBNAl -seropositive referent serum, which was
negative for any other EBNA types, at 37 "C for 60 minutes. The specificity of the referent serum was confirmed
by Western blot analysis using EBV-positive lymphoblastoid cell lines. The sections were then incubated with
EBV-seronegative serum as a source of complement and
with FITC-conjugated goat anti-human C3c (Dakopatts,
Copenhagen, Denmark), at 37 "C for 45 minutes. For detection of EBNA2 and EBV-encoded LMP1, avidin-biotin
immunoperoxidase staining using monoclonal antibodies PE2 (Dako A/S) specific for the EBNA2 and CS1-4
(Dako A/S) recognizing LMP1, were employed on acetone-fixed frozen sections.28The smears of EBV-positive
Raji cells and lymphoblastoid cells were used as positive
controls. The smear of BJAB cells was used as a negative
control. The specificity of the detection methods was confirmed by EBV-seronegative human serum and purified
normal mouse IgGl (MsIgG1; Coulter Cytometry, Hialeah, FL) instead of EBNAl-seropositive referent serum
and mouse monoclonal antibodies, respectively.
Clonotypic analysis of the EBV genomes was performed by our previous method of Southern blot hybridization" with some modifications. Briefly, tissue DNA was
digested with Barn HI endonuclease. The hybridization
probe used was a 3'P-labeled 1.9-kilobase (kb) Xho I subfragment of Bum HI-Dhet derived from the termini of
EBV DNA. In clonotypic proliferation of EBV genomes
and monoclonal proliferation of EBV-infected cells, a single restriction fragment size band was detected.
Serum samples were assayed on admission for antibodies to EBV capsid antigen (VCA)(IgG, IgM, and I@),
early antigens (EA) (IgG and IgA), and EBNA (IgG) by
immunofluorescence.'7~'g~30
Serum antibodies to human
T-lymphotrophic virus type 1and to the human immunodeficiency virus were determined by particle agglutination.
Phenotypic Study
The phenotypic study was performed on frozen or paraffin
embedded sections by avidin-biotin immunoperoxidase
staining using various monoclonal antibodies, biotinylated
2139
antimouse IgG (Vector Laboratories), and peroddase-labeled avidin-biotin complex (Vector Laboratories), subsequently." The monoclonal antibodies used were the following: Leu5b/CD2, Leu4/CD3, Leu3a/CD4,Leu1/CD5, Leu2a/
CD8, anti-interleukin-2 receptorlCD25 (Becton-Dickinson,
Mountain View, CAI, UCHLlICD45RO (Dakopatts), and
MT1 (Bio-Science, Emmenbrucke, Switzerland) for T-cell
phenotypes; PFl and TCRS1 (T Cell Science, Cambridge,
MA) for TCR chains; B4/CD19, Bl/CD20 (Coulter Clone,
Hialeah, FL), L26 (kindly provided by Prof. K. Kikuchi, Sapporo Medical University, Sapporo, Japan), anti-IgG, -IgM,
and -1gD (Coulter Clone) for B-cell phenotypes; B2/CD21
(Coulter Clone) for EBV receptor; NKH-I/CD56 (Coulter
Clone), LeullalCDlG, and Leu71CD57 (Becton-Dickinson)
for NK cells; OKIal(Orth0, Raritan, NJ) for histocompatibility antigen-DR (HLA-DR);and Ki-l/CD30 (Dakopatts).The
number of surface-antigen positive cells was independently
evaluated by 3 observers on highly magnified fields (x400).
Phenotype was defined as greater than 70% positive-staining tumor cells counted in the area involving atypical mononuclear cells as indicated by the serial sections stained with
H&E.
