Yoji Ishida

Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial

BACKGROUND First-line imatinib treatment can be successfully discontinued in patients with chronic myeloid leukaemia after deep molecular response has been sustained for at least 2 years. We investigated the safety and efficacy of discontinuing second-line or subsequent dasatinib after at least 1 year of deep molecular response. METHODS The Dasatinib Discontinuation trial was a prospective multicentre trial done in Japan. Eligible patients taking dasatinib and with confirmed stable deep molecular response were enrolled between April 1, 2011, and March 31, 2012. All patients received dasatinib consolidation therapy for at least 1 year. In those with sustained deep molecular response, dasatinib was discontinued. Patients were followed up every month in year 1 (clinical cutoff), every 3 months in year 2, and every 6 months in year 3 for deep molecular response and immunological profiles. The primary endpoint was the proportion of patients with treatment-free remission at 6 months after discontinuation. Molecular relapse was defined as loss of deep molecular response at any assessment. This study is registered, number UMIN000005130. FINDINGS 88 patients were enrolled in the consolidation phase, 24 were excluded from the discontinuation phase due to fluctuations in BCR-ABL1 transcript levels. One patient was excluded because of positive expression of major and minor BCR-ABL1 transcripts in chronic myeloid leukaemia cells and the detection of minor BCR-ABL1 transcripts during consolidation. Thus, 63 patients discontinued dasatinib treatment. The 25 patients who were excluded from discontinuation continued to receive dasatinib and none showed disease progression. Median follow-up was 20.0 months (IQR 16.5-24.0). Of the 63 patients who discontinued and were not excluded, 30 patients maintained deep molecular response while 33 patients had molecular relapses, all within the first 7 months after discontinuation. The estimated overall treatment-free remission was 49% (95% CI 36-61) at 6 months. No severe treatment-related toxic effects were seen. Treatment was restarted in the 33 patients with relapse; rapid molecular responses were seen in all 33 patients, of whom 29 (88%) regained deep molecular response within 3 months, as did the remaining four by 6 months. INTERPRETATION Dasatinib discontinuation after sustained deep molecular response for more than 1 year is feasible. FUNDING Epidemiological and Clinical Research Information Network (ECRIN).

Absence of pseudomembranes in Clostridium difficile-associated diarrhea in patients using immunosuppression agents.

Objective. Clostridium difficile is a major cause of diarrhea in hospitalized patients. Although pseudomembranes are crucial evidence for diagnosis of C. difficile-associated diarrhea (CDAD), some cases do not show any pseudomembranes. The aim of this study was to verify the hypothesis that pseudomembranes are not generated in immunosuppressed patients because of the absence of immunoreactions. Material and methods. We investigated the endoscopic findings of patients with ulcerative colitis (UC) or who had received hematopoietic stem cell transplantation, and who presented with C. difficile toxin A and had undergone colonoscopy between April 2002 and July 2007 at our institutes. Results. In 4 patients the diagnosis was UC and C. difficile infection, and in another 4 patients the diagnosis was CDAD after hematopoietic stem cell transplantation. None of these cases showed pseudomembranes. Shallow ulcers were found in all four cases with UC. Only non-specific findings were obtained for the CDAD patients after hematopoietic stem cell transplantation. Conclusions. Pseudomembranes, the typical evidence for CDAD, were not detected in any patients using immunosuppressive agents. Additional bacterial examination is therefore essential when UC becomes exacerbated and when patients present with diarrhea after hematopoietic stem cell transplantation, even in the absence of pseudomembranes.

Recombinant human c‐Mpl ligand is not a direct stimulator of proplatelet formation in mature human megakaryocytes

To evaluate the effect of the c‐Mpl ligand on platelet production by megakaryocytes, we investigated proplatelet formation in isolated human megakaryocytes cultured in serum‐free medium, with or without the c‐Mpl ligand, interleukin‐6 and erythropoietin. When interleukin‐6 was added to the culture medium, the percentage of megakaryocytes displaying proplatelets was approximately 1.5‐fold the control value; whereas, in the presence of the c‐Mpl ligand, the percentage of megakaryocytes displaying proplatelets decreased in a dose‐dependent manner and did not increase compared to control values at any dose tested. However, the viability of megakaryocytes after a 4 d culture in the presence of the c‐Mpl ligand was significantly higher than that of the cells cultured without it. The c‐Mpl ligand did not stimulate the proplatelet formation in megakaryocytes in vitro

Impact of human leucocyte antigen mismatch on graft-versus-host disease and graft failure after reduced intensity conditioning allogeneic haematopoietic stem cell transplantation from related donors.

