ALBERT

Description: The past decade has shown a marked increase in the use of high-throughput assays in clinical research into human cancer, including acute myeloid leukemia (AML). In particular, genome-wide gene expression profiling (GEP) using DNA microarrays has been extensively used for improved understanding of the diagnosis, prognosis, and pathobiology of this heterogeneous disease. This review discusses the progress that has been made, places the technologic limitations in perspective, and highlights promising future avenues

Description: Abstract 1689 Poster Board I-715 Introduction The use of the proteasome inhibitor bortezomib has demonstrated activity in multiple myeloma and lymphomas. The HDAC inhibitor romidepsin is being evaluated in CTCL and PTCL, though its activity in B-cell lymphomas is less clear. We hypothesized that the combination of bortezomib and romidepsin would result in synergistic apoptosis in different B-cell NHL cell lines based upon the observed activity of this combination in more mature B-cell malignancies such as myeloma. Experimental Design Daudi, HT, Ramos and SUDHL-4 cell lines were exposed to different concentrations of bortezomib and romidepsin, separately, concurrently, and sequentially. Cell viability was assessed using MTT-assay, induced apoptosis was evaluated using Annexin V and PI staining from 24-48 hours. Apoptosis was also evaluated using western blot analysis of caspases and PARP cleavage. LC3 and HDAC6 level expressions were performed to determine if the effect of the combination was a result of the aggresome or autophagy pathway. Cell cycle studies were also performed to study if there were any changes after treating cells with the combination. Results The combination of bortezomib and romidepsin resulted in synergistic B-cell apoptosis as measured by MTT-assay with combination indices of 〈 0.5. This was associated with increased caspases and PARP cleavage as early as 24 hours after exposure. Order of addition experiments demonstrated definite sequence specificity. When romidepsin was added first, and 6 hours later followed by bortezomib, apoptosis was enhanced, compared to both agents being given concurrently or when bortezomib was administered first. Cell cycle analysis studies demonstrated that pretreatment of cells with romidepsin for 6 hours followed by the addition of bortezomib arrested the cells in G2M phase. HDAC6 expression was significantly reduced following combination therapy, and LC3-I was cleaved to LC3-II in treated cells suggesting that the combination affected aggresome formation and autophagy. Conclusion The combination of romidepsin and bortezomib at low nanomolar concentrations suggests that this may be an important clinical combination to test in patients with relapsed or refractory B-cell malignancies. Sequence of administration data is currently being tested to determine if the effect is a result of autophagy inhibition as is seen in myeloma cell lines. Additional mechanistic studies will be presented with the goals of identifying predictors of response that can then be validated in prospective clinical trials. Disclosures Lechowicz: Gloucester: Consultancy. Kaufman:Millennium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Gloucester: Research Funding; Novartis: Consultancy; BMS: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy. Flowers:Millennium: Research Funding.

Description: Abstract 3701 Poster Board III-637 Background Romidepsin is an anti-neoplastic agent that has been identified as a novel pan-HDAC inhibitor with single-agent activity in T-cell lymphoma. In a combined analysis of 167 patients (pts) with cutaneous T-cell lymphoma (CTCL) from 2 clinical studies (GPI and NCI studies), the overall response rate was 35%, including 10 pts with a complete clinical response (CCR). Median duration of response was 13.8 months and 42% of pts with advanced disease (stage ≥IIB) responded [Demierre et al. J Clin Oncol 27:15s, 2009 (suppl; abstr 8546)]. The most common hematologic abnormalities in these pts included anemia (41%), thrombocytopenia (34%), neutropenia (27%), and lymphopenia (26%). Most hematologic toxicities were Grade 1 or 2, although 'Grade 3 events were observed. These events were reversible and a small portion of the patients discontinued the study drug because of these events (2%). This report details an analysis of platelet counts in pts receiving romidepsin and an investigation into the mechanism of thrombocytopenia in nonclinical studies. Methods Pts with CTCL who received ≥1 prior systemic therapy failure and had an ECOG PS of 0-2 were enrolled in 2 single-arm, open-label, multicenter and international clinical studies. Treatment with QTc prolonging therapies or CYP34A inhibitors was prohibited and pts with significant cardiovascular abnormalities were excluded. Romidepsin at 14 mg/m2 was administered as a 4-hr IV infusion on days 1, 8, and 15 of a 28 day cycle. Nonclinical studies were conducted in mice to investigate the mechanism of romidepsin effects on platelets. Romidepsin was administered to female BALB/c mice at doses of 1 or 4 mg/kg by tail-vein injection on days 1, 5 and 9. Blood samples were collected every 2 days from alternating groups of mice to minimize effects of bleeding on platelet counts. Results In clinical studies, there is a mean decrease in platelet counts during the treatment period of each cycle, and a return to baseline levels or above between cycles observed in both clinical studies as described in the table below. No clinically meaningful change has been observed in the central tendency over 4 cycles of treatment in both studies. In the mouse studies, dose-dependent effects were seen on both WBC and platelet counts. Day 2 WBC counts dropped to 45% and 10% of normal at the 1 and 4 mg/kg doses, respectively. WBC counts remained low until after the dosing period in the 1 mg/kg romidepsin group, but recovered more quickly in the 4 mg/kg group. Day 2 platelet counts were 70% of normal at the 1 mg/kg dose and remained near this level until day 10, followed by recovery to normal at day 15. At the 4 mg/kg dose, profound thrombycytopenia was induced, with platelet counts only 20% of normal on days 4-6. Platelet counts slowly recovered to 70% of normal by day 15. Plasma thrombopoietin levels were normal throughout the experiment for the 1 mg/kg group, and showed a large increase to 275% of normal on day 6 in the 4 mg/kg group, which is the expected response to thrombocytopenia as a signal to increase platelet production and indicates that platelet reduction is not attributable to defective TPO production. Bone marrow megakaryocyte populations are being examined to determine the effects of romidepsin on these platelet-producing cells. Conclusions Following romidepsin administration, a saw tooth pattern is observed in the reduction and recovery of platelets. Recovery of platelets appears to occur more rapidly in humans than in mice; however, the effects are reversible after dosing in clinical studies and in murine models. In the clinical data the recovery pattern suggests that the transient effects are direct and are not effects on bone marrow. Disclosures: Whittaker: Gloucester Pharmaceuticals: Research Funding. Prince:Gloucester Pharmaceuticals: Consultancy. Demierre:Gloucester Pharmaceuticals: Consultancy, Honoraria. Lonial:Gloucester Pharmaceuticals: Honoraria. Kim:Gloucester Pharmaceuticals: Consultancy, Honoraria. Nichols:Gloucester Pharmaceuticals: Employment, Equity Ownership. Nix:Gloucester Pharmaceuticals: Employment.

Description: Precursor T-cell acute lymphoblastic leukemia (T-ALL) in children represents a clinical challenge, because relapses are usually fatal. It is thus necessary to identify high-risk patients as early as possible to effectively individualize treatment. We aimed to define novel molecular risk markers in T-ALL and performed array-based comparative genomic hybridization (array-CGH) and expression analyses in 73 patients. We show that DNA copy-number changes are common in T-ALL and affect 70 of 73 (96%) patients. Notably, genomic imbalances predicted to down-regulate the TGF-β or up-regulate the PI3K-AKT pathways are identified in 25 of 73 (34%) and 21 of 73 (29%) patients, suggesting that these pathways play key roles in T-ALL leukemogenesis. Furthermore, we identified a deletion at 6q15-16.1 in 9 of 73 (12%) of the patients, which predicts poor early treatment response. This deletion includes the CASP8AP2 gene, whose expression is shown to be down-regulated. The interaction of CASP8AP2 with CASP8 plays a crucial role in apoptotic regulation, suggesting a functional link between the clinical effect of the deletion and the molecular mode of action. The data presented here implicate the TGF-β and PI3K-AKT pathways in T-ALL leukemogenesis and identify a subgroup of patients with CASP8AP2 deletions and poor early treatment response.

Description: Abstract 1955 Poster Board I-978 The detection of chromosome abnormalities in mature B-cell neoplasms by conventional cytogenetics remains difficult, mainly due to the low proliferative rate of mature lymphoid cells. The current FISH panel for chronic lymphocytic leukemia (CLL) is designed to detect some of the more common abnormalities of prognostic significance in CLL [i.e., del(6q), del(11q), +12, del(13q), del(17p)]. This CLL FISH panel has improved the detection rate of these markers by making it possible to obtain cytogenetic information from interphase cells; however, as it is limited to only these 5 markers, it cannot detect all abnormalities associated with CLL. More importantly, the impact of other chromosome abnormalities on prognosis and disease progression, with and without the presence of these 5 prognostic markers, is not known. CpG-oligodeoxynucleotides (ODNs) such as DSP30 activate cells of the immune system in a sequence-dependent manner and promote proliferation of CLL cells in vitro [Decker et al. Blood 2000;95:999-1006]. They also upregulate costimulatory molecules and potential target antigens during immunotherapy. The use of DSP30 in combination with interleukin 2 (IL2) has proven effective in increasing the detection of chromosome abnormalities in CLL [Dicker et al. Blood 2006;108:3152-60] and other mature B-cell lymphoid malignancies by conventional cytogenetics [Struski et al. Leukemia 2009;23:617-9], when compared to the well-established B-cell mitogens. In our extensive clinical experience of incorporating DSP30/IL2 into our culture media, this cocktail has significantly increased the detection of chromosome abnormalities in CLL by conventional cytogenetics, from 55% to greater than 80%. We thus decided to investigate if various other lymphoid malignancies would respond to the mitogen activity of DSP30/IL2 as well as or better than CLL. Specifically, we evaluated 812 cases of mature B-cell lymphoid malignancies that were abnormal by flow cytometry, morphology, or cytogenetic analysis. All samples (bone marrow or blood) were cultured for approximately 72 hours using the DSP30/IL2 mitogen cocktail. Of these 812 cases, 746 (91%) provided sufficient mitotic index and quality for a complete cytogenetic analysis and interpretation. Of the CLL cases (n=509), 79 were initially interpreted as normal by conventional cytogenetic analysis, but were later interpreted as abnormal by FISH for deletion 13q only. In view of the known cryptic nature of this deletion in CLL, these cases were not included in the study, leaving a total of 430 CLL cases, and thus bringing the total number of cytogenetically successful study cases to 667. In addition to the 430 CLL cases, there were 14 variant CLLs; 36 diffuse large B-cell lymphomas (DLBCLs); 35 follicular lymphomas; 34 non-Hodgkin lymphomas (not further specified); 29 marginal zone B-cell lymphomas of splenic type (sMZBCL); 27 mantle cell lymphomas (MCLs), of which 8 were blastoid; 16 MZBCL of MALT type; 13 hairy cell leukemias (HCLs); 12 lymphoproliferative disorders (not further specified); 10 lymphoplasmacytic lymphomas (LPLs); 6 Burkitt lymphomas; 3 Hodgkin lymphomas; and 2 B-cell prolymphocytic leukemias (PLLs). Of particular interest is the fact that we detected clonal abnormalities in 100% of HCLs, blastoid MCLs, variant CLLs, and B-cell PLL, as well as in 97% of sMZBCLs, 89% of DLBCLs, and 80% of LPLs This is of great importance since HCLs and LPLs are rarely abnormal by conventional cytogenetics using the more traditional combinations of mitogens making it difficult to identify markers of prognostic significance. In conclusion, our findings demonstrate that the DSP30/IL2 cocktail induces proliferation of various B-cell mature lymphoid disorders and that its mitogenic action is not limited to CLL. We are continuing to develop our understanding of the considerable response of specific lymphoid malignancies to the DSP30/IL2 cocktail by correlating additional clinical data, and hope that the end result will open new avenues in regards to prognostic outcome and therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Description: Higher levels of procoagulant factors and factor XII deficiency may be risk factors for first venous thromboembolism (VTE). We studied associations of coagulation factors IX through XIII with risk of future VTE in 2 general population samples. Using a nested case-control study combining the 21 860 participants of the Atherosclerosis Risk in Communities study and the Cardiovascular Health Study, we determined antigenic levels of these coagulation factors in primarily pre-event blood samples from 462 participants who subsequently developed VTE and 1047 participants who remained free of VTE. Only elevated levels of factors IX and XI were associated with increased risk of VTE after adjustment for age, sex, race, and study. For factor IX, the odds ratio (OR) was 1.4 (95% confidence interval [CI], 1.0-2.0) comparing the top to bottom quintile. The OR for factor XI was higher: 2.0 (95% CI, 1.4-2.9). With further adjustment for body mass index and diabetes, only elevated factor XI remained associated with VTE risk: OR 1.8 (95% CI, 1.3-2.7). Associations were similar by study and whether the thrombosis was idiopathic or secondary. Factor XII deficiency was not related to VTE risk. Among these procoagulant factors, only elevated factor XI was a risk factor for VTE.

