Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia.

Gene targeting studies in mice have shown that the lack of Ikaros activity leads to T-cell hyperproliferation and T-cell neoplasia, establishing the Ikaros gene as a tumor suppressor gene in mice. This prompted us to investigate whether mutations in Ikaros play a role in human hematological malignancies. Reverse transcription-PCR was used to determine the relative expression levels of Ikaros isoforms in a panel of human leukemia/lymphoma cell lines and human bone marrow samples from patients with hematological malignancies. Among the cell lines examined, only BV-173, which was derived from a chronic myelogenous leukemia (CML) patient in lymphoid blast crisis, overexpressed the dominant-negative isoform, Ik-6. In 9 of 17 samples of patients in blast crisis of CML, Ikaros activity had been reduced either by drastically reducing mRNA expression (4 of 17) or by overexpressing the dominant-negative isoform Ik-6 (5 of 17). Significantly, expression of Ikaros isoforms seemed normal in chronic phase CML patients and patients with other hematological malignancies. In some cases, overexpression of the dominant-negative Ik-6 protein was confirmed by Western blot analysis, and Southern blot analysis indicated that decreases in Ikaros activity correlated with a mutation in the Ikaros locus. In summary, these findings suggest that a reduction of Ikaros activity may be an important step in the development of blast crisis in CML and provide further evidence that mutations that alter Ikaros expression may contribute to human hematological malignancies.

Extramedullary T lymphoid blast crisis representing an additional translocation, t(6;8)(q25;q22) in a patient with Philadelphia-positive chronic myelogenous leukemia after allogeneic bone marrow transplantation.

A patient with extramedullary crisis from chronic myelogenous leukemia after allogeneic bone marrow transplantation is reported. A pathological neck lymph node observed after transplantation revealed pre-T lymphoblastic phenotype, and the fluorescence in situ hybridization (FISH) analysis showed recipient type sex chromosomes and bcr/abl fusion gene. The cells represented an additional translocation, t(6;8)(q25;q22). No rearrangements of the T-cell receptor (TCR) beta, gamma or delta chain genes were observed. The absence of TCR rearrangement indicated the clonogenic involvement of pluripotent hematopoietic stem cells by Philadelphia chromosome. Bone marrow specimens at that time showed donor type sex chromosomes and no bcr/abl-positive cells by FISH.

[Three cases of malignant lymphoma accompanied by renal cell carcinoma].

Three cases of malignant lymphoma (ML) accompanied by renal cell carcinoma (RCC) are reported. From September 1997 through August 2000, we treated 85 patients with ML. Among these patients, three had accompanying RCC (clear cell type): case 1, a 57-yr-old man with gamma/delta-T cell lymphoma; case 2, a 25-yr-old man with Grade 3 follicular lymphoma; case 3, a 64-yr-old man with MALToma of the right orbit. Renal cell carcinoma is a relatively rare disease, but several reports have indicated that, for some reason, the incidence of concurrent RCC and ML is higher than expected. It is possible that the two malignancies share some common background factors, such as genetic mutation, immunological abnormality, or an immunomodulatory effect of the first tumor. The patient in case 2 was thought to have an abnormal immunological background from his medical history, which included bronchial asthma, idiopathic thrombocytopenic purpura, and mesangial proliferative glomerulonephritis (non-IgA type). Therefore the combination of ML and RCC in this patient may have been due to immunological impairment.

[Clinical application of reticulated platelet analysis using the matic-modified thiazole orange method and flow cytometry].

Reticulated platelet (RP) analysis using thiazole orange (TO) and a flow cytometer is a convenient and promising method for estimating platelet kinetics in patients with thrombocytopenia. Among many different modifications of the TO method, a novel protocol reported by Matic in 1998 seems to be superior because it allows RP analysis using a sample of whole blood very quickly and accurately. Therefore, we used this method to analyze platelet kinetics in patients with idiopathic thrombocytopenic purpura (ITP) and hemophagocytic syndrome (HPS), as well as patients with hematopoietic malignancies (HM) after intensive chemotherapy. The proportion of RP was increased in the patients with ITP and HPS, and furthermore showed a negative linear correlation with the platelet count. The change in the proportion of RP occurred about one week before the change in the circulating platelet count in the patients with HM after chemotherapy. This modification of the TO method by Matic is expected to become a standard protocol for RP analysis.

Cutaneous lymphocyte antigen-positive T cells may predict the development of acute GVHD: alterations and differences of CLA+ T- and NK-cell fractions.

