ALBERT

Description: CD26 is a transmembrane glycoprotein with intrinsic dipeptidyl peptidase IV (DPPIV) activity as well as costimulatory activity of mitotic signals triggered by the CD3/TCR complex. Based on the expression level of CD26, CD4+ and CD8+ T cells can be divided into 3 (high/intermediate/low or negative) subsets. The significance of CD26 has been studied mainly on CD4+ T cells, and CD26highCD4+ T cells are considered to represent effector memory T cells of a typical Th1 phenotype producing IL2 and IFNg. Furthermore, we reported a significant decrease of this subset in CML patients under imatinib therapy in comparison to those under IFNa therapy and normal volunteers. In contrast, the role of each subset of CD8+ T cells has not yet been clarified. Multi-parameter flow cytometry analysis was performed to characterize CD8+ T cells differentially expressing CD26 in combination with intracellular detection of effector molecules such as perforin (P) and granzyme B (Gr). The capacity to secrete effector cytokines such as IFNg following short-term stimulation was also assessed. As a result, according to the expression level of CD26, we could clearly categorize CD8+ T cells as follows: CD26highCD8+ T cells are defined as central memory T cells which has a phenotype of CD45RO+CD28+CD27+ IFNg+Gr−P+/−, CD26intCD8+ T cells as naïve T cells of CD45ROCD28+ CD27+ IFNg−Gr−P−, and CD26lowCD8+ T cells as effector memory/effector T cells of CD45RO−/+ CD28−CD27−IFNg++Gr++P++, respectively. We next investigated the effects of imatinib on 3 distinct subsets during CD8+ T cell differentiation program. Peripheral blood mononuclear cells were primed with anti-CD3/CD28 MAb and subjected to the grading doses of imatinib for short term culture, followed by flow cytometory. CFSE labeling was used for monitoring cell proliferation. Intriguingly, we found that imatinib dose-dependently inhibits activation, cytokine production and proliferation of CD26highCD8+ central memory T cell subsets in a differentiation stage-specific manner. Finally, we compared the absolute number of peripheral blood CD26highCD8+ T cell subsets between 20 patients with CML in imatinib-induced CCR and 20 normal volunteers, clearly indicating a significant decrease of this subset in CML patients (22.30/ml vs 45.60/ml, p

Description: Ligation of inhibitory receptors renders natural killer (NK) cells inactive against autologous tumors. Recently, the proteasome inhibitor bortezomib was shown to sensitize tumors to autologous NK-cell cytotoxicity in vitro. Here, we show bortezomib augments the antitumor effects of syngeneic NK-cell infusions in tumor-bearing animals; this effect is further enhanced in regulatory T cell (Treg cell)–depleted hosts. In vitro, bortezomib-treated tumors had higher tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and perforin/granzyme-mediated caspase-8 activity, which enhanced their susceptibility to NK-cell lysis. Bioluminescence imaging of mice with established tumors showed treatment with bortezomib and syngeneic NK cells reduced tumor growth and prolonged survival compared with controls receiving bortezomib or NK cells alone. In contrast, tumor progression was not delayed when animals received bortezomib and perforin-deficient NK cells, showing drug-induced augmentation in NK-cell cytotoxicity was mediated through perforin/granzyme. Furthermore, tumor growth was slower in bortezomib-treated recipients when host Treg cells were eradicated with anti-CD25 antibody before infusing NK cells compared with mice without Treg-cell ablation (tumor doubling time, 16.7 vs 4.9 days, respectively; P = .02). These findings suggest that depletion of Treg cells followed by bortezomib-induced tumor sensitization to autologous NK cells could be used as a novel strategy to treat cancer.

