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Most cancers evolve over time as patients initially responsive to therapy acquire resistance to the same drugs at relapse. Cancer stem cells have been postulated to represent a therapy-refractory reservoir for relapse, but formal proof of this model is lacking. We prospectively characterized leukemia stem cell populations (LSCs) from a well-defined cohort of patients with acute myelogenous leukemia (AML) at diagnosis and relapse to assess the effect of the disease course on these critical populations. Leukemic samples were collected from patients with newly diagnosed AML before therapy and after relapse, and LSC frequency was assessed by limiting dilution analyses. LSC populations were identified using fluorescent-labeled cell sorting and transplantation into immunodeficient NOD/SCID/interleukin 2 receptor γ chain null mice. The surface antigen expression profiles of pretherapy and postrelapse LSCs were determined for published LSC markers. We demonstrate a 9- to 90-fold increase in LSC frequency between diagnosis and relapse. LSC activity at relapse was identified in populations of leukemic blasts that did not demonstrate this activity before treatment and relapse. In addition, we describe genetic instability and exceptional phenotypic changes that accompany the evolution of these new LSC populations. This study is the first to characterize the evolution of LSCs in vivo after chemotherapy, identifying a dramatic change in the physiology of primitive AML cells when the disease progresses. Taken together, these findings provide a new frame of reference by which to evaluate candidate AML therapies in which both disease control and the induction of more advanced forms of disease should be considered.

Acute myeloid leukemia (AML) can develop after an antecedent myeloid malignancy (secondary AML [s-AML]), after leukemogenic therapy (therapy-related AML [t-AML]), or without an identifiable prodrome or known exposure (de novo AML). The genetic basis of these distinct pathways of AML development has not been determined. We performed targeted mutational analysis of 194 patients with rigorously defined s-AML or t-AML and 105 unselected AML patients. The presence of a mutation in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 was >95% specific for the diagnosis of s-AML. Analysis of serial samples from individual patients revealed that these mutations occur early in leukemogenesis and often persist in clonal remissions. In t-AML and elderly de novo AML populations, these alterations define a distinct genetic subtype that shares clinicopathologic properties with clinically confirmed s-AML and highlights a subset of patients with worse clinical outcomes, including a lower complete remission rate, more frequent reinduction, and decreased event-free survival. This trial was registered at www.clinicaltrials.gov as #NCT00715637.

High BAALC expression levels are associated with poor outcome in cytogenetically normal acute myeloid leukemia (CN-AML) patients. Recently, miR-3151 was discovered in intron 1 of BAALC. To evaluate the prognostic significance of miR-3151 expression levels and to gain insight into the biologic and prognostic interplay between miR-3151 and its host, miR-3151 and BAALC expression were measured in pretreatment blood of 179 CN-AML patients. Gene-expression profiling and miRNA-expression profiling were performed using microarrays. High miR-3151 expression was associated with shorter disease-free and overall survival, whereas high BAALC expression predicted failure of complete remission and shorter overall survival. Patients exhibiting high expression of both miR-3151 and BAALC had worse outcome than patients expressing low levels of either gene or both genes. In gene-expression profiling, high miR-3151 expressers showed down-regulation of genes involved in transcriptional regulation, posttranslational modification, and cancer pathways. Two genes, FBXL20 and USP40, were validated as direct miR-3151 targets. The results of the present study show that high expression of miR-3151 is an independent prognosticator for poor outcome in CN-AML and affects different outcome end points than its host gene, BAALC. The combination of both markers identified a patient subset with the poorest outcome. This interplay between an intronic miR and its host may have important biologic implications.