Acute GVHD (aGVHD) is a serious complication after allogeneic SCT (allo-SCT). However, an adequate immunological index is not yet available for assessing its severity. We analyzed the fraction of cutaneous lymphocyte antigen (CLA)+ cells in peripheral blood T and natural killer (NK) cells in 33 patients and evaluated its association with aGVHD. The CLA+ T-cell fraction often increased 3-7 days before the onset of aGVHD, and the maximum percentage of CLA+ T cells in grades II-IV aGVHD cases was significantly higher than that in grade 0 or I aGVHD (P<0.01). When the cutoff value of the maximum CLA+ T-cell percentage was set at 20%, any higher percentage was a significant risk for the development of severe aGVHD (P<0.0001). The maximum CLA+ T-cell percentage was significantly correlated with a high body temperature, low percutaneous oxygen saturation, and fibrinogen/fibrin degradation product D-dimer level. The post-allo-SCT CLA+ T cells exhibited a high ability to produce IL-2 and IFN-gamma, and may be the effectors and immunological markers for aGVHD. The CLA+ NK-cell-fraction steadily increased 2-4 weeks after allo-SCT but was not influenced by aGVHD. The CLA+ T-cell percentage may predict the development of severe aGVHD in clinical settings.

Proliferation, migration and platelet release by megakaryocytes in long-term bone marrow culture in collagen gel.

The proliferation of megakaryocytes, their migration, and their platelet release processes were observed in long-term bone marrow culture in collagen gel. Megakaryocytes proliferated for more than 6 months, not only with myeloid cells but also with sinusoid-like capillaries. The megakaryocyte count decreased at 2 weeks of culture, increased to more than 400 at 6 weeks, and then decreased to about 100. Megakaryocyte colonies appeared after 2 weeks of culture; the number increased to more than 10 at 4 weeks of culture, and was maintained at that level. Morphologically, most fully mature megakaryocytes extended several long antennae-like processes, with periodic constrictions, demarcation membranes (DM), and platelet fields. Pro-platelets were released from these processes primarily by a "pinching off" mechanism, but some megakaryocytes released pro-platelets by the dissociation of DM, without extending long processes. Mature megakaryocytes migrated like ameba in collagen gel, and a few migrated to the abluminal side of the capillary or into the capillary; in both cases releasing pro-platelets into the capillary lumina. These observations were very similar to those noted in vivo.

Long-term maintenance of multilineal hemopoiesis in collagen gel culture.

We examined the long-term maintenance of multilineal hemopoiesis in a collagen gel culture of mouse bone marrow cells. When cells were inoculated into the gel, stromal cells formed foci that were composed of sinusoidlike capillary structures, fibroblastic cells, adipocytes and macrophages. Many small hemopoietic foci similar to granulocyte-macrophage colonies (CFU-GM) appeared within a week and disappeared after two weeks. Several large hemopoietic foci appeared after two to three weeks of culture, without a second challenge of marrow cells. These large hemopoietic foci were composed mainly of myeloid cells. Megakaryocytes and mast cells were also observed. When erythropoietin (EPO) was added to the culture at the beginning, the erythroid focus appeared after 3 weeks and the number of megakaryocytes was greater than that in the culture without EPO. However, when EPO was added to the cultures after 6 or 12 weeks, erythroid cells appeared after 1 week and the number of megakaryocytes increased. This hemopoiesis lasted more than 6 months.

Murine spleen stromal cell line SPY3-2 maintains long-term hematopoiesis in vitro.

The hematopoietic microenvironment (HIM) of mouse spleen predominantly induces the differentiation of hematopoietic progenitors into erythroid lineage in vivo. However, the mechanisms of this phenomenon have not been fully explored because of the lack of an adequate in vitro system mimicking the spleen hematopoiesis. To reconstruct the HIM of mouse spleen in vitro, we established spleen stromal cell lines from a three-dimensional (3D) spleen primary culture in collagen gel matrix. Of these, SPY3-2 cells were negative for preadipocytic and endothelial markers, had a fibroblastoid morphology, and were not converted to adipocytes in the presence of 1 mumol/L hydrocortisone. They supported the maintenance and multilineal differentiation of hematopoietic progenitor cells for more than 8 weeks in vitro. The differentiated hematopoietic cells in the coculture medium were predominantly monocytes rather than granulocytes. Furthermore, erythropoiesis was predominantly induced in the presence of 2 U/mL erythropoietin and continued for more than 12 weeks. The number of burst-forming units-erythroid (BFU-E) was increased 10 times after 3 weeks of coculture, which was followed by pronounced production of erythroid cells in the coculture after week 4. SPY3-2 expressed high levels of c-kit ligand and low levels of granulocyte macrophage colony-stimulating factor and interleukin-3, and these molecules were all involved in this long-term erythropoiesis. Thus, the clonal SPY3-2 cell line will provide a novel HIM in vitro analogous to that of mouse spleen in vivo. These results suggest that 3D collagen gel culture may facilitate the establishment of functioning stromal cell lines of hematopoietic organ.

