Studies on nasal T/natural killer (NK)–cell lymphoma have been hampered by its tendency to cause necrosis. Thus, the establishment of cell lines of this neoplasm would seem to be valuable. This study attempted to establish cell lines from primary lesions of this tumor, and successfully obtained 2 novel Epstein-Barr virus (EBV)–positive cell lines, SNK-6 and SNT-8, by means of high-dose recombinant interleukin 2. Flow cytometry showed that SNK-6 had an NK-cell phenotype, CD3CD4CD8CD19CD56+T-cell receptor (TCR) α/β TCR γ/δ, whereas SNT-8 was CD3+CD4CD8CD19CD56+TCR α/β TCR γ/δ+. These were consistent with immunophenotypes of their original tumors, and the cell lines had monoclonal EBV clones identical to ones in their original tumors. Thus, the cell lines developed from cells forming the primary lesions. Genotypic analysis showed that SNK-6 had unrearranged TCR and immunoglobulin heavy-chain genes, supporting the conclusion that SNK-6 was of NK-cell lineage. On the other hand, SNT-8 had rearranged TCR β-, γ-, and δ-chain genes, and together with its phenotype, SNT-8 proved to be a γδ T-cell line. This is the first report of the establishment of cell lines from primary lesions of nasal T/NK cell lymphomas, and the results demonstrated that there are at least 2 lineages, NK- and γδ T-cell, in this neoplasm. Moreover, it has been suggested that nasal T/NK cell lymphomas of these lineages may belong to the same clinicopathologic entity because both types of cases shared common clinical and histopathologic features.

Nasal T/natural killer (NK)–cell lymphoma is a distinct clinicopathologic entity characterized by progressive necrotic lesions in the nasal cavity, nasopharynx, and palate.1 It has been called angiocentric lymphoma, lethal midline granuloma, or polymorphic reticulosis.2 It is a relatively rare disease associated with a poor prognosis and more often seen in Asia than in the United States and Europe.3-14 Nasal T/NK-cell lymphomas can exhibit a broad histologic spectrum and usually show invasion of the vascular walls, which is associated with prominent ischemic necrosis of both tumor cells and normal tissue.

The most characteristic feature of the disease is a consistent and strong association with the Epstein-Barr virus (EBV); indeed, almost all cases are positive for EBV irrespective of ethnic difference,15-22 yet the precise role of the virus in the etiology of the disease is poorly understood. The cellular origin of the nasal T/NK-cell lymphoma has been controversial. It was initially thought to originate from the T-cell lineage based on immunophenotype.23-27 Later studies using combined immunophenotypic and genotypic analysis have suggested that most nasal T/NK-cell lymphomas are of NK-cell lineage, being CD56+, negative for surface CD3 (Leu4), and unassociated with rearrangements of the T-cell receptor (TCR) genes.11,28-30 On the other hand, some studies have reported the existence of T-cell lymphomas that express TCR proteins, that is α/β and γ/δ chains, associated with rearranged TCR genes; these were presumably αβ T-cell lymphomas10,30 and γδ T-cell lymphomas.18,31 In addition, there have been reports of atypical cases in which cell surface antigens were expressed discordantly; these were CD3(Leu4)+CD56+ cases without TCR gene rearrangements,32 and cases with TCR proteins expressed discordantly with their rearrangement status of the TCR genes.10,29,30 Moreover, it has recently been suggested that nasal T/NK-cell lymphomas of NK- and γδ T-cell lineages are derived from activated cytotoxic cells, have common clinical and histopathologic features, and belong to the same clinicopathologic entity.31 Therefore, precise phenotypes of this neoplasm including the T-cell subset in cases of T-cell lymphomas and their relationships to clinicopathologic features need to be studied. However, it is often difficult to obtain a sufficient amount of tissues from such a necrotic lesion. To solve this problem, the propagation of tumor cells in vitro seems to be useful. Recently, a novel NK-cell line, NK-YS, was established from peripheral blood of a patient with a leukemic-state nasal NK-cell lymphoma.33However, because nasal T/NK-cell lymphomas infrequently develop leukemia, a more applicable method for establishing a cell line is needed.

Therefore, in the present study, we aimed to establish cell lines from primary lesions of nasal T/NK-cell lymphomas. We established 2 novel EBV-positive cell lines of NK- and γδ T-cell lineages, SNK-6 and SNT-8. In the present report, we describe immunologic and molecular biologic features of these cell lines.

Patients and nasal tumors

Tumor tissues obtained from 2 Japanese patients with nasal T/NK-cell lymphomas were used in this study. Patient 1 was a 62-year-old man and patient 2 was a 48-year-old woman. Both patients had tumors on their right inferior turbinates through the nasal vestibula. The tumors were composed of granulomatous parts and white necrotic parts (Figure 1). Biopsies were performed at the granulomatous parts, but small necrotic tissues were included. Freshly biopsied tissues were cut into several pieces, and portions of these tissues were used for establishing cell lines. The remaining tissues were fixed in 10% buffered formalin for paraffin sections or frozen in liquid nitrogen for immunophenotyping and molecular analysis.