The impact of human leucocyte antigen (HLA) incompatibility between donor and recipient on graft‐versus‐host disease (GVHD) and graft failure after reduced‐intensity conditioning stem cell transplantation (RICT) remains to be elucidated. We retrospectively analysed outcome in 341 patients who underwent RICT from related donors for haematological malignancies. The overall cumulative incidence of grade II–IV acute GVHD (aGVHD) was 40% for all subjects; 39% in recipients with HLA‐matched donors, 44% in those with one‐locus‐mismatched donors, and 50% in those with two‐ to three‐loci‐mismatched donors. In a Cox regression model adjusted for potential confounders, the tendency for grade II–IV aGVHD (P = 0·01), chronic GVHD (cGVHD) (P = 0·05) and graft failure (P = 0·033) increased with HLA disparity. Use of peripheral blood grafts instead of marrow was a risk factor for cGVHD. Use of antithymocyte globulin was associated with reduced aGVHD and cGVHD. Overall survival (OS) in recipients of two‐ to three‐loci‐mismatched RICT at 2 years (18%) was significantly worse than that in patients who received one‐locus‐mismatched RICT (51%) and HLA‐matched RICT (48%) (P < 0·0001). A two‐ to three‐loci mismatch was identified as an independent risk factor for OS (P < 0·001), but there was no significant difference in OS between HLA‐matched and one‐locus‐mismatched RICT. HLA incompatibility between the donor and recipient is an important risk factor for graft failure, aGVHD, cGVHD and OS after RICT. RICT from a one‐locus‐mismatched donor may represent an effective alternative approach in patients with high‐risk malignancies who lack HLA‐matched related donors.

Clinical and biological significance of CD56 antigen expression in acute promyelocytic leukemia.

The biological significance of CD56 antigen expression in patients with acute promyelocytic leukemia (APL) has been under investigation. We investigated the clinical and biologic features of CD56+APL. In our series, CD56 antigen was positive in 4 of 28 (14%) APL patients. No differences were found regarding age, gender, performance status (PS), initial leukocyte and platelet counts, lactate dehydrogenase (LDH) and fibrinogen (Fbg) levels according to CD56 expression. CD34 antigen was co-expressed in 3 of the 4 patients with CD56+ APL, in contrast to 2 of the 24 patients with CD56- APL (P = .01). Extramedullary relapse occurred in 3 of the 4 patients with CD56+ APL, in contrast to none of the 24 patients with CD56- APL (P = .001). Median remission duration was 4 months in CD56+ APL and was not reached in CD56- APL. The CD56+ population had a shorter remission duration (P < .0001) and disease-free survival (P < .0001). In contrast, no difference was found in overall survival. These results suggested that CD56 expression was associated with the leukemogenetic mutation at the primitive hematopoietic progenitor cell level and extramedullary relapse in APL patients treated with ATRA and chemotherapy.

Deguelin suppresses cell proliferation via the inhibition of survivin expression and STAT3 phosphorylation in HTLV-1-transformed T cells

Adult T-cell leukemia (ATL) is an aggressive malignancy of peripheral T cells infected with human T-cell leukemia virus type 1 (HTLV-1). The prognosis of aggressive ATL patients remains poor because of its resistance to conventional chemotherapy. We examined the effect of deguelin, a naturally occurring rotenoid, on HTLV-1-transformed T-cell lines, KUT-1 and MT-2 cells. We found that deguelin suppressed cell proliferation and induced cell death in these cells. Immunoblot analysis showed the inhibition of survivin expression and signal transducers, and activators of transcription (STAT) 3 phosphorylation of both cells. We also observed the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP) in deguelin-treated cells, indicating that deguelin induces caspase-dependent apoptosis in these cells. Furthermore, proteasome inhibitor MG132 prevented the down-regulation of survivin expression and STAT3 dephosphorylation by deguelin, suggesting that the action mechanism of deguelin involves the degradation of survivin and phosphorylated STAT3 through the ubiquitin/proteasome pathway. Our data indicate that deguelin presents a potent anti-proliferative effect in part via the down-regulation of survivin expression and STAT3 phosphorylation in HTLV-1-transformed cells. Deguelin merits further investigation as a potential chemotherapeutic agent for ATL.

