大嶺 謙

To clarify the clinical features of adult patients with acute leukemia (AL) with 11q23 abnormalities, we performed a retrospective analysis of data from 58 adult Japanese patients: 51 with acute myeloid leukemia (AML), and 7 with acute lymphoblastic leukemia (ALL). The incidences according to fusion partners in AML were: t(9;11), 31.3%; t(11;19), 27.4%; t(6;11), 21.5%. The incidence of patients with t(11;19) was higher than those in the US and Europe, and the incidence of t(4;11) was lower than that in childhood. The results indicated the poor prognosis of AML with 11q23 abnormalities regardless of the fusion partners. AML patients with 11q23 aged < 60 years in the first CR who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) showed a more favorable outcome than those treated without allo-HSCT, although the differences were not statistically significant (P = 0.322 for DFS, P = 0.138 for OS). This result suggests that treatment strategies including allo-HSCT may be considered in the first CR in cases of AML with 11q23 abnormalities. However, further studies involving a large number of cases are required to assess the effect of allo-HSCT on adult AL with 11q23 abnormalities.

We evaluated the long-term outcome of very dose-intensive chemotherapy (TCC-NHL-91) for advanced intermediate-grade lymphoma, in which an eight-cycle regimen with 11 drugs was given with granulocyte colony-stimulating factor (G-CSF) support (total 18 weeks). Fifty-nine patients were treated during February 1, 1991 and March 31, 2001 (median age: 48 years). Forty-three patients (73%) were in a high-intermediate risk or high-risk group (HI/H) according to the age-adjusted International Prognostic Index (aa-IPI). Forty-six patients received 7 or 8 cycles of therapy. Ten of 15 patients over age 60 stopped before 7 cycles. Forty-three patients with an initial bulky mass or a residual mass received involved-field radiation. Overall, 56 patients (95%) achieved complete remission (CR). Grade 4 hematotoxicity was observed in all patients. With a median follow-up of 128 months, the 10-year overall survival (OS) and progression-free survival (PFS) rates were 76% and 61%, respectively. Neither aa-IPI risk factors nor the index itself was associated with response, OS, or PFS. One patient died of sepsis during the therapy and one died of secondary leukemia. This retrospective study suggests that the TCC-NHL-91 regimen achieves high CR, OS, and PFS in patients with advanced intermediate-grade lymphoma up to 60 years old and may be a valuable asset in the management of this disease. Further evaluation and prospective studies of the TCC-NHL-91 are warranted.

Objective. Results of previous studies have suggested that tipifarnib (Zarnestra), a farnesyltransferase inhibitor, is useful for treating various hematological disorders, including chronic myeloid leukemia. However, acquisition of resistance may be a problem for patients being treated with tipifarnib. Methods. We generated a tipifarnib-resistant BCR/ABL-positive cell line, K562/RR, and examined its characteristics. Results. While levels of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and cleaved poly (ADP-ribose) polymerase were significantly increased in K562 cells, the levels were not changed in K562/RR cells with tipifarnib treatment, indicating that induction of apoptosis signaling mediated by tipifarnib is much less in K562/RR cells than in K562 cells. In addition, tipifarnib-mediated induction of cell-cycle blockage was abrogated in K562/RR cells. No mutation of farnesyltransferase alpha and beta genes was found and the level of unprocessed HDJ-2, which is a substrate of farnesyltransferase, was increased by tipifarnib treatment in K562/ RR cells, suggesting that tipifarnib inhibits protein farnesylation in K562/RR cells in the same manner as in K562 cells and that mechanisms independent of farnesyltransferase activity are involved in the acquisition of resistance to tipifarnib in these cells. By DNA microarray analyses using a cDNA microarray comprising 25,000 genes, we identified 5 genes with higher expression levels in K562/RR cells than in K562 cells. These genes include P-globin, calcium channel Caveolin 2, and FEN1, which is involved in DNA replication and repair, and CUGBP2, which may affect expression of cyclooxygenase 2. C onclusion. The results of this study provide useful information for clarification of the mechanisms of resistance to tipifarnib. (c) 2007 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

A 30-year-old man with acute myeloid leukemia who was pancytopenic after undergoing intensive chemotherapy developed pyrexia and severe pain of both lower legs. We immediately started empiric therapy with cefepime, vancomycin, and fluconazole for febrile neutropenia. However, symptoms progressed. After 4 days, Trichosporon was isolated from venous blood cultures. MRI showed hyperintense lesions within both gastrocnemius muscles and demonstrated reactive vasodilatation and interstitial tissue edema, thought to be induced by hyperpermeability of vessel membranes due to the local fungal infection. Amphotericin B was very effective against this organism. Trichosporosis is a rare infectious disease generally occurring in immunocompromized hosts. To the best of our knowledge, this is first reported case of bilateral Trichosporon infection of lower leg muscles. Severe leg pain was one of the most important signs of fungal infection in this patient with hematologic malignancy. © 2005 The British Infection Society.