Fig. 1.

Endoscopic findings.

The patients' right nasal cavities show granulomatous tumors and white necrosis. Left panel, patient 1; right panel, patient 2.

Fig. 1.

Endoscopic findings.

The patients' right nasal cavities show granulomatous tumors and white necrosis. Left panel, patient 1; right panel, patient 2.

Close modal

Immunohistochemistry and in situ hybridization

Immunophenotyping of cells for B-, T-, and NK-cell differentiation antigens was performed on frozen sections as well as on paraffin sections. The antibodies used for the paraffin sections were anti-CD3ε polyclonal antiserum (Dako, Glostrup, Denmark), L26 (anti-CD20, Dako), UCHL-1 (anti-CD45RO, Dako), and anti-CD56 (Novocastra, Newcastle, UK) monoclonal antibodies. The antibodies used for the frozen sections were anti-CD2 (Leu5b) and anti-CD3 (Leu4) monoclonal antibodies (Becton Dickinson, San Jose, CA). A standard peroxidase antiperoxidase complex method followed by diaminobenzidine reaction was used to detect positive signals. Paraffin sections were also used for in situ hybridization with a digoxigenin-labeled oligonucleotide probe complementary to the EBV-encoded RNA (EBER-1): the sequence is 5′-AGACACCGTCCTCACCACCCGGGACTTG TA-3′. Hybridization was detected by the procedure described by Chang and colleagues.34 

Cell culture and morphologic evaluation

The tumor tissues were minced, and strained through a 70-μm cell strainer (Becton Dickinson). The cells obtained through the strainer were then suspended in RPMI 1640 medium supplemented with antibiotics, 10% heat-inactivated human serum, and 700 U/mL of recombinant human interleukin 2 (IL-2). The cells were cultured in a humidified atmosphere at 37°C with 5% CO2 in air. One half of the medium was exchanged with fresh medium twice a week. The cultured cells from patients 1 and 2 have been maintained in the presence of IL-2 for over 20 months and 17 months, respectively, and designated as SNK-6 and SNT-8. Cytocentrifuge smears of the cells were prepared and stained with May-Giemsa for morphologic evaluation.

Flow cytometric analysis

The SNK-6 and SNT-8 cells were analyzed by 2-color immunofluorescence with a flow cytometer (EPICS XL, Beckman Coulter, Hialeah, FL) for the expression of surface markers. The following antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) (Becton Dickinson) were used: anti-CD3, -CD4, -CD8, -CD16, -CD19, -CD21, -CD25, -CD56, -CD57, -HLA-DR, -TCR α/β, and -TCR γ/δ.

Southern blot analysis for TCR and immunoglobulin genes

The SNK-6 and SNT-8 cells were analyzed for rearrangements of the TCR β, γ, and δ chains and the immunoglobulin heavy-chain genes by Southern blotting. DNA (3 μg) extracted from each of the cell lines was digested with a restriction endonucleaseBamHI, EcoRI, or HindIII (TAKARA, Kyoto, Japan), electrophoresed through a 0.7% agarose gel and transferred onto a nylon membrane. Human placenta DNA was used as a germline control. The membrane was subjected to a fluorescein-labeled Cβ1, Jγ1, Jδ1, or Jδ3 probe for the TCR genes, or a Jh probe for the immunoglobulin heavy-chain gene.35-39 The Jδ3 probe was a 1.5-kb XbaI genomic fragment of the Jδ3 region. In addition, DNA was digested with KpnI for the Jγ1 probe, and processed in the same manner. Hybridizations were visualized by a Fluorescein Gene Images System (Amersham, Buckinghamshire, UK) according to the manufacturer's instructions.

Assessment of clonality of EBV

DNA extracted from the cell lines as well as the frozen tumor tissues were digested with BamHI, separated on a 0.7% agarose gel, and transferred to a nylon membrane. A 1.9-kbXhoI subfragment of BamHI-Dhet derived from EBV termini was used as a probe for terminal repeats of the EBV DNA40 according to the methods for detecting signals described above. Raji and B95-8 cells were used as controls for cell expansion with monoclonal EBV infection and polyclonal EBV replication, respectively.

Primary lesions

The tumors in both patients shared common histologic features. The normal structure of the nasal mucosa disappeared and was replaced by tumors, and nonsuppurative necrosis was readily noted. The tumors were composed of pleomorphic, medium- to large-sized atypical lymphoid cells. Infiltration of neoplastic cells to small vessels was observed (Figure 2). In situ hybridization showed that both tumors were positive for EBV. Immunophenotypes of both tumors were CD3ε+ CD20CD56+, whereas the tumors showed a difference in positivity for the surface CD3 (Leu4) antigen. The tumor in patient 1 was CD3 (Leu4), and the tumor in patient 2 was CD3 (Leu4)+. These results indicated that the tumor in patient 1 had a phenotype of NK cells. The results are summarized in Table 1.