Genotypic Study
Genotype of the tumor cells was analyzed by Southern
blot hybridization, as described previo~sly.'~For this
study, we chose the biopsy tissues in which atypical Tlymphocytes had infiltrated prominently. Biopsy samples, including inflammatory cells and necrosis, were
avoided as much as possible. Histology of the samples
was confirmed by immunohistologic methods. Tumor tissues were homogenized and incubated with 300 pg/mL
proteinase K, 0.6% sodium dodecyl sulfate in 150 mM
NaCl, 10 mM Tris-HC1, and 10 mM EDTA (pH 7.51, at
65 "C for 20 minutes, to deactivate DNase. After incubation at 37 "C overnight, the phenol, phenol/chloroform/
isoamyl alcohol, and chloroformlisoamyl alcohol extractions were subsequently performed and DNA was then
recovered by ethanol precipitation. Ten micrograms of
DNA was digested with restriction enzymes such as Bum
HI, Eco RI, and Hind 111, and electrophoresed in 0.8%
agarose gel. The DNA was then transferred to nitrocellulose paper and hybridized with the 32P-labeled DNA
probes for 48 hours at 42 "C. After hybridization, the filters
were washed in 0 . 1 sodium
~
salt citrate (SSC) (20x SSC:
3M NaC1, 0.3M sodium citrate [pH 71) and 0.1% sodium
dodecyl sulfate, and then submitted to autoradiography
for 24 hours. A DNA probe specific for the joining region
of heavy chain (JH) gene was used for Ig gene rearrangement analysis. Rearrangement of the TCR-/? gene was
analyzed using a probe specific for the constant region 2
of the /?-gene (CP2). For analysis of the TCR-y gene, the
Ca/Cb probe specific for the Cy region3' and/or the
M13H60 probe specific for the Jy region3' were used.
Analysis of the TCR-6 gene was performed by a probe
2140
CANCER May 15,1996 / Volume 77 / Number 10
specific for the JS2 region33and/or by the J6S16 probe
specific for the 161 region.34
RESULTS
Clinical and Histologic Findings
The clinical presentation and follow-up results of all 18
patients are listed in Table 1. The median age of the patients was 46 years with a range of 28 to 72 years. There
were 9 male and 9 female patients. The possible primary
site appeared to be midfacial tissues such as the nasal
cavity, palate, nasopharynx, and larynx, with mucosal
swelling, necrotic granulomatous change, and ulcerative
destruction. One patient (Patient 18) was diagnosed with
LMG, probably resulting from progression of lymphomatoid papulosis of 8 years' duration, as reported prev i o ~ s l y Systemic
.~~
symptoms such as prolonged fever
and weight loss, as well as nasal symptoms such as nasal
obstruction andlor bloody rhinorrhea, were most common at the time of diagnosis, as seen in 16 (89%)and 14
(78%)patients, respectively. Cheek and/or orbit swelling,
sore throat, and hoarseness were present in 12 (67%), 12
(67%), and 3 (17%) patients, respectively. Clinical staging
at the time of diagnosis revealed 9 (50%) patients with
Stage I and 8 (44%) patients with Stage 11, according to
the Ann Arbor clas~ification.~~
Of the 18 patients, 15 were
treated with a multiple combined chemotherapy and radiotherapy. Two patients (Patients 3 and 17) were treated
with only cobalt-60 irradiation (40-45 Grays), because of
multiple medical illness. The clinical courses were quite
aggressive; 14 patients died of widespread relapse or dissemination into distant sites, with a median survival of 6
months (range, 1 to 31 months), and the overall 3-year
survival rate was only 9%. The common distant involvement sites during progression of the disease were systemic lymph nodes (11 patients), lungs (11 patients), liver
and/or spleen (9 patients), skin (8 patients), and gastrointestinal tracts (5 patients); it is noteworthy that only 2
patients had bone marrow involvement.
Histologic findings of the biopsies from all patients
corresponded to that of polymorphic reticulosis or angiocentric lymphoma: diffuse infiltrates of pleomorphic large
lymphoid cells and atypical small lymphoid cells with
frequent mitosis, admixing with a large number of inflammatory cells such as granulocytes, macrophages, and
plasma cells, with ischemic necrosis and angiocentric
andlor angioinvasive lesions. The lymphoid proliferation
was classified into two groups, according to the Working
Formulation System of the National Cancer In~titute.'~
Eight patients (Patients 1-3, 5 , 10, 11, 17, and 18) were
classified as diffuse large cell, immunoblastoid polymorphous (LIP)-type, in which pleomorphic large cell infiltration was predominant. The remaining 10 patients (Patients 4, 6-9, and 12-16) were classified as diffuse mixed
(DM) type, showing admixed infiltration of pleomorphic
large lymphoid cells and atypical small lymphoid cells
(Table 1). Areas of necrosis were observed in all cases.