Dasatinib with corticosteroid yields high complete remission (CR) rates in Ph+ ALL with minimal induction death. Optimal post-remission therapy is not known. We report a prospective study evaluating dasatinib/dexamethasone induction then consolidation with reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (alloHCT), autologous HCT (autoHCT), or chemotherapy alone (chemo), followed by dasatinib-based maintenance. Methods: We conducted a phase II, 3-arm, non-randomized trial (NCT01256398, Support: U10CA180821, U10CA180882, U10CA180861, U24CA196171) at 17 US centers with primary objectives to determine 3-year overall survival (OS) and disease-free survival (DFS) of adults ≥18 years (yrs) old with untreated Ph+ ALL treated with dasatinib/dexamethasone induction, CNS prophylaxis with systemic and intrathecal methotrexate, consolidation with alloHCT, autoHCT, or chemo, then dasatinib maintenance. Induction (Course I) used dasatinib 140 mg orally daily and dexamethasone 10 mg/m2/day orally or IV days 1-7. If ≤20% lymphoblasts in marrow at Day 15, Course II continued dasatinib with 7 days of dexamethasone. If >20% lymphoblasts, patients (pts) also received vincristine and daunorubicin. Pts not in CR/CRi received a second induction (Course III) with dasatinib, cyclophosphamide, vincristine, daunorubicin, and dexamethasone. CNS prophylaxis (Course IV) used dasatinib, IV vincristine and IV, oral, and intrathecal methotrexate. Course V consisted of HCT or chemo. Pts 18-70 yrs old underwent RIC alloHCT if they had a HLA-matched donor; otherwise they underwent autoHCT. AlloHCT conditioning used fludarabine 30 mg/m2/day IV day -7 through -3, alemtuzumab 20 mg/day IV day -7 through -3, and melphalan 140 mg/m2 IV once on day -2. GVHD prophylaxis with tacrolimus began day -2. Pts undergoing autoHCT received etoposide 10 mg/kg/day (age >65, 5 mg/kg/day) CIV for 4 days and cytarabine 2 g/m2 (age >65, 1 g/m2) IV every 12 hours for 8 doses then G-CSF for mobilization. AutoHCT conditioning was melphalan 100 mg/m2/day IV on days -2 and -1. Pts >70 yrs old or unable to undergo HCT received etoposide/cytarabine alone. Dasatinib maintenance (Course VI) began day 30 of Course V and continued for 12 months and until 2 consecutive negative BCR-ABL1 RT-PCR assays 3 months apart or relapse. BCR-ABL1 isoform and ABL1 kinase domain (KD) mutation determination were done locally. Results: From 12/15/2010 to 11/14/2014, 64 eligible pts enrolled. Median age was 60 yrs (22-87); median WBC 23.2 x 103/μl (0.3-454). The dominant BCR-ABL1 isoform was p190 in 59%, p210 in 17%, and not reported in 23%. The CR rate was 97% with no induction deaths. Fifty-five pts completed CNS prophylaxis (Course IV). Twenty pts underwent protocol-specified alloHCT, 7 autoHCT, and 9 chemo. Nine pts underwent off-study alloHCT and 5 skipped Course V. Dasatinib maintenance was tolerable with 72%, 80%, and 80% receiving >50% of planned maintenance doses in alloHCT, autoHCT, and chemo arms, respectively. With a median follow up of 48 months (37-78) for survivors, 3-yr OS and DFS were 55% (median OS 45 months) and 43% (median DFS 30 months), respectively. For pts undergoing consolidation with protocol-specified alloHCT, autoHCT, or chemo, 3-yr OS was 75%, 71%, and 55% respectively with median OS not reached (NR) for all groups. DFS at 3-yrs was 55%, 43%, and 46% respectively. Median DFS for alloHCT was 42 months vs 15 months for autoHCT and 34 months for chemo (Log-Rank P=0.4). Outcomes were similar with inclusion of off-study alloHCT and chemo patients skipping Course V. Relative to p190 BCR-ABL1 isoform, p210 was associated with shorter median OS (NR vs 16 months, P=0.04) and median DFS (35 months vs 10 months, P Conclusions: Dasatinib and dexamethasone followed by alloHCT, autoHCT, or chemo yield similar 3-yr survival comparable to approaches using intensive induction chemotherapy. Pts with the p210 BCR-ABL1 isoform had dismal outcomes and may benefit from alternate approaches. T315I mutation was the major cause of relapse and should be addressed in future studies. Disclosures Wieduwilt: Amgen: Research Funding; Reata Pharmaceuticals: Equity Ownership; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Leadiant: Research Funding; Shire: Research Funding; Merck: Research Funding. Uy:GlycoMimetics: Consultancy; Curis: Consultancy. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria. Liedtke:Caelum: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Research Funding; Genentech/Roche: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BlueBirdBio: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Devine:Kiadis Pharma: Consultancy. Larson:BristolMyers Squibb: Consultancy, Research Funding; Ariad/Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding.