Fig. 2.

Histopathology.

Primary lesions show invasion of tumor cells to small blood vessels. (A) Patient 1. (B) Patient 2. (Hematoxylin-eosin, final magnification × 140).

Fig. 2.

Histopathology.

Primary lesions show invasion of tumor cells to small blood vessels. (A) Patient 1. (B) Patient 2. (Hematoxylin-eosin, final magnification × 140).

Close modal
Table 1.

Summary of phenotypes of primary tumors

CaseCD2 (Leu5b)CD3 (Leu4)CD3εCD20CD45ROCD56EBER-1Developed cell line
− − SNK-6 
− SNT-8 
CaseCD2 (Leu5b)CD3 (Leu4)CD3εCD20CD45ROCD56EBER-1Developed cell line
− − SNK-6 
− SNT-8 

Morphology and phenotypes of the cell lines

The morphologies of the SNK-6 and SNT-8 cells were similar to each other, except for their sizes. The SNK-6 cells were composed of medium to large cells, whereas the SNK-8 cells included small cells as well as medium to large ones (Figure 3). Large cells in both cell lines had large nuclei and abundant basophilic cytoplasm. Nuclei as well as cytoplasm became smaller and pycnotic as the cell sizes became smaller. Both cells had a number of cytoplasmic vacuoles, but scarcely any azurophilic granules.

Fig. 3.

Morphology.

Established cell lines show rich cytoplasmic vacuoles and no azurophilic granules. (A) SNK-6. (B) SNT-8. (Wright-Giemsa, final magnification × 440)

Fig. 3.

Morphology.

Established cell lines show rich cytoplasmic vacuoles and no azurophilic granules. (A) SNK-6. (B) SNT-8. (Wright-Giemsa, final magnification × 440)

Close modal

Flow cytometry showed that the SNK-6 cells were CD3 CD4CD8CD16CD19CD21CD25+CD56+CD57HLA-DR+, and TCR α/β TCR γ/δ (Figure4A). Thus, SNK-6 was shown to have an NK-cell phenotype. On the other hand, the SNK-8 cells were CD3+CD4CD8CD16CD19CD21CD25+CD56+CD57HLA-DR+, and TCR α/β TCR γ/δ+ (Figure 4B).

Fig. 4.

Expression of cell surface markers for subset of T cells and NK cells by established cell lines.

(A) SNK-6. (B) SNT-8.

Fig. 4.

Expression of cell surface markers for subset of T cells and NK cells by established cell lines.

(A) SNK-6. (B) SNT-8.

Close modal

Southern blot analysis for TCR and immunoglobulin genes

Neither of the cell lines showed a rearrangement of the immunoglobulin heavy-chain gene. In terms of the TCR genes, the SNK-6 cells showed no rearrangements of the TCR β-, γ-, and δ-chain genes, supporting that SNK-6 is of NK-cell lineage (Figure5). On the other hand, the SNT-8 cells showed rearrangements of the β-, γ-, and δ-chain genes. The rearrangements of the γ-chain gene were initially not detected by the Jγ1 probe hybridized with BamHI, EcoRI, orHindIII digest (Figure 5B). Then, we evaluated hybridization of the probe with the KpnI digest, because it has been reported that the Vγ9-JγP rearrangement frequently observed in rearranging γ loci can be detected with the KpnI digest, but the BamHI rearranged band has virtually the same size as the germline bands and the EcoRI and HindIII bands are in germline configuration in this rearrangement.41,42 As expected, a 9-kb germline band derived from a KpnI fragment including the JγP gene changed to a 12-kb band probably by assignment of the Vγ9 gene to the JγP gene (Figure 5B). With respect to the δ-chain gene, deletion was initially found in the Jδ1 region in the SNT-8 cells (Figure 5C), suggesting that rearrangements of the α-chain gene or deletion of the δ-chain gene occurred in the cells.43 Subsequently, we sought rearrangements of the δ-chain gene by hybridizing the blots with the Jδ3 probe, because the SNT-8 cells showed expression of the TCR γ/δ receptor by flow cytometry. As a result, the Jδ3 probe successfully detected rearranged bands, indicating that the deletion of the Jδ1 gene was a consequence of the usage of the Jδ3 gene in rearrangements of the δ locus. (Figure 5D).

Fig. 5.

Southern blot analysis.

Southern blot analysis shows no rearrangements of TCR genes in SNK-6 and rearrangements of the β-, γ-, and δ-chain genes in SNT-8. Panels show hybridization with the following probes: (A) the Cβ1 probe, (B) the Jγ1 probe, (C) the Jδ1 probe, (D) the Jδ3 probe. Human placenta DNA was used as a germline control. The letters above the pictures are BamHI (B), EcoRI (E),HindIII (H), and KpnI (K) digest. Note that the rearrangements of the γ-chain gene in SNT-8 cannot be detected by the BamHI, EcoRI, orHindIII digest. The lack of a 5-kb HindIII band hybridizing with the Jγ1 probe in SNT-8 is due to a polymorphism for a HindIII cleavage site.41 

Fig. 5.