Description: The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. Using liquid chromatography–tandem mass spectrometry, we analyzed the protein composition of microparticles (MPs) originating from the cell surface of EPC cultures. Our data revealed that the conventional methods for isolating mononuclear cells lead to a contamination with platelet proteins. Notably, platelets readily disintegrate into platelet MPs. These platelet MPs are taken up by the mononuclear cell population, which acquires “endothelial” characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n = 526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. Our study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits in clinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

Description: Abstract 18 Introduction: Long-term survival in pediatric relapsed AML is only 20-30%. Optimal reinduction therapy is unknown, and there is a concern about cardiotoxicity with repeated anthracycline use at relapse. Preclinical in vitro and animal studies, and limited clinical data suggest that liposomal daunorubicin (DaunoXome®, DNX) is less cardiotoxic. These considerations lead to a phase III study, in the setting of the International BFM Study Group. Materials and methods: FLAG was randomised against FLAG/DNX in the 1st reinduction course. The conventional 5-days FLAG only was recommended as the 2nd course. DNX was dosed at 60 mg/m2/day on days 1, 3 and 5. After induction, allogeneic stem cell transplantation was generally recommended, but time-to-transplant could be bridged by high- or low-intensive consolidation therapy. Primary endpoint of the study was early treatment response, based on bone marrow examination shortly before reinduction course 2, and defined as either good (≤20% leukemic blasts) or poor (〉20% leukemic blasts). This endpoint was chosen because of its prognostic value in earlier relapsed AML BFM-trials, and because compliance with an extended protocol guideline was likely to be suboptimal within the context of a highly multinational and multicenter AML Relapse protocol. However, secondary endpoints were defined, including the CR2 rate determined after 2 courses, long-term survival, and toxicity. Patients with AML M3 and those 〉18 years of age at initial diagnosis were ineligible. The study opened in most countries in 2002/2003. The study closed for accrual on April 1, 2009 when the required 360 fully eligible and evaluable patients had been randomized. Early and late relapsed AML was defined as a relapse within or after 1 year from initial diagnosis, but this only influenced treatment in that early relapsed AML patients were eligible for haploidentical SCT, while late relapsed AML patients were eligible for autologous SCT, if a matched or partly mismatched transplant was not possible. Thirteen groups from 20 countries and 〉100 centers have enrolled patients, with informed consent and after approval of the study by regulatory authorities. Data are presented according to intention-to-treat, with a median follow-up of 2.7 years for patients at risk. Results: Overall 4-year probability of survival (pOS) was 35% SE 2%, the overall CR2 rate 62%. The good early responders had a 4-year pOS of 45% SE 3% versus 10% SE 3% for poor responders (p

Description: Abstract 634 FLT3, a transmembrane receptor tyrosine kinase constitutively activated via mutation in blasts of patients (pts) with AML, is an important therapeutic target. Blasts from approximately 25% of pts have a length or internal tandem duplication (ITD) mutation in the juxtamembrane region or tyrosine kinase domain (TKD1) of FLT3, which is associated with reduced disease-free survival and overall survival (OS), particularly in pts with normal cytogenetics. Blasts from 5–10% of pts have a point mutation (typically D835Y) in the tyrosine kinase domain (TKD); the effect of this mutation on prognosis is uncertain. Midostaurin (PKC412) is a multi-targeted kinase inhibitor with demonstrated clinical activity in FLT3-mutant (FLT3–mut) and FLT3-wild-type (FLT3–wt) AML (peripheral blood blast reduction in 70% and 30% of pts, respectively) but rarely produces complete remissions). Preclinical studies demonstrated synergy between FLT3 inhibitors and chemotherapy. We conducted a Phase 1b trial to investigate the feasibility of administering daunorubicin (60 mg/m2 IV, days 1–3) and cytarabine (100 mg/m2 IVCI, days 1–7) induction and high-dose cytarabine post-remission therapy (3 gm/m2 over 3h every 12h, days 1, 3, and 5 for 3 cycles) plus oral midostaurin at 100 mg or 50 mg each twice daily on days 8–21 (sequentially) or days 1–7, 15–21 (concomitantly) with all chemo cycles in newly diagnosed pts under age 61 with de novo AML. Whereas 100 mg of midostaurin plus induction chemotherapy was poorly tolerated due to nausea and vomiting, the 40 pts who received 50 mg of midostaurin orally twice daily ( 20 each on the sequential and concomitant schedules; 27 FLT3–wt; 13 FLT3–mut [9 with an ITD]), tolerated the combination well. Median midostaurin exposure was 133 days (range 21–975) for the FLT3–mut pts and 90 days (range 7–1016) for FLT3–wt pts. Maintenance therapy with midostaurin was allowed with investigator discretion and was received by 5 pts (3 FLT3–mut, 2 FLT–wt). The median ages for the FLT3–wt and FLT3–mut pts were 50 years (range 25–60) and 46 years (range 20–65), respectively. 77% of the FLT3–mut pts displayed normal, 15% adverse and 8% other intermediate cytogenetics compared with 18.5%, 26%, and 26%, respectively, for FLT3-wt (also 18.5% favorable; 11% unknown). Complete response occurred in 32/40 (80%) of all pts (20/27 [74%] of FLT3–wt patients, 12/13 [92%] of FLT3–mut pts). Patients were censored at the last date they were known to be alive with a median post treatment follow-up for FLT3-mut pts of 1059 days and 1086 days for FLT3-wt. Even accounting for their differing cytogenetics and ages, the OS of the FLT3–mut subgroup was expected to be inferior to that of the FLT3–wt subgroup. However, we report that the 1 and 2 year OS for the pts with FLT3–mut AML was 85% and 62%, respectively, and was comparable to that of the FLT3–wt subgroup (81% and 59%, respectively). Although based on small numbers and not stratified for type of FLT3 mutation (TKD, ITD, ITD length, location, or allelic ratio), these long-term results suggest that combination therapy with a FLT3 inhibitor and chemotherapy might be effective enough to obviate the perceived need for allogeneic stem cell transplantation for FLT3–mut AML pts in first complete remission. Moreover, these data support the rationale for the ongoing international phase 3 study of induction, post-remission intensification, and maintenance with midostaurin (50 mg po bid) or placebo. Disclosures: Stone: Novartis: Research Funding, ad hoc consultancy; Cephalon: ad hoc consultancy. Off Label Use: midostaurin with chemothereapy for AML. Paquette:Novartis: Honoraria, Research Funding, Speakers Bureau. Schiller:Novartis: Research Funding, Speakers Bureau; Millenium: Research Funding, Speakers Bureau; Genzyme: Research Funding; Vion: Research Funding; Centocor: Research Funding; Eli Lilly: Research Funding; Celgene: Research Funding. Schiffer:Novartis: Consultancy, Research Funding; Genzyme: Consultancy. Ehninger:Novartis: Honoraria, Research Funding. Cortes:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Wyeth: Research Funding. Kantarjian:Novartis: Research Funding. DeAngelo:Bristol-Myers Squibb: Speakers Bureau; Celgene: Speakers Bureau; Enzon: Speakers Bureau; Novartis: Speakers Bureau. Huntsman-Labed:Novartis: Employment, Equity Ownership. Dutreix:Novartis: Employment, Equity Ownership. Rai:Novartis: Employment, Equity Ownership. Giles:Novartis: Research Funding; Merck: Research Funding; Bristol-Myers Squibb: Research Funding; Vion: Research Funding.

Description: Abstract 2168 Poster Board II-145 The deregulated tyrosine kinase associated with t(9;22) fusion protein BCR-ABL is efficiently targeted by tyrosine kinase inhibitors (Imatinib, Nilotinib and Dasatinib). It is generally believed that most patients have residual disease. The inability to eliminate the refractory leukemia stem cell in chronic myeloid leukemia (CML) is not well understood. The refractory stem cell is present despite effective inhibition of the BCR-ABL kinase in these cells, and dissociation between kinase inhibition and cell death implies lack of dependency on BCR-ABL aberrant kinase activity, unlike the one seen in more mature CML cells. Thus the oncogene addictive dependency of these leukemia stem cells may be overcome by intrinsic and/or extrinsic factors/pathways. One such pathway involved in hematopoeitic stem cell maintenance is the Wnt/β-catenin signaling pathway. BCR-ABL signaling is known to directly activate the Wnt/β-catenin pathway. The loss of function of a negative regulator of the Wnt/β-catenin pathway, known as GSK3β is associated with CML progression from chronic phase to the blast phase. Lastly, the loss of β-catenin in murine models impairs the self renewal capacity of both the normal and the BCR-ABL+ leukemia stem cell. Thus this pathway is important during various stages of the disease. We explored the effect of Wnt inhibition using a novel Wnt pathway inhibitor (AG-214, University of Michigan). AG-214 is structurally related to a previously reported Wnt-inhibitor. AG-214 is able to antagonize β-catenin/TCF in luciferase reporter assays, and expression of Wnt targets in colon cancer cell lines. We utilized a serum free culture system with 5 added cytokines and treated both CML blast crisis CD34+ as well as chronic phase CD34+ cells with AG-214. Our in vitro experiments show that primary blast crisis CD34+ cells are induced to undergo apoptosis at an IC-50 of approximately 2 μM. Furthermore, combination of 1.25 μM of AG-214 with 2 μM Imatinib achieved greater than 50% apoptosis. Analysis of chronic phase CML CD34+ cells showed that targeting of Wnt/β-catenin pathway requires higher concentration of the Wnt-inhibitor (〉2.5 μM), and that the addition of Imatinib can cooperate to enhance the apoptotic response. Furthermore, chronic phase progenitors (Lin-CD38+/CD34+) are more sensitive to lower concentrations of AG-214 whereas 5 μM is required to induce significant apoptosis of ∼70% in the primitive leukemia stem cell (Lin-/CD38-/CD34+) population, and addition of 2 μM Imatinib increased their apoptotic response to ∼84%. Normal CD34+ cells do not undergo significant apoptosis with 5 μM of AG-214 (∼10%) even when combined with 2 μM Imatinib (∼20%). Targeting of the Wnt/β-catenin pathway enhances apoptosis in both blast crisis and chronic phase CML progenitors and leukemia stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Although well characterized in the mouse, the role of Notch signaling in the human T-cell receptor αβ (TCR-αβ) versus TCR-γδ lineage decision is still unclear. Although it is clear in the mouse that TCR-γδ development is less Notch dependent compared with TCR-αβ differentiation, retroviral overexpression studies in human have suggested an opposing role for Notch during human T-cell development. Using the OP9-coculture system, we demonstrate that changes in Notch activation are differentially required during human T-cell development. High Notch activation promotes the generation of T-lineage precursors and γδ T cells but inhibits differentiation toward the αβ lineage. Reducing the amount of Notch activation rescues αβ-lineage differentiation, also at the single-cell level. Gene expression analysis suggests that this is mediated by differential sensitivities of Notch target genes in response to changes in Notch activation. High Notch activity increases DTX1, NRARP, and RUNX3 expression, genes that are down-regulated during αβ-lineage differentiation. Furthermore, increased interleukin-7 levels cannot compensate for the Notch dependent TCR-γδ development. Our results reveal stage-dependent molecular changes in Notch signaling that are critical for normal human T-cell development and reveal fundamental molecular differences between mouse and human.

Description: During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

Description: The ability of CD8+ T cells to engage a diverse range of peptide–major histocompatibility complex (MHC) complexes can also lead to cross-recognition of self and nonself peptide-MHC complexes and thus directly contribute toward allograft rejection or autoimmunity. Here we present a novel form of cross-recognition by herpes virus–specific CD8+ cytotoxic T cells that challenges the current paradigm of self/non-self recognition. Functional characterization of a human leukocyte antigen (HLA) Cw*0602-restricted cytomegalovirus-specific CD8+ T-cell response revealed an unusual dual specificity toward a pp65 epitope and the alloantigen HLA DR4. This cross-recognition of HLA DR4 alloantigen was critically dependent on the coexpression of HLA DM and was preferentially directed toward the B-cell lineage. Furthermore, allostimulation of peripheral blood lymphocytes with HLA DRB*0401-expressing cells rapidly expanded CD8+ T cells, which recognized the pp65 epitope in the context of HLA Cw*0602. T-cell repertoire analysis revealed 2 dominant populations expressing T-cell receptor beta variable (TRBV)4-3 or TRBV13, with cross-reactivity exclusively mediated by the TRBV13+ clonotypes. More importantly, cross-reactive TRBV13+ clonotypes displayed markedly lower T-cell receptor binding affinity and a distinct pattern of peptide recognition, presumably mimicking a structure presented on the HLA DR4 allotype. These results illustrate a novel mechanism whereby virus-specific CD8+ T cells can cross-recognize HLA class II molecules and may contribute toward allograft rejection and/or autoimmunity.

Description: Recent studies suggested that JAK2V617F mutation is frequent in patients with splanchnic vein thrombosis (SVT) but not in patients with other venous thromboembolic events (VTE). However, whether screening for the JAK2V617F mutation in VTE patients is justified remains unclear. Therefore, we performed a systematic review to assess the frequency of JAK2 mutation in VTE patients and the role of JAK2V617F mutation in the diagnosis of myeloproliferative neoplasms. MEDLINE and EMBASE databases were searched. Two reviewers independently performed study selection and extracted study characteristics. Pooled odds ratios of case-control studies and weighted mean proportion of the prevalence of JAK2V617F mutation of uncontrolled series were calculated. Twenty-four studies involving 3123 patients were included. Mean prevalence of JAK2 mutation was 32.7% (95% confidence interval, 25.5%-35.9%) in SVT patients. JAK2 mutation was associated with increased risk of SVT (odds ratio, 53.98; 95% confidence interval, 13.10-222.45). Mean prevalence of JAK2 mutation in other VTE patients was low (range, 0.88%-2.57%). Presence of JAK2V617F mutation in SVT patients was associated with a subsequent diagnosis of myeloproliferative neoplasm in many patients. JAK2 mutation is strongly associated with SVT, and routine screening of JAK2 mutation appears to be indicated in these patients.

Description: Abstract 3441 Poster Board III-329 Background CLL is characterized by the progressive accumulation of monoclonal B lymphocytes. One theory to explain how CLL cells avoid elimination through immune surveillance mechanisms is through a defect in the ability of T-cells to form immunological synapses with antigen-presenting tumor B-cells (Ramsay et al JCI 2008). Lenalidomide is an immunomodulatory agent with clinical activity in the treatment of B-cell malignancies. Recent laboratory studies showed that lenalidomide not only stimulates T- and natural killer (NK)-cell-mediated ADCC, it also restores the T-cell-mediated ability to form immunological synapses with CLL tumor cells. Since NK cells also exert cytotoxicity through immune synapse formation, here we explore how lenalidomide affects NK-cell-mediated cytotoxicity mechanisms and whether this activity is altered in the presence of rituximab since published studies showed that lenalidomide-pretreated B-cells have a down-regulated surface CD20 expression. Further, we investigated the molecular events associated with immune synapse formation and the effect of lenalidomide. Methods Immune synapse formation was assessed in NK cells (from healthy donors PBMCs) co-cultured with either B-CLL cells derived from pts or with K562 cells (positive control). Cells were fixed and the ability to form synapses was assessed via immunohistochemisty co-staining for either F-actin and CD2, or F-actin and perforin (a cytolytic protein found in NK cells). Synapse formation was visualized by microscopy and measured via relative mean fluorescent intensity. Activity of RhoA, Rac1, Cdc42 were measured using Rho GTPases assay kits. Inhibition of lenalidomide-mediated immune synapse activity was assayed using the cell permeable Rho inhibitor C3 (0.5 mM). Flow cytometry was used to measure changes in surface CD20 and CD54 (ICAM-1) expression in B-CLL samples from 3 pts after treatment with lenalidomide. Results Lenalidomide induced the formation of immunological synapses between NK cells and primary B-CLL cells (p

Description: The antiphospholipid syndrome (APS) is an acquired thrombophilia, characterized by the occurrence of venous and arterial events. This article examines the laboratory and key clinical aspects of APS. Particular focus is given to anti–beta 2-glycoprotein I (β2GPI) antibodies in view of their recent inclusion in the APS classification criteria. The clinical utility of using the β2GPI enzyme-linked immunosorbent assay, in conjunction with the established lupus anticoagulant assays and cardiolipin enzyme-linked immunosorbent assay, for diagnosing and risk stratifying patients suspected of having APS is discussed. The relative importance of the various assays in diagnosing obstetric APS (early and late gestation miscarriages) is explored. The implications of recent epidemiologic findings for possibly understanding the underlying pathophysiologic mechanisms of obstetric APS are highlighted. Insights into which patients with obstetric APS may be at most risk of thrombotic complications are presented.

Description: To study B-cell development from bone marrow (BM), we generated recombination-activating gene 1 (Rag1)–targeted mice lacking mature lymphocytes. B-cell development can be induced in such mice by B cell–specific restoration of a functional Rag1 transcription unit. Follicular and marginal zone B cells populated the spleen when Rag1 expression was permitted. Notably, the peritoneal cavity was dominated by bona fide B-1a cells, as judged by surface markers and functional properties. These BM-derived B-1a cells exhibited a polyclonal VDJ repertoire with substantial N nucleotide insertions. Nevertheless, physiologic frequencies of phosphatidylcholine-specific B cells were detected. Importantly, the BM of young and 5-month-old mice was indistinguishable with regard to the potential to generate B-1a cells.

