Riko Kawano

Expression of FoxP3, a key molecule in CD4+CD25+ 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.

Low-grade follicular lymphoma with t(14;18) presents a homogeneous disease entity otherwise the rest comprises minor groups of heterogeneous disease entities with Bcl2 amplification, Bcl6 translocation or other gene aberrances.

Follicular lymphomas (FL) are morphologically classified into grades 1, 2, 3a and 3b by the World Health Organization. Bcl2, Bcl6 and CD10 are phenotypic markers of FL while the Bcl2 t(14;18) and Bcl6 t(3q27) gene translocations are common genetic changes. However, to date, there has been no integrated analysis based on phenotype, grade and genotype from large numbers of FL cases. We graded 261 cases of FL and determined their phenotypes and gene alterations. According to the antigen markers and gene alterations of 147 cases, we classified FL into typical and the others types. The typical group, which includes 69% cases of FL, is characterized by low histological grade (grade 1, 2), coexpression of BCL2 and CD10 and Bcl2 gene translocation. The rest comprises a small part of low-grade FL without Bcl2 gene translocation and high-grade (grade 3a, 3b) FL. These FLs include some heterogeneous disease entities. They are characterized by high histological grade (87%), no definite expression of BCL2 or CD10 and several kinds of gene aberrances including Bcl2 translocation, Bcl6 translocation, Bcl2 amplification or other unknown gene abnormality. Our findings indicate that typical FL presents a homogeneous disease entity whereas the rest comprises heterogeneous diseases entities.

Clinicopathological features of cutaneous lesions of adult T-cell leukaemia/ lymphoma.

Background  Adult T‐cell leukaemia/lymphoma (ATLL) is a human malignancy associated with human T‐cell leukaemia virus type I (HTLV‐I). ATLL frequently involves the skin.

Expression of chemokines and chemokine receptors in cutaneous CD30+ lymphoproliferative disorders.

Background  Little is known about the mechanisms involved in skin‐specific homing in CD30+ cutaneous lymphoproliferative disorders (CLPD). Chemokine/chemokine receptor interactions have been implicated in the homing of lymphoma cells to various tissue sites.

Prognostic significance of hepatocyte growth factor and c-MET expression in patients with diffuse large B-cell lymphoma

The expression and prognostic significance of hepatocyte growth factor (HGF) and its receptor c‐MET (MET proto‐oncogene) was analysed in 96 cases of diffuse large B‐cell lymphoma (DLBCL). Tissue sections were immunohistochemically stained for HGF and c‐Met. The prognosis of HGF‐positive and c‐Met‐positive cases was significantly worse than negative cases (HGF: P = 0·0036; c‐Met: P = 0·0002). In addition, in the low‐risk international prognostic index group, HGF‐negative and c‐Met‐negative cases had a significantly better prognosis than positive cases (HGF: P = 0·0009; c‐Met: P < 0·0001). Our results suggest that HGF/c‐MET is a useful clinical marker of prognosis for patients with DLBCL.

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.

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.

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‐κB. Caspase‐3‐ and ssDNA‐positive apoptotic cells were frequently detected in HNL, but were rare in NSL. However, bcl‐2‐ and NF‐κB‐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.

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.

No correlation between immunoglobulin heavy chain rearrangements and the prognosis of angioimmunoblastic T-cell lymphoma.

To the Editor: Angioimmunoblastic T-cell lymphoma (AILT), originally described by Frizzera et al. in 1974, is a systemic disease of uncertain etiology (1, 2). Recently, AILT is recognized as a T-cell lymphoma by modern classification systems, including the WHO classification (3). DNA clonal analysis studies of AILT cases have shown heterogeneous Tcell receptor (TCR) and immunoglobulin heavy chain (IgH) gene rearrangements. Some cases show co-existing rearrangements of TCR and immunoglobulin genes (4– 7). In a previous study of ours (8) Southern blot analysis did not identify any correlation between IgH or TCR clonality and prognosis of patients with AILT. However, Southern blot analysis can not detect a monoclonal population of either B or T cells when they constitute <5% of the cells present in tissue samples. On the other hand, PCR analysis can detect smaller monoclonal populations of <2%. We therefore designed a retrospective molecular study using PCR analysis of 45 archival cases of AILT to assess the incidence of clonal IgH rearrangements by and the prognostic significance of B-cell clonality in AILT. All AILT samples were described previously (8). The patients were aged between 42 and 81 yrs (median age; 63 yrs), and their 5-yr survival rate was 45%. This study was carried out in accordance with the principles of the Helsinki declaration and was approved by the ethics committee of each institution. DNA preparations were made from whole sections of 45 frozen lymph nodes. TCRc PCR analysis was performed using the consensus primers TCR-GV1 and J-Mix. IgH PCR analysis was performed using the consensus primers complementary to the framework 2 portion of the VH region (FR2B) and the JH region (CFW1) from genomic DNA. Survival curves were calculated with the Kaplan–Meier method, and differences in survival rates were tested for significance with Log-Rank test. Differences were considered statistically significant if the P-value was <0.05. Clonal gene rearrangements for IgH were detectable in 11 of 45 cases (24.4%) and all cases (100%) were clonal for TCR. There was no correlation between IgH rearrangements and the prognosis of AILT (P = 0.94; Fig. 1), nor did Epstein–Barr virus infection examined previously (8) correlate with IgH rearrangements. Frizzera et al. (9) reported detecting TCR and IgH rearrangements in 85% and 7% of AILT cases respectively by using Southern blot analysis. Furthermore, 7% showed coexisting rearrangements of TCR and IgH, indicating the presence of either two different clonal populations or one clone of indefinite lineage, carrying both TCR and IgH rearrangements. In our study using PCR, we detected TCR and IgH rearrangement in 100% and 24.4%. Feller et al. (10) reported that IgH rearrangements of AILT cases were associated with increased overall survival, although this finding was not statistically significant. On the other hand, our previous study (8) showed there was no correlation between survival and Ig or TCR rearrangements of AILT cases. In conclusion, our data obtained from PCR showed no relationship between survival and IgH rearrangements of AILT cases. It is thus possible that AILT is a lymphoma with a clinical behavior that varies irrespective of IgH rearrangements.