Southern blot analysis.

Southern blot analysis shows no rearrangements of TCR genes in SNK-6 and rearrangements of the β-, γ-, and δ-chain genes in SNT-8. Panels show hybridization with the following probes: (A) the Cβ1 probe, (B) the Jγ1 probe, (C) the Jδ1 probe, (D) the Jδ3 probe. Human placenta DNA was used as a germline control. The letters above the pictures are BamHI (B), EcoRI (E),HindIII (H), and KpnI (K) digest. Note that the rearrangements of the γ-chain gene in SNT-8 cannot be detected by the BamHI, EcoRI, orHindIII digest. The lack of a 5-kb HindIII band hybridizing with the Jγ1 probe in SNT-8 is due to a polymorphism for a HindIII cleavage site.41 

Close modal

Clonality of EBV

Southern blot analysis for the number of EBV terminal repeats showed a monoclonal band in each of the SNK-6 cells, the SNT-8 cells, and their original tumors. In addition, the bands in the cell lines corresponded to the bands in their respective original tumors (Figure6).

Fig. 6.

Clonality of EBV.

Southern blot analysis for clonality of EBV as evidence for monoclonal expansions of EBV-positive cells in primary tumor in patient 1 (lane 1), SNK-6 cells (lane 2), primary tumor in patient 2 (lane 3), and SNT-8 cells (lane 4). Controls are B95-8 cells (B) and Raji cells (R) for cells with monoclonal EBV expansion and polyclonal EBV replication, respectively.

Fig. 6.

Clonality of EBV.

Southern blot analysis for clonality of EBV as evidence for monoclonal expansions of EBV-positive cells in primary tumor in patient 1 (lane 1), SNK-6 cells (lane 2), primary tumor in patient 2 (lane 3), and SNT-8 cells (lane 4). Controls are B95-8 cells (B) and Raji cells (R) for cells with monoclonal EBV expansion and polyclonal EBV replication, respectively.

Close modal

The present study established 2 novel cell lines of T/NK-cell lineage from the primary lesions of nasal T/NK-cell lymphomas by means of a simple technique using high-dose IL-2. Established cell lines had monoclonal EBV clones identical to those in their respective original lesions. Moreover, the flow cytometric phenotypes of the cell lines corresponded with the immunohistochemical phenotypes of the primary lesions. Therefore, the cell lines were demonstrated to have developed from cells that formed the nasal T/NK-cell lymphomas in the patients. As far as we know, this is the first report describing the establishment of cell lines from primary lesions of the nasal T/NK-cell lymphoma.

The establishment of tumor cell lines seems to be valuable for the study of the nasal T/NK-cell lymphoma because the tendency of the disease to cause necrosis has hampered detailed analyses of the tumor. Recently, a novel NK-cell line NK-YS was established from the peripheral blood of a patient with a leukemic-state nasal NK-cell lymphoma.33 However, an association of leukemia with this disease is not common. Thus, we believe that a technique to establish a cell line from a primary lesion is more applicable to the disease. We have tried to date to establish cell lines from 5 primary lesions obtained from 5 independent patients and have succeeded in obtaining the 2 cell lines and in maintaining 2 other cells for over 1 month. Therefore, we believe that the technique described here will be generally useful for establishing cell lines from more cases of nasal T/NK-cell lymphomas.

The establishment of tumor cell lines appears to have advantages over biopsies for phenotypic and genotypic studies. In phenotypic studies, the expression of cell surface antigens can be analyzed more accurately by flow cytometry for suspended cells than by immunohistochemistry on tissue sections because most monoclonal antibodies for cell surface antigens were originally developed for flow cytometry, and the results of immunohistochemistry are occasionally difficult to interpret. Moreover, a cytoplasmic molecule such as CD3ε in NK cells, which is not expressed on the cell surface,30,44,45 may lead to false-positive results by immunohistochemistry, and can be assessed accurately by flow cytometry. With respect to genotypic studies, propagation of a clonal population of tumor cells appears to generate a clear result, whereas analysis of a biopsied tissue is associated with potentially equivocal findings because of the presence of nontumorous cells.46 Furthermore, the availability of a large amount of DNA allows us to repeat analysis and to try various DNA probes to confirm the reproducibility of our results.

