ALBERT

Description: Abstract 3283 Donor-derived regulatory T cells (Treg) and natural killer (NK) cells can respectively improve stem cell transplant (SCT) outcome by reducing graft versus host disease (GVHD) severity and exerting a graft-versus-leukemia effect. High frequencies of donor Treg are associated with less GVHD, and low doses of interleukin-2 (IL-2) can expand both NK and Treg after allogeneic SCT. To explore the feasibility of improving the quality of peripheral blood SCT donations, we evaluated the safety and the tolerability of ultra-low dose IL-2 administration to volunteers with the aim of preferentially expanding Treg and NK cells. Twelve healthy volunteers (mean age 34 years; range 22–57) received 0.1 or 0.2 million U/m2/day IL-2 subcutaneously for 5 days (NIH protocol 11-H-0268). Blood samples were collected before and 1, 2, 3, 4, 7 and 28 days after IL-2 injection. Samples were analyzed by multiplex techniques including whole transcriptome gene expression with HumanGene 1.0ST microarrays; serum levels of 69 cytokines and chemokines by Luminex assay; and lymphocyte phenotyping by flow cytometry, to comprehensively characterize the cellular and molecular immune response to IL-2 (“IL-2 immunome”). Treg subsets were determined within the CD4+ T cell population using FoxP3, Helios, CD45RA and CD31 to identify thymus-derived natural Treg (nTreg), induced Tregs (iTreg) and their recent thymic emigrants (RTE). NK cell subsets were determined within CD56+CD3- population using NKG2A, KIR2DL1, KIR2DL2/3, KIR3DL1 and CD57 to identify CD56bright, CD56dim NKG2A+KIR-, and CD56dim KIR+CD57+ cells. All subjects tolerated ultra-low dose IL-2 with minimal adverse events (mainly grade 1–2 injection site reactions). The fraction of FoxP3+Treg in CD4 rose significantly above baseline peaking at 4 days (3.7% vs 5.8%; p=0.0004) after the first dose of IL-2. Treg subset analysis demonstrated that the fraction of nTreg and RTE nTreg in CD4 expanded significantly in the lower dose cohort compared to the higher dose cohort (p=0.004 and p=0.005 respectively). %CD56bright NK significantly increased at 7 days (p=0.008), whereas CD56dimNKG2A+KIR-, and CD56dimKIR+CD57+ NK cells remained at baseline. The Ki67 proliferation marker further verified a significant in vivo expansion of CD56bright NK cells with ultra-low dose IL-2. Cytokine and chemokine profiling demonstrated significant increase circulating level of IP-10 (P=0.0018) through day 2 to 4 after IL-2 injections. In contrast, circulating levels of IL-2, IL-6, IL-10, IL-15 and IL-17 remained unchanged after IL-2 injection. Gene expression microarray studies revealed significant changes in 24 genes (P value 〈 0.1 corrected by false discovery rate (FDR) for multiple testing), including up-regulation of IL-2RA and FOXP3 as early as 2 days after IL-2 injections. Gene Set Analysis (GSA) revealed significant changes (P value 〈 0.1 after FDR) in innate immune response pathways, including Toll-like receptor signaling and interferon signaling. This is the first study to show that ultra-low dose IL-2 could be safely administrated to healthy volunteers to expand thymic-derived natural Treg and CD56bright NK cells. These results raise the possibility of using ultra-low dose IL-2 to boost Treg and NK cells in stem cell donors. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Eltrombopag promotes hematopoiesis in patients with severe aplastic anemia by stimulating stem and progenitor cells. Eltrombopag can be discontinued safely in robust responders with maintenance of hematopoiesis.

