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The aim of this article is to help develop a common understanding of the current status, challenges, and future perspectives of real-world data (RWD) and real-world evidence (RWE) in Japan. RWD and RWE are very widely used terms, but standardized definitions are lacking. Given broad and growing applications of RWD/RWE from the perspective of clinical development and medical affairs, the PhRMA Japan Medical Affairs Committee Working Group 1 have proposed the following definitions: “RWD are the data relating to patient health status and/or the delivery of health care routinely collected from a variety of sources” and “RWE is the evidence derived from analysis of RWD.” The key challenges for RWD and RWE in Japan include restricted access and linkage of RWD, as well as a lack of universally accepted methodological approaches, which reduces the potential for patient and healthcare benefits. These challenges for RWD/RWE are by no means unique to Japan and similar challenges exist for countries in Europe and the USA. The quality of data and analysis, study design, and the transparency of reporting should be discussed more to ensure credibility and acceptance by decision-makers as the demand for RWD and RWE increases. The future developments around Japan’s RWD and RWE are expected to include improved RWD access, data linkage, and increased acceptance by decision-makers, all supported by innovative technology. Improvements in RWD access and database linkage will enable both public and private sectors to assemble more comprehensive health information in Japan. Supplementary Information The online version contains supplementary material available at 10.1007/s40801-021-00266-3.

Limited data are available regarding the prevalence of thrombotic/cardiovascular disease and treatment patterns for patients with coronavirus disease 2019 (COVID-19) in Japan. In this study we describe patients hospitalized for COVID-19 in Japan.This retrospective database study analyzed the Japan Medical Data Vision database (416 acute care hospitals) for patients hospitalized for COVID-19 during the identification period from 1 January 1 to 30 September 2020.Among 9282 eligible patients, 832 (9%) had developed thrombotic disease including myocardial infarction, ischemic stroke, deep vein thromboembolism and pulmonary embolism. Intriguingly, 171(1.8%) had two thrombotic events and 25 (0.3%) had three or four thrombotic events at the same time. The data also showed that arterial thrombotic events accounted for 77% of total thrombotic events. Anticoagulant and/or antiplatelet medication was provided to 3312 patients. Even with antithrombotic medication, 21.2% of patients suffered from thrombotic diseases.Patients with COVID-19 could experience thrombotic complications in every blood vessel. Further optimization of medication is crucial for preventing thrombotic complications and improving prognosis.

High-dose chemotherapy and autologous stem cell transplantation is too toxic for elderly patients with relapsed or refractory (DLBCL). Therefore, tolerable and efficient salvage regimens for elderly patients are greatly needed. In this study, therapy with rituximab, gemcitabine, dexamethasone, and cisplatin (R-GDP) will be performed every 4 weeks, and an interim evaluation will be performed after the completion of the 3rd course. If a complete response (CR) is achieved at the time of interim evaluation, 1 course of R-GDP therapy and 2 courses of monotherapy with rituximab will be additionally performed. If a partial response (PR) is achieved, 3 courses of R-GDP therapy will be additionally conducted. In patients without a PR or CR by the time of the interim evaluation, treatment will be discontinued. Treatment will also be discontinued at any point if disease progression is observed during protocol treatment. After the completion of the final course of R-GDP therapy, final effects of the regimen will be evaluated. A primary endpoint is the efficacy of R-GDP therapy (CR and response rates). This is the first multicenter phase II clinical study of R-GDP therapy to examine post-treatment activities of daily living in addition to the safety and efficacy of treatment in elderly patients with relapsed or refractory transplantineligible DLBCL.

We have designed and evaluated a simple and practical system for CD3+ (T-lymphocyte) and CD19+ (B-lymphocyte) cell counting on-chip using a PDMS microfluidic chip which captures these cells using a pillarbased filtration technique. Conventional pillar-based or membrane-based cell separation methods suffer from the problem of cell clogging, thereby reducing the separating and capturing efficiency of the device. To address these issues, we proposed a unique and simple design of a microfluidic chip with an escape route at the pillar-based cell filtration area in the chip which prevents cell clogging. Currently, the diagnosis, monitoring and treatment of immunodeficiency disorder require quantifying the leukocyte population, especially T-lymphocytes and Blymphocytes, in whole blood. We evaluated the performance of our microfluidic chip by obtaining the ratio of T-lymphocytes and Blymphocytes from whole blood and compared the results to those obtained using a flow cytometer. We confirmed that the cell capture area of the microfluidic chip remains clog-free. We achieved 99.8% efficiency in capturing leukocytes using the microfluidic chip, and the ratio of CD3+ and CD19+ had a coefficient of r = 0.9876 and a coefficient of variation (CV) of 2.77%, from 1 μL of whole blood sample.

More than 40% of Japanese patients with multiple myeloma (MM) are over 75 years of age at diagnosis. Regardless of the treatment benefits, complications and relapses obstruct long-term survival. We conducted a phase II, open-label, single-arm, multicenter clinical trial to assess the efficacy and safety of alternating bortezomib-dexamethasone (Bd) and lenalidomide-dexamethasone (Ld) (Bd/Ld) treatment in MM patients aged over 75 years (MARBLE trial). Patients received Bd therapy from days 1 to 35 and Ld therapy from days 36 to 63. For Bd therapy, patients were administered bortezomib 1.3 mg/m

High-dose methotrexate (Hd-MTX) therapy has recently been applied to the treatment of adult acute lymphoblastic leukemia (ALL) based on pediatric protocols; however, its effectiveness for adult ALL has not yet been confirmed in a rigorous manner. We herein conducted a randomized phase III trial comparing Hd-MTX therapy with intermediate-dose (Id)-MTX therapy. This study was registered at UMIN-CTR (ID: C000000063). Philadelphia chromosome (Ph)-negative ALL patients aged between 25 and 64 years of age were enrolled. Patients who achieved complete remission (CR) were randomly assigned to receive therapy containing Hd-MTX (3 g/m2) or Id-MTX (0.5 g/m2). A total of 360 patients were enrolled. The CR rate was 86%. A total of 115 and 114 patients were assigned to the Hd-MTX and Id-MTX groups, respectively. The estimated 5-year disease-free survival rate of the Hd-MTX group was 58%, which was significantly better than that of the Id-MTX group at 32% (P=0.0218). The frequencies of severe adverse events were not significantly different. We herein demonstrated the effectiveness and safety of Hd-MTX therapy for adult Ph-negative ALL. Our results provide a strong rationale for protocols containing Hd-MTX therapy being applied to the treatment of adult ALL.

Summary Background Fungal infections are a major complication of neutropaenia following chemotherapy. Their early diagnosis is difficult, and empirical antifungal treatment is widely used, and uses of less toxic drugs that reduce breakthrough infection are required. Objective We conducted a multicentre, open‐label, randomised, non‐inferiority trial to compare the safety and efficacy of intravenous itraconazole (ivITCZ) and liposomal amphotericin B (LAmB) as empirical antifungal therapy in patients with haematological malignancies with neutropaenia and persistent fever. Methods Patients with haematological malignancies who developed fever refractory to broad‐spectrum antibacterial agents under neutropaenia conditions were enrolled. Patients were randomised for treatment with LAmB (3.0 mg/kg/d) or ivITCZ (induction: 400 mg/d, maintenance: 200 mg/d). Results Observed overall favourable response rates of 17/52 (32.7%) and 18/50 (36.0%) in the LAmB and ivITCZ groups, with a model‐based estimate of a 4% difference (90% CI, −12% to 20%), did not fulfil the statistical non‐inferiority criterion. In the LAmB group, there were two cases of breakthrough infection and five cases of probable invasive fungal disease, whereas in the itraconazole group, neither breakthrough infection nor probable invasive fungal disease occurred. Patients in the ivITCZ group had significantly fewer grade 3‐4 hypokalaemia‐related events than LAmB group patients (P

Acute promyelocytic leukemia (APL) in human immunodeficiency virus (HIV)-infected individuals is very rare. There is currently no consensus regarding the use of anti-cancer drugs with highly active anti-retroviral therapy (ART) in these patients due to their small number. We herein report two cases of APL with HIV-infected patients. Both cases received all-trans-retinoic acid-containing chemotherapies and achieved complete remission. ART was continued throughout the treatment course. The clinical course of these cases suggests that it is preferable to perform standard chemotherapy for APL with ART if patients have an adequate performance status.