In the present study, the results of phenotypic and genotypic analysis completely agreed with each other in the SNK-6 cells. The SNK-6 cells had an NK-cell phenotype CD3(Leu4)CD4 CD8CD19CD56+TCR α/β TCR γ/δ and unrearranged TCR genes. On the other hand, the SNT-8 cells had a γδ T-cell phenotype CD3(Leu4)+CD4CD8CD19TCR α/β TCR γ/δ+ and showed rearrangements of the γ- and the δ-chain genes of the TCR, yet it was also CD56+ and associated with a rearranged β-chain gene. Because it was reported that peripheral γδ T-cell lymphomas frequently expressed CD5631,47-49 and showed rearrangements of the β-chain gene,47,49,50 SNT-8 was considered to be a tumor cell line of γδ T-cell lineage. Hence, the nasal T/NK-cell lymphoma in case 2 proved to be a γδ T-cell lymphoma. On the basis of previous reports, NK-cell is the predominant phenotype in nasal T/NK-cell lymphomas, and T cell is a minor phenotype.11,18,28,30 32 Therefore, SNT-8 should be useful for the study on the nasal T/NK-cell lymphoma of the γδ T-cell subset.

The genotypic studies of the SNT-8 cells gave us 2 lessons in evaluating rearrangements of the TCR genes in T-cell lymphomas. First, for analysis of the TCR γ-chain gene by using the Jγ1 probe,KpnI digest should be analyzed, when BamHI,EcoRI, or HindIII digest does not show a rearrangement.41,42 Second, when no signal is detected by the Jδ1 probe, another probe for the δ-chain gene should be applied to determine whether it is deleted or rearranged.39,43 However, to determine the subset of T-cell lymphomas, expression of the α/β or γ/δ TCR should be confirmed, because rearrangements of the γ-chain gene are commonly found in T-cell lymphomas irrespective of the type of TCR that is expressed51 and rearrangements of the β-chain gene are also found in γδ T-cell lymphomas as described above. In addition, consistency between the genotype and the phenotype seems necessary. Reviewing previously reported cases with nasal T-cell lymphomas with these considerations, we found only a small number of cases that can be clearly categorized as αβ or γδ T-cell lymphoma. Two of the 4 cases with nasal T-cell lymphomas reported by Chiang and coworkers29,30 (case 2 in reference 29 and case 9 in reference 30), a case in a series of 10 cases of T-cell lymphomas by Harabuchi and colleagues10 (case 17), and a CD3+ TCR α/β+ sinonasal case by Kanavaros and associates12 appeared to be αβ T-cell lymphomas. Interestingly, in contrast to most γδ T-cell lymphomas, 3 of these 4 cases were negative for CD56 and the other one was only weakly positive for CD56. On the other hand, one γδ T-cell case was reported by Kanavaros and coworkers,18 and 2 more cases were recently reported by the same group.31 Independently, 7 cases of possible γδ T-cell lymphomas were reported by Harabuchi and colleages10; these cases were positive for the δ-chain protein and negative for the β-chain protein, whereas 2 of them were CD3, and none of them showed simultaneous rearrangements of the γ- and δ-chain genes. In addition, our case 2 was a γδ T-cell lymphoma. Therefore, it may be that the γδ T-cell subset is relatively common in the nasal T-cell lymphoma. The establishment of more cell lines appears to contribute to clarifying the features of the subset of the nasal T-cell lymphoma.

In the present study, the NK- and the γδ T-cell lymphomas formed similar tumors characterized by granulomatous appearance and necrosis in the nasal mucosa. Moreover, both tumors consisted of pleomorphic medium- to large-sized cells, which showed angioinvasion and were positive for EBV and CD56+. Recently, Arnulf and coworkers studied 11 cases of nonhepatosplenic peripheral γδ T-cell lymphomas31; 3 of these cases presented in the nasal cavity and the other cases were in other extranodal sites, including the larynx, lung, gastrointestinal tract, or skin. Interestingly, all of the 3 nasal cases were EBV positive, and 2 of them were CD56+, whereas only a few cases in the other sites were EBV positive, and none of them was CD56+. They concluded that nasal T/NK-cell lymphomas, whatever their NK- or γδ T-cell origin, have common clinical and histopathologic features, are associated with EBV, and are derived from activated cytotoxic cells, and thus most likely belong to the same clinicopathologic entity. The present results appear to support their conclusions.

Although the etiology of the nasal T/NK-cell lymphoma is largely unknown, it has been recognized that the disease occurs in a consistent and strong association with EBV.15-22 Likewise, SNK 6 and SNT-8 harbored monoclonal EBV clones. However, the mechanisms by which T/NK cell lineages are affected by the virus are poorly understood. In terms of the infecting route, CD21 is the only known receptor for the virus to date.52,53 Kaneko and colleagues showed that CD21 was expressed on normal NK cells activated with IL-2 as well as on isolated tumor cells from a nasal NK-cell lymphoma.54However, 2 EBV-positive NK-cell lines, SNK-6 in the present study and NK-YS,33 did not express the CD21 antigen. Likewise, 5 EBV-positive T-cell lines, SNT-8 in the present study and 4 cell lines reported previously,55 were negative for the CD21 antigen. With respect to the tumorigenicity of the virus, the latent membrane protein LMP-1 is known to possess transforming activity for rodent as well as human cells,56,57 and may also play a role in the nasal T/NK-cell lymphoma. Cytogenetic studies have shown frequent abnormalities in chromosome 6 in nasal T/NK-cell lymphomas,58,59 and there has also been a report that abnormalities of chromosome 6 were additionally recognized in cells isolated from patients with chronic active EBV infection and that the abnormalities might be caused by the integration of the virus to chromosome 6.60 Therefore, to clarify a role of the EBV in the etiology of the nasal T/NK-cell lymphoma, virologic, molecular biologic, and cytogenetic studies seem to be necessary, warranting the use of the established cell lines for future studies and necessitating further efforts to develop more cell lines from nasal T/NK-cell lymphomas.

