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Aims: Most primary gastrointestinal lymphomas are of B-cell origin and T-cell origin is very rare. Recent studies have suggested that human T-cell lymphotrophic virus type 1 (HTLV-1) may be involved in the development of primary gastric T-cell lymphoma. We analysed 31 patients with primary gastric T-cell lymphoma in south-west Japan, an area endemic for HTLV-1, and determined their phenotypes, genotypes, and HTLV-1 status. Methods and results: Here we present 31 cases of primary gastric T-cell lymphoma in a HTLV-1-endemic area in Japan and analyse the clinical status, histology, phenotype and virus status. The median age at onset of primary gastric T-cell lymphoma was 57 years with a gender ratio of M:F = 1.58:1. Six of the 31 primary gastric T-cell lymphoma cases had HTLV-1 proviral DNA (five males, one female), nine of the 31 cases were positive for anti-adult T cell leukaemia antibody, without examination of HTLV-1 proviral DNA (five males, four females), eight were non-HTLV-1-associated primary gastric T-cell lymphoma (four males, four females) and the other eight cases were unknown. Primary gastric T-cell lymphoma usually presented as a large ulcerated tumour at the corpus to the antrum and histologically consisted of anaplastic large cell type (n = 2), pleomorphic large cell type (n = 3), pleomorphic medium and large cell type (n = 14), pleomorphic medium cell type (n = 11), and angioimmunoblastic T-cell lymphoma type (n = 1). There were no clear macroscopic and microscopic differences between HTLV-1-associated and non-HTLV-1-associated primary gastric T-cell lymphoma. Most patients died within 2 years of diagnosis, and both types of primary gastric T-cell lymphoma (with and without HTLV-1) were associated with poor prognosis. Cytotoxic marker analysis showed that HTLV-1-associated lymphomas were negative for TIA-1, while non-HTLV-1-associated lymphomas were positive for TIA-1. Conclusions: Our results suggest that in HTLV-1-endemic areas, patients with HTLV-1-associated primary gastric T-cell lymphoma should be managed carefully and that TIA-1 seems to be useful for identifying the aetiology of this lesion.

Accessory cell tumour: a clinicopathological study of 16 aggressive tumours containing EBV-positive Hodgkin and Reed–Sternberg-like giant cells Aims: Lymph nodes contain non-lymphoid accessory cells including follicular dendritic cells and interdigitating dendritic cells. Functionally, these cells belong to the category of immune accessory cells involved in antigen presentation to B or T-lymphocytes. Neoplastic proliferation of these cells is very uncommon. We present here the clinicopathological features of 16 cases of accessory cell tumour. Methods and results: We performed electron microscopic and immunohistochemical examinations, and used in-situ hybridization for EBV-encoded RNA (ISH-EBV) to detect the EBV genome in 11 cases, and Southern blot analysis to assess EBV clonality in two cases. Tumour cells were composed of oval-to-spindle cells arranged in diffuse, vague storiform, fascicular and sometimes whorled patterns in a background of small lymphocytes. In all cases, binucleated or multinucleated Hodgkin and Reed–Sternberg-like giant cells were encountered. Staining for CD68 was positive in all cases. CD21, CD35, Ki-M4p, Ki-FDC1p, and S100 exhibited variable reactivity. ISH-EBV yielded positive labelling in seven of 11 cases, of which five exhibited EBV only in Hodgkin and Reed–Sternberg-like giant cells. Southern blot analysis showed clonality of EBV terminal repeats (EBV-TR) in the two cases examined. Electron microscopic examination showed that many of the tumour cells had numerous interwoven long villous cell processes connected by occasional desmosomes. Many tumours were very refractory to chemotherapy and radiation, with a few exceptions, and half of the cases classified initially as stage IV. A short survival time, of 10 months or less, was observed in seven of 16 patients. Conclusions: Our study identified more aggressive behaviour of accessory cell tumours. Our results suggest that EBV may potentially induce activation of accessory cells to form Hodgkin and Reed–Sternberg-like giant cells, which correspond with poor prognosis.

