Hirokazu Nagai

Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes

We previously reported that the hypermethylation of the p15INK4B gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15INK4B gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15INK4B gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15INK4B methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15INK4B-methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15INK4B methylation were detected in both patients. Furthermore, X-chromosome inactivation (XCI) pattern of each colony was simultaneously determined by MSP-based human androgen receptor gene analysis (HUMARA-MSP), and all p15INK4B-methylated colonies showed the same XCI pattern, which was dominant among the colonies, while p15INK4B-unmethylated colonies showed both patterns of XCI, in each of the two patients. We then examined the methylation status of the p15INK4B gene of granulocyte (PB-PMN) fractions from 10 patients with available peripheral blood cells. In all four patients with p15INK4B-methylated BM-MNCs, their PB-PMNs showed p15INK4B methylation. These results suggest that p15INK4Bmethylation in hematopoietic cells in MDS patients is restricted to the MDS clone but not necessarily to blast cells.

Mutations and aberrant DNA methylation of the PROX1 gene in hematologic malignancies

The homeobox gene PROX1 is related to the Drosophila prospero gene, which is expressed in the developing central nervous system and lens‐secreting cone cells. We found that the PROX1 gene had missense and nonsense mutations in 4 of 29 hematologic cell lines analyzed. Decreased mRNA expression was also observed in half of these cell lines by RT‐PCR. The restoration of PROX1 gene expression after treatment with the demethylating agent 5‐aza‐2′‐deoxycytidine, as well as bisulfite sequencing analysis, indicated that gene silencing is caused by DNA hypermethylation at intron 1. Such hypermethylation was also seen in primary lymphomas (56.3%, 18/32) in a tumor‐specific manner. These findings indicate that the profile of the PROX1 gene corresponds to that of a candidate tumor‐suppressor gene.

hSNF5/INI1 gene mutations in lymphoid malignancy

hSNF5/INI1 is one of the components of the SWI/SNF multiprotein complex that is necessary for the transcriptional activation of several genes and functions by altering chromatin structure. This gene has been thought to be one of the tumor suppressor genes (TSGs) because deletions or mutations were reported in malignant rhabdoid tumors and atypical teratoid and rhabdoid tumors. To evaluate the hSNF5/INI1 gene as a TSG in lymphoid malignancies, we performed a mutational analysis in 23 patients with non-Hodgkin lymphoma (NHL), 24 with acute lymphoblastic leukemia (ALL), 24 with multiple myeloma (MM), 24 with adult T-cell lymphoma/leukemia (ATLL), and 19 with lymphoid cell lines, by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis. Nonsense and missense mutations were found in 1 NHL case and 2 cell lines. Mutations from this NHL case proved to be somatic in origin. These data indicated that alterations in the hSNF5/INI1 gene might be involved in the pathogenesis of lymphoid malignancies.

The CDKN2 gene alterations in various types of adult T‐cell leukaemia

The CDKN2 gene, encoding the cyclin‐dependent kinase‐4 inhibitor p16, is a putative tumour‐suppressor gene because it is frequently altered in many malignant tumours. We analysed the CDKN2 gene in 44 cases of adult T‐cell leukaemia (ATL) by Southern blot analysis, polymerase chain reaction‐mediated single‐strand conformation polymorphism (PCR‐SSCP) analysis, and direct sequencing. Southern blot analysis detected a homozygous deletion of the CDKN2 gene in 5/44 patients (11.4%). Mutational analysis by the PCR‐SSCP method and direct sequencing showed one nonsense mutation at codon 72 (nucleotide 232), and two missense mutations at codon 43 (nucleotide 146) and codon 97 (nucleotide 309, 3/44, 6.8%). Therefore we found changes in the CDKN2 gene, including point mutations, in 18.2% of the patients with ATL. Interestingly, most of these patients had acute type ATL. Our results suggest that the CDKN2 gene is inactivated not only by homozygous deletion but also by point mutation, and these alterations contribute to the aggressiveness of ATL.

Aplastic anaemia with 13q–: a benign subset of bone marrow failure responsive to immunosuppressive therapy

Summary. In an attempt to determine the pathological significance of a long arm deletion of chromosome 13 (13q–) in bone marrow failure syndrome, we reviewed the clinical records of nine patients who were initially diagnosed with aplastic anaemia due to bone marrow hypoplasia without dysplasia. Six patients responded to immunosuppressive therapy and the other three improved with steroids. None of the patients developed acute leukaemia (follow up: 54–129 months) and the estimated 5‐year survival was 78%. These findings indicate that pancytopenia with 13q– represents bone marrow failure of a benign nature, similar to aplastic anaemia without karyotypic abnormalities, rather than preleukaemia.

Identification and mapping of novel tumor suppressor loci on 6p in diffuse large B-cell non-Hodgkin's lymphoma.

Allelic deletions have been thought to be indicators of the presence of tumor suppressor genes (TSGs). As indicated by this allelotype study using 39 highly informative microsatellite markers distributed among all autosomal chromosomes, frequent loss of heterozygosity (LOH) has been found at 6p in B‐cell non‐Hodgkins lymphoma. To identify the commonly deleted regions (CDRs), we performed fine deletion mapping using 26 highly polymorphic microsatellite markers on 6p. The most frequent LOH occurred at D6S1721, where 9 of 18 of the informative cases (50%) had allelic losses. Seventeen of 32 cases (53%) exhibited LOH at least at one locus on 6p. Ten of these 17 cases showed interstitial deletions, and their LOH patterns indicated two CDRs on 6p; one between D6S1721 and D6S260 (at 6p23–24), and the other between D6S265 and D6S291 (at 6p21). The genetic distance of both CDRs was 6 cM. The CDKN1A (p21) gene is reported to be located within the interval of the CDR at 6p21, but no mutation of the gene was found in these 32 patients. These data suggested that these two loci might harbor novel putative TSGs responsible for the pathogenesis of malignant lymphoma. We have constructed a contig of yeast artificial chromosome (YAC) clones spanning the most frequent CDR at 6p23–24. This YAC contig can be used for fine physical mapping of the region and cloning of candidate TSGs. Genes Chromosomes Cancer 25:277–283, 1999.