Description: Abstract 54 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell (HSC) progenitors. SAA is treated with immunosuppressive therapy (IST) or allogeneic HSC transplantation (HSCT), with a successful outcome after either treatment in a majority of patients. However, 20–40% of patients without a suitable donor for HSCT and a suboptimal response to IST may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Several lines of evidence support the concept that signaling through mpl also influences expansion and maintenance of primitive HSCs and multi-potent progenitor cells. Eltrombopag, is a small molecule TPO receptor agonist that stimulates mpl, and increases platelet counts in patients with chronic immune thrombocytopenic purpura (ITP). It is approved for the treatment of chronic ITP (Promacta®). We conducted a non-randomized, pilot phase II study of eltrombopag in SAA patients who remained severely thrombocytopenic at least six months after one or more rounds of IST (clinical trials.gov identifier NCT00922883). Consecutive patients fulfilling the inclusion criteria received eltrombopag 50mg daily with dose escalation every two weeks to a maximum dose of 150mg daily. Primary end points assessed after three months of treatment were changes in peripheral blood counts (platelets, hemoglobin, absolute neutrophil counts), with hematologic response criteria defined a priori for each lineage. Secondary endpoints included the incidence of bleeding events, and health related quality of life. Patients who achieved hematologic responses were maintained on eltrombopag through an extended access protocol. We completed our planned accrual of twenty five patients, and 22 are evaluable for response to date. Median age was 45 years old (range 18–77 years), and the median time from the last course of IST was 13 months (range 6–54 months). Median follow up time was 9 months (range 1–24 months). Nine of twenty two patients (41%) achieved hematologic responses: seven of twenty-two patients (32%) achieved platelet responses with transfusion independence for eight weeks or greater; six patients had improved hemoglobin levels after starting treatment (mean hemoglobin increase of 3.8 g/dL) and 4 patients who were previously dependent on red blood cell transfusions have achieved transfusion-independence. Five neutropenic patients had increased neutrophil counts after treatment with eltrombopag (mean increase 660 cells/uL). Plasma TPO levels did not predict for hematologic response to eltrombopag. Serial bone marrow biopsies performed on patients with hematologic responses demonstrated normalization of trilineage hematopoiesis and cellularity in three of four responders receiving a year or more of therapy, with no increase in reticulin fibrosis (Figure 1). These results represent the first evidence that TPO stimulation can expand the HSC pool in humans, with clinically meaningful trilineage hematologic improvements in patients with SAA, resulting in transfusion-independence and improved quality of life with a simple daily oral regimen. Updated response data on the full 25 patients will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for severe aplastic anemia.

Description: Clinical observations and laboratory evidence link bone marrow failure in myelodysplastic syndrome (MDS) to a T cell–mediated immune process that is responsive to immunosuppressive treatment (IST) in some patients. Previously, we showed that trisomy 8 MDS patients had clonally expanded CD8+ T-cell populations that recognized aneuploid hematopoietic progenitor cells (HPC). Furthermore, microarray analyses showed that Wilms tumor 1 (WT1) gene was overexpressed by trisomy 8 hematopoietic progenitor (CD34+) cells compared with CD34+ cells from healthy donors. Here, we show that WT1 mRNA expression is up-regulated in the bone marrow mononuclear cells of MDS patients with trisomy 8 relative to healthy controls and non–trisomy 8 MDS; WT1 protein levels were also significantly elevated. In addition, using a combination of physical and functional assays to detect the presence and reactivity of specific T cells, respectively, we demonstrate that IST-responsive MDS patients exhibit significant CD4+ and CD8+ T-cell responses directed against WT1. Finally, WT1-specific CD8+ T cells were present within expanded T-cell receptor Vβ subfamilies and inhibited hematopoiesis when added to autologous patient bone marrow cells in culture. Thus, our results suggest that WT1 is one of the antigens that triggers T cell–mediated myelosuppression in MDS.

