Detection of Tax-specific CTLs in lymph nodes of adult T-cell leukemia/lymphoma patients and its association with Foxp3 positivity of regulatory T-cell function.

Human T-cell lymphotropic virus type (HTLV)-1 Tax is a viral protein that has been reported to be important in the proliferation of adult T-cell leukemia/lymphoma (ATLL) cells and to be a target of HTLV-1-specific cytotoxic T lymphocytes (CTLs). However, it is not clear how Tax-specific CTLs behave in lymph nodes of ATLL patients. The present study analyzed the immunostaining of Tax-specific CTLs. Furthermore, ATLL tumor cells are known to be positive for forkhead box P3 (Foxp3)and to have a regulatory T (Treg)-cell-like function. The association between T-reg function and number and activity of Tax-specific CTLs was also investigated. A total of 15 ATLL lymphoma cases with human leukocyte antigen (HLA)-A24, for which Tax has a high affinity, were selected from the files of the Department of Pathology, School of Medicine, Kurume University (Kurume, Japan) using a polymerase chain reaction (PCR) method. Immunostaining was performed for cluster of differentiation (CD) 20, CD3, CD4, CD8, T-cell intracellular antigen-1 and Foxp3 in paraffin sections, and for Tax, interferon γ and HLA-A24 in frozen sections. In addition, the staining of Tax-specific CTLs (HLA-A24-restricted) was analyzed by MHC Dextramer® assay in frozen sections. In addition, the messenger RNA expression of Tax and HTLV-1 basic leucine zipper factor were also evaluated by reverse transcription-PCR. Immunohistochemical staining of Tax protein in lymphoma tissue revealed the presence of positive lymphoma cells ranging from 5 to 80%, and immunohistochemical staining of HLA-A24 revealed the presence of positive lymphoma cells ranging from 1 to 95%. The expression of Tax and HLA-A24 was downregulated by viral function. Foxp3, a marker for Treg cells, was expressed in 0-90% of cells. Several cases exhibited Tax-specific CTL (HLA-A24-restricted)-positive cells, and there was an inverse correlation between Tax-specific CTLs and Foxp3. However, neither Tax nor HLA-A24 expression was associated with CTL or Foxp3. Our study indicated the possibility that ATLL cells, which expressed Tax, target of CTL, evade the CTL-mediated immune control by expression of Foxp3 as a Treg function.

Composite lymphoma of peripheral T-cell lymphoma and Hodgkin lymphoma, mixed cellularity type; pathological and molecular analysis.

Composite lymphomas (CLs) are defined as two unrelated lymphomas occurring at the same time within the same tissue. The incidence of these tumors is low. Of all possible combinations between lymphomas, the least frequent are the ones combining peripheral T-cell lymphoma (PTCL) and Hodgkin lymphoma (HL). We recently identified five cases of CL composed of PTCL and classical HL, mixed cellularity type. We investigated histological and clinical features of these cases. Immunostaining was performed on paraffin sections. PTCL cells were positive for CD8 and TIA-1 in four of the five cases. Hodgkin and Reed-Sternberg (HRS) cells were positive for CD30 and weakly positive for PAX5 in all cases, positive for CD15 in three of five cases, positive for CD20 in one of five cases, and negative for EBER. Monoclonal rearrangement of the T-cell receptor (TCR) and immunoglobulin heavy chain (IGH) genes was confirmed by polymerase chain reaction (PCR) using whole paraffin sections. We concluded more precisely the monoclonality of the IGH rearrangement of HRS cells based on single-cell PCR for IGH and DNA sequencing analysis after laser microdissection of single cells in one case. HL can occur in CD8-positive and TIA-1-positive PTCL. Clinicians should recognize the possibility of these CL.

Six Cases of CD20-Positive Adult T-Cell Leukemia.

Adult T-cell leukaemia/lymphoma (ATLL) is a neoplasm originating in mature CD4+ peripheral T cells. However, rare cases of CD20+ ATLL have been reported. Here, we describe six cases of CD20+ ATLL diagnosed in our department. The median age was 79 years (range, 54-90 years); two patients were men, and four were women. Elevated lactate dehydrogenase was observed in four cases. All cases were lymphoma type and positive for human T-lymphotropic virus-1 (HTLV-1). HTLV-1 proviral DNA was detected in four cases. The Ann Arbor stage was I, II, or IV in one patient each and III in three patients. The clinical course was poor in almost all cases. Tumour cells were large in all cases, and flow cytometry revealed CD20+ lymphoma cells in five of six cases. Immunohistochemistry revealed lymphoma cells positive for CD20, CD3, CD4, and CCR4 and negative for CD8, CD79a, and PAX5 in all cases. CD20 expression was lower than that in normal B cells. One case was initially misdiagnosed as diffuse large B-cell lymphoma. Thus, combined use of an antibody panel and molecular genetic studies is important to avoid misdiagnosing ATLL as B-cell lymphoma.