In vitro induction of apoptosis for nasal angiocentric natural killer cell lymphoma-derived cell line, NK-YS, by etoposide and cyclosporine A.

Epstein-Barr virus (EBV)-associated nasal T/natural killer (NK) cell lymphoma has often been reported in Asian countries and has been recently confirmed as a novel clinicopathological entity. The prognosis of advanced stage disease is quite poor and an effective chemotherapeutic modality is strongly advocated. We have established the novel cell line NK-YS, which preserves the original characteristics of EBV-associated nasal angiocentric T/NK cell lymphoma. Using this cell line, we investigated the induction of apoptosis by apoptosis-inducing agents, and expression of P-glycoprotein (P-gp), p53 and bcl-2 proteins. NK-YS showed resistance towards apoptosis-inducing agents and expressed bcl-2 and P-gp but not p53. To overcome this drug resistance, we added cyclosporine A (CsA) and these agents to culture media as a P-gp antagonist. The combination of CsA and etoposide or CsA and doxorubicin induced apoptotic cell death. These results indicated that P-gp-mediated drug resistance is an essential mechanism of drug resistance of the NK-YS cell line. Combined therapy of conventional anti-cancer agents with CsA may have an important place in the establishment of a curative therapy for disseminated nasal angiocentric NK cell lymphoma.

Antisense oligodeoxynucleotides to latent membrane protein 1 induce growth inhibition, apoptosis and Bcl-2 suppression in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells, but not in EBV-positive natural killer cell lymphoma cells.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) is essential for immortalization of B cells by EBV, protects the infected cells from apoptotic cell death and induces Bcl-2 expression. Suppression of LMP-1 expression by antisense oligodeoxynucleotides (AS-oligo) to LMP-1 inhibits proliferation, promotes apoptosis and suppresses Bcl-2 expression in EBV-transformed B cells. However, the function of LMP-1 expression in EBV-positive natural killer (NK) cell lymphoma cells has not been reported previously. We examined the function of LMP-1 in two EBV-positive NK cell lymphoma cell lines (NK-YS and YT) through suppressing LMP-1 expression by AS-oligo to LMP-1. The AS-oligo to LMP-1 suppressed LMP-1 mRNA and protein expression in two EBV-positive NK cell lymphoma cell lines, as well as in an EBV-transformed B-cell line (CMG-1). Proliferation was inhibited, apoptosis was induced and Bcl-2 expression was suppressed in CMG-1 cells, but none of these events were observed in NK-YS or YT cells. These results suggest that proliferation, inhibition of apoptosis and Bcl-2 expression in EBV-positive NK cell lymphoma cells are not directly regulated by LMP-1 as in EBV-transformed B-cell lines, but are probably mediated through other signal transducing systems.

Lymphomatous polyp of mantle cell type in the duodenum complicated by gastric cancer: a case of trisomy 3 and t(11;14)(q13;q32).

We experienced a rare case of a lymphomatous polyp of mantle cell type forming a polypoid mass lesion in the duodenum bulbous together with advanced gastric cancer. A total gastrectomy was performed, and the specimen revealed atypical small- to medium-sized lymphoid cells with indented nuclei, which infiltrated the Peyer's patch and formed a nodular mass in the lamina propria and submucosa of the duodenum. The lymphoma cells also infiltrated the lymphoid follicle of the gastric mucosa, spleen, and regional lymph node with a typical mantle zone pattern. Flow cytometric analysis of the single cells of the lymph node and immunohistochemistry of a paraffin-embedded specimen revealed that the lymphoma cells expressed surface CD5, CD19, CD20, and nuclear cyclin D1. Chromosomal analysis of this single cell suspension revealed that these lymphoma cells have trisomy 3 in conjunction with t(11;14)(q13;q32), which is frequently seen in mucosa-associated lymphoid tissue lymphomas (MALToma) in the stomach and is also reported in mantle cell lymphoma as a secondary genetic alteration. Our report suggests that trisomy 3 may be a common chromosomal abnormality in lymphomatous polyps of mantle cell type.