Description: This study found that MS-275, a novel synthetic benzamide histone deacetylase inhibitor (HDACI), blocked Akt/mTOR signaling in acute myelogenous leukemia (AML) HL60 and acute promyelocytic leukemia (APL) NB4 cells, as assessed by decreased levels of the phosphorylated (p)-Akt, p-p70 ribosomal S6 kinase (p70S6K), and p-S6K by Western blot analysis. Interestingly, further inactivation of mTOR by rapamycin analogue RAD001 (everolimus) significantly enhanced MS-275-mediated growth inhibition and apoptosis of these cells in parallel with enhanced upregulation of p27 kip1 and downregulation of c-Myc. In addition, RAD001 potentiated the ability of MS-275 to induce differentiation of HL60 and NB4 cells, as measured by expression of CD11b cell surface antigens, as well as reduction of nitroblue tetrazolium. Importantly, RAD001 potentiated the ability of MS-275 to induce expression of the myeloid differentiation-related transcription factor CCAAT enhancer binding protein e in these cells in association with enhanced acetylation of histone H3 on its promoter. Furthermore, RAD001 (5 mg/kg) significantly enhanced the effects of MS-275 (10 mg/kg) to inhibit proliferation of HL60 tumor xenografts in nude mice without adverse effects. Taken together, concomitant administration of a HDACI and a mTOR inhibitor may be a promising treatment strategy for the individuals with a subset of human leukemia.

Description: Abstract 4763 INTRODUCTION Peripheral blood involvement (leukemic presentation) is considered as a part of bone marrow involvement and detected about 18% of bone marrow (BM) involvement of follicular lymphoma (FL). It is not known whether leukemic presentation is an adverse prognostic factor in rituximab era. METHOD We retrospectively evaluate prognostic value of peripheral blood involvement in patients with follicular lymphoma received rituximab containing regimen as an initial therapy from October 2000 to January 2009. Leukemic presentation was defined by morphologic identification of an abnormal lymphoid population in the peripheral blood. RESULT Total 129 patients were treated with rituximab containing initial therapy. Bone marrow involvement was detected in 39 /108 (36.1%) patients and leukemic presentation was identified in 8 / 39 (20.5%) of patients with BM involvement. Leukemic presentation shows significant poorer progression free survival than BM involvement without peripheral blood involvement. (2yr PFS 23.4% vs 73.3% p=0.015) Multivariate analysis conducted by Cox proportional hazard analysis including five variables of FLIPI revealed Hb

