Kyoko Haraguchi

Hes1 immortalizes committed progenitors and plays a role in blast crisis transition in chronic myelogenous leukemia

Hairy enhancer of split 1 (Hes1) is a basic helix-loop-helix transcriptional repressor that affects differentiation and often helps maintain cells in an immature state in various tissues. Here we show that retroviral expression of Hes1 immortalizes common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in the presence of interleukin-3, conferring permanent replating capability on these cells. Whereas these cells did not develop myeloproliferative neoplasms when intravenously administered to irradiated mice, the combination of Hes1 and BCR-ABL in CMPs and GMPs caused acute leukemia resembling blast crisis of chronic myelogenous leukemia (CML), resulting in rapid death of the recipient mice. On the other hand, BCR-ABL alone caused CML-like disease when expressed in c-Kit-positive, Sca-1-positive, and lineage-negative hematopoietic stem cells (KSLs), but not committed progenitors CMPs or GMPs, as previously reported. Leukemic cells derived from Hes1 and BCR-ABL-expressing CMPs and GMPs were more immature than those derived from BCR-ABL-expressing KSLs. Intriguingly, Hes1 was highly expressed in 8 of 20 patients with CML in blast crisis, but not in the chronic phase, and dominant negative Hes1 retarded the growth of some CML cell lines expressing Hes1. These results suggest that Hes1 is a key molecule in blast crisis transition in CML.

Host-Residual Invariant NK T Cells Attenuate Graft-versus-Host Immunity

Invariant NK T (iNKT) cells have an invariant TCR-α chain and are activated in a CD1d-restricted manner. They are thought to regulate immune responses and play important roles in autoimmunity, allergy, infection, and tumor immunity. They also appear to influence immunity after hemopoietic stem cell transplantation. In this study, we examined the role of iNKT cells in graft-vs-host disease (GVHD) and graft rejection in a mouse model of MHC-mismatched bone marrow transplantation, using materials including α-galactosylceramide, NKT cells expanded in vitro, and Jα18 knockout mice that lack iNKT cells. We found that host-residual iNKT cells constitute effector cells which play a crucial role in reducing the severity of GVHD, and that this reduction is associated with a delayed increase in serum Th2 cytokine levels. Interestingly, we also found that host-residual iNKT cause a delay in engraftment and, under certain conditions, graft rejection. These results indicate that host-residual iNKT cells attenuate graft-vs-host immunity rather than host-vs-graft immunity.

Notch Activation Induces the Generation of Functional NK Cells from Human Cord Blood CD34-Positive Cells Devoid of IL-15

The development of NK cells from hematopoietic stem cells is thought to be dependent on IL-15. In this study, we demonstrate that stimulation of human cord blood CD34+ cells by a Notch ligand, Delta4, along with IL-7, stem cell factor, and Fms-like tyrosine kinase 3 ligand, but no IL-15, in a stroma-free culture induced the generation of cells with characteristics of functional NK cells, including CD56 and CD161 Ag expression, IFN-γ secretion, and cytotoxic activity against K562 and Jurkat cells. Addition of γ-secretase inhibitor and anti-human Notch1 Ab to the culture medium almost completely blocked NK cell emergence. Addition of anti-human IL-15-neutralizing Ab did not affect NK cell development in these culture conditions. The presence of IL-15, however, augmented cytotoxicity and was required for a more mature NK cell phenotype. CD56+ cells generated by culture with IL-15, but without Notch stimulation, were negative for CD7 and cytoplasmic CD3, whereas CD56+ cells generated by culture with both Delta4 and IL-15 were CD7+ and cytoplasmic CD3+ from the beginning and therefore more similar to in vivo human NK cell progenitors. Together, these results suggest that Notch signaling is important for the physiologic development of NK cells at differentiation stages beyond those previously postulated.

Immunotherapy against metastatic renal cell carcinoma with mature dendritic cells

Objective:  We performed a clinical trial of immunotherapy using autologous mature dendritic cells (DC) pulsed with autologous tumor lysate, for patients with metastatic renal cell carcinoma (RCC).