The authors thank Mrs Aya Kasuya for her technical assistance.

Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (no. 11670207 and 09253103) and Grants-in-Aid for Education and Research Promotion Program 1999 from Tokyo Medical and Dental University.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Jaffe
ES
Chan
JKC
Su
IJ
et al
Report of the workshop on nasal and related extranodal angiocentric T/natural killer cell lymphomas: definitions, differential diagnosis, and epidemiology.
Am J Surg Pathol.
20
1996
103
111
2
Harris
NL
Jaffe
ES
Stein
H
et al
A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.
Blood.
84
1994
1361
1392
3
Lipford
EH
Margolick
JB
Longo
DL
Fauci
AS
Jaffe
ES
Angiocentric immunoproliferative lesions: a clinicopathologic spectrum of post-thymic T-cell proliferations.
Blood.
72
1988
1674
1681
4
Weiss
LM
Picker
LJ
Grogan
TM
Warnke
RA
Sklar
J
Absence of clonal beta and gamma T-cell receptor gene rearrangements in a subset of peripheral T-cell lymphomas.
Am J Pathol.
130
1988
436
442
5
Frierson
HF
Innis
DJ
Mills
SE
Wick
MR
Immunophenotypic analysis of sinonasal non-Hodgkin's lymphomas.
Hum Pathol.
20
1989
636
642
6
Aozasa
K
Ohsawa
M
Tajima
K
et al
Nation-wide study of lethal midline granuloma in Japan: frequencies of Wegener's granulomatosis, polymorphic reticulosis, malignant lymphoma and other related conditions.
Int J Cancer.
44
1989
63
66
7
Ho
FCS
Choy
D
Loke
SL
et al
Polymorphic reticulosis and conventional lymphomas of the nose and upper aerodigestive tract: a clinicopathologic study of 70 cases, and immunophenotypic studies of 16 cases.
Hum Pathol.
21
1990
1041
1050
8
Ratech
H
Burke
JS
Blayney
DW
et al
A clinicopathologic study of malignant lymphomas of the nose, paranasal sinuses, and hard palate, including cases of lethal midline granuloma.
Cancer.
64
1989
2525
2531
9
Ferry
JA
Sklar
J
Zukerberg
LR
Harris
NL
Nasal lymphoma: a clinicopathologic study with immunophenotypic and genotypic analysis.
Am J Surg Pathol.
15
1991
268
279
10
Harabuchi
Y
Imai
S
Wakashima
J
et al
Nasal T-cell lymphoma causally associated with Epstein-Barr virus: clinicopathologic, phenotypic, and genotypic studies.
Cancer.
77
1996
2137
2149
11
Emile
J-F
Boulland
M-L
Haioun
C
et al
CD5− CD56+ T-cell receptor silent peripheral T-cell lymphomas are natural killer cell lymphomas.
Blood.
87
1996
1466
1473
12
Kanavaros
P
Briere
J
Lescs
M-C
Gaulard
P
Epstein-Barr virus in non-Hodgkin's lymphomas of the upper respiratory tract: association with sinonasal localization and expression of NK and/or T-cell antigens by tumour cells.
J Pathol.
178
1996
297
302
13
Nakamura
S
Katoh
E
Koshikawa
T
et al
Clinicopathologic study of nasal T/NK cell lymphoma among the Japanese.
Pathol Int.
47
1997
38
53
14
Cheung
MMC
Chan
JKC
Lau
WH
et al
Primary non-Hodgkin's lymphoma of the nose and nasopharynx: clinical features, tumor immunophenotype, and treatment outcome in 113 patients.
J Clin Oncol.
16
1998
70
77
15
Harabuchi
Y
Yamanaka
N
Kataura
A
et al
Epstein-Barr virus in nasal T-cell lymphomas in patients with lethal midline granuloma.
Lancet.
335
1990
128
130
16
Ho
FCS
Srivastava
G
Loke
SL
et al
Presence of Epstein-Barr virus DNA in nasal lymphomas of B and T cell type.
Hematol Oncol.
8
1990
271
281
17
Weiss
LM
Gaffey
MJ
Chen
YY
Frierson
HF
Frequency of Epstein-Barr viral DNA in “Western” sinonasal and Waldeyer's ring non-Hodgkin's lymphomas.
Am J Surg Pathol.
16
1992
156
162
18
Kanavaros
P
Lescs
MC
Briere
J
et al
Nasal T-cell lymphoma: a clinicopathologic entity associated with peculiar phenotype and with Epstein-Barr virus.
Blood.
81
1993
2688
2695
19
Borisch
B
Hennig
I
Laeng
RH
Waelti
ER
Kraft
R
Laissue
J