Ocular adnexal mucosa-associated lymphoid tissue lymphoma with polyclonal hypergammaglobulinemia.

PURPOSE To determine the characteristics of patients with primary ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma associated with polyclonal hypergammaglobulinemia. DESIGN Case series study. METHODS Among 81 Japanese patients with primary ocular adnexal MALT lymphoma, seven patients (9%) were diagnosed with polyclonal hypergammaglobulinemia. Patient clinical data included a history of autoimmune disease and dissemination. Peripheral blood collected from all patients was analyzed for serum levels of rheumatoid factor, soluble interleukin-2 receptor (sIL-2R), and immunoglobulins at the time of diagnosis and after each treatment. RESULTS Seven patients with polyclonal hypergammaglobulinemia had elevated serum levels of rheumatoid factor, sIL-2R, immunoglobulin G (IgG), and immunoglobulin E (IgE) at the time of diagnosis. One patient had Sjogren syndrome. Six patients (86%) had a dissemination of the MALT lymphoma or lymphadenopathy at the time of diagnosis. Histopathologic examination of the patients with lymphadenopathy revealed not only MALT lymphoma but also secondary follicles. None of the seven patients showed improvement in serum levels of IgG, rheumatoid factor, or sIL-2R in spite of complete regression of the ocular lesions after radiotherapy. After administration of cyclophosphamide/doxorubicin/vincristine/prednisone and/or rituximab to three patients, all three showed improved serum levels of IgG, rheumatoid factor, and sIL-2R. CONCLUSIONS Patients with ocular adnexal MALT lymphoma and polyclonal hypergammaglobulinemia have elevated serum levels of rheumatoid factor, sIL-2R, and IgE, and high dissemination or lymphadenopathy. These unique characteristics may correlate with the systemic immunologic imbalances.

Remission induction therapy containing rituximab markedly improved the outcome of untreated mature B cell lymphoma

Many controlled clinical trials have proven that rituximab improves the clinical outcome of patients with mature B cell lymphoma. This study was conducted to assess the contribution of rituximab in the actual clinical practice. Patients with newly diagnosed mature B cell lymphoma treated at 20 National Hospital Organization hospitals from January 2000 to December 2004 were consecutively registered. Rituximab was approved in September 2002 for indolent B cell lymphoma and in September 2003 for aggressive B cell lymphoma in Japan. The patients were divided into two groups depending on whether they received induction therapy containing rituximab. The endpoint was to evaluate the rituximab benefit based on 2‐year progression‐free survival (PFS) and 2‐year overall survival (OS). A total 1126 patients received chemotherapies. Of these, 762 were diagnosed as diffuse large B cell lymphoma (DLBCL) and 215 as follicular lymphoma (FL). PFS and OS were markedly improved in the rituximab group compared with the non‐rituximab group in patients with DLBCL (both P < 0·001) and in patients with FL (P < 0·001 and P = 0·003 respectively). Rituximab, when used for remission induction therapy, significantly improved the clinical outcome of the mature B cell lymphoma patient in actual clinical practice.

Actual status of AIDS-related lymphoma management in Japan

Recently, with the widespread use of highly active antiretroviral therapy (HAART), the occurrence of opportunistic infections as an acquired immunodeficiency syndrome (AIDS)-defining illness (ADI) has declined dramatically [1]. Decreases in the incidence of AIDSrelated lymphoma (ARL) are not as evident compared with other ADI, so lymphoma has now become one of the most common ADIs [2]. Lymphoma has also become a more common cause of mortality due to AIDS. Management of ARL is starting to represent a critical problem in the total care of the Human Immunodeficiency virus (HIV)-infected

Novel interleukin-2 dependent T-cell line derived from adult T-cell leukemia not associated with human T-cell leukemia virus type I.

A novel interleukin‐2 (IL‐2)‐dependent T‐cell line, WHN2, was established from a patient with adult T‐cell leukemia (ATL) not associated with human T‐cell leukemia virus type I (HTLV‐I). Neither the original leukemic cells nor the WHN2 cells showed proviral integration in their cellular DNAs by Southern blot analysis. The surface phenotype showed that both the original leukemic cells and the WHN2 cells had a common phenotype of ATL, i.e., positive for CD2, CD4, human leukocyte antigen DR (HLA‐DR) and CD25, but negative for CD8, a characteristic of helper/inducer T‐cells. Most of the chromosomal abnormalities of the original leukemic cells were maintained in the WHN2 cell line. Furthermore, Southern blot analysis of the T‐cell receptor β‐chain gene rearrangement revealed that the original leukemic cells and WHN2 cell line had identical patterns, suggesting that the WHN2 cell line was truly derived from the original leukemic cells. Dose‐dependent growth on IL‐2 was demonstrated, and at the maximal stimulation, the number of cells doubled within three days. This IL‐2‐dependent growth was inhibited by the simultaneous existence of anti‐IL‐2 receptor a and β chain antibodies. These results indicate that the character of the WHN2 cell line is similar to that of the cell lines derived from ATL associated with HTLV‐I. Thus, the HTLV‐I‐negative ATL cell line, WHN2, should be useful in the comparative study of the pathogenesis of ATL associated with or without HTLV‐I.