Activated janus kinase 3 expression not by activating mutations identified in natural killer/T-cell lymphoma.

Janus Kinase 3 (JAK3) is a non-receptor tyrosine kinase, predominantly expressed in hematopoietic cells, that plays an essential role in hematopoiesis during T cell development. JAK3 somatic-activating mutations were identified in extranodal natural killer/T cell lymphomas (ENKTL) in recent cases in Singapore. We hypothesized these mutations might play an important role in the pathogenesis of T and NK cell neoplasms in other areas of the world. We performed JAK3 exon13 sequencing for different types of T and NK cell neoplasms including ENKTL (59 cases total). We identified four mutations in three (5.0%) cases. All of the mutations were from ENKTL cases (15.8%). Among the four newly found mutations, three are silent mutations and one introduces a stop codon, which was not an activating mutation as in the cases in Singapore. We detected four (30.8%) cases positive for phosphorylated JAK3 expression among 13 NKTCL cases when we performed JAK3 (phospho Y785) immunostaining on sections of ENKTL samples. It seems that phosphorylated JAK3 expression does not necessarily harbor exon 13 mutations. The mechanism responsible for activating expression of the gene will be a topic for further research.

Oncogene associated cDNA microarray analysis shows PRAME gene expression is a marker for response to anthracycline containing chemotherapy in patients with diffuse large B-cell lymphoma.

CHOP (cyclophosphamide, adriamycin, vincristine, and prednisolone) therapy achieves a response in more than 60% patients with diffuse large B-cell lymphomas (DLBCLs). However, DLBCL shows a heterogeneous response to chemotherapy, and some patients are refractory to CHOP therapy. This difference in response to therapy is most likely due to differences in biological characteristics. We used cDNA microarray analysis to identify genes differentially expressed in anthracycline containing chemotherapy-resistant DLBCLs (7 patients) compared with anthracycline containing chemotherapy-sensitive DLBCLs (6 patients). Nine genes on the cDNA chip showed increased expression in anthracycline containing chemotherapy-resistant patients. We chose the preferentially expressed antigen of melanoma (PRAME) gene because it showed the highest expression in anthracycline containing chemotherapy-resistant DLBCLs on the cDNA chip, and it has been linked to prognosis of hematological malignancies. We also examined the relationship between PRAME gene expression and progression-free survival (PFS) in 45 patients with DLBCL. The progression-free survival of PRAME-positive patients (n=12) was significantly worse than that of PRAME-negative patients (n=33) (p=0.0373). Our results therefore indicate that PRAME expression in DLBCL correlates with response to anthracycline containing chemotherapy.

Bcl2-negative follicular lymphomas frequently have Bcl6 translocation and/or Bcl6 or p53 expression.

Bcl2 is an important protein involved in the pathogenesis of follicular lymphoma (FL). However, approximately 10% of FL cases do not express Bcl2. The present study was designed to compare gene aberrations, prosurvival gene expression, apoptosis and proliferation rates in Bcl2-positive and -negative FL cases. Bcl2 translocation and Bcl6 translocation were detected and compared using fluorescence in situ hybridization (FISH). A tendency for Bcl6 translocation to occur was found more frequently in Bcl2-negative FL than in the Bcl2-positive cases. The expression of Bcl-X, BAX, p53, Bcl6 was analyzed by immunohistochemistry. Bcl2 family proteins Bcl-X and BAX were expressed similarly in the two FL types. In some cases of Bcl2-negative FL there was high expression of Bcl6 or p53 but no such Bcl2-positive FL cases were detected. Furthermore, there was an inverse relationship between the expression of Bcl6 and p53. These results indicate that the Bcl6 translocation occurs more frequently in Bcl2-negative FL. Furthermore, other prosurvival proteins such as p53 and Bcl6 may play an important role in the pathogenesis of Bcl2-negative FL.

Epstein-Barr virus genome level, T-cell clonality and the prognosis of angioimmunoblastic T-cell lymphoma.