Evidence for local immunosuppression and demonstration of c-myc amplification in pyothorax-associated lymphoma Aims: Pyothorax-associated lymphoma (PAL) develops in the pleural cavity of patients with a long history of pyothorax. Epstein–Barr virus (EBV) is also involved in PAL, similar to lymphomas in immunodeficient patients. Here we examined T-lymphocyte subsets as well as c-myc and REL gene amplification in PAL tissues. Methods and results: We determined the number and distribution of CD4+ and CD8+ T-lymphocytes, to evaluate T-cells in the host immune reaction in seven cases of PAL. As controls, we also studied 10 cases of extranodal diffuse large B-cell lymphoma (DLBL) and 10 cases of nodal DLBL. Chromosomal imbalances in PAL were determined by using comparative genomic hybridization (CGH) analysis. The mean numbers of CD4+ and CD8+ and their ratio were significantly lower in PAL than in nodal DLBL. CGH analysis of PAL showed amplification of the 8q24 chromosomal region. In addition, c-myc amplification was found in four cases of PAL by Southern blot analysis. Conclusions: Our results suggest that the development of PAL may involve a local immunosuppressive environment and that amplification of c-myc might promote tumour progression, as has been described in the development of Burkitt’s lymphoma.

CD10 and Bcl10 expression in diffuse large B-cell lymphoma: CD10 is a marker of improved prognosis Aims: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin’s lymphoma, is clinically and pathologically heterogeneous. The Bcl10 gene was recently isolated from the breakpoint region of t(1;14)(p22;q32) in mucosa-associated lymphoid tissue (MALT) lymphomas, and is considered to be an apoptosis-associated gene. CD10 is considered to be a marker of follicular centre B-cell differentiation. To assess the clinical significance and roles of CD10 and Bcl10 in DLBCL, we analysed 138 cases, using immunohistochemical methods. Methods and results: CD10 expression was limited to the cytoplasm, whereas Bcl10 expression was detected in the cytoplasm and/or nuclei. CD10 expression was detected in 39 of 138 cases (28.2%), cytoplasmic Bcl10 in 68 cases (49.2%), and nuclear Bcl10 in 34 cases (24.6%). Nuclear Bcl10 was detected in 14 of 28 cases (50%) of extranodal DLBCL, but only 20 of 110 cases (18.2%) of nodal DLBCL. Cytoplasmic Bcl10 was detected in 19 of 28 cases (67.8%) of extranodal DLBCL and 49 of 110 cases (44.5%) of nodal DLBCL. CD10 expression closely correlated with improved survival (68% overall survival (OS) vs. 48% OS), but not with site of disease. A high International Prognostic Index (IPI) was considered to be a poor prognostic factor associated with a shorter OS. CD10 expression was detected in 27 of 84 cases (32.1%) with low-risk IPIs, and in 12 of 54 cases (22.2%) with high-risk IPIs. In the low-risk group, cases expressing CD10 carried a better prognosis than CD10− cases (93% OS vs. 71% OS), whereas this was not the case in the high-risk group (25% vs. 20%). Conclusions: Bcl10 expression was associated with extranodal DLBCL, but not with prognosis. CD10 expression was closely associated with improved survival, but not with risk as predicted by IPI. Overall, our results suggest that CD10 expression may be useful, in combination with clinical parameters, for determining the prognosis of DLBCL.

Aims: Hodgkin's disease (HD) is characterized by the presence of Hodgkin and Reed–Sternberg (H–RS) cells against a hyperplastic background of reactive cells such as lymphocytes, histiocytes, plasma cells, eosinophils, neutrophils, and stromal cells. There is ample evidence to suggest that proliferation and survival of HD-derived cells is due to cytokine signalling. Recently, high expression of interleukin (IL)-13 was described in HD-derived cell lines. Here we investigated the possible involvement of IL-13 in the pathophysiology, especially autocrine pathways of H–RS cells. Methods and results: The expression of IL-13 and IL-13 receptor (IL-13R) was determined by immunostaining and reverse transcriptase-polymerase chain reaction in 39 cases of HD, including 17 cases with nodular sclerosis (NS) type, 19 cases with mixed cellularity (MC), and three cases with lymphocyte predominance (LP) type. Expression of IL-13 was confined to H–RS cells and a few lymphocytes. IL-13R was expressed in H–RS cells, lymphocytes, histiocytes, fibroblasts, and endothelial cells. H–RS cells of MC and NS types frequently expressed both IL-13 and IL-13R. However, the number of IL-13-positive H–RS cells was statistically higher in NS-type than in MC-type, but the number of IL-13R was similar. IL-13R-positive fibroblasts were frequently encountered in NS-type. H–RS cells of LP type rarely expressed IL-13. Conclusions: Our results suggest that IL-13 might be involved in autocrine pathways of H–RS cells and fibrosis at least in NS-type. Our results also indicated that in addition to the morphological and phenotypic differences, the neoplastic cells of LP type might be functionally different from H–RS cells of MC- and NS-types.