Description: Abstract 4427 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell progenitors. SAA is treated with immunosuppression or allogeneic stem cell transplantation (SCT), with a successful outcome in a majority. However, 20–40% of patients without a suitable donor for SCT do not respond to immunosuppression and may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Eltrombopag, a small molecule TPO mimetic that binds to mpl, increases platelet counts in healthy subjects, and in patients with chronic immune thrombocytopenic purpura. Both TPO and eltrombopag stimulate more primitive multilineage progenitors and stem cells in vitro. Patients with SAA and thrombocytopenia have very elevated TPO levels; nevertheless, we asked whether pharmacologic doses of eltrombopag could stimulate hematopoiesis in these patients without other options. We are conducting a pilot phase II study of eltrombopag in SAA patients with severe thrombocytopenia refractory to immunosuppressive therapy. Consecutive eligible adult patients were treated with oral eltrombopag at an initial dose of 50 mg daily, with escalation to a maximum dose 150 mg daily, with the goal of maintaining a platelet count of 〉20,000/uL above baseline. Treatment response was measured after three months and was defined as platelet count increases to 20,000/uL above baseline, or stable platelet counts with transfusion-independence for a minimum of 8 weeks. Nine patients have been enrolled and six are evaluable for response to date. Two patients did not respond to treatment. Three patients achieved platelet responses by 12 weeks of treatment, and all have sustained their responses (median follow up 10 months). Four patients exhibited improved hemoglobin levels 12 weeks after starting treatment (median hemoglobin increase of 2.1 g/dL) and two patients who were previously dependent on packed red blood cell transfusions have achieved transfusion-independence. Three neutropenic patients exhibited increased neutrophil counts after treatment with eltrombopag (median increase 0.46K cells/uL). These results provide evidence that eltrombopag can improve platelet counts in patients with severe refractory thrombocytopenia, and perhaps more surprisingly, have a clinically relevant impact on erythropoiesis and myelopoiesis. Updated data will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for thrombocytopenia in refractory severe aplastic anemia patients.

Description: In a subgroup of patients with myelodysplastic syndrome (MDS) an immune mechanism is believed to cause bone marrow failure. In these individuals immunosuppressive treatment (IST) can restore marrow function. We previously reported a 68% response rate in a phase I/II study of alemtuzumab in 31 patients with low to int-2 risk MDS (Sloand etal JCO Dec 10, 2010:5166-5173). Patients were enrolled according to their predicted probability of response based on an algorithm that incorporated HLA-DR15 positivity, age and transfusion burden (Blood 2003 Oct 15; 102(8):3025-7). We now report updated efficacy data on our cohort of 40 patients and long term follow up data on responders. The first 37 patients received 10mg of alemtuzumab for 10 days intravenously. Alemtuzumab was administered subcutaneously in the subsequent 3 patients. Primary endpoints were changes in peripheral blood counts. Secondary endpoints included improvement in the transfusion requirements (in transfusion- dependent patients), duration of response, and late effects of treatment, relapse and survival. Thirty nine patients were evaluable. One patient discontinued treatment after one dose because of severe hypotension and was excluded from analysis. Median follow-up time was 25 months (3-64 months). Median age was 56 years (23-71 years). The overall response rate (ORR) was 64% (25/39) with 21% of patients having a complete response (8/39). Twenty of 29 (69%) int-1 patients; 4 of 7 (57 %) int-2 patients and 1 of 3 (33%) low risk patients responded following alemtuzumab treatment. Median time to response was 3 months with median duration of response 9 months (range 1-69 months). Eight of 11 patients who relapsed regained their response with addition of cyclosporine and one patient was lost to follow up. The median duration of response in patients rescued with cyclosporine is 37 months (1-42 months). The median duration of response to immunosuppressive treatment (alemtuzumab +/- cyclosporine) is 22.2 months (range 3-69 months). All responding patients who were previously transfusion dependent became transfusion independent after alemtuzumab treatment. One of three patients who received subcutaneous alemtuzumab administration responded. The median survival for all patients was 34 months (range 1-72 months) (figure 1a) with median survival of 36 months (6-72 months) in responding group compared to 16 months (1-56 months) in non-responding group (p