Recurrent idiopathic iridocyclitis after autologous peripheral blood stem-cell transplantation followed by G-CSF administration for acute lymphoblastic leukemia.

We describe a patient who experienced a recurrence of idiopathic iridocyclitis on day 12 after autologous peripheral blood stem-cell transplantation (auto-PBSCT) followed by G-CSF administration for acute lymphoblastic leukemia (ALL). Autologous SCT has been reported to be effective and safe in achieving dose intensification of chemotherapeutic drugs for the treatment of hematopoietic malignancies, but its therapeutic effect on autoimmune diseases is not definite. The findings from the present case suggest that auto-PBSCT followed by G-CSF administration for patients with a history of some kind of autoimmune disorders may induce exacerbation or recurrence of its symptoms after hematopoietic recovery.

Cutaneous lymphocyte antigen is expressed on memory/effector B cells in the peripheral blood and monocytoid B cells in the lymphoid tissues.

Cutaneous lymphocyte antigen (CLA) is expressed on a subpopulation of human memory T cells and is involved in the primary step of their skin homing. T cells and some B cells in the peripheral blood express CLA, but the pathophysiologic roles of CLA(+) B cells have not yet been clarified. We examined the relationships among CLA expression in B cells and immunoglobulin heavy chain subtype, the localization of CLA(+) B cells in the peripheral lymphoid tissues, and their functional binding to E-selectin. CLA was expressed on class-switched, memory B cells in the peripheral blood and tonsils as revealed by flow cytometry. Immunohistochemical staining of the lymph nodes with various types of inflammation or reactive hyperplasia showed CLA on the monocytoid B cells, which correspond to memory cells. The functional study revealed that CLA on B cells bound to E-selectin transfectants. E-selectin was detected on some of the high endothelial venules in the monocytoid B-cell-rich lymph nodes. These findings suggest that CLA is also expressed on a subset of memory/effector B cells, in addition to a subset of memory T cells. Such B cells were located in the lymph nodes or tonsils and rarely in chronic dermatitis. Therefore, CLA seems to be related to memory/effector B-cell trafficking to the lymph nodes or tonsils. According to the multistep theory, mechanisms involved in the second or third step might be different between CLA(+) B and T cells.

Common clonal origin of lymphocytes and plasma cells in splenic lymphoma with villous lymphocytes.

In two-thirds of patients with splenic lymphoma with villous lymphocytes (SLVL) a small amount of M-protein can be detected in association with the presence of plasma cells in the peripheral blood (PB) and/or bone marrow (BM). However, it is not known whether lymphoma cells and plasma cells originate from the same clone. In this report we describe a case of SLVL which was characterized by the presence of marked monoclonal gammopathy (IgG-kappa 90 g/l) and increased plasma cells in the BM. In an attempt to elucidate the origin of lymphoma cells and plasma cells, we performed morphological, cytogenetic and molecular studies on PB mononuclear cells (PBMNC) without plasma cells and BMMNC containing 10% plasma cells from this patient. Immunofluorescence showed that lymphoma cells and plasma cells were positive for cytoplasmic gamma heavy and kappa light chains. Well-developed endoplasmic reticulum was observed in the cytoplasmic organelles of PBMNC using an electron microscope. The mean IgG concentration in the 3 d supernatant cultures of PBMNC was 374 +/- 24 microg/l. More than 50% PBMNC differentiated into plasmacytoid cells in 6 d of liquid culture with IL-3 and IL-6. Analysis by two-colour FISH revealed that karyotypic abnormalities of monosomy X and trisomy 17 existed simultaneously in both lymphoma cells and plasma cells. JH gene rearranged bands from PBMNC and BMMNC by Southern blot hybridization were identical, whereas DNAs from PBMNC failed to hybridize with the Cmu probe. These observations strongly suggest that lymphoma cells and plasma cells originate from the same clone, and that plasma cells, as well as lymphoma cells, which have undergone class switch recombination, could produce IgG type M-protein in this case.

High-dose chemotherapy and autologous peripheral blood stem cell transplantation for treatment of unspecified peripheral T-cell lymphoma presented with hepatosplenomegaly and hypercytokinemia syndrome: report of three cases.