Description: Historically, graft-versus-host disease (GVHD) beyond 100 days after hematopoietic cell transplantation (HCT) was called chronic GVHD, even if the clinical manifestations were indistinguishable from acute GVHD. In 2005, the National Institutes of Health (NIH) sponsored a consensus conference that proposed new criteria for diagnosis and classification of chronic GVHD for clinical trials. According to the consensus criteria, clinical manifestations rather than time after transplantation should be used in clinical trials to distinguish chronic GVHD from late acute GVHD, which includes persistent, recurrent, or late-onset acute GVHD. We evaluated major outcomes according to the presence or absence of NIH criteria for chronic GVHD in a retrospective study of 740 patients diagnosed with historically defined chronic GVHD after allogeneic HCT between 1994 and 2000. The presence or absence of NIH criteria for chronic GVHD showed no statistically significant association with survival, risks of nonrelapse mortality or recurrent malignancy, or duration of systemic treatment. Antecedent late acute GVHD was associated with an increased risk of nonrelapse mortality and prolonged treatment among patients with NIH chronic GVHD. Our results support the consensus recommendation that, with appropriate stratification, clinical trials can include patients with late acute GVHD as well as those with NIH chronic GVHD.

Description: Chronic immune activation is a major cause for progressive immunodeficiency in human immunodeficiency virus type-1 (HIV) infection. The underlying trigger, however, remains largely unknown. HIV single-stranded RNA is a potent immune activator by triggering Toll-like receptor (TLR) 7/8. Thus, we hypothesized that sustained TLR7 triggering induces chronic immune activation and thereby contributes to progressive immunodeficiency. We used the synthetic compound R848 or a mixture of uridine-rich HIV single-stranded (ss) RNA oligonucleotides—both are potent TLR7/8 agonists—to explore the effects of sustained TLR7 triggering on the murine lymphoid system. Sustained TLR7 triggering induced an immunopathology reminiscent of progressive lymphoid destruction in HIV disease; we observed lymphopenia, elevated proinflammatory cytokines, splenomegaly, contracted lymphoid subsets, and lymphoid microarchitecture alteration with reduced marginal zone B-lymphocytes. Upon exposure to inactivated vesiculo-stomatitis virus, antibody production was abolished, although splenic lymphocytes were activated and total IgG was elevated. Our data imply that HIV itself may directly contribute to immune activation and dysfunction by stimulating TLR7. Thus, manipulation of TLR7 signaling may be a potential strategy to reduce chronic hyper-immune activation and, thereby, disease progression in HIV infection.

Description: Polymorphisms in folate pathway genes may influence the susceptibility to acute lymphoblastic leukemia (ALL). DNA was isolated from 245 pediatric ALL patients (cases) and from 500 blood bank donors (controls). Polymorphisms in methylene-tetrahydrofolate reductase (MTHFR 677C〉T, 1298A〉C), methionine synthase (MTR 2756A〉G), methionine synthase reductase (MTRR 66A〉G), methylenetetrahydrofolate dehydrogenase (MTHFD1 1958G〉A), nicotinamide N-methyltransferase (NNMT IVS −151C〉T), serine hydroxymethyl transferase (SHMT1 1420C〉T), thymidylate synthase (TS 2R3R), and the reduced folate carrier (RFC1 80G〉A) were detected. In ALL patients, an increased occurrence was observed of the RFC1 80AA variant (odds ratio [OR] = 2.1; 95% confidence interval [CI] = 1.3-3.2; P = .002) and the RFC1 80A allele (OR = 1.5; 95% CI, 1.1-2.1; P = .02). Likewise, the NNMT IVS −151TT genotype showed a 2.2-fold increased ALL risk (OR = 2.2; 95% CI, 1.1-4.6; P = .04). A 1.4-fold reduction in ALL risk was observed for (heterozygous or homozygous) carriers of the TS 2R allele and the MTHFR 677T allele (OR = 0.7; 95% CI, 0.5-1.0; P 〈 .05). Furthermore, interactions between NNMT and MTHFR 677C〉T and RFC1 were observed. NNMT IVS −151CC/MTHFR 677CT + TT patients exhibited a 2-fold reduction in ALL risk whereas RFC1 80AA/NNMT IVS −151CT + TT subjects had a 4.2-fold increase in ALL risk (P = .001). For the first time, we associate the RFC1 80G〉A and NNMT IVS −151C〉T variants to an increased ALL susceptibility.

Description: Abstract 4311 Objectives Oral mucositis is a common complication of high-dose chemotherapy and radiotherapy followed by hematopoietic stem-cell support (HSCT). We evaluated the efficacy and safety of calcium phosphate mouth rinse (Caphosol) for prevention and reduction of severity and duration of oral mucositis in patients treated with HSCT. Methods In 2009 23 patients were treated. Three received allogeneic stem cells (ALLO): 2- Hodgkin disease (HD), 1-myeloma multiplex (MM) and twenty autologous transplantation (AUTO): 8-non-Hodgkin lymphomas (NHL); 7- HD; 3-MM. For ALLO conditioning regimens were composed of: fludarabine 150mg/m 2 and melphalan 140mg/m 2. AUTO were treated with: BEAM: carmustine, etoposide, cytarabine and melphalan (HD and NHL) and melphalan 200mg/m 2 (MM). The source of hematopoietic stem cells was peripheral blood. Caphosol was prepared according to the manufacturer‘s instructions and administered 4 times daily, starting from the day before the beginning of chemotherapy till the end of hospitalization. Control group was composed of patients, who had been treated with HSCT previously, before “palifermin and caphosol era”. The groups were comparable according to number of patients, their age, type of disease, transplant and conditioning regimen. Oral mucositis was assessed with the use of the five-grade World Health Organization (WHO) oral-toxicity scale. Oral mucositis was evaluated daily after the beginning of transplantation procedure until discharge from the BMT Unit. The number of days with painkillers or antibiotics were estimated. Total parenteral nutrition was given according to the standards [Martin-Salces M, De Paz R, Canales MA et al (2008) Nutritional recommendations in hematopoietic stem cell transplantation. Nutrition 24: 769-775]. Treatment with antibiotics was started when neutropenic fever occurred. Safety was assessed on the basis of the incidence of adverse events. Statistical analysis was performed using Wilcoxon‘ test for analysis of differences between groups. Results Oral mucositis grade 2-4 was not observed. In patients treated with calcium phosphate mouth rinse oral mucositis grade 2-4 was not observed. Nobody had to receive opioid analgesics or total parenteral nutrition. 30% patients developed the first degree of oral mucositis 4-5 days' duration. In the control group OM was observed in all cases, 50% patients had III- IV degree. Median duration of OM was 10 and 12 days (range 5- 20) for auto- and allogeneic patients, respectively. As compared with control group, treatment with calcium phosphate mouth rinse was associated with significant reduction of the incidence of oral mucositis in II- IV degrees (0 percent versus 50 percent, p 〈 0.001), duration of oral mucositis (4/ 5 days vs. 10/12 days, p 〈 0.001), duration of pain-killers‘ treatment (0- 22 days vs. 0- 3 days, p 〈 0.001) and number of days with antibiotics‘ treatment (0- 7 days vs. 7- 20 days, p= 0.002). These differences were observed in both types of transplantation. No side effects of calcium/phosphate oral rinse were observed. Conclusion In comparison with control group, treatment with Caphosol was associated with the significant reduction of the incidence of oral mucositis in II- IV degrees, duration of oral mucositis, pain-killers‘ treatment and number of days with antibiotics‘ treatment. Calcium phosphate mouth rinse is a very promising medicine for prevention of oral mucositis for patients treated with high dose chemotherapy supported with hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: Acute lymphoblastic leukemia (ALL) diagnosed in the first month of life (congenital ALL) is very rare. Although congenital ALL is often assumed to be fatal, no studies have been published on outcome except for case reports. The present study reports the outcome of 30 patients with congenital ALL treated with the uniform Interfant-99 protocol, a hybrid regimen combining ALL treatment with elements designed for treatment of acute myeloid leukemia. Congenital ALL was characterized by a higher white blood cell count and a strong trend for higher incidence of MLL rearrangements and CD10-negative B-lineage ALL compared with older infants. Induction failure rate was 13% and not significantly different from that in older infants (7%, P = .14), but relapse rate was significantly higher in congenital ALL patients (2-year cumulative incidence [SE] was 60.0 [9.3] vs 34.2 [2.3], P 〈 .001). Two-year event-free survival and survival of congenital ALL patients treated with this protocol was 20% (SE 9.1%). Early death in complete remission and treatment delays resulting from toxicity were not different. The survival of 17% after last follow-up, combined with a toxicity profile comparable with that in older infants, justifies treating congenital ALL with curative intent. This trial was registered at www.clinicaltrials.gov as no. NCT 00015873, and at www.controlled-trials.com as no. ISRCTN24251487.

Description: The role of hematopoietic cytokines in lineage commitment remains uncertain. To gain insight into the contribution of cytokine signaling to myeloid lineage specification, we compared granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF) signaling in Ba/F3 cells expressing both the G-CSF and M-CSF receptors and in lineage-negative murine marrow cells. G-CSF and M-CSF serve as prototypes for additional cytokines that also influence immature myeloid cells. G-CSF specifically activated signal transducer and activator of transcription 3 and induced Src homology region 2 domain-containing phosphatase 2 (SHP2) phosphorylation, whereas M-CSF preferentially activated phospholipase Cγ2, and thereby extracellular signal-regulated kinase (ERK), to stabilize c-Fos and stimulate CCAAT/enhancer-binding protein (C/EBP)α(S21) phosphorylation. In contrast, activation of Jun kinase or c-Jun was similar in response to either cytokine. Inhibition of ERK prevented induction of c-Fos by M-CSF and reduced C/EBPα phosphorylation and formation of colony-forming unit–monocytes. SHP2 inhibition reduced ERK activation in G-CSF, but not M-CSF, and reduced colony-forming unit–granulocytes, underscoring divergent pathways to ERK activation. Phorbol ester mimicked the effect of M-CSF, activating ERK independent of SHP2. In summary, M-CSF activates ERK more potently than G-CSF, and thereby induces higher levels of c-Fos and phospho-C/EBPα(S21), which may directly interact to favor monopoiesis, whereas G-CSF activates signal transducer and activator of transcription 3 and SHP2, potentially shifting the balance to granulopoiesis via gene induction by C/EBPα homodimers and via effects of SHP2 on regulators besides ERK.

Description: To investigate the cell of origin linking follicular (FL) and transformed (t-FL) lymphomas, we analyzed the somatic hypermutation (SHM) pattern of the variable region of the immunoglobulin heavy gene (IgH-VH) in 18 sequential FL/t-FL samples and a father (donor) and son (recipient), who developed FL and t-FL, after transplantation. Genealogic trees showed a pattern compatible with a common progenitor cell (CPC) origin in 13 cases. The identification of the t-FL clonotype in the previous FL sample and of the putative CPC sequence in both the FL/t-FL biopsies showed that the intraclonal diversity of FL and t-FL germinal centers (GCs) is more intricate than previously described, and all 3 clonotypes (CPC, FL, t-FL) may occur simultaneously within the same lymph node. On the basis of the father/son model, this CPC must be long-lived, providing a possible explanation for the incurable nature of this disease.

Description: Hematogenous metastasis is promoted by interactions of tumor cells with leukocytes, platelets, and the endothelium in the local intravascular microenvironment. Here we show that the activation of the microvascular endothelium results in recruitment of monocytes to metastatic tumor cells and promotes the establishment of the metastatic microenvironment. This inflammatory-like endothelial response was observed in microvascular endothelial cells only. Microarray analysis of microvascular endothelial cells cocultured with tumor cells in the presence of leukocytes and platelets revealed a specific gene expression profile. Selectin-mediated interactions of tumor cells with platelets and leukocytes activated endothelial cells and induced production of C-C chemokine ligand 5 (CCL5). Inhibition of CCL5-dependent monocyte recruitment during the early phase of metastasis by a CCL5 receptor antagonist strongly reduced tumor cell survival and attenuated metastasis. Collectively, these findings demonstrate that the endothelial expression of CCL5 contributes to the formation of a permissive metastatic microenvironment.

Description: Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P 〈 .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3′ untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca2+ levels after activation with ADP (P 〈 .004). These data provide novel insights into key hubs within platelet signaling networks.

Description: Abstract 3453 Poster Board III-341 Introduction Increasing insight into the biology of CLL suggests a relevant interplay between tumor cells and the microenvironment. Preclinical data point to the potency of Lenalidomide to favourably modulate such interactions and remarkable clinical activity has been demonstrated in monotherapy trials in pretreated and also in previously untreated CLL patients. However, tumor flare and tumor lysis have been observed as problems and the optimal dose and schedule is still under investigation. In addition, the potential for interaction with effective standard treatment for CLL is unknown. We used a combination of Fludarabine and Rituximab as a backbone to establish a tolerable Lenalidomide dose in a dose escalation design. Study design The study treatment follows two phases: In the induction phase the maximal tolerated dose (MTD) of Lenalidomide in combination with FR should be determined. The protocol combines 6 cycles of Fludarabine (40mg/m2 po d1-3) and Rituximab (375mg/m2 iv day 4 on cycle 1 and 500mg/m2 iv day 1 on cycles 2-6), both in a 28 day cycle. Lenalidomide is given at a starting dose of 2,5 mg daily (day 7-21 of cycle 1) and with dose escalation steps to 5, 10, 15, 20 and 25mg of Lenalidomide from day 1-21 of the following cycles, if toxicity of the combination permits. In a second phase, Rituximab (375mg/m2 iv) at 2, 4 and 6 months after the last cycle is combined with Lenalidomide (day 1-28 of 28 day cycles) at the last tolerated dose for 6 months of maintenance. 40 patients are planned for this study. We herewith present the planned interim analysis for dose finding and safety endpoints for the induction phase of the first 10 patients recruited into the study. Results Mean age of patients was 70 years (range 59-76). Six of 10 patients had stage Rai III/IV and mean WBC count was 159 G/L. Seven of 10 patients had at least one high risk feature from CD38 and FISH analysis or by mutation status. Of the 60 planned cycles 46 (77%) are currently evaluable for this analysis. No systematic toxicity determining MTD was found. 50% of patients proceeded through dose escalation steps as planned. Two patients have already tolerated 25mg of Lenalidomide with their FR cycles. Regarding toxicities, grade 3 and 4 neutropenia was expected in this combination and observed in 7/10 patients. However it was not used as a dose limiting toxicity per se. Still, one 75 year old patient was dose reduced because of febrile neutropenia in the previous cycle. Overall three infectious episodes were observed on treatment. Two patients experienced thrombotic events one of which was then taken off study because of Richter transformation, which might in hindsight have been present from study onset. Surprisingly, 5/10 patients experienced significant skin toxicity, mostly in the form of skin rashes, which was deemed dose limiting in two patients. It was, however, clearly associated with Pneumocystis prophylaxis in one patient, who then went on to receive the full Lenalidomide dose without further rashes. No tumor lysis or flare reaction was observed in the 10 patients reported. Preliminary efficacy data show that all patients achieved at least a PR after 3 cycles of therapy (except for the patient with the Richter transformation). Of the 3 patients currently evaluable after 6 cycles of treatment one CR and two very good PRs were observed before starting the maintenance phase of the study. Conclusions The combination of Lenalidomide with Fludarabine and Rituximab seems clinically feasible and no tumor lysis or flare reaction have been observed, possibly due to the Lenalidomide step up design, as well as the initial tumor load reduction by the chemo-immunotherapy backbone. No clear dose dependent toxicity has emerged as dose limiting for Lenalidomide escalation in this combination. However, 50% of patients had to be dose-limited due to not clearly dose-dependent skin and vascular toxicities. A regime of thromboprophylaxis as well as a delayed start of prophylaxis against pneumocystis have since been amended to improve the management of the patients. In addition, and remarkably, the regimen shows clinical efficacy despite controversial in vitro reports, suggesting a potential negative interaction between Lenalidomide and Rituximab. This might be due to the Lenalidomide pause before each Rituximab cycle or may reflect a difference between in vitro and in vivo. Disclosures Egle: Roche: Research Funding, Speakers Bureau. Off Label Use: Lenalidomide treatment in CLL, Rituximab maintenance in CLL. Greil:Roche: Honoraria, Research Funding; Celgene: Research Funding.