We developed a highly efficient and clogging-free cells separation microfluidic chip based on their size and deformability. Currently, cells separation is categorized either by hydrodynamic or using filtration. Hydrodynamic cell separation manipulates flow for separation process based on cell size. Whereas filtration technique with microstructure constriction to separate the cells based on size and deformability which is known in relation to cell phenotypes. However, size based separation techniques suffer from clogging and fouling issues.

Mantle cell lymphoma (MCL) is a B cell malignancy characterized by aberrant expression of cyclin D1 due to a t(11;14) translocation. MCL is refractory to conventional chemotherapy, and treatment remains challenging. We investigated the efficacy of the histone deacetylase (HDAC) inhibitor vorinostat combined with one of several B-cell receptor (BCR) signaling inhibitors on MCL cell death and the underlying mechanisms, using MCL cell lines. The Bruton’s tyrosine kinase inhibitor PCI-32765 and the spleen tyrosine kinase inhibitor R406 showed synergistic effects with vorinostat on growth inhibition. Treatment with PCI-32765 or R406 alone induced 27.3 ± 2.1 or 25.1 ± 3.2 % apoptosis. When combined with vorinostat, these apoptotic fractions significantly increased to 50.8 ± 4.9 and 63.1 ± 5.0 %, respectively. Activation of caspase-3 and poly-(ADP-ribose) polymerase cleavage were markedly increased. We performed gene expression profiling following treatment with the combination of vorinostat and individual BCR signaling inhibitors using a microarray, and differentially expressed genes were identified. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the nuclear factor (NF)-κB signaling pathway was significantly enriched following treatment with the combination of vorinostat and R406. Protein expression analysis confirmed the down-regulation of NF-κB1/p105 and cyclin D1, suggesting inhibition of the NF-κB pathway. Taken together, the combination of an HDAC inhibitor and a BCR signaling inhibitor may be a novel therapeutic strategy for MCL.

The objective of this prospective clinical trial (JALSG-STIM213, UMIN000011971) was to evaluate treatment-free remission (TFR) rates after discontinuation of imatinib in chronic myeloid leukemia (CML). CML patients who received imatinib treatment for at least 3 years and sustained deep molecular response for at least 2 years were eligible. Molecular recurrence was defined as loss of major molecular response (MMR). Of the 68 eligible patients, 38.2% were women, the median age was 55.0 years, and the median duration of imatinib treatment was 97.5 months. The 12-month TFR rate was 67.6%. Patients who lost MMR were immediately treated with imatinib again; all re-achieved MMR. Three-year treatment-free survival (TFS) was estimated as 64.6% using the Kaplan–Meier method. Undetectable molecular residual disease (UMRD) was defined as no BCR-ABL1 in > 100,000 ABL1 control genes using international scale polymerase chain reaction. UMRD at the study baseline was found to be predictive of continuation of TFR. Our findings suggest that CML patients who meet all the eligibility criteria that have commonly been used in the TFR trials are able to discontinue imatinib use safely. TFR may thus be valuable as a new goal for CML treatment in Japan.

Stromal cells and osteoblasts play major roles in forming and modulating the bone marrow (BM) hematopoietic microenvironment. We have reported that FGF2 compromises stromal cell support of normal hematopoiesis. Here, we examined the effects of FGF2 on the leukemia microenvironment. In vitro, FGF2 significantly decreased the number of stromal-dependent and stromal-independent G0-leukemia cells in the stromal layers. Accordingly, CML cells placed on FGF2-treated stromal layers were more sensitive to imatinib. Conversely, FGF2 increased the proliferation of osteoblasts via FGFR1 IIIc, but its effects on osteoblast support of leukemia cell growth were limited. We next treated a human leukemia mouse model with Ara-C with/without systemic FGF2 administration. BM sections from FGF2-treated mice had thickened bone trabeculae and increased numbers of leukemia cells compared to controls. Leukemia cell density was increased, especially in the endosteal region in FGF2/Ara-C -treated mice compared to mice treated with Ara-C only. Interestingly, FGF2 did not promote leukemia cell survival in Ara-C treated spleen. Microarray analysis showed that FGF2 did not alter expression of many genes linked to hematopoiesis in osteoblasts, but modulated regulatory networks involved in angiogenesis and osteoblastic differentiation. These observations suggest that FGF2 promotes leukemia cell growth in the BM by modulating osteoblast functions.

Prospero-related homeobox 1 (PROX1) is important for embryonic organ formation and differentiation, and changes in PROX1 activity were recently associated with cancer. To address the PROX1 roles in tumorigenesis, we established cells stably overexpressing PROX1 using the human cervical cancer cell line, HeLa. Overexpression of PROX1 reduced cell proliferation and the rate of tumor formation as compared with controls. Comparison of gene expression profiles between PROX1-overexpressing and mock-transfected cells revealed that the expression of protein kinase C βII (PRKCB2) is down-regulated in PROX1-overexpressing cells. A PRKCB inhibitor suppressed cell growth of control cells more than PROX1-expressing cells. Analysis of the 5'-promoter of PRKCB revealed that a region between -110 bp and the first exon contains two Sp1 binding sites and is important for transcriptional regulation of PRKCB. The inhibition of Sp1 transcription factor resulted in down-regulation of PRKCB2 protein levels. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, restored PRKCB2 mRNA expression in PROX1-expressing cells, suggesting that the 5'-promoter of PRKCB is methylated in these cells. Actually, it was found that a CpG island in this region, in particular a CpG site overlapping with the distal Sp1 site, was hypermethylated and direct Sp1 binding to this region was inhibited in PROX1-overexpressing cells. Thus, the suppressive effect of PROX1 on cell growth and tumor formation might be partially mediated by PRKCB2 via altered methylation of its promoter.

Acute respiratory distress syndrome (ARDS) is a crippling disease with no effective therapy characterized by progressive dyspnea. Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for ARDS because MSCs can attenuate inflammation and repair the damaged tissue by differentiating into several cell types. Macrophages participate in the development of ARDS; however, MSCs only weakly modulate macrophage function. The chemokine CCL2 is a potent inducer of macrophage recruitment and activation, and its expression is elevated in patients with ARDS. We established MSCs that are stably transduced by a lentiviral vector expressing 7ND, a dominant-negative inhibitor of CCL2, to enhance the therapeutic function of MSCs. 7ND-MSCs retained the innate properties of MSCs and produced a large amount of 7ND. Many 7ND-MSCs were detected in bleomycin-treated lungs (immunostaining 24 hours after injection), suggesting that MSCs could work as a drug delivery tool. Mice treated with 7ND-MSCs showed significantly milder weight loss, lung injury, collagen content, accumulation of inflammatory cells and inflammatory mediators that were induced by bleomycin, and subsequent survival benefit. No evidence of 7ND-MSC–induced toxicity was observed during or after treatment. Thus, inhibiting the effects of macrophages may greatly enhance the ability of MSCs to effect lung repair in ARDS.

We report the case of a 68-year-old woman with acute thrombotic thrombocytopenic purpura (TTP) that developed after pneumococcal vaccination. She was found in a confusional state with high fever 15 days after vaccination. Laboratory data showed hemolytic anemia and thrombocytopenia, and blood smear showed fragmented erythrocytes. TTP was diagnosed based on the clinical presentation, and was subsequently confirmed by the absence of ADAMTS13 activity and the presence of inhibitor against ADAMTS13 in serum. She was successfully treated using plasma exchange and corticosteroids, and no recurrence has been identified. This appears to represent the first report of TTP following pneumococcal vaccination.

Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for reconstituting the hematopoietic microenvironment by improving engraftment in stem cell transplantation. However, the availability of conventional bone marrow (BM)-derived MSCs (BMSCs) is limited. Recent studies showed that a large number of MSCs can be easily isolated from fat tissue (adipose tissue-derived MSCs [ADSCs]). In this study, we extensively evaluated the hematopoiesis-supporting properties of ADSCs, which are largely unknown. In vitro coculture and progenitor assays showed that ADSCs generated significantly more granulocytes and progenitor cells from human hematopoietic stem cells (HSCs) than BMSCs. We found that ADSCs express the chemokine CXCL12, a critical regulator of hematopoiesis, at levels that are three fold higher than those with BMSCs. The addition of a CXCL12 receptor antagonist resulted in a lower yield of granulocytes from ADSC layers, whereas the addition of recombinant CXCL12 to BMSC cocultures promoted the growth of granulocytes. In vivo cell homing assays showed that ADSCs facilitated the homing of mouse HSCs to the BM better than BMSCs. ADSCs injected into the BM cavity of fatally irradiated mice reconstituted hematopoiesis more promptly than BMSCs and subsequently rescued mice that had received a low number of HSCs. Secondary transplantation experiments showed that ADSCs exerted favorable effects on long-term HSCs. These results suggest that ADSCs can be a promising therapeutic alternative to BMSCs.

Introduction The prognosis of Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL) has been dramatically improved since the introduction of tyrosine kinase inhibitors (TKIs). Although allogeneic hematopoietic cell transplantation (allo-HCT) is a major treatment option, the role of autologous peripheral blood stem cell transplantation (auto-PBSCT) has been reconsidered, especially in patients who achieved early molecular remission. Methods and analysis This is a multicenter exploratory study for Ph + ALL patients aged between 55 and 70 years who achieved complete molecular remission within 3 cycles of chemotherapy. The target sample size is 5, and the registration period is 2 years. The primary endpoint is Day100- mortality after transplantation, and the secondary endpoints are survival, relapse rate, nonrelapse mortality, and adverse events.This study is divided into 3 phases: peripheral blood stem cell harvest, transplantation, and maintenance. Chemomobilization is performed using a combination of cyclophosphamide (CPM), doxorubicin, vincristine (VCR), and prednisolone (PSL). As a preparative regimen, the LEED regimen is used, which consists of melphalan, CPM, etoposide, and dexamethasone. Twelve cycles of maintenance therapy using a combination of VCR, PSL, and dasatinib are performed.In association with relapse, the minimal residual disease (MRD) of BCR-ABL chimeric gene and T-cell subsets are analyzed both before and after auto-PBSCT. Ethics and dissemination The protocol was approved by the institutional review board of Nagoya University Hospital and all the participating hospitals. Written informed consent was obtained from all patients before registration, in accordance with the Declaration of Helsinki. Results of the study will be disseminated via publications in peer-reviewed journals. Trial registration Trial registration number UMIN000026445.

In response to diverse stresses, the tumor suppressor p53 differentially regulates its target genes, variably inducing cell-cycle arrest, apoptosis or senescence. Emerging evidence indicates that p53 plays an important role in regulating hematopoietic stem cell (HSC) quiescence, self-renewal, apoptosis and aging. The p53 pathway is activated by DNA damage, defects in ribosome biogenesis, oxidative stress and oncogene induced p19 ARF upregulation. We present an overview of the current state of knowledge about p53 (and its target genes) in regulating HSC behavior, with the hope that understanding the molecular mechanisms that control p53 activity in HSCs and how p53 mutations affect its role in these events may facilitate the development of therapeutic strategies for eliminating leukemia (and cancer) propagating cells.

Several ETS transcription factors, including ELF4/MEF, can function as oncogenes in murine cancer models and are overexpressed in human cancer. We found that Elf4/Mef activates Mdm2 expression; thus, lack of or knockdown of Elf4/Mef reduces Mdm2 levels in mouse embryonic fibroblasts (mef's), leading to enhanced p53 protein accumulation and p53-dependent senescence. Even though p53 is absent in Elf4(-/-) p53(-/-) mef's, neither oncogenic H-Ras(V12) nor c-myc can induce transformation of these cells. This appears to relate to the INK4a/ARF locus; both p19(ARF) and p16 are increased in Elf4(-/-) p53(-/-) mef's, and expression of Bmi-1 or knockdown of p16 in this context restores H-Ras(V12)-induced transformation. Thus, ELF4/MEF promotes tumorigenesis by inhibiting both the p53 and p16/Rb pathways.

The current first-line therapy of chronic myeloid leukemia (CML) is based on tyrosine kinase inhibitors. Diagnostic kits that are capable of identifying at least a 4.5 log reduction in the international scale % BCR-ABL/ABL (IS % BCR-ABL/ABL) are needed for use in the clinical evaluation of deeper molecular response to treatment and to determine the timing of dose selection or treatment interruption during the course of treatment. In this study, we evaluated the performance of a new diagnostic kit, ODK-1201, designed to be capable of performing reverse transcription and quantitative PCR in a single tube for the quantitation of major BCR-ABL mRNA. The kit demonstrated excellent assay performance (limit of detection 0.0007 %) and a broader detection range of BCR-ABL mRNA in peripheral blood compared to a commercially available kit for CML (Amp-CML). ODK-1201 was also shown to be as sensitive as Ipsogen and Molecular MD kits in the same assay. Results obtained in this study indicate evidences that ODK-1201 was capable of identifying at least a 4.5 log reduction in the IS % BCR-ABL/ABL.

A lateral-inhibitive shifting model is proposed as a computational model of geometric illusions. This model is used to implement typical geometric illusions on a computer. Furthermore, this shifting model is used to naturally derive the Riemann metric and the Gaussian curvature distribution in the visual space as the psychological features for describing an optical illusion. ©2002 Wiley Periodicals, Inc. Electron Comm Jpn Pt 3, 86(4): 1–10, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecjc.10060

Histone acetylation has been shown to affect chromatin structure and gene expression. The mitogen-activated protein (MAP) kinase pathway is activated by a number of cytokines and plays critical roles in hematopoietic cell survival, proliferation, and differentiation. We focused on the part of the MAP kinase cascade and granulocyte colony-stimulating factor (G-CSF)in histone acetylation at one of the critical myeloid differentiation-associated genes, myeloperoxidase (MPO). G-CSF caused rapid acetylation of histone H3 and H4 at the promoter of MPO as revealed by chromatin immunoprecipitation. In addition, CBP and p300 were recruited to the promoter in response to G-CSF. Furthermore, we showed that rapid histone acetylation induced by G-CSF is MAP kinase-dependent. These results illustrate how myeloid-differentiating signals via G-CSF may be coupled with histone acetylation during the process of gene expression.

Objective: There are limited treatment options for relapsed/refractory acute myeloid leukemia patients or previously untreated elderly (� 60 years) patients with acute myeloid leukemia. In Phase II studies from the USA and Europe, single-agent clofarabine demonstrated activity and acceptable toxicity in elderly patients with previously untreated acute myeloid leukemia. This Phase I, multicenter study assessed the maximum-tolerated dose, safety, pharmacokinetics and efficacy of clofarabine in Japanese adults with acute myeloid leukemia. Methods: Intravenous clofarabine (20, 30 and 40 mg/m 2 /day) was administered for 5 days to Japanese adult patients with relapsed or refractory acute myeloid leukemia or elderly patients with newly diagnosed acute myeloid leukemia. Results: Fourteen patients, median age of 67.5 (59‐72) years, were enrolled in this study. Eleven out of 14 patients had relapsed/refractory acute myeloid leukemia. Three patients received clofarabine at 20 mg/m 2 ,s ix at 30 mg/m 2 and five at 40 mg/m 2 . Frequently reported treatment-related adverse events included thrombocytopenia (100%), anemia (93%), neutropenia (86%), nausea (86%), alanine aminotransferase increase (71%), headache (71%) and febrile neutropenia (57%). Three patients experienced reversible dose-limiting toxicities; two had increased alanine aminotransferase with 30 and 40 mg/m 2 and one had Grade 3 elevation of serum amylase with 40 mg/m 2 . The maximum-tolerated dose was 30 mg/m 2 /day. Cmax and exposure area under the curve0224h increased with increasing dose and were proportional to dose through the tested dose range. Among the 14 assessable patients, four (29%) achieved complete remission and two (14%) complete remission without platelet recovery. The overall remission rate was 43%. Conclusions: These results demonstrate safety and preliminary, promising activity of clofarabine in Japanese patients with acute myeloid leukemia. Further investigation is warranted.