Angiocentric and angioinvasive lesions, which were also
evident in all 18 patients, were prominent in tissues with
LIP type and relatively mild in tissues with DM type.
EBV Findings
The EBV findings of these patients are summarized in
Table 2. EBER transcripts were detected in most tumor
cells with intense nuclear staining from 16 of 18 patients
(Fig. la). Of these 16 patients, EBV genomic DNA was
identified in the great majority of the tumor cells with
granular staining of the nuclei from all of 11 patients
tested, EBV-encoded LMPl was expressed in most tumors
cells with weak and/or intense membrane staining from
all of 9 patients tested, and EBNAl was detected in a great
majority of the atypical cells with nuclear fluorescence
from all of 11 patients tested (Figs. lb, lc, and Id). EBNA2
was identified in a few tumor cells from 3 patients. Southern blot analysis using EBV-terminal region probe
showed a single hybridization band in all 7 EBV-positive
cases tested (Fig. 2). In 2 patients (Patients 17 and 181,
EBER transcripts, EBV genomic DNA, and any EBV-encoded proteins were never detectable.
The serum levels of VCA IgG and FA IgG antibodies
were high in all patients tested, except for one (Patient
18) in whom EBV was not detected in situ. The VCA IgA
and EL4 IgA antibodies were positive in sera from eight
and five patients, respectively; EBV DNA was detected in
tumor cells from all patients. Antibodies to human Tlymphotrophic virus type 1 and to the human immunodeficiency virus were not detectable in any patients.
Phenotypic and Genotypic Findings
Phenotypic findings are summarized in Table 3. Expression of pan-B antigens such as L26, CD19, CD20, and/or
surface immunoglobulins was not detectable on the tumor cells in any patients. Conversely, the vast majority
of atypical mononuclear cells in biopsy specimens from
all patients expressed pan-T antigens such as MT1,
CD45R0, and/or CD2 (Fig. 3a). The other pan-T antigen,
CD5, was absent in 5 of 11 patients tested. The T-helper/
inducer phenotype CD4 was determined in 11 of 13 patients tested. The T cytotoxidsuppressor phenotype CD8
was negative in all 13 patients tested. The HLA-DR antigen was present in all 13 patients tested. The CD30 was
positive in only one patient. The interleukin-2 receptor
CD25 was expressed in only two tumors. The CD21, which
defines the CSdIEBV-receptor, was not detected in any
tumor cells.
Ten patients underwent genotypic analysis of Ig
heavy chain, TCR-/3, -6,and - y chains. The rearrangement
bands of the TCR-/3, -6, and/or - y genes were shown in
all ten tumors; however, only germline bands were seen
using the JH gene fragment as a probe (Table 3 and
Fig. 4).
M
F
41
57
22
50
32
42
28
38
39
59
64
55
30
36
38
72
52
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Nasal obstruction, cheek swelling,
fever, weight loss
Nasal obstruction, bloody rhinonhea,
cheek swelling, fever, weight loss
Nasal obstruction, sore throat, fever,
weight loss
Nasal obstruction, sore throat
Nasal obstruction, sore throat, cheek
swelling, fever, weight loss