The clinical impact of FLT3-internal tandem duplications (ITDs), an adverse prognostic marker in adults aged < 60 years with primary cytogenetically normal acute myeloid leukemia (CN-AML), requires further investigation in older patients. In CN-AML patients aged ≥ 60 years treated on Cancer and Leukemia Group B frontline trials, we found that FLT3-ITD remained associ-ated with shorter disease-free survival (P < .001; hazard ratio = 2.10) and overall survival (P < .001; hazard ratio = 1.97) in multivariable analyses. This impact on shorter disease-free survival and overall survival was in patients aged 60-69 (P < .001, each) rather than in those aged ≥ 70 years. An FLT3-ITD–associated gene-expression signature revealed overexpression of FLT3, homeobox genes (MEIS1, PBX3, HOXB3), and immunotherapeutic tar-gets (WT1, CD33) and underexpression of leukemia-associated (MLLT3, TAL1) and erythropoiesis-associated (GATA3, EPOR, ANK1, HEMGN) genes. An FLT3-ITD–associated microRNA-expression signature included overexpressed miR-155 and underexpressed miR-144 and miR-451. FLT3-ITD identifies older CN-AML patients with molecular high risk and is associated with gene- and microRNA-expression signatures that provide biologic insights for novel therapeutic approaches.

We compared the treatment-related mortality, relapse rate, disease-free survival (DFS), and overall survival (OS) by cytogenetic risk group of 261 patients with acute myeloid leukemia in first complete remission (CR1) and 299 patients in CR2 in undergoing matched unrelated donor hematopoietic stem cell transplantation (HSCT). For patients in first CR, the DFS and OS at 5 years were similar for the favorable, intermediate, and unfavorable risk groups at 29% (95% confidence interval [CI], 8%-56%) and 30% (22%-38%); 27% (19%-39%) and 29% (8%-56%); and 30% (95% CI, 22%-38%) and 30% (95% CI, 20%-41%), respectively. For patients in second CR, the DFS and OS at 5 years were 42% (95% CI, 33%-52%) and 35% (95% CI, 28%-43%); 38% (95% CI, 23%-54%) and 45% (95% CI, 35%-55%); and 37% (95% CI, 30%-45%) and 36% (95% CI, 21%-53%), respectively. Cytogenetics had little influence on the overall outcome for patients in first CR. In second CR, outcome was modestly, but not significantly, better for patients with favorable cytogenetics. The graft-versus-leukemia effect appeared effective, even in patients with unfavorable cytogenetics. However, treatment-related mortality was high. Matched unrelated donor HSCT should be considered for all patients with unfavorable cytogenetics who lack a suitable HLA-matched sibling donor.

CALGB 9511 used pegaspargase (PEG-ASP) in lieu of the native enzyme. The aim was to compare differences in overall survival (OS) and disease-free survival (DFS) between patients who did and did not achieve asparagine depletion, defined by enzyme levels greater than 0.03 U/mL plasma for 14 consecutive days after at least 1 of 4 planned PEG-ASP administrations. Samples were available from 85 eligible patients. On univariate analyses, the 22 patients who did not achieve asparagine depletion had inferior OS (P = .002; hazard ratio [HR] = 2.37; 95% CI = 1.38-4.09) and DFS (P = .012; HR = 2.21; 95% CI = 1.19-4.13). After adjusting for age, performance status, leukocyte count, and karyotype in a proportional hazards model, both the OS and DFS HRs decreased to 1.8 (P = .056; 95% CI = 1.0-3.2 and P = .084; 95% CI = 0.9-3.6, respectively). We conclude that effective asparagine depletion with PEG-ASP is feasible as part of an intensive multiagent therapeutic regimen in adult acute lymphoblastic leukemia and appears associated with improved outcomes.