We report here three cases of peripheral T-cell lymphoma unspecified (PTCL-US), which presented with bone marrow infiltration and hepatosplenomegaly and were successfully treated with high-dose chemotherapy (HDCT) and autologous peripheral blood stem cell transplantation (auto-PBSCT). The patients were all characterized by cytokine-induced symptoms such as fever, anasarca, cytopenia, poor general condition, and disseminated intravascular coagulation syndrome. Laboratory data showed extremely high levels of soluble interleukin-2 receptor, beta(2)-microglobulin, and ferritin. All three patients were negative for anti-adult T-cell leukemia antibody. In one patient, hemophagocytosis was revealed by a histological examination of the bone marrow. The International Prognostic Index was high for all three patients, and they all achieved complete remission after the intensive chemotherapy for remission induction. During complete remission, they were treated with HDCT [modified interleukin-converting enzyme regimen] followed by auto-PBSCT. The recovery of hematopoiesis after auto-PBSCT was prompt and sustained engraftment was obtained. No serious adverse effects other than myelosuppression were noted. One patient died due to cerebrovascular disease without relapse 18 months after auto-PBSCT. The other two patients are still alive and have not suffered from relapse. Our observations suggest that auto-PBSCT following HDCT may be an effective and safe therapeutic modality for high-risk PTCL-US patients characterized by hepatosplenomegaly and cytokine-induced syndrome.

Reduction of hematopoietic cell-specific tyrosine phosphatase SHP-1 gene expression in natural killer cell lymphoma and various types of lymphomas/leukemias : combination analysis with cDNA expression array and tissue microarray.

To investigate the lymphomagenesis of NK/T lymphoma, we comprehensively and systematically analyzed the expression pattern of the human NK/T cell line (NK-YS) genome by cDNA expression array and tissue microarray. We detected significant changes in the gene expression of NK-YS cell line: an increase in 18 and a decrease in 20 genes compared to normal NK cells or peripheral blood mononuclear cells. Among these genes, we found a strong decrease in hematopoietic cell specific protein-tyrosine-phosphatase SH-PTP1 (SHP1) mRNA by cDNA expression array and reverse transcriptase-polymerase chain reaction. Further analysis with standard immunohistochemistry and tissue microarray, which used 207 paraffin-embedded specimens of various kinds of malignant lymphomas, showed that 100% of NK/T lymphoma specimens and more than 95% of various types of malignant lymphoma were negative for SHP1 protein expression. On the other hand, SHP1 protein was strongly expressed in the mantle zone and interfollicular zone lymphocytes in reactive lymphoid hyperplasia specimens. In addition, various kinds of hematopoietic cell lines, particularly the highly aggressive lymphoma/leukemia lines, lacked SHP1 expression in vitro, suggesting that loss of SHP1 expression may be related to not only malignant transformation, but also tumor cell aggressiveness. SHP1 expression could not be induced in either of two NK/T cell lines by phorbol ester, suggesting that genetic impairment or modification with methylation of SHP1 DNA could be one of the critical events in the pathogenesis of NK/T lymphoma. This evidence strongly suggests that loss of SHP1 gene expression plays an important role in multistep tumorigenesis, possibly as an anti-oncogene in the wide range of lymphomas/leukemias as well as NK/T lymphomas.

Characterization of a novel human natural killer-cell line (NK-YS) established from natural killer cell lymphoma/leukemia associated with Epstein-Barr virus infection.

A novel cell line was established from a patient with a leukemic-state nasal angiocentric natural killer (NK) cell lymphoma with systemic skin infiltration. The morphology of the leukemic cells was large-granular-lymphocyte (LGL), and their immunophenotype was CD2+, CD3-, CD5+, CD7+, CD16-, CD56+, and CD57-. The presence of Epstein-Barr viral (EBV) genome was shown in specimens from the patient's nose, skin, and peripheral blood by in situ hybridization using an EBV-encoded small RNA-1 probe or by Southern blotting using a terminal-repeat probe of the EBV genome. Leukemic cells were cocultured with a mouse stromal cell line (SPY3-2) in the presence of 100 U/mL recombinant human interleukin-2 and a novel stromal cell-independent cell line, NK-YS, was established. The NK-YS cells showed LGL morphology and expressed surface CD2, CD5, CD7, CD25, CD56, and CD95. The NK-YS cells retained cytotoxicity against K562 and Jurkat cells. A Southern blotting using a terminal-repeat probe of EBV showed that NK-YS and fresh leukemic cells had a clonal EBV genome, whereas the T-cell receptor beta and gamma chain genes of NK-YS were not rearranged. In an immunocytochemical analysis, the NK-YS cells showed a type-II latent infection of EBV. The NK-YS cells preserved the original characteristics of NK cell lymphoma/leukemia and will be a useful tool for the study of biological characteristics of EBV-associated nasal angiocentric NK cell lymphoma/leukemia.