Description: Abstract LBA-2 Background: In a large proportion of patients that have completed 6 to 12 months of treatment with a vitamin K antagonist (VKA) for their acute episode of venous thromboembolism (VTE) the question arises whether to stop or continue this treatment. Continuation implies the need for regular laboratory control and subsequent dose adjustments. Furthermore, the risk of bleeding persists. New oral anticoagulants hold the promise of simple fixed-dose regimens without the need for monitoring and could make continuation of therapy more attractive. The Einstein-Extension study was therefore designed to assess the relative efficacy and safety of rivaroxaban, a direct oral factor Xa inhibitor, versus placebo in patients who had completed 6 to 12 months of anticoagulant treatment for their acute episode of VTE. Patients in whom there was a clear indication for continued anticoagulant treatment were not eligible. Study Design: This randomized, double-blind, placebo-controlled, superiority study evaluated therapy with rivaroxaban 20 mg once-daily for an additional 6 or 12 months. The primary efficacy outcome was symptomatic recurrent VTE (i.e., the composite of recurrent DVT, non-fatal PE, and fatal PE). The principal safety outcome was major bleeding. Also the occurrence of clinically relevant non-major bleeding (e.g. nose bleeds, large skin hematomas, and macroscopic hematuria) was recorded. The study was event-driven requiring a minimum of 30 confirmed recurrent events. All outcomes were adjudicated by an independent blinded committee. Results: A total of 1197 patients were randomized between February 2007 and May 2009 by 280 study sites in 28 countries. The intention-to-treat population consisted of 602 rivaroxaban and 594 placebo patients. Baseline characteristics and risk factors for VTE were comparable between the two groups. The mean duration of study treatment was 190 days in both groups. During the treatment period, symptomatic recurrent VTE events occurred in 42 (7.1%) of the placebo treated patients and in 8 (1.3%) of the rivaroxaban recipients (hazard ratio, 0.18; 95 % CI, 0.09 – 0.39; p〈 0.0001). After the stop of study medication, 6 symptomatic recurrent VTE events occurred in each group during the one month observational period. Major bleeding did not occur in placebo patients and was observed in 4 (0.7%) rivaroxaban recipients (p=0.106). None of these bleeding events were fatal or in a critical site. Clinically relevant non-major bleeding was noted in 7 (1.2%) and 32 (5.4%) of the placebo and rivaroxaban recipients, respectively. Two (0.3%) patients in the placebo group died versus 1 (0.2%) in the rivaroxaban group. No patients were observed to have an alanine aminotransferase (ALT) rise above 3 times the upper limit of normal (xULN) combined with a total bilirubin above 2 xULN. Conclusion: A fixed dose of 20 mg of rivaroxaban once-daily is associated with an 82% relative risk reduction in the recurrence of VTE in patients who had completed a 6 to 12 month course of anticoagulant therapy for their index event. Based on the estimated cumulative incidence rates, approximately, 15 patients need to be treated to prevent one recurrent VTE event. This clinically relevant reduction in recurrence was associated with a low incidence of major bleeding (0.7%). This oral once-daily regimen provides the clinician with a simple option for patients in whom continued anticoagulant treatment is indicated. Disclosures: Buller: Bayer Healthcare: Research Funding.

Description: BLyS and its major receptor BAFF-R have been shown to be critical for development and homeostasis of normal B lymphocytes, and for cell growth and survival of neoplastic B lymphocytes, but the biologic mechanisms of this ligand/receptor-derived intracellular signaling pathway(s) have not been completely defined. We have discovered that the BAFF-R protein was present in the cell nucleus, in addition to its integral presence in the plasma membrane and cytoplasm, in both normal and neoplastic B cells. BAFF-R interacted with histone H3 and IKKβ in the cell nucleus, enhancing histone H3 phosphorylation through IKKβ. Nuclear BAFF-R was also associated with NF-κB/c-Rel and bound to NF-κB targeted promoters including BLyS, CD154, Bcl-xL, IL-8, and Bfl-1/A1, promoting the transcription of these genes. These observations suggested that in addition to activating NF-κB pathways in the plasma membrane, BAFF-R also promotes normal B-cell and B-cell non-Hodgkin lymphoma (NHL-B) survival and proliferation by functioning as a transcriptional regulator through a chromatin remodeling mechanism(s) and NF-κB association. Our studies provide an expanded conceptual view of the BAFF-R signaling, which should contribute a better understanding of the physiologic mechanisms involved in normal B-cell survival and growth, as well as in the pathophysiology of aggressive B-cell malignancies and autoimmune diseases.

Description: Abstract 432 Elotuzumab is a humanized monoclonal IgG1 antibody directed against CS1, a cell surface glycoprotein, which is highly and uniformly expressed in multiple myeloma (MM). Elotuzumab induces significant antibody-dependant cytotoxicity (ADCC) against primary myeloma cells in the presence of either autologous or allogeneic peripheral lymphocytes (PBMC), which is significantly enhanced when PBMC effector cells were pretreated with lenalidomide (Tai et al., Blood 112:1329, 2008). The primary objective of the phase 1 portion of the study is to evaluate the maximum tolerated dose (MTD) of elotuzumab in combination with lenalidomide and low dose dexamethasone in patients with relapsed MM. The study is also evaluating safety, pharmacokinetics (PK) and clinical response. Lenalidomide (25 mg PO) is given on Days 1-21 of a 28-day cycle. Elotuzumab in three escalating dose cohorts (5, 10 and 20 mg/kg) is administered by IV infusion on Days 1, 8, 15 and 22 of the 28-day cycle in the first two cycles and then on Days 1 and 15 of each subsequent cycle. Dexamethasone is given weekly at 40 mg PO. Initially, patients received 6 cycles of treatment unless withdrawn earlier due to disease progression or unacceptable. toxicity. The protocol was amended to allow for patients in the 10 and 20 mg/kg cohorts to receive treatment for up to 12 months following enrollment of the last patient. Key entry criteria: age ≥ 18 years; MM with at least one relapse; measurable disease M-protein component in serum and/or in urine; and prior lenalidomide treatment, if any, more than 6 weeks of first dose. To date, 24 patients with a median age of 60 years have been enrolled in the study and 23 patients have received study drug. The median time from initial diagnosis of MM was 5 years and patients had received a median of 3 prior MM treatments. Patients had been previously treated with thalidomide (58%), bortezomib (67%) or lenalidomide (21%) and 42% were refractory to their most recent MM therapy. Patients have been treated in the 3 cohorts; 3 patients each in the first two cohorts (5 and 10 mg/kg elotuzumab) and 17 patients (7 in dose-escalation phase and 10 in the expansion phase) in the third cohort (20 mg/kg). No dose limiting toxicities were identified during the dose-escalation phase of the study and no MTD was established. One patient discontinued in the first cycle due to grade 4 allergic reaction resulting from elotuzumab infusion in the expansion phase of the study. Additional SAEs (1 of each) included grade 2 atrial fibrillation (related to lenalidomide/dexamethasone) and unrelated grade 4 ruptured diverticulum, grade 3 neutropenic fever and grade 3 diarrhea.. Other common grade 3 or 4 AEs included neutropenia (25%) and thrombocytopenia (25%), which were managed by dose withholding or dose reduction of lenalidomide. Approximately 25% of patients experienced grade 1 or 2 chills and/or pyrexia associated with elotuzumab infusion. The best clinical response (IMWG criteria) in the 13 patients who have received at least two cycles of treatment is shown in the table below. Preliminary PK analysis of elotuzumab suggests a serum half-life of 10-11 days at 10 and 20 mg/kg. Elotuzumab at all three doses resulted in near complete saturation of CS1 sites on plasma cells and NK cells in bone marrow and NK cells in the peripheral compartment. In conclusion, the combination of elotuzumab with lenalidomide and low-dose dexamethasone has a manageable adverse event profile and compared to historical data for lenalidomide and high-dose dexamethasone, the preliminary efficacy data (≥ PR of 92%) are very encouraging. Additional safety, efficacy and PK/PD data will be presented at the meeting. Disclosures: Lonial: Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Off Label Use: Lenalidomide/dexamethasone in combination with elotuzumab in patients with relapsed/refractory multiple myeloma. Vij:Celgene: Research Funding, Speakers Bureau. Harousseau:Celgene France: Advisory Board; Janssen Cilag France: Advisory Board; Celgene: Honoraria; Janssen Cilag: Honoraria; Novartis: Honoraria; Amgen: Honoraria. Facon:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Kaufman:Celgene: Consultancy, Research Funding; Millennium: Consultancy; Genzyme: Consultancy; Merck: Research Funding. Mazumder:Celgene: Speakers Bureau; Millennium: Speakers Bureau. Leleu:Celgene: Research Funding, Speakers Bureau. Fry:Facet Biotech: Employment. Singhal:Facet Biotech: Employment. Jagannath:Millennium: Advisory Board; Merck: Advisory Board.

Description: This phase 3 prospective randomized trial evaluated the efficacy and long-term safety of erythropoietin (EPO) with or without granulocyte colony-stimulating factor plus supportive care (SC; n = 53) versus SC alone (n = 57) for the treatment of anemic patients with lower-risk myelodysplastic syndromes. The response rates in the EPO versus SC alone arms were 36% versus 9.6%, respectively, at the initial treatment step, 47% in the EPO arm, including subsequent steps. Responding patients had significantly lower serum EPO levels (45% vs 5% responses for levels 〈 200 mU/mL vs ≥ 200 mU/mL) and improvement in multiple quality-of-life domains. With prolonged follow-up (median, 5.8 years), no differences were found in overall survival of patients in the EPO versus SC arms (median, 3.1 vs 2.6 years) or in the incidence of transformation to acute myeloid leukemia (7.5% and 10.5% patients, respectively). Increased survival was demonstrated for erythroid responders versus nonresponders (median, 5.5 vs 2.3 years). Flow cytometric analysis showed that the percentage of P-glycoprotein+ CD34+ marrow blasts was positively correlated with longer overall survival. In comparison with SC alone, patients receiving EPO with or without granulocyte colony-stimulating factor plus SC had improved erythroid responses, similar survival, and incidence of acute myeloid leukemia transformation.

Description: AML1-ETO is the chimeric protein product of the t(8;21) in acute myeloid leukemia. The ETO portion of the fusion protein includes the eTAFH domain, which is homologous to several TATA binding protein–associated factors (TAFs) and interacts with E proteins (E2A and HEB). It has been proposed that AML1-ETO–mediated silencing of E protein function might be important for t(8;21) leukemogenesis. Here, we determined the solution structure of a complex between the AML1-ETO eTAFH domain and an interacting peptide from HEB. On the basis of the structure, key residues in AML1-ETO for HEB association were mutated. These mutations do not impair the ability of AML1-ETO to enhance the clonogenic capacity of primary mouse bone marrow cells and do not eliminate its ability to repress proliferation or granulocyte differentiation. Therefore, the eTAFH-E protein interaction appears to contribute relatively little to the activity of AML1-ETO.

Description: Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLex). It is thought that multivalent 6-sulfo SLex expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLex in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin–binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLex epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLex. N-glycans containing potential 6-sulfo SLex epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLex epitopes on O-glycans that are important for its recognition by L-selectin.

Description: Abstract 3619 Poster Board III-555 Granulocyte colony-stimulating factor (G-CSF) controls neutrophil production in the bone marrow under steady state conditions and during demand-driven hematopoiesis occurring in response to infection. STAT3 is a principal signaling molecule activated by the G-CSF receptor (G-CSFR). We previously reported that STAT3 has an important role in demand-driven granulopoiesis, although its cellular and molecular mechanisms have been unclear. To address this, we investigated STAT3 function in emergency granulopoiesis stimulated by G-CSF administration or infection with Listeria monocytogenes, which is restrained by the G-CSF response pathway in vivo. Our results show that STAT3-deficiency renders hematopoietic stem cells and myeloid progenitors refractory to the proliferation-inducing effects of G-CSF or Listeria monocytogenes infection. STAT3-deficient myeloid progenitors have a cell autonomous defect in G-CSF-responsive cell cycle progression and undergo delayed granulocyte maturation relative to wild type cells. To define STAT3 target pathways in granulocytic progenitors, we investigated the expression of CCAAT enhancer binding protein (C/EBP) beta, a transcription factor that is necessary for G-CSF-driven emergency granulopoiesis. We found that STAT3 directly regulates G-CSF-responsive C/EBPbeta expression by binding to Cebpb promoter. Moreover, we show that STAT3 and C/EBPbeta co-regulate c-Myc during emergency granulopoiesis. These results place STAT3 as a crucial G-CSF-responsive signal transducer during demand-driven granulopoiesis, through its regulation of critical transcription factors in developing granulocytes. Disclosures: Zhang: Amgen: Research Funding. Nguyen-Jackson:Amgen: Research Funding. Watowich:Amgen, Inc: Research Funding.

Description: Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is thought to promote tumor angiogenesis mostly through its protease-inducing function and more recently by its ability to increase tumor cell expression of vascular endothelial growth factor (VEGF). In this study, we present evidence that EMMPRIN can promote angiogenesis by a direct effect on endothelial cells through a paracrine regulation of the VEGF/VEGF-receptor (VEGFR) system. Using human microvascular endothelial cell line–1 endothelial cells, we show that EMMPRIN selectively increased the soluble VEGF isoforms (121 and 165), but not the matrix-bound VEGF 189 form. In addition, EMMPRIN up-regulated the expression of VEGFR-2 without an effect on VEGFR-1. This increase in VEGFR-2 was responsible for the observed EMMPRIN stimulation of the migratory and tube formation capacity of endothelial cells. EMMPRIN′s effects, which were matrix metalloproteinase and urokinase-type plasminogen activator independent, were mediated primarily through hypoxia-inducible factor-2α expression, also up-regulated by EMMPRIN. VEGFR-2 increase was also observed in vivo in a mouse model of xenograph tumors overexpressing EMMPRIN. These results suggest that in addition to increasing protease production, EMMPRIN may contribute to the formation of a reactive stroma also through the up-regulation of hypoxia-inducible factor-2α, VEGFR-2, and the soluble forms of VEGF in endothelial cells, thus directly regulating the angiogenic process.