Nasal obstruction, cheek swelling,
fever, weight loss
Nasal obstruction, weight loss, cheek
aND orbit swelling, fever
Nasal obstruction, hoarseness, sore
throat, fever, weight loss
Nasal obstruction, sore throat, cheek
swelling, fever, weight loss
Nasal obstruction, cheek swelling,
sore throat, fever, weight loss
Bloody rhinorrhea, cheek swelling,
sore throat, fever, weight loss
Nasal obstruction, cheek swelling,
fever, weight loss
Nasal stiffness, cheek swelling, fever,
weight loss
Nasal obstruction, sore throat, fever,
weight loss
Nasal obstruction, cheek swelling,
sore throat, fever, weight loss
Nasal obstruction, cheek swelling,
sore throat, fever, weight loss
Sore throat, hoarseness
Skin nodules, sore throat, hoarseness,
fever, weight loss
Initial symptoms and signs
Skin
Larynx
Nasal cavity,
Nasopharynx
Nasal cavity,
Nasopharynx
Nasal cavity,
Palate
Nasal cavity,
Palate
Nasal cavity,
Palate
Nasal cavity,
Palate
Nasal cavity,
Palate
Nasal cavity
Nasal cavity
Nasal cavity
Nasal cavity,
Palate
Nasal cavity
Nasal cavity,
Nasopharynx
Nasal cavity
Nasal cavity
Nasal cavity
Primary sites
~
LIP
LIP
DM
DM
DM
DM
DM
LIP
LIP
DM
DM
DM
DM
DM
LIP
LIP
LIP
LIP
0
Histology
Iv
I
I
I
I1
I
I
I
I1
II
11
I1
I
II
11
II
Clinical
stage
Lung
Lymph nodes, spleen, lung, s h ,
GI tracts
No involvement
Lymph nodes, lung
Lymph nodes, liver, spleen, lung,
skin, GI tracts, bone marrow
No involvement
Skin
Lymph nodes, liver, spleen, lung,
GI tracts
Liver, spleen, lung
No involvement
Lymph nodes, spleen, lung, skin
No involvement
Lymph nodes, liver, spleen, lung,
skin, GI tracts, bone marrow
Lymph nodes, liver, spleen, skin,
GI tracts
Lymph nodes, lung
Lymph nodes, liver, spleen, lung,
skin
Lymph nodes, liver, spleen, lung,
skin, GI tracts
Lymph nodes
Involvement of distant sites after
diagnosis
Radiation
Mit, C, P
MEPP, MACOPP,
radiation
MACOPP. radiation
MEPP, radiation
MACOPP, radiation
MEPP, MACOPP,
radiation
MACOPP, radiation
VEPA, MEPP,
radiation
MEPP, MACOPP,
radiation
VEPA. radiation
VEPA
VEPA, radiation
MEPP, radiation
C, P, radiation
Radiation
VEMP, radiation
COPP, radiation
Therapy
None
None
Under therapy
None
6
12
None
None
4
45
6
None
10
None
15
None
Npe
None
Complete
remission (mo.)
3, died
2, died
5. alive
6, died
9, alive
24, died
4, died
4, died
17, died
48, alive
18. died
6, dead
31, died
18, alive
6. died
1, died
2, died
6, died
Prognosis
(mo.)
Working Formulation system; LIP large cell, immunoblastic, palymorphylous; D M d i h s e mixed small aND large; ma: months; G I gastrointestinal; COPP vincristine, cyclophosphamide, predonisolone, procarcazine; VEMP: vincristine, cyclophosphamide, predonisolone,
~-mercapropurine;MEPP: miroxanetrone, etoposid, cisplatin, predonisolone; C, P cyclophosphamide. predonisolone; VEPA: vincristine, cyclophosphamide, predonisolone, doxorubicin; MACOPP vincristine, cyclophosphamide, predonisolone, doxorubicin, mithouexate,
p i i ~ $ "-.
i & i e ,-.n,4nnirnlnno
pt=uuLy~ulys~~.
pepieomyck, Mix, i , p &jnane~ol,e, ~. p~q--L---
M
F
M
M
F
M
M
M
F
F
F
M
M
F
F
F
SeX
Patient
no.