Aims Hepatosplenic gammadelta T-cell lymphoma (TCL) is a rare, aggressive subset of peripheral TCL that presents with hepatosplenomegaly and cytopenia. Epstein-Barr virus (EBV) infection and activated cytotoxic molecules (granzyme and perforin) are uncommon in hepatosplenic gammadelta CTL. EBV infection and activated cytotoxic molecules are occasionally detected in non-hepatosplenic gammadelta TCL. We describe the clinicopathological features of three Japanese cases who were not immunodeficient. Methods and results All cases showed gammadelta T-cell type (CD2+, CD3+, T-cell receptor (TCR)delta-1+, betaF1-). Two cases expressed natural killer (NK) cell-associated antigens (CD8-, CD16+, CD56+; CD8-, CD16-, CD56+), and one expressed CD8 (CD8+, CD16-, CD56-). All cases expressed cytotoxicity-associated molecules (perforin, granzyme B, TIA-1 and Fas ligand). However, perforin and Fas ligand were not detected in one case. In-situ hybridization analysis with EBER probes revealed strong nuclear positivity in all neoplastic cells. In addition, two cases showed clonal bands of the EBV terminal repeat (TR) gene. Cytologically, instead of the presence of monomorphic medium-sized cells, our three cases showed pleomorphic medium-sized and large cells. Conclusions Our gammadelta TCL cases were clinicopathologically considered to be compatible with hepatosplenic gammadelta T-cell lymphoma. However, with regard to EBV association, activated cytotoxic profile and cytological features they resembled non-hepatosplenic gammadelta TCL. EBV may play a role in this disease by inducing cellular activation.

Aims Epstein–Barr virus (EBV) is associated with numerous reactive and neoplastic lymphoproliferative disorders. In vitro, EBV infection can transform B-lymphocytes and induce phenotypic alterations. This study presents the clinicopathological features of four cases with malignant lymphoma, which showed phenotypic and/or genotypic alterations during the course of the disease. Methods and results To determine the type of EBV genotype, we performed polymerase chain reaction (PCR) for lymphocyte-defined membrane antigen (LYDMA) of EBV, subtype A/B and latent membrane protein (LMP)-1 deletion. In addition, we analysed the terminal repeat (TR) band of EBV and receptor genes (T-cell receptor gene, TCR; immunoglobulin gene heavy chain, IgH) for EBV-infected cell clonality. Double staining of cell markers (B, T-lymphocytes; histiocytes), and in-situ hybridization (ISH) for EBV were performed using tissues obtained during the course of the disease. The first case showed genotypic and phenotypic alterations of T-cell type to B-cell type. The first TCR rearrangement and T-cell markers (CD3+, CD4+, CD8−) were lost and IgH rearrangement and B-cell markers (CD19+, CD20+) were identified. During the course of the disease, EBV-TR bands changed in size; however, the EBV genotype type B, LMP1 deletion type, and single LYDMA band remained the same. The initial T-cell lymphoma clone was considered to be different from the latter B-cell lymphoma clone. The second case showed phenotypic alterations. The first B-cell marker (CD19+, CD20+, CD68−) changed to histiocytic markers (CD19−, CD20−, CD68+). However, IgH rearrangement and EBV-TR bands remained the same throughout the course of the disease and EBV genotype type A, LMP1 deletion type, and single LYDMA band remained unchanged. The third case showed phenotypic alterations. The B-cell marker (CD20+) was lost; however, IgH rearrangement of PCR and EBV genotype remained the same. In the second and third cases, the initial lymphoma clones were considered to be same as the latter clones. The last case showed lineage alterations from Hodgkin's disease to natural killer (NK) cell leukaemia. However, EBV genotype did not change. The second case and Hodgkin's disease showed LMP expression, but the first and third cases showed no LMP, and EBNA2 was not detected in all cases. Conclusions We report the genotypic and/or phenotypic alterations in four patients with EBV-associated lymphoma/leukaemia. However, EBV genotype did not change in all four. These findings suggest that EBV might induce the cell marker and lineage alteration in vivo, as in vitro.