BACKGROUND AND OBJECTIVES: Angioimmunoblastic T-cell lymphoma (AILT) is a peripheral T-cell tumor of unknown etiology with variable biological and clinical presentations. Previous clonality studies have shown heterogeneous clonal restrictions of B- and T-cell populations in this tumor. AILT is characterized by the presence of increased numbers of Epstein-Barr virus (EBV) infected cells. The aim of this study was to clarify the correlation between clonality, EBV and prognosis. DESIGN AND METHODS: Frozen material from 59 cases of AILT was used for DNA isolation and gene analysis by Southern blotting. A real-time polymerase chain reaction was used to quantify the amount of EBV-DNA in the tissue. Survival data were retrieved from clinical records. RESULTS: Clonal T cells were found in 15/50 and clonal B-cells in 2/50 tumors, using Southern blot analysis. Bands of EBV-W were found in 10/50 tumors. Survival rate did not correlate with either T-cell clonality (p=0.84), or presence of EBV-infected cells (p=0.84). The EBV-DNA copy number in EBV-infected tissue did not correlate with disease progression (p=0.87). The survival rate and clinical status according to the international prognostic index (IPI) did not correlate with T-cell clonality status or EBV infection. INTERPRETATION AND CONCLUSIONS: AILT remains a heterogeneous disease with clinical behavior that varies irrespective of the genomic parameters investigated.

A "floral" variant of nodal marginal zone lymphoma.

We describe 6 cases of a specific variant of nodal marginal zone lymphoma with "floral" lymph follicles in patients ranging in age from 18 to 66 years. All 6 patients had lymphadenopathy, either local (n = 5) or systemic (n = 1), and good performance status (0), and none had fever, weight loss, or night sweating. They all underwent excisional biopsy. Histologically, all lesions had a distinctive morphology, with proliferation of medium-sized atypical lymphoid cells in the marginal zone, hyperplastic lymph follicles with enlarged germinal centers, and a thickened mantle zone. In places, folliculolysis was observed. On immunohistochemical staining, the atypical lymphoid cells showed a B-cell phenotype (CD20 +), IgM positivity in 2 of 5 cases, and negativity for CD5, CD10, CD23, CD43, bcl-6, and IgD. Polymerase chain reaction examination for immunoglobulin heavy chain in 5 cases showed monoclonality in all. Five patients did not receive adjuvant chemotherapy and had no recurrences. The patient with systemic lymphadenopathy received chemotherapy and had a complete response without relapse. This variant should be differentiated from the usual nodal marginal zone lymphoma because of its specific clinical and pathological features.

CXCR3-positive B cells found at elevated frequency in the peripheral blood of patients with MALT lymphoma are attracted by MIG and belong to the lymphoma clone.

Chemokine receptors mediate the migration of lymphocytes through binding of their ligands. CXCR3 is expressed in Th1 T cells; however, CXCR3 was recently reported in B-cell mucosa-associated lymphoid tissue (MALT)-type lymphoma and splenic marginal zone lymphoma. To investigate whether CXCR3-positive B lymphocytes in peripheral blood (PB) migrate to MALT and spleen, and whether the lymphoma clone is present in PB, we studied 16 cases of MALT lymphoma. In MALT cases, CXCR3-positive B lymphocytes in PB could migrate to MIG, the CXCR3 ligand. Immunohistochemical analysis showed that MALT lymphoma cells expressed CXCR3, whereas epithelial glands and/or stromal cells expressed MIG. In the PCR analysis for VH gene rearrangements, MALT lymphoma showed monoclonal or oligoclonal bands. In addition, in 8 of 16 MALT cases, the VH gene rearrangement of MALT lymphoma had the same bands as the CXCR3-positive B lymphocytes in PB. In 4 cases, the same clones of DNA sequences were confirmed in MALT lymphoma and CXCR3-positive B lymphocytes of PB. The findings support the theory that CXCR3-positive B lymphocytes in PB of MALT patients belong to the lymphoma clone and migrate to MIG-expressing mucosa-associated lymphoid tissue. It seemed to be associated with the dissemination of MALT lymphoma.

[Registration of hematological disorders by the Kyushu Hematology Organization for Treatment (K-HOT) Study Group].

The Kyushu Hematology Organization for Treatment (K-HOT) Study Group was organized in 1999 to study hematological disorders diagnosed in the participating institutions in the Kyushu district. We registered all new patients with hematological disorders and from February 2000 to the end of 2003, a total of 2908 patients had been registered. They include non-Hodgkin's lymphoma in 803 patients, leukemia in 556, multiple myeloma (MM) in 276, myelodysplastic syndrome in 273, and adult T-cell leukemia/lymphoma (ATL) in 269 followed in a decreasing order by idiopathic thrombocytopenic purpura, aplastic anemia, and other benign hematological disorders and myeloproliferative disorders. The annual incidence of MM is estimated to be much higher than that previously reported. It is also confirmed that ATL is still one of the frequently encountered lymphoid malignancies in the Kyushu district.