Association of the subtype 2 of the Epstein-Barr virus with T-cell non-Hodgkin's lymphoma of the midline granuloma type.
Blood.
82
1993
858
864
20
Arber
DA
Weiss
LM
Albujar
PF
Chen
YY
Jaffe
ES
Nasal lymphomas in Peru, high incidence of T-cell immunophenotype and Epstein-Barr virus infection.
Am J Surg Pathol.
17
1993
392
399
21
Chan
JKC
Yip
TTC
Tsang
WYW
et al
Detection of Epstein-Barr viral RNA in malignant lymphoma of the upper aerodigestive tract.
Am J Surg Pathol.
18
1994
938
946
22
Gorp
JV
Weiping
L
Jacobse
K
et al
Epstein-Barr virus in nasal T-cell lymphomas (polymorphic reticulosis/midline malignant reticulosis) in western China.
J Pathol.
173
1994
81
87
23
Ishii
Y
Yamanaka
N
Ogawa
K
et al
Nasal T-cell lymphoma as a type of so-called “lethal midline granuloma.”
Cancer.
50
1982
2336
2344
24
Jaffe
ES
Pathologic and clinical spectrum of post-thymic T-cell malignancies.
Cancer Invest.
2
1984
413
426
25
Yamanaka
N
Harabuchi
Y
Sambe
S
et al
Non-Hodgkin's lymphoma of Waldeyer's ring and nasal cavity.
Cancer.
56
1985
768
776
26
Chan
JKC
Ng
CS
Lau
WH
Lo
STH
Most nasal/nasopharyngeal lymphomas are peripheral T-cell neoplasms.
Am J Surg Pathol.
11
1987
418
429
27
Lippman
SM
Grogan
TM
Spier
CM
et al
Lethal midline granuloma with novel T-cell phenotype as found in peripheral T-cell lymphoma.
Cancer.
59
1987
936
939
28
Petrella
T
Delfau-Larue
M-H
Caillot
D
et al
Nasopharyngeal lymphomas: further evidence for a natural killer cell origin.
Hum Pathol.
27
1996
827
833
29
Chiang
AKS
Srivastava
G
Lau
PWF
Ho
FCS
Differences in T-cell-receptor gene rearrangement and transcription in nasal lymphomas of natural killer and T-cell types: implications on cellular origin.
Hum Pathol.
27
1996
701
707
30
Chiang
AKS
Chan
ACL
Srivastava
G
Ho
FCS
Nasal T/natural killer (NK)-cell lymphomas are derived from Epstein-Barr virus-infected cytotoxic lymphocytes of both NK- and T-cell lineage.
Int J Cancer.
73
1997
332
338
31
Arnulf
B
Copie-Bergman
C
Delfau-Larue
M-H
et al
Nonhepatosplenic γδ T-cell lymphoma: a subset of cytotoxic lymphomas with mucosal or skin localization.
Blood.
91
1998
1723
1731
32
Suzumiya
J
Takeshita
M
Kimura
N
et al
Expression of adult and fetal natural killer cell markers in sinonasal lymphomas.
Blood.
83
1994
2255
2260
33
Tsuchiyama
J
Yoshino
T
Mori
M
et al
Characterization of a novel natural killer-cell line (NK-YS) established from natural killer cell/lymphoma leukemia associated with Epstein-Barr virus infection.
Blood.
92
1998
1374
1383
34
Chang
KL
Chen
YY
Shibata
D
Weiss
LM
Description of an in situ hybridization methodology for detection of Epstein-Barr virus RNA in paraffin-embedded tissues, with a survey of normal and neoplastic tissues.
Diagn Mol Pathol.
1
1992
246
255
35
Sims
JE
Tunnacliffe
A
Smith
WJ
Rabbitts
TH
Complexity of human T-cell antigen receptor β-chain constant- and variable-region gene.
Nature.
312
1984
541
545
36
Quertermouse
T
Murre
C
Dialynas
D
et al
Human T-cell γ chain genes: organization, diversity, and rearrangement.
Science.
231
1986
252
255
37
Tkachuk
DC
Griesser
H
Takihara
Y
et al
Rearrangement of T-cell δ locus in lymphoproliferative disorders.
Blood.
72
1988
353
357
38
Korsmeyer
SJ
Arnold
A
Bakhshi
A
et al
Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins.
J Clin Invest.
71
1983
301
313
39
Takihara
Y
Tkachuk
D
Michalopoulos
E
et al
Sequence and organization of the diversity, joining, and constant region genes of the human T-cell δ-chain locus.
Proc Natl Acad Sci U S A.
85
1988
6097
6101
40
Raab-Traub
N
Flynn
K
The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation.
Cell.
47
1986
883
889
41
Huck
S
Lefranc
M-P
Rearrangements to the JP1, JP, and JP2 segments in the human T-cell rearranging gamma gene (TRGγ) locus.