The Polycomb group protein Bmi1 is a transcriptional silencer of the Ink4a-Arf locus, which encodes the cell cycle regulator p16(Ink4a) and the tumor suppressor p19(Arf). Bmi1 plays a key role in oncogenesis and stem cell self-renewal. We report that phosphorylation of human Bmi1 at Ser³¹⁶ by Akt impaired its function by triggering its dissociation from the Ink4a-Arf locus, which resulted in decreased ubiquitylation of histone H2A and the inability of Bmi1 to promote cellular proliferation and tumor growth. Moreover, Akt-mediated phosphorylation of Bmi1 also inhibited its ability to promote self-renewal of hematopoietic stem and progenitor cells. Our study provides a mechanism for the increased abundance of p16(Ink4a) and p19(Arf) seen in cancer cells with an activated phosphoinositide 3-kinase to Akt signaling pathway and identifies crosstalk between phosphorylation events and chromatin structure.

Hodgkin's lymphoma is frequently associated with mast cell infiltration that correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear. Here, we report that mast cells promote the growth of Hodgkin's tumor by modifying the tumor microenvironment. A transplantation assay shows that primary murine mast cells accelerate tumor growth by established Hodgkin's cell lines, and promote marked neovascularization and fibrosis. Both mast cells and Hodgkin's cells were sensitive to bortezomib, but mast cells were more resistant to bortezomib. However, bortezomib inhibited degranulation, PGE(2)-induced rapid release of CCL2, and continuous release of vascular endothelial growth factor-A from mast cells even at the concentration that did not induce cell death. Bortezomib-treated mast cells lost the ability to induce neovasculization and fibrosis, and did not promote the growth of Hodgkin tumor in vivo. These results provide further evidence supporting causal relationships between inflammation and tumor growth, and demonstrate that bortezomib can target the tumor microenvironment.

Introduction: Imatinib have dramatically changed the natural history of chronic myeloid leukemia (CML) leading to significant improvement in clinical outcome and survival rates. Recently, treatment free remission (TFR) is one of the goals in CML treatment, and some prospective trials suggest that imatinib therapy may be safely discontinued in CML patients with deep and sustained molecular responses (Mahon Lancet Oncol 2010, Ross Blood 2013, Rousselot JCO 2014). The purpose of this study was to confirm TFR in Japanese CML patients and to define prognostic biomarkers of successful TFR after stopping imatinib. Methods: Japanese CML patients on imatinib treatment in confirmed deeper molecular response (DMR) for at least two year (>4 log reduction on imatinib therapy for >24 months confirmed by four consecutive PCR tests) and under imatinib treatment for at least 3 years were eligible. Patients treated with other tyrosine kinase inhibitors or who received stem cell transplantations were excluded. MR4.5 was confirmed at the beginning of this study using Ipsogen BCR-ABL1 M-BCR IS-PCR kit in a central laboratory (Sysmex, Kobe, Japan). Primary endpoint was the major molecular remission (MMR) rate at 12 months after stopping imatinib. Molecular recurrence of CML was defined as loss of MMR according to A-STIM criteria (Rousselot JCO 2014). Results: From November 2013 to March 2014, 77 CML patients in chronic phase from 26 institutions were enrolled in this study. Nine were excluded (consent withdrawal n=1, not eligible n=8). Of the eligible 68 patients, 38.2% were female. Median age was 55.0 years (range, 23 to 84), and 13.2% and 16.2 % were high-risk according to EUTOS and Sokal Scores. Thirteen patients were treated with interferon prior to imatinib therapy. Median duration of imatinib treatment was 8 years (range, 3-12 years). The duration of imatinib treatment was less than 5 years in 12%, 5-8 years in 34% and > 8 years in 54% of pts. Time to MMR was 11.5 months (25%-75%, 7.5-22.7 months) and time to DMR (not detected by PCR) was 30.6 months (25%-75%, 17.6-59.9 months). Among the 68 patients, 46 patients (67.6%, 95%CI: [56.5% to 78.8%]) remained without molecular recurrence the first 12 months according to A-STIM criteria, defined as loss of MMR. Moreover, 43 patients (63.2%, 95%CI: [51.8% to 74.7%]) remained without molecular recurrence the first 12 months according to STIM criteria, defined as two consecutive loss of MR4.5 with 1 log increase. On the other hand, 22 patients who lost MMR were treated again with imatinib and all patients achieved MMR within 6 months. Time to 2nd MMR was 40 days. Although there was a trend for a better TFR rate for patients treated longer with Imatinib (Figure 1), no significant difference could be observed for molecular relapse within 12 months according to clinical characteristics including age, sex, Sokal risk score, prior IFN, and time to MMR/DMR (Table 1). Ten patients (15%) showed "withdrawal syndrome" which is transitory musculoskeletal pain within several weeks after imatinib discontinuation, and all patients except one recovered without any treatments. Conclusion: According to the A-STIM criteria, around 70% of patients with deep and sustained molecular responses could safely stop imatinib. TFR in this prospective Japanese clinical study was higher than previously reported, probably because there were much more patients who treated with imatinib for longer duration than previous studies. We will report prognostic factors in the exploratory research of JALSG-STIM213 study including T/NK-cell profiling in peripheral blood, BIM deletion polymorphism, and ABCG2 421C/A polymorphism at this ASH meeting. Table 1. Table 1. Figure 1. Figure 1. Disclosures Takahashi: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Otsuka: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Astellas: Speakers Bureau; Masis: Consultancy; Sysmex: Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Hatta:CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; Celgene K.K.: Honoraria. Usuki:Fuji Film RI Pharma: Other: personal fees; Fujimoto Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Shionogi: Other: personal fees; MSD: Other: personal fees, Research Funding; Nippon Shinyaku: Other: personal fees, Research Funding; Astellas: Research Funding; Chugai Pharmaceutical: Other: personal fees; Takeda Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Other: personal fees, Research Funding; SymBio Pharmaceutical: Other: personal fees, Research Funding; Sanofi: Other: personal fees, Research Funding; Novartis: Other: personal fees, Research Funding; Boehringer Ingelheim: Other: personal fees, Research Funding; Celgene: Other: personal fees, Research Funding; Sumitomo Dainippon Pharma: Other: personal fees, Research Funding; Taiho Pharmaceutical: Other: personal fees, Research Funding; Shire: Research Funding; GlaxoSmithKline: Other: personal fees, Research Funding; Bristol-Myers Squibb: Other. Kobayashi:Gilead Sciences: Research Funding. Naoe:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; Pfizer Inc.: Research Funding; Toyama Chemical CO., LTD.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Celgene K.K.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding.

PAX5 is a transcription factor required for B-cell development and maintenance. PML is a tumor suppressor and a pro-apoptotic factor. A fusion gene, PAX5-PML, was found in acute lymphoblastic leukemia (ALL) with chromosomal translocation t(9;15)(p13;q24), but no functional analysis has been reported. Here, we demonstrate that PAX5-PML had a dominant-negative effect on both PAX5 and PML. PAX5-PML dominant negatively inhibited PAX5 transcriptional activity in the luciferase reporter assay and suppressed the expression of the PAX5 transcriptional targets in B-lymphoid cell line. Surprisingly, PAX5-PML hardly showed DNA-binding activity in vitro although it retained the DNA-binding domain of PAX5. Additional experiments, including chromatin immunoprecipitation (ChIP) assay, suggested that PAX5-PML bound to the promoter through the association with PAX5 on the promoter. On the other hand, coexpression of PAX5-PML inhibited PML sumoylation, disrupted PML nuclear bodies (NBs), and conferred apoptosis resistance on HeLa cells. Furthermore, treatment with arsenic trioxide (ATO) induced PML sumoylation and reconstitution of PML NBs, and overcame the anti-apoptotic effect of PAX5-PML in HeLa cells. These data suggest the possible involvement of this fusion protein in the leukemogenesis of B-ALL in a dual dominant-negative manner and the possibility that ALL with PAX5-PML can be treated with ATO.

Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for reconstituting the hematopoietic microenvironment by improving engraftment in stem cell transplantation. However, the availability of conventional bone marrow (BM)-derived MSCs (BMSCs) is limited. Recent studies showed that a large number of MSCs can be easily isolated from fat tissue (adipose tissue-derived MSCs [ADSCs]). In this study, we extensively evaluated the hematopoiesis-supporting properties of ADSCs, which are largely unknown. In vitro coculture and progenitor assays showed that ADSCs generated significantly more granulocytes and progenitor cells from human hematopoietic stem cells (HSCs) than BMSCs. We found that ADSCs express the chemokine CXCL12, a critical regulator of hematopoiesis, at levels that are three fold higher than those with BMSCs. The addition of a CXCL12 receptor antagonist resulted in a lower yield of granulocytes from ADSC layers, whereas the addition of recombinant CXCL12 to BMSC cocultures promoted the growth of granulocytes. In vivo cell homing assays showed that ADSCs facilitated the homing of mouse HSCs to the BM better than BMSCs. ADSCs injected into the BM cavity of fatally irradiated mice reconstituted hematopoiesis more promptly than BMSCs and subsequently rescued mice that had received a low number of HSCs. Secondary transplantation experiments showed that ADSCs exerted favorable effects on long-term HSCs. These results suggest that ADSCs can be a promising therapeutic alternative to BMSCs.

Hematopoietic stem cells (HSCs) can remain quiescent or they can enter the cell cycle, and either self-renew or differentiate. Although cyclin C and cyclin dependent kinase (cdk3) are essential for the transition from the G0 to the G1 phase of the cell cycle in human fibroblasts, the role of cyclin C in hematopoietic stem/progenitor cells (HSPCs) is not clear. We have identified an important role of cyclin C (CCNC) in regulating human HSPC quiescence, as knocking down CCNC expression in human cord blood CD34+ cells resulted in a significant increase in quiescent cells that maintain CD34 expression. CCNC knockdown also promotes in vitro HSPC expansion and enhances their engraftment potential in sublethally irradiated immunodeficient mice. Our studies establish cyclin C as a critical regulator of the G0/G1 transition of human HSPCs and suggest that modulating cyclin C levels may be useful for HSC expansion and more efficient engraftment.

Stem cells of acute myeloid leukemia (AML) have been identified as immunodeficient mouse-repopulating cells with a Lin(-)CD34(+)38(-) phenotype similar to normal hematopoietic stem cells. To identify the leukemia-propagating stem cell fraction of Philadelphia chromosome-positive (Ph(+)) leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n = 3) or Ph(+) acute lymphoblastic leukemia (n = 3) into NOD/SCID/IL-2Rgammac(-/-) mice. Engrafted cells were almost identical to the original leukemia cells as to phenotypes, IGH rearrangements, and karyotypes. CD34(+)CD38(-)CD19(+), CD34(+)38(+)CD19(+), and CD34(-)CD38(+)CD19(+) fractions could self-renew and transfer the leukemia, whereas the CD34(-)CD38(+)CD19(+) fraction did not stably propagate in NOD/SCID mice. These findings suggest that leukemia-repopulating cells in transformed Ph(+) leukemia are included in a lineage-committed but multilayered fraction, and that CD34(+) leukemia cells potentially emerge from CD34(-) populations.

SummaryThe importance of the p53 protein in the cellular response to DNA damage is well known, but its function during steady-state hematopoiesis has not been established. We have defined a critical role of p53 in regulating hematopoietic stem cell quiescence, especially in promoting the enhanced quiescence seen in HSCs that lack the MEF/ELF4 transcription factor. Transcription profiling of HSCs isolated from wild-type and p53 null mice identified Gfi-1 and Necdin as p53 target genes, and using lentiviral vectors to upregulate or knockdown the expression of these genes, we show their importance in regulating HSC quiescence. Establishing the role of p53 (and its target genes) in controlling the cell-cycle entry of HSCs may lead to therapeutic strategies capable of eliminating quiescent cancer (stem) cells.

Fms-like tyrosine kinase 3 (FLT3) is expressed in hematopoietic progenitor cells. An internal tandem duplication (ITD) of FLT3 (FLT3/ITD) is the most frequent mutation in human adult acute myeloid leukemia (AML). FLT3/ITD contributes to the constitutive activation of FLT3 itself and its downstream signal components, mitogen-activated protein kinase and signal transducers and activators of transcription 5 (STAT5), and enables interleukin (IL)-3-dependent cell lines to grow autonomously. In the present study, we showed the specific association of FLT3/ITD with Lyn, which led to the phosphorylation of Lyn in vivo. We also demonstrated that FLT3/ITD receptors displayed a higher affinity to bind to Lyn than wild-type FLT3 receptors in vitro and that this affinity was relative to the intensity of tyrosil phosphorylation of the receptor. Both treatment with small interfering RNA (siRNA) targeting Lyn and the Src family kinase inhibitor PP2 suppressed the IL-3-independent growth of FLT3/ITD-expressing 32D cells (FLT3/ITD-32D), reducing the constitutive phosphorylation of Lyn and STAT5. PP2 treatment of mice transplanted with FLT3/ITD-32D cells blocked the onset of tumors and decreased the size of established tumors. These results demonstrate that Lyn is an important component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.

Objective: Exploration of the pathogenesis of B-cell lymphomas, including mantle cell lymphoma (MCL), has led to the development of new therapeutic strategies. We focused on the aberrant activation of histone deacetylase (HDAC) and the B-cell receptor (BCR) signaling pathway, which are both novel targets of lymphoma therapy. We evaluated the effects of combined treatment with an HDAC inhibitor and a BCR signaling inhibitor on MCL. Methods: Using the MTT assay, we investigated the cytotoxic effects of BCR signaling pathway inhibitors (fostamatinib, ibrutinib, enzastaurin and idelalisib) alone or in combination with vorinostat, a potent HDAC inhibitor, using MCL cell lines. Apoptosis was determined with flow cytometry using annexin V/propidium iodide staining and detection of cleaved caspase-3 and its substrate poly-(ADP-ribose) polymerase (PARP). Gene expression profiling was analyzed with a cDNA microarray, and the lists of differentially expressed genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The expression of mRNA and protein was validated with quantitative real-time PCR and western blotting, respectively. Results: Among the BCR signaling inhibitors we examined, fostamatinib, a spleen tyrosine kinase inhibitor, and ibrutinib, a Bruton’s tyrosine kinase inhibitor, significantly inhibited cell growth of Jeko-1 cells when combined with vorinostat. Single treatment with fostamatinib and ibrutinib induced 25.1 ± 3.2% and 27.3 ± 2.1% apoptosis, respectively. When combined with vorinostat, the apoptotic fraction was significantly increased to 63.1 ± 5.0% and 50.8 ± 4.9%, respectively. The expression of activated caspase-3 and PARP cleavage were increased, suggesting that the combination of vorinostat and these BCR signaling inhibitors induced caspase-dependent apoptosis. Cyclin D1, which is overexpressed in MCL, was markedly down-regulated by both combination treatments. To elucidate the underlying mechanism, we performed gene expression profiling and found that numerous genes involved in multiple cellular pathways were differentially expressed following combination treatment compared to each agent alone. KEGG pathway analysis identified the NF-κB signaling pathway, which regulates cyclin D1, as a significantly enriched pathway among the down-regulated sets of genes. We validated the expression of genes involved in this pathway and found that the expression of NF-κB1/p105 was strongly down-regulated by the combination treatment. Conclusion: The combination of vorinostat and a BCR signaling inhibitor enhanced apoptosis and induced synergistic growth inhibition of MCL cells, probably by down-regulating the NF-κB signaling pathway and suppressing cyclin D1 expression. Disclosures Naoe: Otsuka Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding. Nagai:Chugai Pharmaceutical Co.LTD: Speakers Bureau; Symbio Pharmaceutical Co. LTD: Research Funding; Mundi Pharma: Research Funding.