Description: Introduction: Inotuzumab ozogamicin (CMC-544) is an antibody-targeted chemotherapeutic agent composed of a monoclonal antibody which targets the CD22 antigen, conjugated to calicheamicin, a potent cytotoxic antitumor antibiotic. Since CD22 is expressed on more than 90% of B-lymphoid malignancies, CMC-544 may be useful for treating patients (pts) with B-cell non-Hodgkin lymphoma (B-NHL). In a phase I study in the US and EU, CMC-544 showed definite clinical activity in pts with relapsed/refractory B-NHL (both follicular and diffuse large) with clinically manageable thrombocytopenia as the main toxicity. In this phase 1 study, the safety, tolerability, efficacy and pharmacokinetics (PK) of CMC-544 was evaluated in Japanese pts with relapsed or refractory B-cell NHL. Methods: CMC-544 was administered IV once every 28 days ± 2 days (1 cycle). A dose escalation was planned using 1.3 mg/m2 and 1.8 mg/m2, the maximum tolerated dose (MTD) which was previously determined in non-Japanese pts. Pts were allowed to enroll in the study if they had relapsed or refractory CD22+ B-NHL. Tumor responses were evaluated by investigator’s assessment according to the International Workshop Criteria for NHL. Results: Enrollment for this study is complete and included 13 pts (6 women, 7 men, median age [range] of 49 yrs [43–72]). All of the 13 pts who were enrolled had follicular lymphoma and had received at least one regimen of rituximab alone or rituximab-containing chemotherapy in their prior treatments. The median number of prior treatment regimens was 1 (range: 1–13). In dose escalation, no pts had dose limiting toxicities and the tolerability in the MTD previously determined in non-Japanese pts (1.8mg/m2) was confirmed for Japanese pts. 3 pts and 10 pts were treated with 1.3 mg/m2 and 1.8 mg/m2 of CMC-544, respectively. The median number of CMC-544 treatment cycles was 3 (range: 2–8). The most common drug-related adverse events (AEs, all grades ≥ 35% pts) included thrombocytopenia (100%), leukopenia (92%), neutropenia (85%), elevated AST (85%), anorexia (85%), lymphopenia (85%), nausea (77%), elevated ALT (54%), malaise (46%), and headache (46%). Grade 3/4 AEs ≥ 15% pts were: thrombocytopenia (54%), lymphopenia (31%), neutropenia (31%), and leukopenia (15%). 7 pts discontinued treatment due to AEs; 1 pt because of grade 2 rash, 1 pt because of grade 2 urticaria and 5 pts because of AEs which required treatment delays of 〉3 wks (2 pts with prolonged thrombocytopenia, 1 pt with prolonged thrombocytopenia and neutropenia, 1 pt with neutropenia and elevated alkaline phosphatase, and 1 pt with prolonged neutropenia and elevated total bilirubin). PK analyses demonstrated that maximum serum concentration (Cmax) and area under the curve (AUC) of CMC-544 increased in a dose dependent manner. Both parameters increased with the second dose in the second cycle. At all dose levels, terminal half-life (t1/2) was prolonged, and total clearance (CL) was decreased in the second cycle. Overall, the PK profile was similar to that of the previous study with non-Japanese pts. 7 pts had CRs (CR + CRu), 4 pts had PRs, and 2 pts had stable disease. The objective response rate (ORR) was 85% (11/13). Conclusions: The tolerability of CMC-544 for Japanese pts with relapsed or refractory follicular B-NHL who had been pretreated with rituximab was confirmed at 1.8 mg/m2 administered once every 28 days. This is the same dose level as the MTD for non-Japanese pts. The PK profile of CMC-544 in Japanese pts was similar to that in non-Japanese pts. Based on the acceptable safety profiles and a high preliminary ORR, CMC-544 should be considered for further investigations in Japanese pts with relapsed or refractory B-NHL who have been pretreated with rituximab.

Description: Abstract 2969 Poster Board II-945 The roles of MLL holocomplex in embryogenesis, hematopoiesis and tumor suppression MLL is an epigenetic transcriptional regulator that serves critical roles in multiple developmental and homeostatic processes. It is essential for proper maintenance of Hox gene expression during embryogenesis and hematopoiesis, and regulates expression of cyclin-dependent kinase inhibitors (CDKIs) in endocrine tissues such as the pancreas. Conversely, mis-regulation of MLL-dependent transcriptional pathways is associated with various pathologies. Activating mutations of MLL result in constitutive expression of Hox genes leading to acute leukemia, whereas loss of MLL transcriptional function through mutations of menin, an essential MLL-associated cofactor, leads to decreased expression of CDKIs, hyper proliferation of endocrine cells, and development of multiple endocrine neoplasias. Thus, MLL functions in critical growth regulatory transcriptional circuits that are subject to perturbations in various malignancies. MLL is translated as a large precursor protein that subsequently undergoes proteolytic processing into two fragments (MLLN and MLLC) that self-associate through non-covalent interaction to form an intra-molecular complex. MLL is processed by the Taspase I endopeptidase, which specifically cleaves sites that are evolutionally conserved with MLL2 and Drosophila TRX. However the biological requirement for processing remains unknown. MLLN appears to comprise a targeting sub-unit that contains several motifs involved in DNA binding (AT hooks, CXXC domain) and chromatin recognition (PHD fingers, bromo domain). By contrast, MLLC has features of a transcriptional effector sub-unit that possesses a potent transactivation domain and a methyltransferase (SET) domain specific for lysine 4 of histone H3, an epigenetic mark associated with transcriptionally active states. The SET domain also associates with accessory factors (WDR5, RBBP5 and ASH2L) that promote optimal substrate recognition and enzymatic activity. Thus, the MLL intra-molecular complex can be conceptualized as comprised of an MLLC effecter sub-unit tethered to the MLLN targeting sub-unit by non-covalent association. This model has prompted the hypothesis that MLL may serve functionally distinct roles in transcriptional regulation dependent on the conditional association or disassociation of the MLLC subunit. Our biochemical study shows that MLL indeed takes two different forms: an MLLN/MLLC holocomplex and an MLLN complex without MLLC. We examined the roles of the two distinct complexes using a genetic approach employing knock-in mice with targeted mutations of the MLL processing sites. Mice engineered to constitutively express the MLLN subunit without MLLC (thus produce only the MLLN complex) displayed embryonic lethality, hematopoietic defects and transcriptional defects that phenocopy MLL deficiency. By contrast, mutations that disable proteolytic processing, which can assume the MLLN/MLLC holocomplex but not the MLLN complex, had no adverse consequences, demonstrating the lack of an essential role for the MLLN complex. The intra-molecular association of MLLN with MLLC is mediated in part by the PHD1 finger of the MLLN subunit. An MLL mutant lacking PHD1, which mimics deletion mutants associated with human T-cell leukemias, is unable to form an MLLN/MLLC holocomplex. Hence, this oncogenic mutation likely results in loss of function of MLL, suggestive of a potential tumor suppressor role for MLL in the lymphoid lineage similar to that of menin in the endocrine tissues. Our results support the critical roles of the MLLN/MLLC holocomplex in embryogenesis and hematopoiesis and a possible tumor suppressor function in the lymphoid lineage. Disclosures: No relevant conflicts of interest to declare.