FEBS Lett.
224
1987
291
296
42
Triebel
F
Faure
F
Graziani
M
et al
A unique V-J-C-rearranged gene encodes a protein expressed on the majority of CD3+ T cell receptor-α/β− circulating lymphocytes.
J Exp Med.
167
1988
694
699
43
Chien
Y
Iwashima
M
Kaplan
K
Elliott
J
Davis
M
A new T cell receptor gene located within the alpha locus and expressed early in T cell differentiation.
Nature.
327
1987
677
682
44
Lanier
LL
Chang
C
Spits
H
Phillips
JH
Expression of cytoplasmic CD3ε proteins in activated human adult natural killer (NK) cells and CD3γ, δ, ε complexes in fetal NK cells.
J Immunol.
149
1992
1876
1880
45
Chan
JKC
Tsang
WYW
Ng
FCS
Pau
MY
Discordant CD3 expression in lymphomas when studied on frozen and paraffin sections.
Hum Pathol.
26
1995
1139
1143
46
Cossman
J
Zehnbauer
B
Garrett
CT
et al
Gene rearrangements in the diagnosis of lymphoma/leukemia: guidelines for use based on a multiinstitutional study.
Am J Clin Pathol.
95
1991
347
354
47
Wong
KF
Chan
JKC
Matutes
E
et al
Hepatosplenic γδ T-cell lymphoma: a distinctive aggressive lymphoma type.
Am J Surg Pathol.
19
1995
718
726
48
Cooke
CB
Krenacs
L
Stetler-Stevenson
M
et al
Hepatosplenic T-cell lymphoma: a distinct clinicopathologic entity of cytotoxic γδ T-cell origin.
Blood.
88
1996
4265
4274
49
Gaulard
P
Bourquelot
P
Kanavaros
P
et al
Expression of the alpha/beta and gamma/delta T-cell receptors in 57 cases of peripheral T-cell lymphomas: identification of a subset of γ/δ T-cell lymphomas.
Am J Pathol.
137
1990
617
628
50
Mastovich
S
Ratech
H
Ware
R
Moore
JO
Borowits
MJ
Hepatosplenic γδ T-cell lymphoma: an unusual case of a γδ T-cell lymphoma with a blast-like terminal transformation.
Hum Pathol.
25
1994
102
108
51
Medeiros
LJ
Bagg
A
Cossman
J
Molecular genetics in the diagnosis and classification of lymphoid neoplasms.
Surgical Pathology of the Lymph Nodes and Related Organs: Major Problems in Pathology.
Jaffe
ES
16
1995
58
97
Saunders
Philadelphia
52
Moore
MD
Cooper
NR
Tack
BF
Nemerow
G
Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes.
Proc Natl Acad Sci U S A.
84
1987
9194
9198
53
Tanner
J
Weis
J
Fearon
D
Whang
Y
Kieff
E
Epstein-Barr virus gp350/220 binding to the B lymphocytes C3d receptor mediates adsorption, capping, and endocytosis.
Cell.
50
1987
203
213
54
Kaneko
T
Fukuda
J
Yoshihara
T
et al
Natural killer (NK) cell lymphoma: report of a case with activated NK cells containing Epstein-Barr virus and expressing CD21 antigen, and comparative studies of their phenotype and cytotoxicity with normal NK cells.
Br J Haematol.
91
1995
355
361
55
Imai
S
Sugiura
M
Oikawa
O
et al
Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection.
Blood.
87
1996
1446
1457
56
Wang
D
Liebowitz
D
Kieff
E
An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells.
Cell.
43
1985
831
840
57
Fahraeus
R
Rymo
L
Rhim
JS
Klein
G
Morphological transformation of human keratinocytes expressing the LMP gene of Epstein-Barr virus.
Nature.
345
1990
447
449
58
Tien
H-F
Su
I-J
Tang
J-L
et al
Clonal chromosomal abnormalities as direct evidence for clonality in nasal T/natural killer cell lymphomas.
Br J Haematol.
97
1997
621
625
59
Wong
KF
Chan
JKC
Kwong
YL
Identification of del(6)(q21q25) as a recurring chromosomal abnormality in putative NK cell lymphoma/leukemia.
Br J Haematol.
98
1997
922
926
60
Ohshima
K
Suzumiya
J
Ohga
S
Ohgami
A
Kikuchi
M
Integrated Epstein-Barr virus (EBV) and chromosomal abnormality in chronic active EBV infection.
Int J Cancer.
71
1997
943
947

Author notes

Norio Shimizu, Department of Virology, Division of Virology and Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan; e-mail: [email protected].

Sign in via your Institution