~~
Clinical and Histologic Features of 18 Patients with Nasal T-Cell Lymphoma
TABLE 1
tt
tt
tt
tt
tt
tt
tt
t
tt
tt
ti-
6
7
8
9
tt
-
-
tt
-
-
tt
tt
tt
tt
tt
tt
tt
tt
tt
tt
+t
tt
-
t
-
t
t
tt
tt
t
tt
-
-
t
t
tt
tt
t
t
-
-
-
+
-
-
-
5
t
tt
t
2
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
LMPl
-
-
ND
ND
ND
ND
ND
ND
ND
Monoclonal
Monoclonal
Monoclonal
Monoclonal
Monoclonal
Monoclonal
Monoclonal
ND
ND
of EBV
genomes"
C1onotype
160
640
640
640
1280
640
5120
5120
1280
1280
2560
ND
ND
ND
ND
ND
ND
640
<5
<5
<5
<5
<5
<5
<5
<5
ND
ND
ND
ND
ND
ND
<5
10
<5
<5
VCA
<5
<S
5
20
20
10
40
10
20
<5
80
ND
ND
ND
ND
ND
ND
<5
I@
15
160
40
640
80
80
160
20
40
20
320
160
ND
ND
ND
ND
ND
ND
IgG
Serum anti-EBV titers
EA
EBV Epstein-Barr virus; ISH in situ hybridization; EBER: EBV-encoded small nuclear early region: EBNA EBV-encoded nuclear antigen; LMP: latent membrane protein; VCA: viral capsid antigen; W: early antigen; N D not done; -: not detected
positive; t:50-75W ceUs positive; +t: > 75% cells positive.
a Analyzed by Southern blot hybridization with EBV-terminal repeat probe.
15
16
17
18
13
14
10
11
12
tt
ND
tt
ND
tt
ND
ND
ND
ND
5
tt
ND
ND
ND
ND
t
ti
1
2
3
4
Expression of EBV genes and proteins
lmmunohislology
EBV DNA
EBNAl
EBNA2
EBER
ISH
no.
Patient
TABLE 2
EBV Findings of 18 Patients with Nasal T-cell Lymphoma
2 : a few
20
<5
<5
<5
20
10
<5
15
20
20
<5
ND
ND
ND
ND
ND
ND
<5
IgA
cells ( 3 4 % ) cells
10
10
80
80
320
40
40
20
20
20
10
20
ND
ND
ND
ND
ND
ND
EBNA
Nasal T-cell Lymphoma/Harabuchi et al.
2143
FIGURE 1. Expression of Epstein-Barf virus (EBV)-encoded small nuclear early region (EBER) RNA transcripts (a; Patient 3), EBV DNA (b; Patient
13),latent membrane protein (LMP) (c; Patient 8), and EBV-encoded nuclear antigen (EBNA) (d; Patient l o ) , in nasal biopsy sections. In situ hybridization
profiles show signals of the EBER RNA transcripts and EBV DNA in the nuclei of the great majority of tumor cells (a and b). lmmunoperoxidase staining
shows that the LMP is expressed intensively in large lymphoid cells and weakly in numerous atypical lymphoid cells (c). The EBNA immunofluorescence
is detected in the nuclei of most tumors cells (d). (original magnifications, a: x200; b and d: x400; c: x100).
Relationships between Phenotype, Genotype, and EBV
Detection
As shown in Tables 3 and 4, the NK cell phenotype CD56
was expressed in all 9 EBV-positive NTL tested, but was
not detected in EBV-negative patients. Of nine tumors
expressing CD56, five tumors also showed positivity for
CD16, whereas all were negative for CD57. With regard to
expression and gene arrangement of TCR, 7 EBV-positive
patients represented CD3+’-PF1-TCR61+ phenotype with
rearrangement of the TCR-6 gene (Patients 8, 13, and 15)
or of the TCR-y gene (Patients 9, 11, 12, and 16). The
remaining 2 EBV-positive patients (Patients 10 and 14)
displayed CD3+PFl-TCR61- phenotype. Conversely, both
EBV-negative NTL patients (Patients 17 and 18) expr’essed
the TCR-0 chain with a rearranged TCR-P gene.
FIGURE 2. Clonotypic analysis of Epstein-Barr virus (EBV) genomes by
Southern blot hybridization with 32P-labeled 1.9-kilobase Xho I subfragment of Barn HI-Dhet probe after digestion of tissue DNA with Barn HI
endonuclease. Lane B represents 695-8 cells. The lane numbers correspond to the patient numbers in the text. A single restriction band is
detected in all lanes from the 7 patients tested. Conversely, multiple bands
are seen in lane B from 895-8 cells.