Description: Human embryonic stem cells (hESCs) proliferate infinitely and are pluripotent. Only a few reports, however, describe specific and efficient methods to induce hESCs to differentiate into mature blood cells. It is important to determine whether and how these cells, once generated, behave similarly with their in vivo–produced counterparts. We developed a method to induce hESCs to differentiate into mature neutrophils. Embryoid bodies were formed with bone morphogenic protein-4, stem cell factor (SCF), Flt-3 ligand (FL), interleukin-6 (IL-6)/IL-6 receptor fusion protein (FP6), and thrombopoietin (TPO). Cells derived from the embryoid bodies were cultured on a layer of irradiated OP9 cells with a combination of SCF, FL, FP6, IL-3, and TPO, which was later changed to granulocyte–colony-stimulating factor. Morphologically mature neutrophils were obtained in approximately 2 weeks with a purity and efficiency sufficient for functional analyses. The population of predominantly mature neutrophils (hESC-Neu's) showed superoxide production, phagocytosis, bactericidal activity, and chemotaxis similar to peripheral blood neutrophils from healthy subjects, although there were differences in the surface antigen expression patterns, such as decreased CD16 expression and aberrant CD64 and CD14 expression in hESC-Neu's. Thus, this is the first description of a detailed functional analysis of mature hESC-derived neutrophils.

Description: Abstract 2700 Poster Board II-676 Background: Hepatitis B virus reactivation after systemic chemotherapy including rituximab is a well-documented complication. However, no studies have investigated the influence of hepatitis C virus (HCV) infection for hepatic toxicity of diffuse large B-cell lymphoma (DLBCL) patients treated by rituximab containing chemotherapy. The prognostic value of HCV infection in DLBCL in the era of rituximab was also unclear. Herein we conducted a multicenter retrospective analysis to compare the outcome and hepatic toxicity of DLBCL patients with and without HCV infection treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) as an initial therapy. Methods: We analyzed 548 patients: HCV-positive (n=126) or -negative (n=422) patients with CD20-positive DLBCL receiving RCHOP between January 2004 and March 2008. HCV-negative patients treated during same period with HCV-positive patients in each institute were enrolled. Hepatitis B surface antigen positive patients were excluded in this study. For survival analysis, event-free survival (EFS) and overall survival (OS) were compared according to HCV infection. The definition of severe hepatic toxicity was more than Grade 3 transaminases increase according to National Cancer Institute of Canada criteria. The change of serum HCV-RNA levels was examined in 33 HCV-positive patients. Results: Before the treatment, HCV-positive patients had higher age (P