Classification of distinct subtypes of peripheral T-cell lymphoma unspecified, identified by chemokine and chemokine receptor expression: Analysis of prognosis.

WHO classification for malignant lymphoma was recently proposed. However, PTCL is heterogeneous. Chemokines and its receptors are closely associated with the T-cell subtypes. To clarify the T-cell subtype in PTCL, we conducted DNA chips of chemokine, its receptor (R) and cytokines. Angioimmunoblastic T-cell lymphoma (AILD, n=4), anaplastic large cell lymphoma (ALCL, n=4), adult T-cell leukemia lymphoma (ATLL, n=7), NK-cell lymphoma (NKL, n=2) and PTCL, unspecified (PTCL-U, n=6) were analyzed using DNA chips. In addition, immunological stainings were performed in 280 cases. In DNA chip, AILD, ALCL, NKL and ATLL showed a tendency for respective clusters, otherwise, PTCL-U clustered with AILD, ALCL and ATLL. From the gene expression profiling, CCR4, CCR3, MIG, CXCR3 and BLC were selected for immunohistochemistry. ATLL (n=48) expressed CCR4. ALCL (n=26) expressed CCR3, NKL (n=20) expressed MIG, and AILD (n=29) expressed CXCR3 and/or BLC. From the expression patterns, PTCL-U (n=134) were classified into three groups; CCR4 type (CCR4(+), n=42), CCR3 type (CCR3(+), n=31) and CXCR3 type (CXCR3(+) BLC(+/-), n=54). The prognosis was poor for ATLL, intermediate for AILD and favorable for ALCL (P=0.0014). Among PTCL-U, CCR4 type, CXCR3 type and CCR3 type had prognoses equivalent to ATLL, AILD and ALCL, respectively (P<0.0001).

Expression of FoxP3, a key molecule in CD4CD25 regulatory T cells, in adult T-cell leukaemia/lymphoma cells.

Adult T-cell leukaemia/lymphoma (ATLL) is an aggressive neoplastic disease that usually exhibits a CD4(+)CD25(+) phenotype. Regulatory T cells (Treg), which suppress T-cell effector function, are characterized by the co-expression of CD4 and CD25. We analysed the expression of forkhead/winged helix transcription factor (FoxP3), a specific marker that is important for the function of Treg, on ATLL cells from 17 patients (peripheral blood, n = 8; lymph node, n = 9). Real-time polymerase chain reaction and immunostaining detected FoxP3 expression in 10 ATLL cases, but was relatively down-regulated compared with Treg from normal subjects. These results indicate the association of ATLL and Treg.

Apoptosis- and cell cycle-associated gene expression profiling of histiocytic necrotising lymphadenitis.

Cell death is of two types; necrosis and apoptosis. In histiocytic necrotising lymphadenitis (HNL), apoptosis is the main form of cell death. Apoptosis results in the formation of nuclear debris, which is one of the characteristic features of HNL. We previously reported that in HNL it is predominantly CD8-positive cytotoxic T cells that undergo apoptosis; however, the majority of proliferating cells are also CD8-positive T cells. Recent advances in technical and analytical methods have facilitated the parallel quantitation of expression of numerous genes using DNA microarrays. The technology is particularly well suited to compare differences in gene expression between normal tissues and inflammatory disease. To investigate the apoptosis- and cell cycle-associated gene expression in HNL, we analysed five cases each of HNL and non-specific lymphadenitis (NSL), using ready-made microarrays, including cyclins and caspases, and immunohistochemical staining of caspase-3, ssDNA, bcl-2 and NF-kappaB. Caspase-3- and ssDNA-positive apoptotic cells were frequently detected in HNL, but were rare in NSL. However, bcl-2- and NF-kappaB-positive cells were rare in HNL. Gene expression tree analysis of DNA microarrays showed different clustering of HNL and NSL. In comparison with NSL, HNL exhibited diffuse upregulation of these gene profiles, particularly of cyclins and caspases (ratio; cyclin A2, 2.72; caspase-6, 2.43; caspase-3, 2.02); whereas, Mcl-1, which has been shown to delay apoptosis, was downregulated (ratio, 0.71), as confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). Almost all apoptosis-associated genes, especially caspases, were upregulated, and apoptosis inhibitory genes, including bcl-2 by immunohistochemistry, were downregulated in all five cases with HNL. In addition, cell cycle-associated genes were upregulated in all. These findings confirm that both apoptosis and proliferation are simultaneously present in HNL lesions.