Description: Abstract 286 It is well established that deregulation of the PI3K signaling pathway plays an important role in the etiology of human malignancies including those of hematologic origin. In 30–50% of solid tumors, oncogenic activation of the PI3K pathway is the result of gain-of-function mutations in the PI3K p110α isoform or due to the loss-of-function of the PTEN phosphatase that is responsible for PI3K downregulation. In B cell malignancies these mutations are rarely observed in spite of the fact that PI3K pathway activation is commonly observed and often essential for tumor cell growth and survival. In this case, PI3K pathway activation has been shown to result from constitutive B cell receptor (BCR) activation and/or from exposure to survival factors present in the microenvironment. The activation of the PI3K pathway by cell surface receptors is directly mediated by the Class I isoforms (α, β, δ, and γ), however, their relative contribution in B cell tumors is unknown. Interestingly, genetic and pharmacological approaches that specifically inactivate the p110δ isoform have demonstrated its important role in normal B cell signaling in response to multiple factors including antigen, CD40L, BAFF, SDF-1 and CXCR13 making it an attractive target for therapeutic intervention in B cell malignancies. CAL-101 is an oral p110δ specific inhibitor which is currently being evaluated in a phase I clinical trial for the treatment of patients with select hematologic malignancies. This compound is a novel potent p110δ inhibitor with an IC50 of 2.5 nM against purified p110δ and EC50 of 65 nM in p110δ-mediated basophil activation in whole blood. CAL-101 demonstrates 300-, 200-, and 40-fold selectivity over the other class I family members (α, β, and, γ respectively) and no activity against class II and III PI3K family members or other PI3K-related proteins including mTOR and DNA-PK. Furthermore, a kinome-wide screen failed to detect activity against any additional kinases. To investigate the potential role of p110δ in B cell hematologic tumors we screened a wide range of leukemia and lymphoma cell lines for constitutive PI3K pathway activation. The expression of p110δ was observed in 〉90% of these cell lines and in many cases was accompanied by constitutive Akt phosphorylation. In this context, CAL-101 was able to reduce p-Akt levels and block additional downstream effectors such as p-S6, and GSK-3β in cells that represent a range of tumor types including follicular lymphoma, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, and mantle cell lymphoma (MCL). Furthermore, treatment with CAL-101 resulted in growth suppression and induction of apoptosis which was accompanied by PARP and caspase-3 cleavage. Growing evidence suggests that signals from the microenviroment are essential for the expansion, homing, and survival of malignant B cells, in addition to promoting resistance to conventional drug therapy. To investigate the potential role p110δ plays during B cell signaling via interactions with the microenvironment, we examined invoked stimulation of leukemia and lymphoma cell lines with CXCL12, CXCL13, BAFF, or BCR crosslinking in the presence or absence of CAL-101. Stimulation with either chemokines or growth factors resulted in the phosphorylation of Akt which was inhibited by CAL-101 in a dose dependent manner. Moreover, p110δ inhibition with CAL-101 inhibits the chemotaxis of ALL and MCL cell lines to CXCL12. These studies have now been extended to the analysis of primary patient B-ALL and MCL cells to further establish preclinical proof of concept for therapeutic application of CAL-101. In summary, CAL-101 is a potent and selective p110δ kinase inhibitor with broad anti-tumor activity against cancer cells of hematologic origin. Our results demonstrate that selective inhibition of p110δ with CAL-101 inhibits malignant B cell growth, survival, and migration. Furthermore, p110δ inhibition may enhance the effect of cytotoxic drugs or overcome cell mediated drug resistance by inhibiting the protective signals of the microenviroment, providing a rational for combination therapy. These data suggest that p110δ may play an important role in regulating signals between malignant B cells and their microenvironment thereby providing the preclinical rationale for clinical evaluation of CAL-101 alone or in combination with cytotoxics or biologics in patients with hematologic malignancies. Disclosures: Lannutti: Calistoga Pharmaceuticals: Employment. Meadows:Calistoga Pharmaceuticals: Employment. Kashishian:Calistoga Pharmaceuticals: Employment. Steiner:Calistoga Pharmaceuticals: Employment. Johnson:Calistoga Pharmaceuticals: Research Funding. Giese:Calistoga Pharmaceuticals: Employment.

Description: Radioimmunotherapy (RIT) options for T-cell non-Hodgkin lymphomas (T-NHLs) are limited. We evaluated anti-CD45-RIT in human (h) and murine (m) T-NHL. CD45 was highly expressed on hT-NHL patient samples (median, 2.3 × 105 antigen-binding capacity units/cell) and hT-NHL cell lines (3.4 × 105 CD45 antigen-binding capacity units/cell). Biodistribution studies in hTNHL xenografts showed that 131I-labeled BC8 (anti-hCD45) delivered 154% (P = .01) and 237% (P = .002) more radioiodine to tumor sites over control antibodies at 24 hours and 48 hours, respectively. Importantly, tumor sites targeted with 131I-BC8 exhibited 2.5-fold (P = .05), 3.0-fold (P = .007), and 3.6-fold (P = .07) higher 131I retention over the nontarget organs of lungs, liver, and kidneys, respectively (24 hours). Because the clinical use of anti-hCD45 would target both T-NHL and other hematolymphoid tissues, we evaluated the ability of anti-mCD45 to target mT-NHL. mT-NHL grafts targeted with anti-mCD45 correspondingly retained 5.3 (P 〈 .001), 5.4 (P 〈 .001), and 8.7 (P 〈 .001) times the radioactivity in tumor sites compared with nontarget organs of lung, liver, and kidney. 131I-labeled BC8 therapy yielded improved complete remission rates (75% vs 0%, P 〈 .001) and progression-free survivals (median, 23 days vs 4.5 days, P 〈 .001) compared with controls. These data indicate that the high CD45 expression of T-NHL allows reliable tumor targeting and disease control supporting anti-CD45 RIT for T-NHL patients.

Description: The importance of donor-recipient human leukocyte antigen (HLA)-DPB1 matching for the clinical outcome of unrelated hematopoietic stem cell transplantation (HSCT) is controversial. We have previously described an algorithm for nonpermissive HLA-DPB1 disparities involving HLA-DPB1*0901,*1001,*1701,*0301,*1401,*4501, based on T-cell alloreactivity patterns. By revisiting the immunogenicity of HLA-DPB1*02, a modified algorithm was developed and retrospectively tested in 621 unrelated HSCTs facilitated through the Italian Registry for oncohematologic adult patients. The modified algorithm proved to be markedly more predictive of outcome than the original one, with significantly higher Kaplan-Meier probabilities of 2-year survival in permissive compared with nonpermissive transplantations (55% vs 39%, P = .005). This was the result of increased adjusted hazards of nonrelapse mortality (hazard ratio [HR] = 1.74; confidence interval [CI], 1.19-2.53; P = .004) but not of relapse (HR = 1.02; CI, 0.73-1.42; P = .92). The increase in the hazards of overall mortality by nonpermissive HLA-DPB1 disparity was similar in 10 of 10 (HR = 2.12; CI, 1.23-3.64; P = .006) and 9 of 10 allele-matched transplantations (HR = 2.21; CI, 1.28-3.80; P = .004), both in early-stage and in advanced-stage disease. These data call for revisiting current HLA matching strategies for unrelated HSCT, suggesting that searches should be directed up-front toward identification of HLA-DPB1 permissive, 10 of 10 or 9 of 10 matched donors.

Description: Abstract 3727 Poster Board III-663 Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins, leading to regulation of gene transcription and other cellular processes. Entinostat (SNDX-275) is a novel and potent DAC inhibitor that is selective for class I DACs and is currently undergoing pre-clinical and clinical testing in Hodgkin lymphoma (HL). Potent synergistic anti-tumor activity has been observed by combining less potent DAC inhibitors with bortezomib in pre-clinical models. In our efforts to develop more therapeutic options for refractory/resistant B-cell lymphoma, we evaluated the effects of Eentinostat as a single agent and in combination with bortezomib against B-cell non-Hodgkin's lymphoma (NHL) cell lines and primary NHL cells. Studies were conducted in a panel of 12 NHL cell lines representing various subtypes of B-cell lymphoma (i.e. DLBCL/ABC, DLBCL/GCB, Burkitt's, transformed and MCL), which included: rituximab-[chemotherapy]-sensitive cell lines (RSCL, Raji, RL and DHL-4), rituximab-[chemotherapy]-resistant cell lines (RRCL, Raji-4RH, Raji-2R, RL-4RH, and DHL-4 4RH), and primary lymphoma cells isolated from patients with various subtypes of NHL and HL. Patient-derived tumor cells were isolated from fresh specimens by negative selection using magnetic beads. NHL cells and patient-derived primary cells were exposed to entinostat at different doses (0.01 to 100uM) either alone or in combination with CDDP (1 to 100μM), doxorubicin (4 to 16μM), vincristine (1 to 5μM), or bortezomib (1 to 10nM). Anti-tumor activity was measured after a 24 or 48 hr incubation. In cell lines, changes in mitochondrial potential and cell proliferation were determined by alamar blue reduction using a kinetic assay measuring activity at 4 hr intervals for 24 and 48 hrs. For patient-derived primary NHL cells, changes in ATP content (apoptosis) was determined using the cell titer glow assay. Entinostat was highly active in all the cell lines tested including rituximab-[chemotherapy]-resistant cell lines. The IC50 of Entinostat in the majority of the cells tested was 0.5 to 5uM at 48 hrs. Similar findings were observed in primary tumor cells derived from lymphoma patients. In addition, synergistic activity was observed by combining entinostat and bortezomib in both NHL cell lines, as well as in primary NHL/HL tumor specimens. A lesser degree of augmented anti-tumor activity was also observed when entinostat was combined with cisplatin or doxorubicin (but not vincristine). In summary, our data suggests that entinostat is a novel and potent DAC inhibitor with a wide therapeutic spectrum. Entinostat is capable of inducing cell death against various subtypes of B-cell lymphoma cell lines including RSCL, RRCL, as well as patient-derived primary tumor cells and augments the anti-tumor effects of bortezomib and other chemotherapeutic agents. Given the isoform selectivity of entinostat, the results indicate that HDAC1 and 2 may be the key targets of DAC inhibitors in HL and NHL cells. Ongoing studies are evaluating the mechanisms responsible for the synergistic effects of entinostat plus chemotherapy and will be updated at the annual meeting. Current findings strongly suggest that entinostat added to bortezomib and/or other chemo agents may become a novel and potent strategy in the treatment of aggressive and indolent NHL and HL in the future. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4919 Waldenstrom's macroglobulinemia (WM) is characterized by the presence of lymphoplasmacytic cells in the bone marrow and the secretion of IgM monoclonal antibody in the serum. Several conventional therapies are available but the disease remains incurable. Recently, bortezomib (a proteasomal inhibitor) has shown promising anti-WM activity with enhanced responses when combined with traditional therapies. Resistance to bortezomib therapy is an important event that is associated with continued treatment. In order to understand the mechanism of bortezomib resistance in WM we exposed BCWM.1 (a known WM cell line) in vitro to increasing concentrations of bortezomib over prolonged periods of time and isolated the bortezomib resistant clone (BCWM.1/BR). This clone was compared with its parent wild type cell line (BCWM.1/WT). Investigation to understand the susceptibility of BCWM.1/Br cells to various therapeutic agents showed that these cells are resistant to many of the agents such as melaphalan, fludarabine or doxorubicin. Interestingly, verapamil, a broad spectrum inhibitor of multidrug resistance, failed to reverse the resistance induced by bortezomib indicating that bortezomib resistance is not because of an activation of multidrug resistance in these cells. While BCWM.1/WT cells showed an IC50 of 7.8nM when treated for 72h with bortezomib, the BCWM.1/BR cells were not inhibited at any concentration of the compound up to 100nM. Furthermore, the cells with the acquired resistance showed a 4 fold increase in the proteasomal activity as measured by the release of a fluorescent product (7-Amino-4-methylcoumarin (AMC)) from its peptide substrate, suc-LLVY-AMC. Biochemical analysis further revealed that many of the proteasomal components are altered in BCWM.1/BR cells as compared to their parental control cells. Interestingly, protein levels of two of the proteasomal catalytic subunits, PSMB5 and PSMB9 are upregulated in resistant cells suggesting a reason for the enhanced proteasomal activity of these cells. The resistant cells showed an altered gene expression profile that indicates a transformation of the parental wild type cell line to acquire resistance. A comparative analysis of the signal transduction pathways operated in these cells showed that many of the activation and cell survival pathways that are present in BCWM.1 cells are inhibited in the resistant cells. For example, BCWM.1 cells show a constitutive activation of AKT and ERK1/2 which are inhibited in the resistant cells thus making them insensitive to the inhibitors of these pathways. Similarly, HSP27 which was earlier shown to contribute to bortezomib induced resistance was completely inhibited in BCWM.1 resistant cells. Interestingly, there is an increase in Bcl-2 protein in BCWM.1/BR cells as compared to WT cells indicating that the resistant cells might be dependent on Bcl-2 family for their survival. Inhibition of Bcl-2 induced potent apoptosis in BCWM.1/BR cells. Thus the results presented here indicate that acquired bortezomib resistance in BCWM.1 cells alters their proteasomal activity, cellular signaling pathways to make them resistant to many of the known therapies but these cells retain the Bcl-2 mediated pathway for targeting thus inhibitors of Bcl-2 may be used in therapy against bortezomib-resistant WM. Disclosures Chanan-Khan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Abstract 4848 Background Myelodysplastic syndrome(MDS) belong to the most common hematological diseases however epidemiological data on MDS are sparse. Until 2008 there were no data about epidemiology of MDS in Poland. Methods From 03.2008-05.2009 we have registered 966 patients in Polish MDS Registry. We have included only alive patients of various time of diagnosis. Patients from 22 centers were diagnosed according to WHO 2001 criteria. Results There were 508(53%)males and 458(47%) females. Median age at diagnosis was 70(range 19-99). Under 50 were 83(9%) cases with preponderance of females- 51 cases( males 32cases), between 50-70 there were 353(41%) cases, half of the patients-432(50%) were above 70( 247 males and 185 females).Prior chemotherapy and/or radiotherapy had 37((3,8%) patients. Distribution of MDS subtypes was as follows: RA-170(20%) cases, RARS-58(7%), RCMD-244(28%), RCMD-RS-18(2%), RAEB-1-120(14%), RAEB-2-169(19,5%), 5q- -40(4,6%), MDS-U-44(5%).In 103(10%) subtype was not done. Karyotype was available in 276(28%) cases. Cytogenetic risk groups were: low risk-182(68%), intermediate-52(20%) and high risk-33(12%). The most frequent cytogenetic results were: normal karyotype 44%, isolated 5q deletion 19%, complex karyotype 6%, 5q deletion + another one change 3% and 5q deletion with at least 2 changes 3%. According to IPSS risk groups low risk was found in 61( 22%) of cases, intermediate-1 -130(48%), intermediate-2-47(17%) and high risk in 31(11,5%). Median values of Hb was 9,1 g/dL, plts 129 G/L, ANC 1,7 G/L. RBC transfusion dependent were 429(44%) patients and platelet transfusion dependent were 100( 11%) pts. At least 2 U/month RBC transfusion requirement was 140(14%) patients. Serum ferritin level was assessed in 530 cases-171 of them( 32%) had higher than 1000μg/L level. Conclusions We have observed predominance of females among MDS patients under 50. Half of the patients had RA or RCMD subtype. Isolated 5 q deletion was the most frequent cytogenetic abnormality. Forty four percentage of patients was RBC transfusion dependant. Serum ferritin level was significantly elevated in 32% of assessed patients at the moment of MDS diagnosis. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4915 Multiple studies have highlighted the critical role of mutation and loss of p53 function in multiple myeloma (MM) when acquiring a more aggressive phenotype and refractoriness to treatment. Therefore, agents capable of overcoming p53 mutational status are important in the context of MM therapeutics. We have previously reported the in vitro and in vivo anti-MM activity of the multi-targeted small molecule inhibitor RGB-286638. Using a human MM cell xenograft model in SCID mice we demonstrated that RGB-286638 inhibited tumor growth and prolonged survival. Our data confirmed suppression of CDK1/cyclin B, CDK4, 6/Cyclin D1, D3, and CDK2/Cyclin E complexes in MM.1S MM cells containing wt-p53, which was correlated with rapid downregulation of Rb phosphorylation, resulting in effective G2/M cell cycle blockage and increased sub-G1phase. RGB-286638 induced dose and time-dependent inhibition of RNA pol II phosphorylation as an early event promptly followed by p53 induction. Moreover, RGB-286638 treatment was associated with p53 phosphorylation at ser 15, indicative of DNA damage followed by apoptosis, evidenced by caspases 8, 9 and 3 cleavage and confirmed by Annexin V/PI staining. All together these data suggested that RGB-286638-induced RNA pol II inhibition triggers cytotoxicity in MM cells via p53-dependent apoptosis. Interestingly, RGB-286638 demonstrated cytotoxic activity even in p53-deficient conventional drug-resistant RPMI 8226/Dox 40 MM cells. RGB-286638 treatment of RPMI 8226/Dox40 MM cells showed increased PARP response associated with enhanced NAD depletion followed by increased ATP consumption. Furthermore, concomitant assessment of RGB-286638-induced ATP depletion versus cytotoxicity demonstrated more than 60% ATP loss preceded cell death in RPMI 8226/Dox40 but not in MM.1S. This data suggests the role of either p53-mediated apoptosis (when active) or PARP-induced NAD/ATP depletion and bioenergetic crisis (when absent). Interestingly, the knockdown of p53 did not rescue MM.1S cells from RGB 286638-induced death, suggesting the existence of alternative p53-independent pathways through which RGB-286638 exerts its cytotoxic activity. Ongoing studies are addressing the molecular effects of p53 silencing in MM cells. In addition, dissecting the mechanism of RGB-286638 p53-independent cytotoxicity in MM cells will provide insights for future therapeutic strategies in patients with aggressive MM and associated mutated/deleted-p53. Disclosures Loferer: GPC Biotech AG: Employment. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Raje:Celgene: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding.