Description: Abstract 786 Murine models have shown infusions of donor NK cells from hematopoietic stem cell transplant donors can prevent GVHD while simultaneously mediating a graft-vs-tumor (GVT) effect. Reduction of GVHD by alloreactive NK cells can be mediated indirectly through the eradication of host antigen presenting cells (APC) or directly by NK cell killing of alloreactive T cells. To assess how the timing of NK cell administration impacts these effects, donor NK cells from B10.d2 (H-2d) mice (1-2×106 cells) were infused into MHC-matched BALB/c (H-2d) recipients following lethal irradiation (950cGy) at one of the following time-points: 1) two days prior to a T cell replete (TR) HCT to target host APC or 2) at the time of a TR-HCT or 3) five days following a TR-HCT to target both host APC and in vivo primed alloreactive donor T cells. We also evaluated whether donor NK cells given on the day of a T cell deplete (TD) HCT could be used to prevent GVHD in mice that subsequently received a delayed donor lymphocyte infusion (DLI) given four days following HCT. Administration of donor NK cells two days prior to allogeneic TR-HCT did not result in a reduction of GVHD (figure). In contrast, the administration of NK cells given either at the time of a TR-HCT or five days following a TR-HCT reduced the incidence of GVHD (GVHD incidence 70% (p=0.2) and 40% (p=0.01) respectively) compared to controls that did not receive NK cells following a TR-HCT (GVHD incidence 100%). Similarly, mice that received a TD-HCT followed by a DLI on day four had a significantly lower incidence of GVHD when NK cells were infused on the day of transplantation compared to controls that did not receive donor NK cells (GVHD incidence 40% vs 100% respectively, p=0.01). Using bioluminescence imaging, we next investigated the impact of the timing of NK cell infusions on GVT effects in tumor bearing mice. Luciferase transduced RENCA tumors were injected intravenously into BALB/c mice ten days prior to allogeneic HCT. Tumor progression was significantly delayed in recipients of a TR-HCT when NK cells were infused on day five compared to when NK cells were infused two days prior to or at the same day of a TR-HCT (tumor doubling times 22.9 days, 7.6 days and 8.5 days respectively; p=0.03) (figure). This delay in tumor progression correlated with a significant improvement in overall survival; recipients of a TR-HCT given NK cells on day five had significantly longer survival compared to recipients of TR-HCT that did not receive NK cells (median survival 54±14 days vs 44±9; p=0.008), whereas infusion of NK cells prior to or concomitant with a TR-HCT did not significantly prolong survival (median survival 49±4 days and 50±12 days respectively; p=0.23). A comparable delay in tumor progression and longer survival was observed in mice that received a TD-HCT followed by a DLI on day four when NK cells were infused at the time of transplant compared to controls not receiving NK cells (tumor doubling time 19.7 days vs 7.2 days and survival 62±16 days vs. 50±9 days respectively; p=0.07). In conclusion, these results show that the timing of adoptive donor NK cell transfer has a critical impact on the ability of NK cells to prevent GVHD and enhance GVT effects following both T-cell replete and T-cell depleted allogeneic HCT. Following a TR-HCT, a delayed add-back of NK cells maximizes GVHD reducing and anti-tumor effects of adoptively transferred donor NK cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1597 Poster Board I-623 Leukemia stem cells (LSCs) are in a quiescent state, which maybe contributes to their drug-resistant character. It is essential to eradicate LSCs to develop curative therapy for leukemia. To verify molecular mechanisms by which LSCs maintain a dormant state, we examined the activity of the major pro-survival signal pathways in LSCs (CD34+/CD38- compartment) and non-LSCs (CD34+/CD38+ compartment) counterparts from patients with acute myelogenous leukemia (AML, n=3) and acute lymphoblastic leukemia (ALL, n=1) by FACS. Interestingly, LSCs compartment expressed a greater amount of the phosphorylated forms of STAT5 (p-STAT5) than non-LSCs counterparts in all patients. Further studies found that levels of interleukin-1b (IL-1b) were significantly down-regulated in LSCs compared with non-LSCs (p