CD1d expression level in tumor cells is an important determinant for anti-tumor immunity by natural killer T cells

Invariant natural killer T (iNKT) cells are thought to regulate anti-tumor immunity. Human iNKT (i.e. Vα24+ NKT) cells have been reported to recognize CD1d on target cells and show cytotoxicity directly on the target cells in vitro. However, the anti-tumor effect of mouse iNKT (i.e. Vα14+ NKT) cells has been repeatedly reported to be dependent on the activity of natural killer (NK) cells via interferon-γ, with no evidence of direct cytotoxicity. In the present study, we report that in vitro cytolysis of EL-4 mouse lymphoblastic lymphoma cells by Vα24+ NKT cells and in vivo eradication of these cells are both dependent on the level of CD1d expression on the tumor cell surface. These observations possibly suggest that direct cytotoxicity of tumor cells by iNKT cells is common to both humans and mice, and that the high expression level of CD1d may be a predictor whether the tumor is a good target of iNKT cells.

CD25 expression on residual leukemic blasts at the time of allogeneic hematopoietic stem cell transplant predicts relapse in patients with acute myeloid leukemia without complete remission

Abstract Recent studies have shown that CD25 expression at the time of diagnosis of acute myeloid leukemia (AML) may be associated with an unfavorable outcome. We focus on patients with AML without complete remission (CR) and examine the clinical correlation between surface CD25 expression at the time of transplant and subsequent transplant outcomes. We observed a significant difference in overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) between CD25 positive (+) (n = 22) and negative (−) groups (n = 44) (2-year OS; CD25 (+) group: 5% vs. CD25 (−) group: 40%, p < 0.0001, 2-year DFS; 5% vs. 29%, p < 0.0001, 2-year CIR; 77% vs. 52%, p = 0.03). Multivariate analysis showed that CD25 expression was an independent adverse factor for OS (p = 0.002) and relapse (p = 0.001). Patients with AML with residual CD25 positive blasts at the time of transplant may require additional therapy before or after transplant to improve survival.

Clinical impact of hematogones on outcomes of allogeneic hematopoietic stem cell transplantation

Increased levels of normal B cell precursors, termed hematogones (HGs), are observed in regenerating bone marrow after chemotherapy or hematopoietic stem cell transplantation (HSCT). Recent reports suggest that emergence of HGs is associated with better outcomes following allogeneic HSCT (allo-HSCT). We reviewed the emergence of HGs and the clinical features of 192 patients after allo-BMT. Patients undergoing allo-BMT from related donors were more likely to develop HGs at day 30 compared to unrelated donors. Furthermore, patients undergoing allo-BMT from HLA-mismatched donors were less likely to develop HGs at day 30. The emergence of HGs at day 30 was an independent prognostic factor among patients who underwent BMT. We found no difference in the relapse rate between HG-positive (+) and HG-negative (−) patients undergoing BMT. HG (−) patients had high non-relapse mortality, grade II to IV acute graft-versus-host-disease (GVHD), fungal infection, and lower IgG levels compared to HG (+) patients. The emergence of HGs at day 30 among patients undergoing BMT may be a very useful indicator of subsequent survival outcomes or acute GVHD in common clinical practice.

Clinical impact of CD25 expression on outcomes of allogeneic hematopoietic stem cell transplant for cytogenetically intermediate-risk acute myeloid leukemia