The transcription factor GATA-1 plays a significant role in erythroid differentiation and association with CBP stimulates its activity by acetylation. It is possible that histone deacetylases (HDACs) repress the activity of GATA-1. In the present study, we investigated whether class I and class II HDACs interact with GATA-1 to regulate its function and indeed, GATA-1 is directly associated with HDAC3, HDAC4 and HDAC5. The expression profiling and our previous observation that GATA-2 interacts with members of the HDAC family prompted us to investigate further the biological relevance of the interaction between GATA-1 and HDAC5. Coexpression of HDAC5 suppressed the transcriptional potential of GATA-1. Our results demonstrated that GATA-1 and HDAC5 colocalized to the nucleus of murine erythroleukemia (MEL) cells. Furthermore, a portion of HDAC5 moved to the cytoplasm concomitant with MEL cell erythroid differentiation, which was induced by treatment with N,N'-hexamethylenebisacetamide. These observations support the suggestion that control of the HDAC5 nucleocytoplasmic distribution might be associated with MEL cell differentiation, possibly through regulated GATA-1 transactivation.

Background: Bendamustine combined with rituximab (BR) is standard chemotherapy for relapsed or refractory follicular lymphoma (FL), mantle cell lymphoma, and MALT lymphoma. The relative dose intensity (RDI) was proposed as an index of the dose and administration interval agents. We investigated the influence of RDI maintenance and therapeutic effects in patients with FL, mantle cell lymphoma, and MALT lymphoma. Patients and methods: Patients received bendamustine at 90 mg/m2 on days 1 and 2 and rituximab at 375 mg/m2 on day 1 every 28 days for up to 6 cycles. We retrospectively examined RDI, progression free survival (PFS), antitumor effect, and treatment safety. Results: Fourteen patients were enrolled, and 9 patients were received BR four or more cycles between Dec 2010 and Nov 2013. The median patient age was 75 years (range, 55 to 81 years), and 66.6% of patients were 65 years of age or order. All patients (100%) were previously treated with rituximab-containg chemotherapy. The overall response rate was 55.5%, with complete response and partial response rates of 44.4 and 11.1%, respectively. Median PFS was 28.4 weeks in those who received >70% of the RDI and 47.3 weeks in those who received Conclusions: These data were suggested that PFS was extended by controlling a myelosuppression even if they could not maintain RDI

Introductions Diffuse large B-cell lymphoma (DLBCL) is one of the aggressive type of non-Hodgkin lymphoma (NHL) and is known to have a good drug-sensitivity and prognosis. More than 40% of patients with DLBCL are older than 70 years old at diagnosis, and age is known as a poor prognostic factor. However, dose modification is frequently necessary due to their comorbidities and organ function decline, and this leads to poor outcome of their treatment. Therefore, it is important to optimize treatment strategy and chemotherapy for the elderly with DLBCL to improve their outcome. Aims The aim of this study is to unveil underlying problems in the treatment against elderly patients with DLBCL and develop a new therapeutic approach to overcome them. Methods The medical records of all new elderly patients (70 years old< ) with DLBCL between 2003 April and 2011 October at Nagoya Medical Center were reviewed for a retrospective analysis , to identify characteristics and treatment response of those patients. Survival of patients intensively treated (82 pts) was evaluated using Kaplan-Meier method, and analyzed using Log-rank test. Results We experienced new 93 patients aged over 70 years (41.9 % of all DLBCL cases). Median observation period of was 52.7 months. R-CHOP and R-CHOP-like regimes were administrated into 78 and 4 patients respectively. Four patients treated without rituximab due to patients’ decision. CR was obtained in 70 patients (CR rate 85.3%), and 3-yr OS and PFS were 72.2% and 72.1% respectively. Eleven patients died during induction chemotherapy or didn’t obtain CR and eventually died. The median doses of ADR, CPM, and VCR were 69.51±15.2%, 70.23±20.0% and 59.1±28.1% of original regimen, respectively.% of VCR to original CHOP regimen was significant different between age 70-79 and age more than 80 (p Disclosures: No relevant conflicts of interest to declare.

e19521 Background: Burkitt lymphoma (BL) progresses very rapidly, but highly intensive chemotherapy (e.g. CODOX-M/IVAC, hyper-CVAD/MA) has been shown to be a promising strategy. In this time, AIDS-related lymphoma is treated with similarly to non-AIDS lymphoma, nevertheless it is not clear whether the highly intensive regimens are feasible and beneficial for AIDS-related Burkitt (AIDS-BL) or not. The purpose of the current study is to compare the toxicity of AIDS-BL to non-AIDS-BL retrospectively. Methods: We collected the data of all patients (pts) with BL between 2004 and 2011 at our institution. Chart review was performed for all identified pts. The effects of treatment and the clinical variables on overall survival (OS) were assessed. Results: We identified 25 Burkitt lymphomas, and a total of 108 cycles of highly intensive chemotherapy were administered. Nine of these 25 were AIDS-BL (median age, 51; range, 36 to 68) and 16 were non-AIDS-BL (median age, 60; range, 27 to 75). As chemotherapeutic regimens, 15 (60%) were treated with CODOX-M/IVAC, 8 (32%) with hyper-CVAD/MA, and 2 (8%) with EPOCH. Of 12 pts (48%) treated with rituximab-contain regimens. Completion rate of planned chemotherapy was 64%. The median follow-up was 34.3 months. Three-year OS was 73.6%. There were no significant differences in OS between chemotherapeutic regimens (p=0.94) and between AIDS-BL and non-AIDS-BL (p=0.39). The overall incidence of grade 3/4 febrile neutropenia was 64.8% (70 of 108 cycles), 60.5% for HIV-infected and 67.7% for HIV-negative case, respectively. Grade 4 thrombocytopenia was frequently observed in AIDS-BL (72.1% vs. 40.0%), and incidence of grade 3/4 anemia was not different (79.1% vs. 70.8%). Conclusions: This study showed that clinical outcomes, including toxicities, of AIDS-BL were similar to non-AIDS-BL when BL treated with highly intensive chemotherapy.

Abstract 4912 Bendamustine, a kind of alkylating agent, has demonstrated a favorable anti-tumor activity both alone and in combination with rituximab in low grade B cell lymphoma. Because the kinase cascades, including B-cell receptor signaling, are necessary for the proliferation and survival of the lymphoma cells, the kinase inhibitors have been developed as molecular target agents of malignant lymphomas. We investigated the cytotoxic effect of bendamustine, both as a single agent and in combination with these new kinase inhibitors in human lymphoid cell lines. We used three B-cell lines, BALL-1 (acute lymphoblastic leukemia), SKLY16 (B-cell lymphoma) and DHL4 (diffuse large B cell lymphoma), and T-cell line THP-6 in this study. These cells were treated with bendamustine alone or combined with enzastaurin (a PKC-β inhibitor), CAL-101 (a p110δ PI3K inhibitor), PCI-32765 (a Btk inhibitor) or R406 (a Syk inhibitor) for 48 hours. Drug-induced cytotoxicity was evaluated using the MTT assay. Cell viability in each experiment was normalized using untreated controls. The treatment with bendamustine alone decreased cell viability in a dose-dependent manner on these three B-cell lines. In contrast, T-cell line THP-6 showed the resistance to bendamusitine. For the combination study, cells were exposed to bendamustine (10μM) and one of these kinase inhibitors at various concentrations simultaneously for 48 hours. CAL-101 and PCI-32765 did not enhance the cytotoxic effect on all of B-cell lines. In BALL-1 and SKLY16 cells, the combination with enzastaurin or R406 resulted in a higher cytotoxic activity than that induced by bendamustine alone. In DHL4 cells, the treatment combined with R406 inhibited cell growth effectively, but not with enzastaurin. To evaluate whether these drug combinations are synergistic, a combination index (CI) was calculated and normalized isobolograms were constructed from non-constant ratio drug combinations using Calcusyn software. The CI values were less than 0. 7 in SKLY16 cells treated with bendamustine and enzastaurin, indicating that these produced synergistic cytotoxic effects in cell line-dependent manner. Our results show that enzastaurin might potentiate the cytotoxic activity of bendamustine in vitro and be a good candidate for the combination with bendamustine. Disclosures: No relevant conflicts of interest to declare.

