Hideki Tsushima

Long-term efficacy of imatinib in a practical setting is correlated with imatinib trough concentration that is influenced by body size: a report by the Nagasaki CML Study Group

Imatinib has dramatically improved long-term survival of chronic myelogenous leukemia (CML) patients. To analyze its efficacy in a practical setting, we registered most of CML patients in Nagasaki Prefecture of Japan. Of these, 73 patients received imatinib as an initial therapy. The overall survival rate of these patients was 88.7% at 6 years, and the cumulative complete cytogenetic response rate was 82.5% at 18 months. These results are comparable with the data of other reports including the IRIS study; however, the administered imatinib dose was smaller in our study than that in other reports. To address these discrepancies, we measured the trough concentration of imatinib among 35 patients. Although 39% of the patients were administered less than 400 mg/day, the trough level was comparable to those of previous reports. The trough level of imatinib showed a significant relationship with its efficacy, and was clearly related to dose of imatinib administrated and dose of imatinib divided by body surface area (BSA). Considering the smaller BSA of Japanese patients as compared to those of foreign origin, the results suggest that a lower dose of imatinib could maintain enough trough level and provided excellent results for the treatment of CML in our registry.

Treatment of relapsed adult T-cell leukemia/lymphoma after allogeneic hematopoietic stem cell transplantation: the Nagasaki Transplant Group experience

Adult T-cell leukemia/lymphoma (ATL) relapse is a serious therapeutic challenge after allogeneic hematopoietic stem cell transplantation (allo-SCT). In the present study, we retrospectively analyzed 35 patients who experienced progression of or relapsed persistent ATL after a first allo-SCT at 3 institutions in Nagasaki prefecture (Japan) between 1997 and 2010. Twenty-nine patients were treated by the withdrawal of immune suppressants as the initial intervention, which resulted in complete remission (CR) in 2 patients. As the second intervention, 9 patients went on to receive a combination of donor lymphocyte infusion and cytoreductive therapy and CR was achieved in 4 patients. Of 6 patients who had already had their immune suppressants discontinued before the relapse, 3 patients with local recurrence received local cytoreductive therapy as the initial treatment, which resulted in CR for more than 19 months. Donor lymphocyte infusion-induced remissions of ATL were durable, with 3 cases of long-term remission of more than 3 years and, interestingly, the emergence or progression of chronic GVHD was observed in all of these cases. For all 35 patients, overall survival after relapse was 19.3% at 3 years. The results of the present study suggest that induction of a graft-versus-ATL effect may be crucial to obtaining durable remission for ATL patients with relapse or progression after allo-SCT.

Improvement of criteria for refractory cytopenia with multilineage dysplasia according to the WHO classification based on prognostic significance of morphological features in patients with refractory anemia according to the FAB classification.

In the criteria of refractory cytopenia with multilineage dysplasia (RCMD) according to the WHO (World Health Organization) classification, the frequency threshold concerning dysplasia of each lineage was defined as 10%. To predict overall survival (OS) and leukemia-free survival (LFS) for patients with refractory anemia (RA) according to the French-American-British (FAB) classification, we investigated prognostic factors based on the morphological features of 100 Japanese and 87 German FAB-RA patients, excluding 5q-syndrome. In the univariate analysis of all patients, pseudo-Pelger–Huet anomalies ⩾10% (Pelger+), micromegakaryocytes ⩾10% (mMgk+), dysgranulopoiesis (dys G) ⩾10% and dysmegakaryopoiesis (dys Mgk) ⩾40% were unfavorable prognostic factors for OS and LFS (OS; P<0.001, LFS; P<0.001). The prognostic effects of the morphological features were similar in both Japanese and German patients. However, dys Mgk ⩾10% was not correlated with OS and LFS. In the multivariate analysis, mMgk+ and dys Mgk⩾40% were adverse prognostic factors for OS for all patients, and dys G ⩾10% and dys Mgk⩾40% were adverse prognostic factors for LFS for all patients. On the basis of the present analysis, we propose the following modified morphological criteria for RCMD. Modified RCMD should be defined as FAB-RA, excluding 5q-syndrome with dys G ⩾10%, dys Mgk⩾40% or mMgk+.

Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2.

We have established an erythropoietin-dependent human leukemia cell line, AS-E2, from a patient with acute myeloid leukemia. These cells have many characteristics of late erythroid progenitor cells, they are positive for CD36, Glycophorin A, and CD71 but negative for CD41, and positive for benzidine and PAS staining. These cells express GATA-1 and have low affinity erythropoietin (EPO) receptor on their surface. Interestingly, AS-E2 cells are strictly dependent on EPO for their growth and survival; other cytokines including GM-CSF, stem cell factor, or IL-3 cannot support the growth of this cell line. These features are similar to late erythroid lineage cells, like normal BFU-E or CFU-E, and we have demonstrated that EPO stimulation induces the tyrosine phosphorylation of several proteins in AS-E2 cells including the EPO receptor and JAK2 kinase. This new cell line is a useful reagent to study biological and molecular events during the late stages of erythropoiesis, and to understand transforming events in human erythroid cells.