Approximately 50% of adult patients with acute myeloid leukemia (AML) do not show clonal chromosome aberrations at diagnosis, and although this group shows an intermediate prognosis, a minority of patients eventually become long-term survivors. [1]. Th is emphasizes the need for new molecular markers that can be used to predict disease aggressiveness and to determine the leukemic response to treatment, including allogeneic hematopoietic stem cell transplant (allo-HSCT) [2]. Recent data have shown that molecular analysis of the presence of mutated FMS-like tyrosine kinase-3 (FLT3), nucleophosmin 1 (NPM1), CCAAT/ enhancer-binding protein alpha (CEBPA), mixed lineage leukemia (MLL) or neuroblastoma RAS viral oncogene homolog (N-Ras) may be useful for this purpose [3]. However, molecular analyses may be costly and not always available, as not all the mutations are detected by sequencing. As an alternative approach, we may be able to focus on distinct clinical features that depend on specifi c genetic aberrations. For example, patients with a FLT3-internal tandem duplication (ITD) mutation tend to have higher white blood cell count and blast count [4], and moreover a high percentage of these patients may have high CD25 (the α -chain of the interleukin-2 [IL-2] receptor) antigen expression as observed with fl ow cytometry [5 – 7]. Recent studies have shown that CD25 is highly expressed in chemotherapy-resistant, cell cycle-quiescent leukemic stem cells, and high CD25 expression may be associated with an unfavorable outcome after conventional chemotherapy, including autologous and allogeneic transplant [5 – 7]. Th us, evaluation of CD25 expression may be an alternative, cost-eff ective non-molecular tool for cytogenetically intermediate (CI)-AML. Here we report the clinical impact of surface expression of the CD25 antigen on transplant outcomes in a total of 40 patients with CI-AML who underwent allo-HSCT in a single institution. In this study, intermediate-risk AML included patients with a normal or indeterminate karyotype. Bone marrow and peripheral blood samples from 40 patients with CI-AML were available for analysis of CD25 expression at the initial diagnosis. Th ese patients eventually underwent allo-HSCT in our institution between January 2001 and January 2013. We list the chemotherapies, conditioning regimens and outcomes (Supplementary Table I to be found online at http://informahealthcare.com/doi/ abs/10.3109/10428194.2014.974044). Patients with French – American – British (FAB) classifi cation M3 were excluded. Flow cytometry analyses were performed in-house using standard immunofl uorescence methods with monoclonal antibodies directed against CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD13, CD14, CD19, CD20, CD25, CD33, CD34, CD38, CD41, CD56 and human leukocyte antigen (HLA)-DR antigens, and were considered to be positive if at least 20% of leukemic blasts expressed the antigen [5]. Th e cut-off level of CD25 depends on the report, for example 10% [6,7] and 20% [5]. Generally the cut-off level of 20% has been known as the standard, and we consider this as the best cut-off level to exclude false-positive results. Transplant procedures have been described in detail elsewhere [8]. Myeloablative conditioning mainly included busulfan (3.2 mg/kg for 4 days) and cyclophosphamide (60 mg/kg for 2 days). Th e main preparative regimen for the reduced-intensity procedure consisted of fl udarabine (30 mg/m 2 for 6 days), melphalan (40 mg/m 2

Severe Hypoxemia in a Healthy Donor for Allogeneic Hematopoietic Stem Cell Transplantation after Only the First Administration of Granulocyte-Colony Stimulating Factor

Background: Granulocyte-colony stimulating factor (G-CSF) is widely used to mobilize peripheral blood stem cells (PBSCs) in healthy donors. A few reports have shown that some healthy donors developed acute respiratory distress syndrome or capillary leak syndrome after more than several rounds of G-CSF administration or leukapheresis. Case Report: We report the case of a healthy donor for allogeneic stem cell transplantation who developed severe hypoxemia 1 h after only the first administration of G-CSF. The donor was administered 10 μg/kg G-CSF (lenograstim) subcutaneously for PBSC mobilization. 1 h after the first administration of G-CSF, the donor suddenly presented with dry cough and dyspnea. The oxygen saturation by pulse oximetry (SpO2) in the room air was 88%. An electrocardiogram and chest radiography revealed no abnormalities. We excluded other causes of severe hypoxemia and diagnosed the donor with hypoxemia due to G-CSF administration, which was subsequently terminated. The donor was administered 2 l/min oxygen via a nasal cannula and 100 mg hydrocortisone intravenously. He subsequently recovered, and SpO2 in the room air returned to 98% 10 h after hypoxemia. Conclusion: These respiratory symptoms might be related to anaphylactoid or hypersensitivity reaction. The donors should be observed for at least 1 h after the first administration of G-CSF.

Successful HLA-identical Unrelated Allogeneic Bone Marrow Transplantation with a very Low Dose of Stem Cells for a Patient with Chronic Myeloid Leukemia in Blast Crisis

The important factors affecting engraftment after bone marrow transplantation (BMT) include infused stem cell dose [1,2], HLA matching [3], stem cell source [4] and T-cell-depletion of graft [5]. BMT with a low dose of marrow cells is at a high risk for graft failure [1,2]. Here we describe a patient who underwent an allogeneic BMT with a very low dose of infused nucleated cells for chronic myeloid leukemia (CML) in blast crisis and achieved delayed engraftment and durable molecular remission.