Identification of S-phase cells with PC10 antibody to proliferating cell nuclear antigen (PCNA) by flow cytometric analysis.

We estimated the expression of proliferating cell nuclear antigen (PCNA) in HeLa S3 cells by flow cytometry with monoclonal antibody (MAb) PC10. HeLa cells were fixed with six different fixation procedures: 15-min and 30-min acetone, 15-min acetone followed by 15-min methanol (acetone/methanol), 30-min methanol, 15-min methanol followed by 15-min acetone (methanol/acetone), and a mixture of acetone and methanol. The fixed cells were applied to MAb PC10 against PCNA and then treated with FITC. With five fixation procedures except for acetone/methanol, PCNA was expressed in almost all cells with similar shapes and different FITC intensity levels on PCNA/DNA bivariate cytograms, whereas acetone/methanol fixation allowed PCNA detection in S-phase cells with a cytogram that showed a horseshoe-like pattern with a peak level at mid-S-phase. Flow cytometric dual parameter analysis of PCNA/BrdU was carried out in HeLa cells to confirm detection of PCNA in S-phase cells with acetone/methanol fixation. The population of cells stained for both parameters, i.e., S-phase cells, was obviously discriminated from that of the non-S-phase cell in PCNA/BrdU bivariate cytograms. These results strongly suggest that PCNA used with acetone/methanol fixation would be equal to BrdU as an S-phase marker.

Estimation of S-phase fraction in tumor tissue sections by immunohistochemical staining of PCNA.

We describe a method for measuring the size of the S-phase fraction in human tumor tissue sections using an antibody to PCNA (PC10). Although treatment with Triton X-100 before fixation extracted a large amount of PCNA from the cells even in frozen tissue sections, PCNA remained exclusively in S-phase cells. Immunohistochemical staining of PCNA after the detergent treatment allowed estimation of the S-phase fraction in solid tumors. The validity of the method was directly proven by double staining of bromodeoxyuridine (BrdU) and PCNA in HeLa cells. The PCNA-positive cells were identical with BrdUrd-positive cells after the detergent treatment. In contrast, almost all HeLa cells in the exponentially growing phase were positive for PC10 without treatment with Triton X-100.

Platelet demand modulates the type of intravascular protrusion of megakaryocytes in bone marrow

Megakaryocytes (MKs) generate platelets via intravascular protrusions termed proplatelets, which are tandem arrays of platelet-sized swellings with a beaded appearance. However, it remains unclear whether all intravascular protrusions in fact become proplatelets, and whether MKs generate platelets without forming proplatelets. Here, we visualised the sequential phases of intravascular MK protrusions and fragments in living mouse bone marrow (BM), using intravital microscopy, and examined their ultrastructure. The formation of intravascular protrusions was observed to be a highly dynamic process, in which the size and shape of the protrusions changed sequentially prior to the release of platelet progenitors. Among these intravascular protrusions, immature thick protrusions were distinguished from proplatelets by their size and the dynamic morphogenesis seen by time-lapse observation. In ultrastructural analyses, the thick protrusions and their fragments were characterised by a peripheral zone, abundant endoplasmic reticulum and demarcation membrane system, and random microtubule arrays. Proplatelets were predominant among BM sinusoids in the physiological state; however, during an acute thrombocytopenic period, thick protrusions increased markedly in the sinusoids. These results strongly suggested that BM MKs form and release two types of platelet progenitors via distinct intravascular protrusions, and that platelet demand modulates the type of intravascular protrusion that is formed in vivo.

A combination chemotherapy with low doses of cytarabine and etoposide for high risk myelodysplastic syndromes and their leukemic stage: A pilot study

Even now, no definitely effective therapy is inducted to high risk myelodysplastic syndromes (MDS) and their leukemic stage (MDS‐AML) except bone marrow transplantation.