Description: Abstract 2179 Corticosteroids have been in widespread use as anti-inflammatory and immunomodulatory agents for decades, and there is a wealth of information on their immunosuppressive effects in animals and in human cells in vitro. However, there is a paucity of data on the effects of systemically administered corticosteroids on human immune cell subsets. We used modern multiplexed techniques to query the cellular and molecular immune response, or “immunome”, in 20 healthy volunteers at baseline and after systemic (IV) hydrocortisone (HC) administered at both moderate (250 mg) and low (50 mg) doses, to provide insight into how corticosteroids exert their immunomodulatory effects at doses that are commonly used therapeutically. We performed blood draws at baseline, and then 1, 4, 8, 12, and 24 hours, and 7 and 28 days after HC infusion. We assessed whole transcriptome gene expression using HumanGene 1.0 ST microarrays on both peripheral blood mononuclear cells and whole blood; plasma levels of 69 cytokines and chemokines by luminex assay; and high dimensional comprehensive lymphocyte phenotyping by flow cytometry. We observed a global decline in circulating numbers of B and T cells which nadired 4–8 hours after the administration of both 50mg and 250mg HC. Viability and Annexin V staining for apoptosis were consistent with peripheral lympho-depletion occurring as a consequence of redistribution of lymphocytes rather than direct HC-mediated cytotoxicity. Both B and T cell numbers rebounded significantly above baseline 24 hours after HC infusion, and the extent of this increase varied among specific lymphocyte subsets, with CD45+CD3-CD19+ B cell numbers exhibiting the highest rebound over baseline values after both the 50mg (P=0.0060) and 250mg (P=0.0036) HC doses, respectively (Figure 1). In contrast, CD3-CD56+CD16+ NK cell numbers remained stable at all time points after administration of 50mg or 250mg of HC. Microarray studies revealed a steroid-responsive gene expression signature that preceded redistribution of lymphocyte subsets, with 336 genes showing significant variation as early as 1 hour after HC administration (P value cutoff

Description: Abstract 3822 Rigosertib is a multi-kinase inhibitor that selectively induces mitotic arrest leading to apoptosis in cancer cells and blasts, while being non-toxic to normal cells. We analyzed bone marrow (BM) response and overall survival (OS) in 60 patients (pts) with myelodysplastic syndrome (MDS), including 51 patients with refractory anemia and excess blasts (RAEB) and 9 patients with refractory cytopenia and multilineage dysplasia (RCMD) enrolled in 4 independent phase 1/2 clinical trials. These pts were treated with rigosertib administered as a continuous intravenous infusion (CIV) from 2 to 6 days weekly or every other week with BM response initially assessed per protocol by week 4 or 8 and every 8 weeks thereafter. Overall survival (OS) analyses were performed by the method of Kaplan-Meier. OS was related (p=0.04) to FAB/WHO classification (see Table 1) in all MDS pts. Eight pts had hematological improvements. OS was also related to IPSS scoring (p=0.02; Table 2) and to BM blastic response (Table 3; p=0.008) in the 51 RAEB-1,-2,-t pts and in a subset of 38 RAEB-1,-2,-t pts refractory or relapsing after treatment with hypomethylating agents (azacitidine/decitabine) (Table 4; p=0.001). A 49-week OS was found in 15 patients in this last group treated with 3-day rigosertib infusions (1800 mg/day) every other week. Rigosertib infusions were well tolerated without evidence of bone marrow myelotoxicity. These results and the predictive value of BM response to rigosertib for estimating OS survival have led to the initiation of a randomized Phase III survival trial of rigosertib 3-day CIV infusions vs best supportive care in RAEB -1, -2 and-t pts who failed or progressed after receiving hypomethylating agents.Table 1.Overall Survival by FAB/WHO Classification in 60 MDS ptsFAB/WHO ClassificationRCMDRAEB-1RAEB-2RAEB-tP valueN pts9172113Median OS (weeks)988235210.04Table 2.Overall Survival by IPSS Scoring in 51 RAEB-1, -2, -t ptsIPSS ScoringIntermediate-1Intermediate-2High RiskP valueN pts101427Median OS (weeks)Not Reached37280.02Table 3.Overall Survival by BM Blast Response in 51 RAEB-1, -2, -t ptsBM Blast Response≥ 50% Blast DecreaseStable BM ResponseProgressive DiseaseNot AssessedP valueN pts1620510Median OS (weeks)513715110.008Table 4.Overall Survival by BM Blast Response in 38 RAEB-1, -2, -t pts Refractory or Relapsing After Azacitidine/Decitabine TreatmentBM Blast Response≥ 50% Blast DecreaseStable BM ResponseProgressive DiseaseNot AssessedP valueN pts1312310Median OS (weeks)444915100.001 Disclosures: Raza: Onconova Therapeutics Inc: Research Funding. Greenberg:Onconova Therapeutics Inc: Research Funding. Olnes:Onconova Therapeutics Inc: Onconova Therapeutics Inc provided study drug. Silverman:Onconova Therapeutics Inc: Research Funding. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership.