Description: Abstract 4886 Detection of high molecular weight light chain oligomers in urinary exosomes of patients with AL amyloidosis. Background Exosomes are microvesicles that are part of the multivesicular body (MVB) pathway. They are created by the inward budding of the cell surface membrane and contain both surface bound membrane proteins and cytosolic proteins which can be used to identify the cell of origin. Immunoglobulin light chain amyloidosis (AL) occurs as the result of amyloid formation by the misfolding of monoclonal light chains (LC) and deposition of these amyloid fibrils in various soft tissues. This reaction requires the organization of the monoclonal LC's into protofibrils which are then weave into amyloid fibrils. This study was undertaken to determine whether urinary exosomes of glomerular origin contain intermediate species of amyloid formation. Method Urine samples from patients with AL, light chain deposition disease (LCDD), multiple myeloma (MM) and monoclonal clonal gammopathy of undetermined significance (MGUS) were collected. Urinary exosomes were isolated and separated into fractions by gradient centrifugation. Western blots were performed on the urinary exosome fractions using anti-kappa or anti-lambda antibodies. Glomerular fractions were identified using antibodies directed toward podocin. Results Urine samples were collected from 5 patients with AL, 2 from LCDD, 1 from MM and 1 MGUS. On the Western blot, immunoglobulin LC were seen in all exosomal fractions in patients with AL amyloidosis, LCDD, MM but not MGUS which is similar to normal controls (not shown). In patients with AL, oligomeric species were found in the highest concentrations in fraction 4 and 5 (Figure 1). Fraction 4 and 5 were also stained for podocin, a glomerular protein (not shown). The highest molecular weight species was ∼250 kd which corresponds to a LC decamer. High molecular weight species were also identified in 1 of 2 LCDD patients corresponding to a tetramer. The band was identified in fraction 10 which had polycystin-1 expression suggesting a tubular origin. No high molecular weight LC species was found in patients with MM or MGUS. Conclusion Our study found high molecular weight LC species corresponding to the intermediates involved in protofibril formation in urinary exosomes of patients with AL. Smaller (tetramer) high molecular weight LC species were also found in a patient with LCDD but not in patients with MM and MGUS. Not only were the high molecular weight LC species found exclusively in the diseases characterized by deposition of LC aggregates, they were also found in the segments of the nephron where the deposits were expected: glomerulus for AL and tubular epithelium for LCDD. This is consistent with our current understanding of the pathogenic mechanisms of these diseases. We believe urinary exosomes are a powerful tool in the study of diseases involving self-aggregation of monoclonal proteins. It has tremendous potential in both diagnostic and scientific research in this area. Disclosures Gertz: celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 4902 Introduction Cytogenetics and fluorescent-in situ hybridization (FISH) are important outcome predictors in multiple myeloma (MM). There were only few small studies that investigated prognostic implication of FISH and/or conventional karyotyping in Korean MM patients. We investigated the incidences and prognostic significances of chromosomal abnormalities detected by FISH and/or conventional karyotyping among Korean MM patients. Patients and Methods We collected data of patients from Korean Myeloma Registry and performed retrospective analysis. We compared the survival of patients with chromosomal abnormalities and other clinical findings. Results From 2000 to 2009, total of 801 newly diagnosed myeloma patients were enrolled in this study. Median age of patients was 62 years. Median overall survival was 82 months, and median follow up of time was 92 months. Among the patients who had conventional karyotype analysis, 17.1% were complex karyotype, followed by del13q (7.4%), hyperdiploidy (7.6%), hypodiploidy (3.0%), and t(11;14) (3.9%). Among the patients who had FISH analysis, 22.8% were del 13q, followed by t(11;14) (18.2%), t(4;14) (13.7%), del17p (11.8%) and t(14;16) (5.9%). Univariate analyses revealed that complex karyotype (p

Description: Abstract 4873 Angiogenesis plays a significant role in the biology of multiple myeloma (MM). Erythropoiesis stimulating agents (ESAs) have been recently associated with reduced survival in a subset of cancer patients who receive ESAs, including MM but the etiology for this correlation has not been sufficiently explored. It is known that the endothelial cells produce angiogenic factors, promote the growth and survival of MM cells and carry erythropoietin receptors which in hypoxic conditions they transport the signal for their own proliferation and expansion under the influence of the endogenous erythropoietin. The aim of this study was to investigate the possible impact of ESAs administration on post-therapy angiogenesis. We studied 84 newly diagnosed MM patients (47M/37F; median age: 65 years, range: 39-82 years) who underwent conventional anti-myeloma therapy: 62 patients received VAD (53 of whom within the context of the randomized study VAD vs. TVAD, conducted by the Greek Myeloma Study Group) and 22 patients received MP. Fifty-two patients received ESAs for at least 8 weeks (ESA group), while 32 did not receive ESA (non-ESA group). MVD was assessed in bone marrow biopsies at baseline and at the time of best response by using monoclonal antibodies targeting CD34. The number of microvessels expressing the CD34 antibody was counted by two experienced pathologists through a grid at a magnification of 400x and was finally divided to the number of the high power fields used for screening the whole marrow surface. The counts were finally expressed as number of vessels per mm2 area of the involved marrow. Fifteen individuals with normal findings in the bone marrow were used as controls. Furthermore, the following cytokines that are involved in the angiogenesis process in MM were measured in the serum of both patients and controls on the day of the trephine biopsy performance: VEGF, bFGF, TGF-b, IL-6, soluble IL-6R (sIL-6R), IL-1b and TNF-α, using an ELISA methodology (R&D, Minneapolis, MN, USA). Patients characteristics between the ESA and non-ESA groups at baseline were well balanced except of Hb which was, as expected, significantly lower in the ESA-group (p

Description: Abstract 4815 Algae preparations are commonly used in complementary and alternative medicine for presumed anti-oxidant, anti-inflammatory, and anti-cancer properties. In this study, we have examined the effects of extracts from algae and algae components on the proliferation of normal hematopoietic and leukemia cells. To prepare extracts, 1 gram of Dunaliella salina (Dun), Astaxanthin (Ast), Spirulina (C-phycocyanin) (Spir), or Aphanizomenon flos-aquae (AFA) were added to 10 ml of 70% ethanol and incubated at 4°C on a shaker for 24 hours. The slurry was centrifuged at 400 g for 10 minutes at 4°C, and the supernatant was filtered through 413-grade filter paper. Leukemia cell lines were purchased from ATCC and blood or marrow aspirates from normal subjects or patients with leukemia were subjected to Ficoll-Hypaque density gradient centrifugation. CD34+ cells were isolated using Miltenyi Biotec MiniMACS magnetic bead cell separation columns. To determine effects on cell proliferation, increasing concentrations of algae extracts were added in fresh medium to plated cells, and MTT reagent was added followed by detergent and absorbance readings were recorded at 570 nm. Leukemic cell lines such as HL60 and MV4-11 were significantly inhibited by Ast and AFA at concentration of 0.8 mg/mL of extract (p

Description: Abstract 4877 Introduction The CD138negCD20+ cell population has been suggested as the putative multiple myeloma (MM) cancer stem cells, both in MM cell lines and in patient specimens. CD200 is an immunosuppressive membrane glycoprotein reported to be co-expressed with other stem-cell markers in prostate, breast, brain, and colon cancers. Also, CD200 is a negative prognostic factor for MM. It remains unknown whether CD200 is expressed in the CD138negCD20+ MM cell population, the putative MM cancer stem cells. Here we addressed this question by assessing CD200 expression in different subpopulations of MM cells according to their CD138 expression levels. Methods Two MM cell lines (RPMI8226 and NCI-H929) were co-stained with FITC-labeled anti-CD200, PE-labeled anti-CD20, and APC-labeled anti-CD138 antibodies for multicolor flow cytometry analysis. The cells were gated into 4 subpopulations (h1〉h2〉h3〉h4 corresponding to CD138 expression levels: high〉dim〉low〉negative). In each gated cell population, CD200 expression was assessed, and the CD200+ and CD200neg cell subpopulations were further gated for analysis of their CD20 expression levels. Results In the RPMI 8226 cell line, the distribution of gated h1, h2, h3, and h4 populations was 87.51%, 5.86%, 5.26%, and 1.29%, respectively. In the h2 (CD138dim) population, 22.88% of the cells were CD200+. In contrast, CD200 was expressed at much lower levels in the other three populations, ranging from 7.28% (h1), 6.65% (h3), to 0.48% (h4). In CD200+ cells from the gated h1, h2, h3, and h4 population, CD20+ cells were 19.50%, 23.03%, 27.67%, and 18.75%, respectively, while in the CD200neg cells, CD20+ cells were 20.79%, 22.50%, 25.08%, and 28.02%, respectively. In the NCI-H929 cell line, 18.59% of the cells in the h2 population were CD200+, whereas only 1.61%, 1.69%, and 0.47% of the cells in the h1, h3, and h4 populations were CD200+. In each population, there were more CD20+ cells in CD200+ cells than in CD200neg cells, which were 24.52% vs 0.04% (h1), 41.94% vs 11.34% (h2), 11.11% vs 3.06% (h3), and 50.00% vs 2.25% (h4), respectively. Conclusions These results demonstrate that the immunosuppressive molecule CD200 is preferentially expressed in a CD138dim subpopulation of multiple myeloma cells. Depending on cell line, the putative myeloma stem cell marker CD20 is either preferentially, or not preferentially, expressed in the CD200+ cells, suggesting that an immune-resistant subpopulation of MM stem cells might exist, which may have important implications for designing immunotherapeutic approaches to treat this disease. Disclosures Goldenberg: Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Description: Abstract 4841 Myelodysplastic syndromes (MDS) are a group of acquired clonal stem cell disorders that mainly affect the elderly population, characterized by ineffective hematopoiesis and high risk of leukemic transformation. MDS are heterogeneous in terms of morphology, clinical features and survival. An increasing body of work reveals that there might be differences in clinical features between Asian and Western cases. Japanese patients seem to be younger, have a lower frequency of refractory anemia (RA) with ringed sideroblast (RARS) and a higher frequency of RA, according to FAB classification, as well as different prognostic factors such as the frequency of cytogenetic abnormalities. Incidence rates for MDS in Brazil are unavailable. The purpose of the study was to obtain epidemiological data of MDS adult patients who presented from January 2003 to December 2007 in 10 Brazilian tertiary-care hematology centers from different regions of the country. Patient data collected by participating physicians were entered and stored with the use of an internet-based, data collection tool. Blood counts, bone marrow aspiration, trephine biopsy and chromosomal study were recorded. Survival was estimated through Kaplan-Meier method and the difference between survival curves was assessed by means of Log-Rank Test. Death incidence rates were estimated and compared. Statistical analyses of relevant variables were performed. Three hundred and forty three patients with diagnosis of MDS according to FAB/WHO classification were included in this retrospective analysis. The mean age at presentation was 68 years (range 17 to 98). Fifty percent of cases were male. Cigarette smoking, alcohol abuse and pesticide/herbicide exposure were reported in 33.5%, 13.4% and 14.3% respectively. Median hemoglobin was 8.7 g/dL, median neutrophils count was 1,575/mm3 and median platelets count was 97,000/mm3. There was no excess of blasts in 68.4% of cases. Bone marrow biopsy was performed in 78.5% of patients. Lymphoid nodules were seen in 11.3% and any degree of fibrosis in 28.6%. Cytogenetic analysis was performed in 67.8% of cases and showed chromosomal abnormalities in 50.5%. The del(5q) isolated or combined with other alterations were observed in 6.0%. Flow cytometry analysis for CD55 and CD59 was performed in 11,3% and was normal in 97,4%. Near 8% of cases were classified as secondary MDS. The distribution of disease subtypes according to FAB classification was: RA 42,3%, RARS 9,0%, RA with excess of blasts (RAEB) 20,7%, RAEB-t 4,2% and chronic myelomonocytic leukemia (CMML) 3,9%. According to IPSS patients were stratified as low-risk (low risk plus intermediate I) 55,9% and high risk (intermediate II and high risk) 13,1%. In 30,1% no stratification was possible. In 26,5% of cases iron overload was diagnosed although only 28,3% of cases had performed serum ferritin. The follow-up time ranged from 1 to 78 months (mean: 28 months). Thirty-six percent of patients died and the death was MDS-related in 68.3% of cases. The high and low risk survival curves were significantly different (p