DISCUSSION
The 18 patients in the present study, all of whom clinically
presented with LMG and histologically presented polymorphic reticulosis, expressed pan-T phenotypes such
as CD2, CD45R0, andlor MT1. In the genotype study,
rearrangements of the TCR-P, -6 andlor -y genes were
detected in all 10 patients tested, indicating the pre,“ence
ND
ND
ND
ND
ND
ND
ND
ND
t
ND
ND
ND
ND
t
ND
ND
ND
ND
ND
ND
ND
t
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
t
t
t
t
3
t
t
2
t
ND
ND
NO
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
t
t
4
ND
ND
t
-
ND
ND
ND
ND
ND
t
-
ND
ND
ND
t
t
t
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
ND
-
+
t
t
t
t
t
7
t
t
t
t
t
6
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
t
t
t
5
t
t
R
R
G
G
-
-
R
G
R
G
t
t
9
t
t
8
t
R
ND
G
G
10
Patient no.
t
R
R
G
G
11
t
R
R
G
G
12
t
G
G
R
G
-
t
-
-
t
t
t
ND
ND
ND
ND
-
-
t
R
G
R
G
t
+
ND
t
t
t
t
15
t
t
t
t
t
ND
t
14
ND
13
t
R
R
G
G
t
t
t
ND
t
t
t
16
TCR: T-cell receptor: N K natural killer; EBV Epstein-Ban virus: IL-2: interleukin-2; HLA-DRhistocompatibility antigen-DR; Ig: immunoglobulin; EBEREBV encoded small nuclear early region; ND: not done: R: rearrangement pattern: G: germline pattern.
Phenotype
MTl (pan-T cell)
CD45RO (pan-T cell)
CD2 (pan-T cell)
CD5 (pan-T cell)
CD3 (TCR complex)
OF1 (TCR-P chain)
TCRdl (TCR-d chain)
CD4 (helperlinducer T-cell)
CDB (suppressorlcytotoxic T)
CD16 (NK cell)
CD56 (NK cell)
CD57 (NK cell)
CD21 (EBV receptor)
CD25 (IL-2 receptor)
CD30 (activated T- or B-cell)
HL4-DR
Genotype
TCR-beta gene
TCR-gamma gene
TCR-delta gene
Ig heavy chain gene
EBER RNA transcripts
1
TABLE 3
Phenotypic and Genotypic Features, and EBV Detection in 18 Patients with Nasal T-cell Lymphoma
-
R
ND
G
G
-
-
t
R
ND
G
G
t
-
-
-
t
t
t
t
18
t
t
t
t
t
t
17
Nasal T-cell Lymphoma/Harabuchi et al.
FIGURE 3. lmmunoperoxidase staining with CD2 (a; Patient 14), CD56
(b; Patient 9), and TCR61 (c; Patient 10) from frozen nasal biopsy sections.
Positive membrane staining is seen in the vast majority of infiltrating
lymphoid cells in each section (original magnifications, a: x200; b and c:
x100).
of a monoclonal proliferation of the 'I'-lineage cells. Furthermore, we found EBER transcripts, which are apparently expressed in human lesions with latent EBV infection,"6in the great majority of tumor cells from 16 of the
18 patients. To the best of our knowledge, this is the first
report documenting a large number of NTL with evidence
of monoclonal proliferation of T-lineage cells and of association with EBV.
The presence of a clonal episome for EBV genomes
in tissues has been thought to be more in keeping with a
2145
pathogenic role for the virus, because infection must have
been established at an early stage of oncogenesis, prior to
clonal expansion of the latently infected cell that subsequently transmits identical viral episomes to all the cell
p r ~ g e n y . ~ ' ,In
~ ' the present and previous studies," we
showed clonotypic EBV genomes in the tissue DNA from
all NTL samples tested. This suggests that EBV may infect
T-lineage cells prior to tumor development and play a
causal role in the lymphomagenesis of NTL, as was previously demonstrated in nasopharyngeal carcinoma, B-cell
lymphoma, and some cases of T-cell lymphoma."'~"S-:i"
Recently, several investigators also demonstrated clonotypic
EBV genomes in angiocentric T-cell lymphoma from nasal
or skin
It has been reported that the B cells latently infected
with EBV express various types of EBNAs and LMP, which
are essential for continued B-cell proliferation and the
latent viral cycle.'6 Transfection with LMPl induces gene
deregulation and morphologic changes, and prevents
programmed cell death by inducing the expression of bcl2 gene product..'" In the present study, EBNAl and I.MPI
were detected in most tumor cells from all patients with
NTI. tested, and EBNA2 was determined in some patients.