Identification of a Novel Cytokine Response Element in the Human IFN Regulatory Factor-1 Gene Promoter

The present study investigates the regulatory mechanisms involved in the cooperation between IFN-γ and TNF-α to promote transcription from IFN regulatory factor-1 (IRF-1). A transient transfection analysis revealed that the region between −218 and −144, where +1 is the transcription start site, as well as previously reported downstream elements, ppκB and IFN-γ activation site/κB, were required for the optimal response to the two cytokines. A subsequent DNase I footprint analysis showed that the region between −171 and −144 was inducibly protected with stimulation by TNF-α, and this protection was significantly enhanced with the combination of IFN-γ and TNF-α. In an EMSA with the protected region as a probe, a TNF-α-inducible complex (C1) and an IFN-γ-inducible complex (C2), but no synergy-specific DNA-protein complexes, were recognized. The C1 complex consisted of a pre-existing factor (p65/p50), whereas the C2 complex consisted of a newly synthesized IRF-1-related factor. A methylation interference assay revealed the critical G residues (from −167 to −151) for the DNA-protein complex formation specific to the cytokine response, and within this region the novel κB sequence, the promoter distal κB (pdκB) element (5′-GGGGAAGTAC-3′), was identified. Because the base substitutions over the pdκB region (from −171 to −144) affected not only the TNF-α-response but also that of IFN-γ, this region might contribute to the cooperative action of the NF-κB subunits with the IRF-1-related factor. Finally, we demonstrated that none of the cis-acting elements, ppκB, pdκB, or IFN-γ activation site/κB, is dispensable for the optimal synergism in response to IFN-γ and TNF-α.

Diagnostic criteria for hypocellular acute leukemia: A clinical entity distinct from overt acute leukemia and myelodysplastic syndrome

In order to establish diagnostic criteria for hypocellular acute leukemia (HL), we have reviewed 32 cases selected on the basis of hypothetical 40% or less cellularity, by focusing on morphology, immunophenotype, karyotype and response to low dose Ara-C (LDAC) regimen and compared them with 40 cases of myelodysplastic syndrome (MDS) and 66 cases of overt acute myeloid leukemia (AML). The onset age ranged from 44 to 75 years (median 67 years). Bone marrow (BM) cellularity ranged from 12.4 to 39.8% (mean 29.8%) in HL, being significantly lower than in MDS (mean 80.7%) or AML (mean 86.4%) (P < 0.001). All reviewed cases characteristically showed smoldering clinical course, bi- or pancytopenia with rare leukemic blasts in the peripheral blood (PB), proliferation of type I leukemic blasts in the BM and markedly reduced background hematopoietic cells with some dysplastic changes in 12/32 cases (37.50/6). Blast percentage (blast %) in the BM ranged from 38.2 to 93.7% (mean 57.3%) in all nucleated cells (ANC). Although a considerable number of cases had blasts with negative or very low myeloperoxidase activity, immunophenotyping revealed that the leukemic blasts in HL had only myeloid markers. Karyotyping revealed non-random chromosome abnormalities in 30% of cases analyzed, which were considerably different from those seen in MDS. With LDAC regimen, a significantly higher CR rate (13/20 cases: 65.0%) was gained in HL than in RAEB/RAEB-t (0%) and overt AML in the elderly cases (27.3%) (P < 0.05). In CR, most cases showed recovery to normocellular BM with an apparent normalization of PB parameters. However, 12 CR cases relapsed 4-12 months later; most of which again showed hypocellular BM. These results indicate that HL is a distinct subtype of AML characterized by slow but distinct proliferation of immature myeloid blasts and by unique hematological features distinct from MDS or overt AML in the elderly. We propose the following diagnostic criteria: (1) pancytopenia with rare appearance of blasts in PB; (2) less than 40% BM hypocellularity; (3) more than 30% blasts in BM-ANC; and (4) myeloid phenotypes of leukemic blasts by MPO staining and/or immunophenotyping.