Frequency and clinical significance of cerebrospinal fluid (CSF) pleocytosis in hemopoietic stem cell (HSC) transplantation were surveyed. Cyclosporine (CSA)- or tacrolimus (FK506)-based regimens were used as graft-vs-host disease (GVHD) prophylaxis in allogeneic HSC transplantation. CSF pleocytosis with or without neurologic symptoms was detected in 12 of 25 patients receiving allogeneic HSC transplants but in none of 11 patients receiving autologous HSC transplants. Of the 12 patients with CSF pleocytosis, only one patient developed leukoencephalopathy later. There was a correlation between CSF cell numbers and trough levels of CSA but not with those of FK506. In patients receiving allogeneic HSC transplants, CSF pleocytosis may be relatively common and may reflect neurologic damage associated with calcineurin inhibitors.

The combination of imatinib and a famesyltransferase inhibitor might be effective for reducing the number of BCR/ABL-positive leukemia cells. In this study, we examined the differences in the mechanisms of the growth inhibitory effect of the combination of imatinib and R115777 (Zarriestra (TM)) among BCR/ABL-positive cell lines. Steel and Peckham isobologram analysis indicated that this combination had a strong synergistic inhibitory effect on growth in all imatinib-resistant cell lines and their parental cell lines. Levels of cleaved caspase 3 were increased by the combination treatment in all cell lines. However, both the level of cleaved PARP and the number of annexin-V-positive cells were much less increased in KCL22 and KCL22/SR cells than in K562, KU812, K562/SR and KU812/SR cells. The combination treatment promoted p27(KIP1) accumulation and induced a significant increase in the percentage of G0/G1 KCL22 and KCL22/SR cells. In other cell lines, the percentage of G0/G1 cells was not increased but rather decreased. The results indicate that induction of apoptosis and blockage of the cell cycle were major mechanisms of the synergistic inhibitory effect of the combination treatment. but the relative importance of these mechanisms differed among cell types. Additional treatment for overriding the G1 checkpoint may be required to eradicate leukemia cells, in which the combination induces cell cycle arrest. (c) 2005 Elsevier Inc. All rights reserved.

Objective. Imatinib, a BCR/ABL tyrosine kinase inhibitor, has shown remarkable clinical effects in chronic myelogenous leukemia. However, the leukemia cells become resistant to this drug in most blast crisis cases. The transcription factor Nrf2 regulates the gene expression of a number of detoxifying enzymes such as gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in glutathione (GSH) synthesis, via the antioxidant response element (ARE). In this study, we examined the involvement of Nrf2 in the acquisition of resistance to imatinib. Since oxidative stress promotes the translocation of Nrf2 from the cytoplasm to the nucleus, we also examined whether ascorbic acid, a reducing reagent, can overcome the resistance to imatinib by inhibiting Nrf2 activity. Results. Binding of Nrf2 to the ARE of the gamma-GCS light subunit (gamma-GCSI) gene promoter was much stronger in the imatinib-resistant cell line KCL22/SR than in the parental imatinib-sensitive cell line KCL22. The levels of gamma-GCSI mRNA and GSH were higher in KCL22/SR cells, a finding consistent with the observation of an increase in Nrf2-DNA binding. Addition of a GSH monoester to KCL22 cells resulted in an increase in the IC50 value of imatinib. In contrast, addition of ascorbic acid to KCL22/SR cells resulted in a decrease in Nrf2-DNA binding and decreases in levels of T-GCSI mRNA and GSH. Consistent with these findings, ascorbic acid partly restored imatinib sensitivity to KCL22/SR. Conclusion. Changes in the redox state caused by antioxidants such as ascorbic acid can overcome resistance to imatinib via inhibition of Nrf2-mediated gene expression. (C) 2004 International Society for Experimental Hematology. Published by Elsevier Inc.