Our previous transcriptase-polymerase chain reaction
analysis showed mHNA of LMPl and EBNAl in the NTL
celk2' EBV may have an immortalizing role for neoplastic
T cells, similar to the direct causal role suggested by detection of clonotypic EBV genomes.
Although molecular studies assessing the clonality of
NI'L cells have been performed, clonal rearrangements
ofTCR genes have not been demonstrated in the majority
of cases and the cases with rearranged TCR genes have
1.12.14.l6,2U,21.~lI
been
In this study, we obtained TCR
gene rearrangements in all 10 patients tested. Although
we cannot resolve this discrepancy, some possible €actors
are the number of probes (we employed five different
specific probes), biopsy specimens (we analyzed specimens in which atypical T cells had infiltrated prominently), and/or differences in racial/geographic distribution of the patients (NTL and its EBV-association are
found frequently in east Asian and Mongolian p e ~ p l e * ~ ~ ' ~ ) .
In the phenotypic study, all EBV-positive NT1, patients tested expressed NK cell phenotype CD56 with rearranged 1CR genes, indicating that ERV-positive NrL
may correspond to NK-like T-cell tumors. The coexpression of CD56 and TCR-y6 found in this study agreed with
a previous report, based on flow cytometric analysis, that
NK-like T-cell lymphoma expressed T C R - y b protein freq ~ e n t l y . ~Recently,
"
it has been reported that NK cells
are restricted to CD56'<:D3 lymphocytes that d o not
rearrange TCH genes nor express TCR proteins, whereas
NK-like'[ cells express CD56, CD3, and TCR proteins, and
rearrange TCR genes."i The speculation, based o n the
CD3' CLX6' phenotype, that NTI. cells may be derived
from NK cellsg~1"~19
can be excluded in the present study
2146
CANCER May 15,1996 I Volume 77 I Number 10
FIGURE 4. Representative Southern blot hybridization profiles of the gene rearrangements of the T-cell
receptor (TCR)-p (a), - y , and -6 chains (b), using 32P-labeledDNA probes after digestion with Barn HI, Eco
RI, and Hind Ill. Rearrangement of the TCR-p gene was analyzed with a probe specific for the Cp2. For
analysis of the TCR-r gene, the CdCb probe specific for the C r region, and/or the M13H60 probe specific
for the J r region was used. Analysis of TCRd gene was performed by a probe specific for the J62 region
and/or by the J6S16 probe specific for the J61 region. Germline bands are indicated by dashes with sizes
and the arrows denote rearranged bands. Lane G represents germline. The lane numbers correspond to the
patient numbers in the text. Rearrangement bands of the TCR-,O, - y , andlor -6 gene are shown in all 10
patients tested.
because rearrangements of TCR genes were detectable in
all patients with NTL tested regardless of expression or
loss of the CD3 or TCR proteins. Previous in vitro studies
demonstrated that activation of T-cell lymphoma cell
lines leads to loss of CD3 or TCR protein^.^^'^^ It is likely
that loss or structural abnormalities of the CD3 antigen
and/or TCR proteins could occur during neoplastic transformation of NK-like T cells. The positivity for CD16 in
some patients may represent an activation of the killing
activity of the
The heterogeneity of the histology
may result from direct effects of neoplastic NK-like T cells
andlor from the effects of cytokines released by the cells.
Alternatively, based on positivity for TCRd protein in
seven patients with EBV-positive NTL, it is suggested that
some cases of EBV-positive NTL could be classified as y5Tcell tumors. Indeed, ybT-celllymphomas have been reported
to express the CD56 p h e n ~ t y p e . ~ ~ , ~ ~genotypic
- ~ ' T h e findings
raise some inconsistencies: the TCR-6 gene was not rearranged in four patients with y-rearrangement, clonal TCR-y gene rearrangement was absent in three patients with 6-
Nasal T-cell Lymphoma/Harabuchi et al.