Description: Abstract 1717 Scientific and clinical evidence support a role for immune dysregulation in the pathogenesis of myelodysplastic syndromes (MDS) in some patients, and immunosuppressive therapy (IST) is considered a standard treatment for selected patients with MDS. Despite a number of studies showing its efficacy in MDS, IST has not been widely adopted because of uncertainty about therapeutic benefit, perceived toxicities of antibody treatment, and confusion about patient selection. To better define the place of IST in MDS, we reviewed all published clinical trials of IST for adult patients with MDS to determine its therapeutic benefit and treatment-related toxicity. We also sought to define clinical and laboratory predictors for response to IST. IST was defined as T cell directed therapies, including the following drugs administered alone or in combination: cyclosporine A (CsA), rabbit or horse anti-thymocyte globulin (ATG), alemtuzumab, sirolimus, tacrolimus, mycophenolate mofetil, or daclizumab. Electronic databases were scanned for all peer-reviewed clinical trial reports concerning IST for MDS from inception through August 1, 2011. Risk of bias for each study was assessed by two independent investigators using the Cochrane Risk of Bias Tool, and the Newcastle-Ottawa Scale. The searches of MEDLINE, Cochrane Clinical Trials Register, SCOPUS, Web of Science and EMBASE yielded a total of 1950 articles, and 1937 were subsequently discarded based on our a priori exclusion criteria. A total of 13 reports from 13 individual clinical trials enrolling 358 patients met criteria for this review. IST was well tolerated with a treatment-related mortality rate of 1.3%. Hematologic improvements (HI), partial response (PR), and complete response (CR) was defined using International Working Group for MDS criteria. The overall response rate (ORR) to IST was 41% (HI 18%, PR 15%, and CR 8%). ORR according to treatment regimen were alemtuzumab 68% (n= 31 patients), CsA 56% (n=111 patients), h-ATG 35%, (n=83 patients), r-ATG/CsA 30% (n= 20 patients), r-ATG 27%, (n=15 patients), and h-ATG/CSA 25% (n=79 patients). Response rates were higher when patients were treated on protocols incorporating specific selection criteria versus unselected patients (ORR 59% vs 41%, p=0.01). Patients with a hypocellular bone marrow had response rates that were equal to individuals with normal or increased marrow cellularity: ORR 63% vs 63%, (p=0.99); PR 25% vs 23%, (p=0.81); CR 15% vs 11%, (p=0.46). Patients younger than 60 years had significantly higher response rates to IST than older individuals (ORR 59% vs 40%, p=0.004), irrespective of the treatment regimen administered. Patients who were transfusion dependent had response rates equal to those who were transfusion independent at the time of treatment (ORR 60% vs 55%, p=0.57). Response rates in patients with low and intermediate cytogenetic risk groups as defined by the International Prognostic Scoring Scale (IPSS) were 51% and 49%, respectively. Response rates varied according to World Health Organization (WHO) Prognostic Scoring Scale (WPSS) morphology categories, with overall response rates of 57% (n=128 patients), 10% (n=10 patients), and 21% (n=39 patients) in individuals with very low, low, and intermediate morphology categories, respectively. Response rates did not vary according to gender or cell lineages involved. These results indicate that the choice of IST may be critical in optimizing responses and that IST should be considered for MDS patients younger than 60 years old, with low and intermediate risk IPSS cytogenetics and very low WHO morphology categories, regardless of bone marrow cellularity and duration of transfusion dependence. CsA Alemtuzumab hATG hATG/CsA rATG rATG/CsA Sirolimus ANOVA Response R% (range) No. R% (range) No. R% (range) No. R% (range) No. R% (range) No. R% (range) No. R% (range) No. P-value CR 3 3/111 23 7/31 5 4/83 10 8/79 20 3/15 15 3/20 0 0/19 0.002 (1–8) (10–41) (1–12) (5–19) (4–48) (3–38) — PR 24 27/111 13 4/31 16 13/83 4 3/79 7 1/15 15 3/20 16 3/19 0.013 (17–33) (4–30) (9–25) (1–11) (0.1–32) (3–38) (3–40) HI 29 32/111 32 10/31 15 12/83 11 9/79 0 0/15 0 0/20 0 0/19

Description: Abstract 973 The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with monosomy 7 MDS and AML. Disclosures: No relevant conflicts of interest to declare.