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 [email protected]), 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 [email protected] 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. 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