Signal transducer and activator of transcription (STAT) proteins are a 7-member family of cytoplasmic transcription factors that contribute to signal transduction by cytokines, hormones, and growth factors. STAT proteins control fundamental cellular processes, including survival, proliferation, and differentiation. Given the critical roles of STAT proteins, it was hypothesized that inappropriate or aberrant activation of STATs might contribute to cellular transformation and, in particular, leukemogenesis. Constitutive activation of mutated STAT3 has in fact been demonstrated to result in transformation. STAT activation has been extensively studied in leukemias, and mechanisms of STAT activation and the potential role of STAT signaling in leukemogenesis are the focus of this review. A better understanding of mechanisms of dysregulation of STAT signaling pathways may serve as a basis for designing novel therapeutic strategies that target these pathways in leukemia cells.

From 2007 to 2011, 66 patients with primary myelofibrosis or myelofibrosis (MF) preceded by essential thrombocythemia or polycythemia vera were enrolled into a prospective phase 2 clinical trial of reduced-intensity allogeneic hematopoietic stem cell transplantation (AHSCT), Myeloproliferative Disorder Research Consortium 101 trial. The study included patients with sibling donors (n = 32) receiving fludarabine/melphalan (FluMel) as a preparative regimen and patients with unrelated donors (n = 34) receiving conditioning with FluMel plus anti-thymocyte globulin (ATG). Patient characteristics in the 2 cohorts were similar. Engraftment occurred in 97% of siblings and 76% of unrelated transplants, whereas secondary graft failure occurred in 3% and 12%, respectively. With a median follow-up of 25 months for patients alive, the overall survival (OS) was 75% in the sibling group (median not reached) and 32% in the unrelated group (median OS: 6 months, 95% confidence interval [CI]: 3, 25) (hazard ratio 3.9, 95% CI: 1.8,8.9) (P < .001). Nonrelapse mortality was 22% in sibling and 59% in unrelated AHSCT. Survival correlated with type of donor, but not with the degree of histocompatibility match, age, or JAK2V617F status. In patients with MF with sibling donors, AHSCT is an effective therapy, whereas AHSCT from unrelated donors with FluMel/ATG conditioning led to a high rate of graft failure and limited survival. This trial was registered at www.clinicaltrials.gov as #NCT00572897.

As a group of more than 100 experts in chronic myeloid leukemia (CML), we draw attention to the high prices of cancer drugs, with the particular focus on the prices of approved tyrosine kinase inhibitors for the treatment of CML. This editorial addresses the multiple factors involved in cancer drug pricing and their impact on individual patients and health care policies, and argues for the need to (1) lower the prices of cancer drugs to allow more patients to afford them and (2) maintain sound long-term health care policies.