Aims Cell death can be divided into necrosis and apoptosis. In histiocytic necrotizing lymphadenitis (HNL), apoptosis is the main form of cell death. Two molecular mechanisms of T-cell-mediated cytotoxicity, one perforin-based and the other Fas-based, have been demonstrated, and both systems induce apoptosis of the target cells. This study was designed to investigate the Fas and perforin pathways in HNL. Methods and results Twelve cases of HNL were analysed using immunohistochemical staining. The Fas and/or FasL-positive cells, including lymphocytes and histiocytes, were frequently encountered in the necrotizing lesions, however, they were rare in the nonpathological regions. The perforin and/or granzyme-positive cells were also confined in the necrotizing lesions. In double staining, CD8-positive lymphocytes occasionally expressed Fas and/or FasL, and histiocytes also expressed FasL and/or Fas. However, CD4-positive lymphocytes rarely expressed FasL and/or Fas. In a flow cytometric analysis, most of the cytotoxic T-cells, which were recognized by cytolytic granules of TIA-1 and considered to be CD8-positive lymphocytes, expressed FasL and Fas. The perforin-positive lymphocytes also expressed FasL and Fas. Conclusion In our previous study, the apoptotic cells were T-cells, especially CD8-positive cells rather than CD4-positive cells. Based on these findings, in Fas and perforin pathways, the CD8-positive cells were considered to be effector and target cells, while histiocytes could possibly be enhancers. As a result, both pathways seemed to induce an abundance of apoptosis and thus induce necrotizing lesions.

Aims: Two molecular mechanisms of T/natural killer (NK) cell-mediated cytotoxicity, one perforin based and the other Fas based, have been demonstrated, and both systems induce cytotoxicity in the target cells. The Fas-based mechanism involves the transducing molecule Fas and its ligand (FasL). In addition, perforin and/or FasL are also expressed in the cytotoxic T/NK cells. This study was thus designed to investigate the Fas and perforin pathways of the cytotoxic T/NK lymphoma cells in the nasal cavity. Methods and results: Eight patients with nasal lymphoma were analysed using immunohistochemical staining methods. Two cases were CD3+ CD56+ (T/NK cell) type, and six were CD3− CD56+ (NK cell) type. All cases showed Epstein–Barr virus genomes by in-situ hybridization. In addition, all cases showed the expression of TIA-1 (GMP-17), which is a marker of cytotoxic T and NK cells. FasL was expressed in the majority of the lymphoma cells and some histiocytes, while Fas was found in lymphoma cells and many non-neoplastic cells. In addition, the expression of perforin was detected in almost all lymphoma cells. In the double stainings, lymphoma cells expressed both FasL and perforin. Based on these findings, both the perforin- and Fas-based pathway of the cytotoxic T/NK lymphoma cells are thus considered to play an important role in the clinical features. Conclusions: Tissue damage is a common morphological feature in nasal T/NK cell lymphoma. The above findings therefore support the theory that tissue damage is due to both the cytotoxicity of T/NK lymphoma cells as well as to angiocentricity.

Twenty-one patients with CD30 (Ki-1) positive lymphoma were studied from a group of 91 patients with adult T-cell leukaemia/lymphoma. The patients were grouped into three types: diffuse CD30 positive anaplastic large cell lymphoma in 11 patients (group 1); pleomorphic type lymphoma with diffuse CD30 expression in five patients (group 2); and pleomorphic type lymphoma with positive CD30 expression in large cells but negative in medium-sized and small cells in five patients (group 3). The patients with diffuse CD30 positive lymphomas (groups 1, 2) frequently presented with extranodal tumours (68.8%) and lymph node enlargement greater than 2 cm in diameter (50%), and rarely with leukaemic changes, bone marrow involvement and hypercalcaemia (one case of each). Patients in group 3 rarely had extranodal tumours, but had frequent leukaemic changes. Expression of intercellular adhesion molecule (ICAM-1; CD54) by the lymphoma cells in 13 patients (81.3%) with diffuse CD30 positive lymphomas, was significantly higher than that in 33 patients (9.1%) with CD30 negative adult T-cell leukaemia/ lymphomas. No positive reaction for epithelial membrane antigen (EMA) was found in the lymphoma cells of CD30 positive cases. The overall survival in patients with diffuse CD30 positive lymphomas was better than that of CD30 negative adult T-cell leukaemia/lymphoma patients, but showed no significant difference. These findings suggest that diffuse CD30 positive adult T-cell leukaemia/lymphoma has unusual clinical and immunohistological findings. It is also speculated that local tumour formation and leukaemic changes in such diffuse CD30 positive cases are influenced by CD54 (ICAM-1) expression by the lymphoma cells.