Hydroxyurea (HU), an inhibitor of DNA synthesis, can also induce haemoglobinization in certain erythroid cell lines. In this study, we report that intracellular peroxides levels were increased in HU-treated murine erythroleukaemia (MEL) cells and that l-acetyl-N-cysteine (LNAC), a potent reducing reagent, had a significant inhibitory effect on the HU-mediated induction of beta-globin, delta-aminolaevulinate synthase mRNA expression and haemoglobinization of MEL cells. In contrast, the addition of LNAC to dimethyl sulphoxide (DMSO)-treated MEL cells had a much smaller effect on the number of haemoglobinized cells. These findings suggest that oxidative stress is involved in HU-mediated induction of erythroid differentiation and that HU induces MEL cell differentiation by a mechanism different to that involved in DMSO-mediated differentiation. Our findings also suggest that the induction of MEL cell differentiation by HU does not involve RAS-MAP (mitogen-activated protein) kinase signalling.

Pancreatic ductal carcinoma (PDC) is one of the most intractable human malignancies. Surgical resection of PDC at curable stages is hampered by a lack of sensitive and reliable detection methods. Given that DNA microarray analysis allows the expression of thousands of genes to be monitored simultaneously, it offers a potentially suitable approach to the identification of molecular markers for the clinical diagnosis of PDC. However, a simple comparison between the transcriptomes of normal and cancerous pancreatic tissue is likely to yield misleading pseudopositive data that reflect mainly the different cellular compositions of the specimens. Indeed, a microarray comparison of normal and cancerous tissue identified the INSULIN gene as one of the genes whose expression was most specific to normal tissue. To eliminate such a "population-shift" effect, the pancreatic ductal epithelial cells were purified by MUC1-based affinity chromatography from pancreatic juice isolated from both healthy individuals and PDC patients. Analysis of these background-matched samples with DNA microarrays representing 3456 human genes resulted in the identification of candidate genes for PDC-specific markers, including those for AC133 and carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7). Specific expression of these genes in the ductal cells of the patients with PDC was confirmed by quantitative real-time polymerase chain reaction analysis. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC that relies on pancreatic juice, which is routinely obtained in the clinical setting.

Lymphoproliferative disease of granular lympho- cytes (LDGL) is characterized by the clonal proliferationoflarge granular lymphocytes of either T- or natural killer cell origin. To better understand the nature of T cell-type LDGL, we purified the CD4(-) CD8(+) proliferative fractions from LDGL patients (n =4) and the surface marker-matched T cells isolated from healthy volunteers (n =4), and compared the expression profiles of 3456 genes using DNA microarray. Through this analysis, we identified a total of six genes whose expression was active in the LDGL T cells, but silent in the normal ones. Interestingly, expression of the gene for interleukin (IL) 1beta was specific to LDGL T cells, which was further confirmed by the examination of the serum level of IL-1beta protein. Given its important role in inflammatory reactions, the disease-specific expression of IL-1beta may have a causative relationship with the LDGL- associated rheumatoidarthritis. Spectratyping analysis of the T-cell receptor repertoire also proved the monoclonal or oligoclonal natureof LDGL cells. These data have shown that microarray analysis with a purified T-cell subset is an efficient approach to investigate the pathological condition of Tcell-type LDGL.

The promoter and enhancer elements of the mouse erythropoietin (mEpo) gene, which have high homology with those of the human erythropoietin (hEpo) gene, were fused with luciferase. ne construct was transfected into erythropoietin-producing hepatoma cell line (Hep3B) cells by lipofectin with lacZ as an internal standard. The wild type (TGATA) showed a 39.5-fold increase in induction by hypoxia. Mouse GATA-2 inhibited the hypoxic induction of the wild-type (m3), promoter-luciferase construct but not the hypoxic induction of the mutant (m4, 5) promoter-luciferasc constructs. N-G-Monomethyl L-arginine (L-NMMA) inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by L-arginine. H2O2 also inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by catalase. Gel shift assays performed on nuclear extracts of 293 cells overexpressing mGATA-1, -2, and -3 revealed that mGATA-1, -2, and -3 bind to the TGATA element of the mEpo promoter. These results indicate that mGATA binds to the TGATA site of the mEpo promoter and negatively regulates mEpo gene expression. Negative regulation of mEpo gene by GATA transcriptional factors is discussed. Int J Hematol. 2002;75:376-381. (C)2002 The Japanese Society of Hematology.