TABLE 4
Expression of TCR and NK Cell Phenotypes and Rearrangement of TCR Genes in Patients with EBV-Positive
and -Negative Nasal I-CeU Lymphoma
-
Expression of NK phenotypes and TCR
Patient no.
8
EBV
CD56
CD16
CD3
TCR-P
Gene rearrangement of TCR
TCR-8
P
Y
t
t
t
-
-
-
t
t
-
12
t
t
t
t
t
t
16
t
t
t
t
9
11
10
t
-
t
t
t
t
13
15
14
17
18
t
-
t
t
-
t
t
t
-
-
2147
ND
t
t
s
-
t
t
ND
ND
ND
ND
TCR: T-cell receptor; N K natural killer: EBV Epstein-Barr Virus; ND: not done; t: detected or positive; -: not detected.
Expression of TCR-8 or -d chain was determined by bFl or TCRdl antibody, respectively.
rearrangement, and TCR-0 gene rearrangement was shown
in six patients. The problem of pseudoclonality, which is
frequently encountered in studies of TCR-y gene rearrangement, may also occur?’ However, three patients with NTL
expressing b-protein showed productive &gene rearrangement. It is reported that the genotype of ybT-cell lymphoma
is markedly different from that of ybT-cell clones derived
from peripheral blood of normal individuals.” The TCR-P
gene rearrangements with y- or 6- rearrangements have frequently been seen in y6T-cell l y m p h o m a ~ . 4 ~ -Therefore,
~~f~’
frequent detection of +-gene rearrangements in patients
expressing the TCR-6 chain supports the possibility that
some NTL associated with EBV may be derived from y6Tcell lineage. Recently, several cases of EBV-positive nasal or
oral T-cell lymphoma with TCR-6 expression and/or y6-gene
rearrangements have been rep~rted.’’~’~-”
Wong et aL4’ did
not find TCR-6 expression in any of their cases of NTL, but
EBV-studies were not available.
In contrast to EBV-positive NTL, both patients in the
present study with EBV DNA-negative NTL had PFl’TCRblphenotype with productive rearrangement of TCR-B gene,
suggesting that these tumors may be derived from crpT-cells.
Clinicopathologic features, except for the initial site, did not
differ between EBV-positive and -negativeNTL. AU EBV-positive NTL originated in the nose andlor nasopharynx, whereas
EBV-negative NTL oripated in the skin or larynx. Therefore,
it is possible that nasal or nasopharyngeal localization may
predispose to the involvement of EBV. High VCA and EA
antibody levels and positivity for EBV IgA antibodies in the
sera may be due tct the anatomic sites at which EBV-positive
NTL developed, similar to how nasopharyngeal carcinoma
shows high EBV antibody levels and positivity for EBV IgA
antibodies:“ supporting this speculation.
The clinical behavior of NTL was aggressive,with a me-
dian survival of only 6 months, although intensive chernotherapy had been administered to most patients. Prolonged
fever of own origin preceding the lymphoma diagnosis
is seen in most patients. Excluding four survivors, all patients
demonstrated widespread dissemination into distant sites
such as systemic lymph nodes, liver, spleen, lung, skin, imd
gastrointestinal tract. These clinical manifestations are similar to those of EBV-associated T-cell lymphoma other than
NTL.37,53Interestingly,bone marrow infiltration developed in
only 2 patients and no leukemic change was observed in (my
patients. This clinical feature seems to be different from that
of human T-lymphotrophic retrovirus type 1-associated
adult T-cell lymphoma, in which bone marrow infiltration
and leukemic change are c0mmon,5~and may represent a
unique homing nature of the NTL cells.
In summary, the expression of the CD56’TCR “silent” or
CD56’TCRyb’ phenotype with rearrangements of the TCIR-y
or -6 gene indicates that EBV-positive NTL may be derived
from the lineage of NK-like T cells or ybT-cells. The detection
of clonotypic EBV genomes and expression of EBV oncogenic
proteins such as LMPl and EBNAl suggests that EBV may
play a role in the lymphomagenesis. Its clinical features have
been characterized as prolonged fever, aggressive course
with widespread dissemination into distant sites, and poor
prognosis. Their findings support the idea that NTL is a idistinct type of non-Hodgkm’s lymphoma.
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