Description: Abstract 2496 Poster Board II-473 Background: HMG-CoA reductase inhibitors (statins) have been used to treat hypercholesterolemia and hyperlipidemia for over 20 years. Statins competitively inhibit HMG-CoA reductase thereby blocking the synthesis of mevalonate. They directly inhibit synthesis of steroid hormones and cholesterol but also indirectly inhibit prenylation and ubiqitination. As all currently marketed statins are lipophillic, with the exception of Pravachol (pravastatin), several therapeutic trials have attempted to exploit these indirect actions to treat both neoplastic (glioblastoma, anaplastic astrocytoma) and degenerative (Alzheimer's, Parkinson's) neurologic disease. We studied the impact of incidentally prescribed statins on high-risk patients with primary central nervous system diffuse large B cell lymphoma (PCNSL). Methods: We used an IRB-approved clinicopathologic database, derived from comprehensive tumor registry data at the Massachusetts General Hospital, to identify all patients diagnosed with primary central nervous system lymphoma (PCNSL) between 1991 and 2007 (n=118). We excluded pediatric patients, patients who did not receive curative treatment and patients who failed to achieve a CR with initial therapy. Automated analysis of billing and medication administration records was used to identify the administration of statins to these patients. We compared the outcome of patients who were receiving statins to controls with PCNSL who did not receive statins. We excluded patients who did not receive statins within the time interval from 6 months prior to 2 years after their PCNSL diagnosis. As only a single statin pt was less than 50 we excluded all pts below this age from the cases and controls. All patients received high dose MTX based therapy. Overall survival (OS) was calculated from the date of first methotrexate. Results: Median age of statin patients was 66 yrs (range 52 – 76); median age of controls was 66 years (range 50 - 86). Nine patients were on atorvastatin, 9 were on simvastatin, 1 each were on pravastatin, rosuvastatin and fluvastatin. At a median follow-up of 47.5 months, concurrent statin therapy was associated with improved OS (62% vs. 37%)(p=0.04 Log-rank test). The median OS for all statin patients 〉 50 years old (n=21) was 60 months versus 37 months for all other patients 〉 50 years old (n=67) (p=

Description: Abstract 2475 Poster Board II-452 Introduction: Non-Hodgkin Lymphoma (NHL) is the fifth most common type of malignancy in Canada. The most common subtype of NHL is diffuse large B-cell lymphoma (DLBCL). Initial standard treatment for DLBCL includes combination immuno-chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). This regimen is typically administered every 21 days for a total of 6 cycles. Febrile neutropenia (FN) is a serious toxicity of lymphoma chemotherapy and patients with this condition must be treated aggressively as it could lead to complications such as prolonged hospitalization and death. Granulocyte-colony stimulating factors such as filgrastim and pegfilgrastim are efficacious in preventing FN, yet their cost-effectiveness has not been evaluated in the publicly funded Canadian healthcare system. Methods: A Markov model was constructed to evaluate the cost-effectiveness (cost-utility) of filgrastim and pegfilgrastim as primary prophylaxis versus no primary prophylaxis against FN in DLBCL patients receiving induction chemotherapy. Health states included in the model were hospitalization for FN, and receiving chemotherapy with no FN. It was assumed that patients in the no primary prophylaxis arm of the model who experienced a FN episode would receive secondary prophylaxis with filgrastim for all subsequent chemotherapy cycles. The time horizon of the model was 18 weeks, the time period over which the six cycles of chemotherapy are administered. The analysis was conducted from the hospital perspective. Costs are reported in 2009 Canadian dollars and outcomes in quality-adjusted life years (QALY). One-way sensitivity analyses were done on model parameters. A probabilistic sensitivity analysis was done to evaluate overall uncertainty in the model. Results: In the base case analysis costs associated with the no primary prophylaxis, filgrastim and pegfilgrastim interventions were $6044, $9450 and $15899, respectively. Quality-adjusted life years associated with the three interventions were 0.198, 0.200, and 0.202 respectively (over the 18-week model time horizon). The incremental cost-effectiveness ratio (ICER) of filgrastim compared to no primary prophylaxis was estimated to be $1.7 million (M)/QALY [95% confidence interval: -14M/QALY (dominated) to $15M/QALY]; for pegfilgrastim compared to filgrastim the ICER was estimated to be $4.4M/QALY [95% confidence interval: -25M/QALY (dominated) to $22.7M/QALY]. All one-way sensitivity analyses yielded ICERs of greater than $1M/QALY. Conclusions: The ICERs for filgrastim and pegfilgrastim when used as primary prophylaxis against FN in DLBCL patients receiving induction chemotherapy are well above the usually accepted cost-effectiveness threshold of $50,000/QALY. Disclosures: Mittmann: Amgen Canada: Unrestriced educational grant.

Description: Abstract 2484 Poster Board II-461 Background: Hematologic (HM) and solid tumor malignancy (STM) patients may be immunocompromised (IC) due to their underlying diseases and the immunosuppressive therapies they received. Availability of a practical and robust algorithm to classify HM and STM patients into IC levels using data from healthcare databases could be valuable for a variety of epidemiologic, health service or outcome research in the field of oncology. Classification of the IC status of patients also permits accurate prediction of the types of supportive care that such patients may need to prevent infectious complications that often accompany treatments for the underlying malignancy. Methods: An expert panel mainly comprised of hematologists and oncologists developed an algorithm to classify HM and STM patients' level of IC into either none/low, medium, or high, using data available in electronic databases. The algorithm was based on the following factors: (1) the type of chemotherapy agents and/or corticosteroids, (2) time since last chemotherapy/corticosteroid treatment, and (3) specific type of HM (for HM patients). Chemotherapy agents were classified into levels of IC, irrespective of dose used. Corticosteroid therapy was classified into levels of IC based on dose and duration of treatment. IC levels were allowed to change monthly to reflect what chemotherapy agents were used, dose and duration of corticosteroid if any, and time since the last IC treatment. In the base case scenario, the patient's IC level (based on treatment) stayed at an assigned level for 6 months after the last treatment and then moved to the next lower level for an additional 6 months. In alternative scenarios, sensitivity analyses were also performed using the 1, 3, 9, and 12 month cutoffs. If the patient received multiple chemotherapy agents/corticosteroid regimens, the most immunocompromising agent determined the IC level during that time period. We applied and tested this algorithm in a study of HM and STM patients diagnosed in 2001-2005 at Kaiser Permanente Northern California (KPNC). Herpes zoster (HZ), a viral disease caused by the reactivation of varicella zoster virus, is associated with impairment of cell-mediated immunity. Therefore, we used incidence of HZ as a proxy for true IC status. The KPNC cancer registry was used to identify cancer diagnoses and the type, stage and grade of the underlying HM. Data on specific chemotherapy agents and/or dose and duration of corticosteroids as well as time since last IC treatment were obtained from KPNC pharmacy databases. Potential episodes of HZ in 2001-2006 were identified from HZ diagnosis codes and antiviral use in various KPNC databases. HZ diagnosis was confirmed by clinical review of patient's medical records. We measured HZ incidence rates in HM and STM patients and examined whether they were correlated with IC level based on our algorithm. Results: In the base case scenario, among the 4,465 patient-years (py) of follow-up in HM patients, 25.3%, 34.4%, and 40.3% of follow-up time was categorized as none/low, medium, or high IC, respectively. The corresponding rates of HZ were 13, 25, and 48/1000 py. Among the 23,072 py of follow-up in STM patients, 74.9%, 8.0%, and 17.1% of follow-up time was categorized as none/low, medium, or high IC, respectively. The corresponding rates of HZ were 10, 20, and 19/1000 py, respectively. The algorithm was not sensitive to changes from 3 to 12 months, but was sensitive to the 1 month cutoff, in the assumption of duration of IC since the last IC treatment. Conclusions: It is feasible and practical to categorize cancer patients into IC levels using electronic pharmacy and cancer registry databases. The correlation between incidence of HZ and levels of IC in both HM and STM patients suggested that the proposed algorithm may appropriately assign IC levels in these patients. Additional testing in other cancer populations may be needed to further validate this algorithm. Disclosures: Tran: Merck & Co., Inc.: Employment. Ray:Merck & Co., Inc.: Investigative. Saddier:Merck & Co., Inc.: Employment. Trigg:Merck & Co., Inc.: Employment. Hayes:Merck & Co., Inc.: Consultancy. Li:Merck & Co., Inc.: Investigative. Rizzieri:Merck & Co., Inc.: Consultancy. Stein:Merck & Co., Inc.: Consultancy. Weber:Merck & Co., Inc.: Consultancy. Serody:Merck & Co., Inc.: Consultancy. Raasch:Merck & Co., Inc.: Consultancy. Habel:Merck & Co., Inc.: Investigative.

Description: Abstract 2456 Poster Board II-433 Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation; the degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been fully evaluated. In the presence of rapamycin, T cell co-stimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype: by intracellular flow, median percentage expression of Foxp3, IFN-γ, and T-bet was 4%, 20%, and 72%, respectively. Phospho-flow cytometry revealed that such Th1/Tc1 cells expressed activated STAT1 and STAT4 in spite of mTOR blockade; STAT activation was abrogated by PI3 kinase inhibition. Rapamycin-resistant human Th1/Tc1 cells (Th1/Tc1.R cells): (1) had increased expression of the autophagy-related gene LC3BII by gene array and protein analysis; (2) preferentially expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) had reduced apoptosis relative to control Th1/Tc1 cells not generated in rapamycin (p=0.04). The anti-apoptotic phenotype of Th1/Tc1.R cells was abrogated by co-incubation with the autophagy inhibitor, 3-methyl adenine. The in vivo effect of the Th1/Tc1.R cells was evaluated using two xenogeneic GVHD (x-GVHD) models. First, in an LPS-induced x-GVHD model, Th1/Tc1.R cells resulted in lethality in 75% recipients; soluble TNF-α receptor therapy with etanercept reduced the frequency of lethality to 15%. Second, using a non-LPS natural history model of x-GVHD, recipients of Th1/Tc1.R cells (relative to recipients of control Th1/Tc1 cells) had increased human T cell engraftment (day 30 post-BMT, p=0.001), increased human T cell cytokine levels, increased human T cell expression of the cytotoxic degranulation molecule CD107 (p=0.05), and increased human T cell infiltration of skin, gut, and liver. In this model, lethality due to x-GVHD was also increased in Th1/Tc1.R cell recipients (lethality increased from 20% to 70%, p=0.04). We conclude that rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, by promoting autophagy rapamycin permits stable expression of T-bet and generates an anti-apoptotic Th1/Tc1 effector phenotype, thereby yielding increased x-GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2437 Poster Board II-414 Introduction: BK virus (BKV) is a polyomavirus that is ubiquitous in humans, infecting over 85% of normal individuals. After initial infection it persists in a latent state in the urothelium from whence it can reactivate causing disease in immunocompromised patients. BKV is a cause of haemorrhagic cystitis after allogeneic SCT and is emerging as one of the major causes of graft loss after renal transplant. Current treatment is limited to reduction of immunosuppression as possible. Aim: To develop a method for production of a T cell product with BKV specificity from normal donors for use in adoptive immunotherapy post hemopoietic stem cell transplantation. Methods: Peripheral blood mononuclear cells (PBMC) or monocyte derived dendritic cells (mo-DC) were pulsed with mixes of overlapping peptides covering the 5 BKV proteins (VP1, VP2, VP2 isoform 3, large T antigen (LTA) and small T antigen (sTA)). Mo-DC were produced by isolating monocytes by adherence to plastic and culturing for 7 days in GM-CSF and IL-4 containing media. On day 6 mo-DC were matured by the addition of TNF. T cells were stimulated on day 1 and 7 with peptide pulsed PBMC or mo-DC and cultured for 21 days with increasing doses of IL-2 from day 7. The cellular product was then analysed for phenotype, BKV specificity and functionality by examining cytokine production and cytotoxicity. Results: Cellular proliferation was seen in all of 10 normal donors with a mean increase of 6.4 fold in total cell number. All cellular products were 〉84% CD3 positive (Mean 96%, SEM 1.3) with CD4 and CD8 ratio varying significantly between individual donors (CD4 range 9.7 to 97.5%, mean 70.79; CD8 range 0.8 to 77.0%, mean 23.3). Cells were of memory phenotype, being CD28+ (mean 86.1%, SEM 6.5), CD45RO+ (mean 84.1%, SEM 5.9) and a variable proportion were of central memory phenotype (CD62L+ mean 21.6%, range 6.9 to 55.0). Cytokine responses to stimulation with BKV peptides could be elicited in 5 of 6 evaluable donors. Multiple cytokines were produced by the responding cells: IFN-γ (mean 29.9% of CD3 cells, range 4.5 to 78.8), TNF (mean 19.9%, range 2.7 to 63) and IL-2 (mean 12.8%, range 1.2 to 37.8). Cytokine responses were seen in both CD4 and CD8 cells and showed significant individual variation. VP1 and LTA specific cells dominated most cultures while a smaller percentage of the cells were specific for VP2, VP2 isoform 3 and sTA. CD8 specificity was mainly confined to a single protein whereas CD4 responses tended to be of lower magnitude but broader specificity. Cultures exhibited cytotoxic activity with the lysis of BKV antigen coated target cells in a pattern that correlated with the presence of CD8 positive cytokine producing cells (up to 78.9% specific lysis at effector to target ratio of 20:1). Discussion: The clinical utility of this product remains to be determined. Potential uses include prophylaxis and therapy of reactivation of BK virus after hemopoietic stem cell or renal transplantation. This method for large-scale expansion of BKV specific CTL could be utilised for analysis of BKV targeted immune responses and epitope identification. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2444 Poster Board II-421 Alloreactive effector T cells are the central to graft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). In GVHD host antigens are never cleared and alloreactive effector T cells are continuously generated over a period of several months or longer, but their suppression and control have proven to be difficult in practice. Using mouse models of GVHD directed against minor histocompatibility antigens (miHAs), we demonstrate that alloreactive effector T cells proliferate and persist upon chronic exposure to alloantigens via reactivation of stem cell transcriptional programs normally expressed in embryonic stem cells and neural stem cells. Many activated stem cell genes in effector T cells were distinct from those in memory T cells and were maintained at high levels upon T cell receptor activation, suggesting a specific role in chronically activated effector T cells. One of these genes, Ezh2, encodes a chromatin modifying enzyme essential to the proliferation, survival and differentiation of stem cells, was upregulated in CD8+ effector T cells upon antigenic stimulation and downregulated when the antigen was withdrawn. Pharmacologically inactivation of EZH2 with 3-Deazaneplanocin A inhibited effector T cell proliferation and survival. Silencing Ezh2 independently validated that Ezh2 was important for regulating effector T cell proliferation and expression of many stem cell genes. To further evaluate whether alloreactive CD8+ effector T cells obtained stem cell-like properties, e.g. the ability to self-renew to continually generate effector cells, we adoptively transferred highly purified miHA H60-specific (H60+) CD8+ effector T cells into secondary allogeneic and congenic recipients, respectively. As compared to congenic recipients, allogeneic recipients had 80-fold more proliferating H60+CD8+ effector T cells. These donor H60+CD8+ effector T cells expressed high levels of CD122, CD69, CXCR3, PD1, IFNγ and Granzyme B, required miHA H60 stimulation to sustain their replication with effector function and expression of stem cell genes, and caused severe GVHD in secondary allogeneic recipients. These results indicate that stem cell transcriptional programs expressed in embryonic and neural stem cells may play important roles in effector T cells. Among these stem cell genes, Ezh2 emerges as an important therapeutic target in modulating alloreactive T cell-mediated GVHD. Disclosures: Zhang: University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding.