Abstract 4694 Introduction: Patients with hematological disease are often immunocompromised due to intensive chemotherapy, including hematopoietic stem cell transplantation (HSCT), and immunosuppressive therapy. Broad-spectrum anti-bacterial drugs were frequently given to those patients as an empiric therapy during a chemotherapy-induced myelosuppressive phase, which causes problematic emergence of drug-resistant strain and microbial substitution. Chemotherapy-induced disturbance of mucosa membrane barrier and skin barrier damage by central venous catheter (CVC) insertion gives pathogens an easy access to blood stream and a chance to develop blood-stream infections (BSIs). Stenotrophomonas maltophilia (S. maltophilia) has been reported to emerge as frequent offenders of immunocompromised patients, especially those with hematological diseases and prolonged neutropenia. S. maltophiliais known to possess a high-level intrinsic resistance to variety of anti-bacterial drugs, including higher-generation cephalosporins and carbapenemes. Objective: The aim of this study is to identify clinical characteristics of S. maltophilia BSIs such as drug sensitivity to properly treat immunocompromised patients with BSIs due to S. maltophilia. Materials and Methods: This retrospective cohort study included all patients who admitted at department of hematology in our institute and from whose blood culuture (BC) or CVC culture (CC) S. maltophilia was isolated during 8 years from September 2003. Samples with positivity within two weeks are counted as a single case. Results: Among 5478 samples of BC and 258 samples of CC submitted to the laboratory, pathogens were isolated from 645 samples (11.8%) of BC and 74 samples (29.8%) of CC. Among positive samples, S. maltophilia was isolated from 52 samples (8.1%) of BC and 9 samples (11.7%) of CC. S. maltophilia was the third most frequent pathogens, next to Staphylococcus epidermidis (23.6% in BC, 29.9% in CC) and Candida (23.6% in BC, 29.9% in CC). This samples occupied 75.8% of 69 BC samples and 52.9% of CC samples among all S. maltophilia positive samples within our institute during the same period. Among these 61 samples, 60 samples (40 cases) were during treatment for hematological malignancies (25 cases AML, 8 cases ALL, 5 cases malignant lymphoma, one case MDS and multiple myeloma). 21 cases were in CR and 6 cases were after allogenic HSCT. Treatment before fever onset was; 6 cases allogenic HSCT, 14 cases AML type, 8 cases ALL type, 3 cases CHOP-like and 3 cases oral agents. CVC was inserted to all patients except a MM patient. CVC culture was submitted in 18 cases of all BC cases and 7 cases (38.9%) were positive. Either BC or CC was positive in 31 cases and 3 cases, respectively. In 7 patients, repeated BC positivity was observed with interval from 22 days to one year 9 months (median 128 days). Most cases were given higher-generation cepharosporins and carbapenems before fever onset, 12 case and 6 cases respectively. Before obtaining samples, cephalosporins and carbapenems were given to 25 cases and 20 cases, respectively. Treatment against S. maltophilia was; 17 cases drawal of CVC, 17 cases minomycline (MINO), 16 cases ceftazidime (CAZ), 10 cases quinolones, 6 cases SMX/TMP (ST) and 6 cases borad-spectrum drugs. Senvitivities of isolated S. maltophilia were; ST 61/61, MINO 61/61, quinolone (levofloxacin:LVFX) 60/61, CAZ 44/61. Only 2 cases (2 patients) died due to S. maltophilia BSIs. Discussion: Our data also shows that patients with hematological malignancies, especially AML and ALL, or after allogenic HSCT, are highly immunocompromised and predisposed to S. maltophilia. Althought high mortality rates around 20% among cancer patients with S. maltophilia infection are reported recently, our series shows only 5% of mortality. Recent resistance to those antbiotics, including ST, was reported but sensitivity test showed our series held high sensitivity to MINO/CAZ/LVFX. Our lower mortality rate could be due to treatment with CAZ/ LVFX. It is reasonable that those patients were still in neutropenic phase and needed be treated aganist other bacterium simultaneously. Although ST was given in only limited cases, none of isolates were resistant to ST. It might be important to identify sensitivities of S. maltophilia isolated in own institutes and choose sensitive agents when S. maltophilia might be a presumable pathogen. Disclosures: No relevant conflicts of interest to declare.

Abstract 3634 Background: A variety of inflammatory cells are present the microenvironment of Hodgkin lymphoma (HL); these cells enhance the survival of lymphoma cells and suppress tumor immunity. HL is frequently associated with the mast cell infiltration that correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear. Aims: To examine whether mast cells can promote the growth of HL by modifying the tumor microenvironment and to determine whether mast cells can be a therapeutic target for HL. Methods: The human HL cell lines, L428, HDLM2, and KMH2, bone marrow-derived mast cells (BMMCs), and spleen-derived mast cells (SPMCs) from C57BL/6 mice were used in our analyses. The proliferative effect of in vitro co-culture was assessed by a colorimetric assay. HL transplantation assays were performed in NOD/SCID mice using HL cells with or without BMMCs. To study the effects of anti-cancer drugs on mast cell functions, BMMCs were treated with or without bortezomib or lenalidomide. Tumor size was measured and histopathological analyses were carried out to determine the effectiveness of the drugs. The expression profile of angiogenesis-related proteins was confirmed using the Angiogenesis Array Kit (R&D Systems, Minneapolis). To analyze the in vitro effects of bortezomib on the BMMCs, VEGF-A, CCL2, and b-hexosaminidase expressions were measured by ELISA and a b-hexosaminidase assay. The statistical significance of inter-group differences was evaluated by Student's t-test. Results: On in vitro co-culture assays, BMMCs weakly induced the proliferation of only KMH2 cells, and SPMCs did not induce the proliferation of any HL cell lines. On the in vivo transplantation assays, HL cells gave rise to tumors in NOD/SCID mice more rapidly when inoculated subcutaneously together with BMMCs than when inoculated HL cells alone. The mean size of tumors derived from inoculated HL cells with BMMCs was significantly greater than that of tumors derived from inoculated HL cells alone (e.g., L428 vs. L428 + BMMC, mean size: 108.39 mm3 vs. 225.19 mm3, respectively, at day 5; p = 0.0026). Microscopically, tumors derived from inoculated HL cells with BMMCs showed increased vasculature and fibrosis, whereas tumors derived from inoculated HL cells alone were generally hypovascularized with less fibrosis and were necrotic in most areas. An antibody array using cell lysates to determine the source of proangiogenic factors showed that HL cells minimally produced proangiogenic factors, but that mast cells produced them abundantly. Next, we examined whether bortezomib can target mast cell functions by inhibiting the secretion of mast cell products. Bortezomib inhibited degranulation of b-hexosaminidase, PGE2-induced rapid release of CCL2, and continuous release of vascular endothelial growth factor-A from mast cells, even at concentrations that did not induce cell death, and profoundly decreased expressions of angiopoietin-1, endoglin, HB-EGF and VEGF-B. On an in vivo transplantation assay in the presence or absence of bortezomib, the mean size of tumors derived from inoculated HL cells plus untreated BMMCs were significantly greater than those of tumors derived from inoculated HL cells plus bortezomib-treated BMMCs (e.g., L428 + intact BMMC vs. L428 + bortezomib-treated BMMC, mean size: 105.6 mm3 vs. 57.7 mm3, respectively, at day 6; p = 0.0255). Microscopically, tumors derived from inoculated HL cells together with intact BMMCs were highly vascularized and fibrotic, whereas tumors derived from inoculated HL cells plus bortezomib-treated BMMCs were generally not. Results from a similar analysis using lenalidomide showed that its effect on BMMCs was much lower than that of bortezomib. Discussion: Mast cells had the ability to promote the growth of HL on in vivo transplantation assay, but not on in vitro co-culture assay, indicating that there may be an indirect event via the promotion of angiogenesis that acts on the tumor microenvironment. Bortezomib effectively inhibited the mast cell-induced growth of Hodgkin's cell tumors in vivo by blocking the release of secretory granules from mast cells, but suppress of mast cells could not have a complete remission. As a treatment strategy for the future, it may be necessary to combine bortezomib with other drugs or irradiation. Conclusions: Mast cells have the ability to promote the growth of HL, and may be a promising target for the treatment of HL. Disclosures: No relevant conflicts of interest to declare.