Expression of myeloperoxidase enhances the chemosensitivity of leukemia cells through the generation of reactive oxygen species and the nitration of protein

Myeloperoxidase (MPO), a pivotal lineage marker for acute myeloid leukemia (AML), has been also shown to have a prognostic value: a high percentage of MPO-positive blasts correlates to favorable prognosis. To understand the relationship between the expression of MPO in leukemia cells and the response to chemotherapeutic agents, we established MPO-expressing K562 leukemia cell lines and then treated them with cytosine arabinocide (AraC). Cells expressing wild-type MPO, but not mutant MPO that could not mature, died earlier of apoptosis than control K562 cells. Reactive oxygen species (ROS) were generated more in leukemia cells expressing MPO, and the generation was abrogated by MPO inhibitors or antioxidants. Tyrosine nitration of cellular protein also increased more in MPO-expressing K562 cells than control cells after treatment with AraC. In clinical samples, CD34-positive AML cells from high-MPO cases showed a tendency to be sensitive to AraC in the colony-formation assay, and the generation of ROS and the nitration of protein were observed only when the percentage of MPO-expressing cells was high. These data suggest that MPO enhances the chemosensitivity of AML through the generation of ROS and the nitration of proteins.

Fas Antigen (CD95) in pure erythroid cell line AS-E2 is induced by interferon-γ and tumor necrosis factor-α and potentiates apoptotic death

Abstract We investigated the expression of Fas antigen (CD95) in the pure erythroid cell line AS-E2 in the presence and absence of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). TNF-α induced apoptosis in AS-E2 cells, whereas IFN-γ did not. In culture containing no IFN-γ or TNF-α, AS-E2 cells expressed little Fas antigen. However, IFN-γ and TNF-α both induced expression of Fas antigen and its mRNA within 24 hours after the stimulation. When anti-Fas monoclonal antibody (IgM) was added to AS-E2 cells after the induction of Fas expression, AS-E2 cells underwent apoptosis as shown by the induction of DNA fragmentation. This apoptotic change was inhibited by an inhibitor of caspase-3-like proteases (Ac-DEVD-CHO) and an inhibitor of CED-3/ICE family proteases (Z-Asp-CH 2 -DCB) but not by an inhibitor of caspase-1-like proteases (Ac-YVAD-CHO), suggesting a role for caspase-3-like proteases in Fas-receptor signaling. Although AS-E2 cells expressed Fas ligand mRNA, treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to suppress IFN-γ– or TNF-α–mediated cytotoxicity. These findings suggest that the late erythroid progenitor cells are negatively regulated by IFN-γ and TNF-α, both of which are capable of inducing functional Fas expression.

The level of MEF but not ELF-1 correlates with FAB subtype of acute myeloid leukemia and is low in good prognosis cases

ETS proteins (such as PU.1, Fli-1 and ETS-1) have been shown to play important roles in normal and abnormal hematopoiesis. We examined the expression of the ELF subfamily of ETS genes (ELF-1, MEF and NERF) in acute myeloid leukemia (AML) cells using Northern blot analysis. ELF-1 and MEF were expressed in all samples, whereas NERF was not. The relative expression (RE) of MEF, but not ELF-1, was significantly lower (P<0.0001) in AML with t(8;21) and t(15;17) compared with AML with normal karyotype. The pattern of MEF expression was not uniform among cells with CD34(+)/CD33(+). It is suggested that the low RE of MEF might be part of a gene expression profile characterizing AML with a good prognosis.

Adult T-cell lymphoma/leukemia with angioimmunoblastic T-cell lymphomalike features : Report of 11 cases

In adult T-cell lymphoma/leukemia (ATLL), the neoplastic lymphoid cells are usually medium-sized to large, often with pronounced nuclear pleomorphism compatible with the diagnosis of diffuse pleomorphic peripheral T-cell lymphoma. We describe here 11 patients with the rare morphologic variant of ATLL, angioimmunoblastic T-cell lymphoma (AILT)-like type. The examined lymph nodes showed proliferation of high endothelial venules and presence of various infiltrating inflammatory cells including plasma cells and eosinophils. The lymphoma cells were medium-to-large size with clear cytoplasm. These findings were suggestive of AILT. However, immunohistochemical features of AILT, namely, CD10 and CXCL13 expression in lymphoma cells and proliferation of CD21-positive follicular dendritic cells, were not detected. Two cases were CXCR3-positive, whereas 9 expressed CCR4, which are usually positive in ATLL. All patients were positive for antiadult T-cell leukemia/lymphoma–associated antigen, which is a specific antibody for human T-cell lymphotropic virus type-I. Southern blot analysis revealed proviral DNA integration in lymphoma cells in 9 patients. The latter was not evident in the first biopsy of 2 patients but in the second biopsy obtained within several months after the first biopsy revealed definite proviral integration. Almost all patients showed aggressive clinical course and poor survival (median survival: 5 mo). This is the first report of ATLL with AILT-like morphologic features.