Description: Abstract 2436 Poster Board II-413 Sensitive in vivo imaging methods have advanced the fields of stem cell transplantation, graft-versus–host disease (GVHD) and graft-versus-tumor responses (GVT). Near-infrared (NIF) fluorescent proteins (FP) appear advantageous for deeper tissue penetration due to minimized absorbance by hemoglobin, water and lipids. Therefore we tested whether a recently published NIF FP (FP635, “Katushka”) could serve as a single reporter for whole body and single cell imaging. To compare signal intensities of eGFP and FP635 we generated fluorescent MOSEC cell lines (mouse ovarian cancer), titrated them in vitro and subcutaneously (s.c.) in vivo in Balb/c nu/nu mice. MOSEC FP635 showed twice the signal intensities compared to MOSEC eGFP in vitro by spectral fluorescence imaging (FLI). In vivo the eGFP signal was attenuated 〉60% in contrast to only 20% for FP635 from subcutaneous sites. However, FP635 signals from deep tissue layers were quenched. To address whether reduced signal attenuation of FP635 may allow sensitive visualization of immune processes by FLI and multiphoton-laser-scanning-microscopy (MPM) we generated transgenic mice in the genetic C57Bl/6 (B6) background, expressing FP635 under the ubiquitin promoter. Transgenic founders were selected upon signal intensities of leukocyte populations measured by flow cytometry in the PerCP channel. Combination of FP635 with colors other than red were possible for multiparameter flow cytometry. Next, eGFP, DsRed and FP635 splenocytes from transgenic donors were titrated as described above. In vitro signal intensities of FP635 splenocytes were 〉5 times lower compared to the other two FPs. FP635 signal absorption in vivo was low (30%) which is consistent with MOSEC titration results. In vivo DsRed detection was most sensitive and signals were similarly attenuated as FP635 in contrast to eGFP (60%). Subsequently, we aimed to visualize FP635 in a model of GVHD, where alloreactive T cells undergo massive expansion. Balb/c nu/nu mice were lethally irradiated and transplanted with 5×106 B6.WT bone marrow cells plus either 2×107 B6.DsRed+Luciferase+ or 2×107 B6.FP635 splenocytes. Sensitivity for DsRed cell detection was superior over FP635 cells. FP635 signal was only weakly detectable in lymph nodes (LN) by ex vivo FLI, where DsRed signals were detectable at earlier timepoints and LNs were even visualized by in vivo FLI. DsRed+ Luciferase+ double transgenic splenocytes allowed direct comparison of bioluminescence imaging (BLI) to FLI. Timely in vivo visualization of immune cells in deep tissues was feasible only by BLI. After whole body imaging the suitability of FP635 for MPM was checked by co-injecting eGFP B cells and either DsRed or FP635 T cells intravenously into RAG-/- mice. As FP635 is a NIF FP we expected to achieve deeper tissue penetration in hemoglobin rich organs, such as the spleen, in single cell microscopy. After 6 weeks of adoptive cell transfer we imaged spleens by MPM. Tissue penetration depths of DsRed or FP635 T cells were compared to eGFP B cells. No advantage in penetration depth of FP635 over DsRed was measured. Photobleaching is an important factor for microscopy, especially if cells are to be tracked over long time. FP635 transfected 293T cells bleached faster (t1/2=108 sec) than 293T cells transfected with eGFP (t1/2〉900 sec) or DsRed (t1/2=411 sec). These experiments indicate that very high expression levels of FP635 need to be achieved for imaging. The signal attenuation of FP635 is low which may increase the sensitivity but in our hands DsRed showed comparable characteristics. Yet, the fast photobleaching of FP635 compared to the broadly established FPs DsRed and eGFP may be disadvantageous for long term microscopic tracking of cells. Our data indicate that BLI is by far superior over FLI in sensitivity and tissue penetration for whole body imaging of immune cells. However, FLI of red or near-infrared clonally selectable tumor cell lines may provide a welcome color addition to study immune cell-tumor interactions in combined models of BLI and FLI. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2430 Poster Board II-407 After transplantation, hematopoietic stem/progenitor cells (HSPCs) home to the marrow, where they engraft and self-renew. To explore the mechanism of this multi-step and dynamic repopulation process, we performed the first in vivo adult vertebrate chemical screen aimed at identifying novel chemical modulators of HSPC repopulation using a novel competitive marrow transplantation assay in zebrafish. To distinguish between the donors, we used ubiquitous GFP or DsRed2 transgenic fish, Tg(β-actin:GFP) and Red GloFish®, for marrow cell isolation. 20,000 GFP+ cells were treated with a chemical and mixed with 80,000 untreated DsRed2+ marrows. This pool of cells was injected retro-orbitally into a transparent adult zebrafish. After a recovery period, the fish was then anesthetized and the region of the kidney (the adult site of hematopoiesis) was examined by fluorescence microscopy. The competition between the two donors was determined by analyzing the ratios of GFP and DsRed2 fluorescence intensity with ImageJ software. Using this assay, we demonstrated that dmPGE2 and/or GSK-3β inhibitor treatment of GFP+ marrows for 3 hrs could dramatically increase repopulation in fish. A chemical library of 480 chemicals with known bioactivities was screened using this in vivo assay. GFP+ marrows were incubated with different chemicals for 3 hrs and ten recipient fish were transplanted for each chemical. By examining engraftment at 4 weeks, we found 10 chemicals that improved HSPC repopulation. Based on the known bioactivity, these chemicals were categorized into several signaling pathways, including prostaglandin metabolism and retinoic acid pathways. Several of the compounds also increased HSC formation in zebrafish embryos, indicating that some pathways might be shared by different developmental stages. To examine whether the bioactivities of these hits are conserved in mammals, CD45.1 mouse whole bone marrow cells were treated with hit compounds for 3 hrs and competitively transplanted into CD45.2 recipients. Peripheral blood was sampled at 3, 6, 12, and 20 week post transplant. Several hits were confirmed to increase long-term chimerism in mice. The retinoic acid pathway has been shown to play an important role in hematopoiesis. Among the six retinoic acid receptor (RAR) agonists in the chemical library, which includes all-trans retinoic acid (ATRA), only two structurally highly related compounds, AM-580 and TTNPB scored positive in the screen. These two compounds have distinctive chemical moieties from ATRA. This structural difference likely leads to stronger agonistic effects on RAR than ATRA and resists degradation. In conclusion, the in vivo chemical screening using zebrafish competitive marrow transplantation provides a successful example of phenotypic screening in whole adult vertebrates. The discovery of novel repopulation modulators should provide a better understanding of signaling events that regulate homing and self-renewal, and may have clinical application in marrow or cord blood transplantation. Disclosures: Zon: FATE Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Stemgent: Consultancy.

Description: Abstract 2398 Poster Board II-375 B-catenin is the central effector molecule of the canonical wnt signaling pathway which governs cell fate and differentiation during embryogenesis as well as self-renewal of hematopoietic stem cells. Deregulation of the pathway has been observed in various malignancies including myeloid leukemias where over-expression of β-catenin is an independent adverse prognostic factor. In the present study we examined the functional outcome of stable β-catenin down-regulation through lentivirus-mediated expression of short hairpin RNA (shRNA). Reduction of the β-catenin levels in AML cell lines and patient samples diminished their in vitro proliferation ability without significantly affecting cell viability. In order to study the role of β-catenin in vivo, we transplanted leukemic cell lines with control or reduced levels of β-catenin in NOD/SCID animals and analyzed the engraftment levels in the bone marrow. We observed that while the immediate homing of the cells was not affected by the β-catenin levels, the bone marrow engraftment was directly dependent on its levels. Subsequent examination of bone marrow sections revealed that the reduced engraftment was partly due to the inability of the cells with lower β-catenin levels to dock to the endosteal niches, a finding that was confirmed in competitive repopulation assays with untransduced cells. When we examined the expression levels of adhesion molecules and integrins in engrafted cells in vivo, we observed a significant down-regulation of CD44 expression, a molecule that participates in the interaction of HSCs with the niche. Gene expression analysis of the components of the wnt signaling pathway showed that the pathway is subject to tight transcriptional regulation with minor expression deviations. We did, however, observe an up-regulation in components that participate in the non-canonical wnt signaling pathways such as the WNT5B ligand. Ongoing experiments in normal cord blood CD34+ cells will determine the in vivo role of β-catenin signaling in normal hematopoietic progenitors. In conclusion, our study showed that β-catenin comprises an integral part in the development and progression of AML in vivo, indicating that manipulation of the wnt pathway may hold a therapeutic potential in the management of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 245 Background: The incidence of MDS in Canada is not known. Diagnosis of MDS is often challenging as dysplastic features on bone marrow may be non-specific, requiring exclusion of other disorders. The province of Manitoba, with a population of 1.2 million, has a cancer registry which has included patients with MDS since 2001. In this province, hematology diagnostic services are centralized at two teaching hospitals and the few bone marrows performed outside are reviewed centrally. This provided us with the opportunity to use registry data and bone marrow records to determine the incidence of MDS. We hypothesized that for an accurate estimate, a proportion of MDS cases would require follow-up data. Methods: Retrospective study to examine all cases of MDS, which included chronic myelomonocytic leukemia (CMML) diagnosed in Manitoba. All adult Manitobans diagnosed with MDS and CMML (excluding RAEB-T), from 01/2006 to 12/2007 were identified from the cancer registry using ICD-O-3 topography code C42.1 and morphology codes 9980/3, 9982/3, 9983/3, 9985/3, 9986/3, 9987/3, 9989/3 and 9945/3. Bone marrow records for the same period were reviewed to identify all cases that had features of MDS. The clinical charts of all these patients were reviewed centrally to exclude those whose clinical course or repeat investigations suggested an alternative diagnosis. Results: A total of 80 patients with newly diagnosed MDS were identified. The age adjusted incidence of MDS was 3.26/100,000. Incidence was higher in men (4.05/100,000) as against women (2.57/100,000). Incidence varied significantly with age at diagnosis: 80yr: 21.93. Eleven cases (13.75%) were not known to the cancer registry but were detected on reviewing the bone marrow data. From the registry, nine cases (11.25%) were excluded as the chart review and follow-up revealed alternative diagnoses. Conclusions: The incidence of MDS for Manitoba is similar to published rates in Europe and the USA. This may be an underestimation of the actual incidence, as elderly patients may not undergo bone marrow examination if the therapeutic intervention is only supportive. Cancer registries that include MDS based on one-time bone marrow reports should include a review process of confirming or excluding the diagnosis of MDS based on follow up investigations and course of illness. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2447 Poster Board II-424 Allogeneic bone marrow transplantation (BMT) represents the most effective treatment for patients with high risk and relapsed hematologic malignancies. One of the major complications of allogeneic BMT is graft-versus-host disease (GvHD), which is caused by donor T cells reacting against host antigens. These same alloreactive donor T cells can provide a beneficial ¡°graft-versus-leukemia¡± (GvL) effect as well resulting in reduction in leukemia relapse. Because regulatory T cells (Tregs) have been shown to suppress GvHD while preserving GvL, their use in the allogeneic transplant setting provides a promising strategy to treat GvHD. However, three major obstacles prevent their routine use in human clinical trials: 1) the low circulating numbers of Tregs in peripheral blood, 2) loss of suppressor activity following ex vivo expansion and 3) the lack of Treg-specific markers to purify ex vivo expanded Tregs. Foxp3 is a forkhead transcription factor which is both exclusively expressed in Tregs and, when overexpressed in conventional effector T cells (Teff), can convert these Teff into functionally suppressive Treg-like T cells. The Foxp3 locus is unmethylated in Tregs while highly methylated and silenced in all other T cells. Several groups have shown that the hypomethylating agent azacitidine (AzaC) induces FOXP3 expression in non-Tregs. Furthermore, we have shown that treatment of anti-CD3/CD28 antibody-coated bead-activated CD4+CD25- T cells with AzaC results in robust and prolonged (〉7 days) expression of FOXP3. AzaC-induced FOXP3 expression is also associated with a potent Treg-like suppressive phenotype in vitro. Thus, we hypothesize that AzaC treatment of mice after allogeneic BMT will dramatically mitigate GvHD while preserving GvL via transcriptional regulation of Foxp3 in alloreactive Tconv. In murine T-cell depleted BMT model (B6¡æBalb/c; 900 cGy TBI) with delayed infusion of conventional T cells (Tconv) (2 ×106) at day 11 post BMT, followed by subcutaneous treatment of AzaC (2 mg/kg at days 15, 17, 19, and 21 post BMT), we found that AzaC dramatically suppressed GvHD caused by allogeneic donor T cells while maintaining donor (H2Kb) engraftment of all lineages. We found that the AzaC group had significantly higher FOXP3+ Tregs than in PBS control group and that these Tregs were derived from donor T cells, suggesting that the suppression of GvHD was mediated by AzaC-induced Tregs. We further tested whether AzaC treatment of mice transplanted with allogeneic T cells preserve GvL while mitigating GvHD. Using the same murine allogeneic BMT model, Click Beetle Red luciferase-expressing A20 leukemia cells (BALB/C derived) were injected with T-cell depleted BM and 10 × 106 Tconv and in vivo bioluminescence imaging was performed to assess tumor burden in vivo post transplant. We found that AzaC treatment mitigated GvHD without abrogating GvL (Fig. 1) or donor engraftment. Thus, the adminstration of hypomethlating agents like AzaC in vivo after allogeneic stem cell transplant dramatically reduces GvHD while maintaining both donor engraftment and a potent GvL effect providing the foundations for future clinical trials. Disclosures: DiPersio: Genzyme Corporation: Honoraria.