Abstract 4295 Background and Purpose: Although cytarabine plus anthracycline (7+3 or 7+5) regimens are commonly used for induction therapy in patients (pts) with acute myeloid leukemia (AML) and there have been improvements in the treatment of AML in younger adults, there is no standard of care in pts with relapsed or refractory (R/R) AML. Treatment options for older pts and those with R/R disease remain limited. Clofarabine (JC0707) is a purine nucleoside analog approved in the United States (US) and European Union for the treatment of pediatric pts with R/R acute lymphocytic leukemia (ALL). In a phase II study from the US, single-agent clofarabine showed activity and acceptable toxicity in pts ≥ 60 years with untreated AML and adverse prognostic factors (Kantarjian, J Clin Oncol 2010;28:549–55). The purpose of this phase I open-label, multi-center study is to assess the safety, tolerability, and pharmacokinetics of clofarabine monotherapy in elderly Japanese pts with newly diagnosed AML for whom standard induction chemotherapy is unlikely to be of benefit or Japanese adult pts with R/R AML. Method: Adult pts (20–74 years) with R/R AML according to World Health Organization (WHO) criteria and elderly pts (60–74 years) with newly diagnosed AML were eligible to participate. Additional inclusion criteria included Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2 and no prior hematopoietic stem cell transplant. The study utilized a standard 3 + 3 dose escalation method; 3 pts enrolled in each dosing cohort, 3 additional pts were added to cohorts where dose-limiting toxicities (DLTs) were observed. The maximum tolerated dose (MTD) was defined as the dose level below that for which 2 DLTs were observed. Based on prior clinical trials in the US, pts were to be treated with clofarabine 20 mg/m2/day (IV over 1 hr), 30 mg/m2/day, or 40 mg/m2/day for one 5 day cycle in cohorts 1, 2 and 3, respectively. Pts with evidence of hematologic response after one cycle could receive up to a maximum of 3 cycles. The primary endpoints of this study were MTD, safety, and pharmacokinetic (PK) parameters. Result: Until June 2011, 14 pts were enrolled and treated in this trial: cohort 1 (n=3), cohort 2 (n=6), and cohort 3 (n=5). Bioanalytical determination of clofarabine concentrations in plasma samples showed an increased concentration with increased dosage. No DLTs were noted in cohort 1 (20 mg/m2). Among the first 3 pts in cohort 2 (30 mg/m2), only 1 patient experienced DLT (reversible, grade 4 elevated ALT). Additionally, 2 pts in cohort 3 (40 mg/m2) experienced DLTs (grade 3 elevated ALT [n=1]; grade 3 elevated amylase [n=1]). Thus, the MTD was determined to be 30 mg/m2. Preliminary safety and efficacy data are available for 9 of these pts and presented herein. Overall, the most common all cause, non-hematologic toxicities were nausea and headache (89% each), rash and elevated ALT and AST (78% each), malaise (56%), pneumonia and hypokalemia (44% each), and elevated bilirubin and vomiting (33% each). Grade 3 or 4 toxicities were primarily hematologic and infectious occurring in 89% and 67% of patients, respectively. Only one patient developed a treatment-related serious adverse event (SAE) (herpes zoster). There were no AE related deaths and no patients discontinued therapy as a result of an AE. Two patients achieved complete remission (CR) and 2 patients achieved CR without platelet recovery (CRp), for an overall response rate (ORR) of 44% (Table 1). Additionally, plasma samples were obtained from all patients for PK evaluation; plasma concentration data from all 14 patients will be presented. Conclusion: Clofarabine monotherapy was well tolerated at doses up to 30mg/m2 and showed preliminary evidence of activity with 44% ORR in elderly newly diagnosed AML or adult Japanese pts with R/R AML, warranting further investigations. Disclosures: Off Label Use: Clofarabine (JC0707) is an investigational agent in Japan; this abstract assesses its use in adult AML patients. Ewesuedo:Sanofi Oncology: Employment. Tabata:Genzyme (a Sanofi company): Employment.

Abstract 5227 BACKGROUND: The homeobox gene Prox1 is a transcription factor related to the Drosophila prospero gene and known to be the master gene which controls the development of lymphatic vasculature. Recent studies have suggested that Prox1 is a strong candidate tumor suppressor gene in several kinds of malignancies. We previously demonstrated that the expression of Prox1 gene was frequently silenced by aberrant DNA methylation in about 60% of diffuse large B cell lymphoma (DLBCL). In this study, we showed that Prox1 regulated Protein Kinase C beta II (PKCβII) expression, which was overexpressed in poor risk DLBCL, and analyzed the inhibitory mechanism of tumor suppression by Prox1. METHODS and RESULTS: We established the Prox1 stable transformants of Hela cells which originally lacked Prox1 expression, and examined them in vitro cell proliferation and in vivo tumor formation using nude mice. Our experiments showed that Prox1 had inhibitory effect on both cell proliferation and tumor formation. cDNA microarray analysis indicated that the expression of PKCβII was highly suppressed by Prox1 overexpression. In Prox1-expressing Hela cells, mRNA expression of PKCβII was undetectable and protein was significantly down-regulated compared with Mock cells. The selective PKCβII inhibitor, enzastaurin was less effective on cell proliferation of Prox1-expressing cells compared with Mock cells. These results showed that PKCβII was required for tumor cell proliferation. We focused on the regulatory mechanism of PKCβII expression by Prox1. The 5'-promoter analysis of PKC-β gene revealed that the proximal region between 110bp of the promoter containing 2 Sp1 binding sites was responsible for the endogenous promoter activity, and Prox1 did not affect that. Mithramycin A, a relatively specific Sp1 inhibitor, and Sp family knockdown by siRNA on Hela cells decreased PKCβII expression, suggesting that Sp family was important for PKCβII transcription. Interestingly, 5-aza-deoxycytidine (5-Az) treatment restored PKCβII mRNA expression in Prox1-expressing Hela cells, suggesting that 5'-promoter region of PKCβII was methylated in these cells. Actually, bisulfite sequencing showed that the predicted CpG island of PKC-β gene, especially around these Sp1 binding sites, was highly methylated in Prox1-expressing Hela cells compared with Mock cells and 5-Az relieved DNA methylation. CONCLUSION: Our study suggested that the decreased PKCβII expression suppressed tumor cell proliferation and Prox1 down-regulated PKCβII expression by enhancing DNA methylation of PKC-β 5'-promoter around Sp1 binding sites. This is the first to report the inverse relationship between Prox1 and PKCβII expression in cancer cells and the novel regulatory mechanism of PKC-β gene expression by Prox1 transcription factor. Disclosures: No relevant conflicts of interest to declare.