Background: Potent inhibitors of BCR-ABL1have improved remission results and altered post-remission therapy for Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Dasatinib plus dexamethasone followed by hematopoietic cell transplantation (HCT) promises high response rates, reduced toxicity, and durable remissions. Methods: We conducted a Phase II trial at 17 U.S. centers with the primary objective being to estimate the 3-year overall survival and disease-free survival of patients with Ph+ ALL treated with dasatinib and dexamethasone for remission induction and intensification, central nervous system (CNS) prophylaxis, consolidation with reduced-intensity conditioning (RIC) allogeneic HCT, autologous HCT, or etoposide and cytarabine, and dasatinib-based maintenance. Eligible patients had untreated Ph+ ALL, were ≥18 years old, and had normal cardiac function. Induction (Course I) used dasatinib 140 mg oral daily and dexamethasone 10 mg/m2/day oral or intravenous (IV) days 1-7. For patients with ≤20% blasts in the Course I, Day 15 bone marrow biopsy, intensification (course II) continued daily dasatinib with another 7 days of dexamethasone. Those with >20% lymphoblasts also received vincristine (VCR) and daunorubicin (DNR). Patients (n=3) not in CR/CRi after Course II received a second induction (Course III) with dasatinib, cyclophosphamide, VCR, DNR, and dexamethasone. After Course II or III, CNS prophylaxis (Course IV) consisted of IV VCR and IV, oral, and intrathecal methotrexate (MTX). Dasatinib was restarted at serum [MTX] 65 years, 5 mg/kg/day) continuous IV for 4 days and cytarabine 2 g/m2 (age >65 years, 1 g/m2) IV every 12 hours for 8 doses (EA) then G-CSF for mobilization. Autologous HCT conditioning used melphalan 100 mg/m2/day on days -2 and -1. Patients >70 years or unable to undergo HCT received EA alone. Dasatinib maintenance (Course VI) began on day 30 of Course V and continued for 12 months and until 2 consecutively negative bone marrow BCR-ABL1RT-PCR assays 3 months apart or until relapse. Dasatinib levels were measured on day 15 of induction. Results: Sixty-six patients enrolled from 12/15/2010 to 11/14/2014; 65 received dasatinib and are evaluable. Median age was 60 years (22-87); 49% were male. Median presenting WBC count was 23.1 x 103/ul (0.3-453.6). No deaths occurred during induction or intensification. CR or CRi occurred 31 patients (48%) by Day 15 of induction and in 62 patients overall (95%; CR 86%). Median dasatinib levels in serum and CSF on Day 15 of induction were 30.3 ng/mL ( Conclusions: Dasatinib with dexamethasone yields CR rates comparable to those reported with tyrosine kinase inhibitors combined with conventional chemotherapy. Post-remission therapy with reduced-intensity allogeneic HCT, autologous HCT, or chemotherapy followed by dasatinib maintenance is feasible. Survival follow up is maturing. Disclosures Stock: Sigma-Tau: Honoraria, Research Funding; Royalties for a chapter in Up to Date: Patents & Royalties; ADC Therapeutics: Honoraria; Amgen: Honoraria; Gilead Sciences: Honoraria. Beumer:Bristol-Myers Squibb: Research Funding. Stone:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Juno Therapeutics: Consultancy; Karyopharm: Consultancy; Sunesis Pharmaceuticals: Consultancy; Agios: Consultancy; Amgen: Consultancy; Celator: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Jansen: Consultancy; Merck: Consultancy; ONO: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Xenetic Biosciences: Consultancy. Larson:Bristol-Myers Squibb: Consultancy; Astellas: Consultancy, Research Funding.

We have recently demonstrated that interleukin (IL)-1 beta levels are elevated in advanced chronic myelogenous leukemia (CML) and that IL-1 inhibitors can suppress CML clonogenic growth. To further assess the clinical implications of increased IL-1 beta expression in CML, we analyzed IL-1 beta and IL-1 receptor antagonist (IL-1RA) levels in leukocyte lysates from a series of CML patients and from normal volunteers. Both IL-1 beta and IL-1RA were measured by enzyme-linked immunosorbent assays (ELISAs), with the lower limits of sensitivity of the assays being 20 pg/mL and 6.5 pg/mL, respectively. The median IL-1 beta level in the 81 CML patients tested was higher (115.8 pg/2.4 x 10(7) cells; range, 0 to 2,000 pg/2.4 x 10(7) cells) than the median level in 25 control samples (10.8 pg/2.4 x 10(7) cells; range, 0 to 95.5 pg/2.4 x 10(7) cells) (P < .01). IL-1 beta was bioactive, as demonstrated with a bioassay based on cytotoxicity to a melanoma cell line (A375). For survival analysis, elevated IL-1 beta levels were defined as those exceeding the mean + 2 SD of normal levels (83 pg/2.4 x 10(7) cells). The survival of the 44 patients with elevated IL-1 beta levels was significantly shorter than that of those who had low IL-1 beta levels (median, 44 v 58 months; P = .049 by Wilcoxon-Gehan method). An association between IL-1 beta and CML prognostic criteria shows that IL-1 beta levels were significantly higher in patients in accelerated/blastic crisis phases of the disease (364.0 pg/2.4 x 10(7) cells) compared with patients in chronic phase (102.0 pg/2.4 x 10(7) cells) (P < .01), and that high IL-1 beta levels correlated with increased blasts in the marrow and peripheral blood (P < .01). In contrast, while IL-1RA levels did not differ between chronic-phase CML patients (median, 471.7 pg/2.4 x 10(5)) and healthy volunteers (median, 454.4 pg/2.4 x 10(5)), patients with accelerated/blast crisis disease had significantly lower levels of IL-1RA (median, 218.7 pg/2.4 x 10(5); P = .03). Finally, although IL-1 beta has been previously shown to increase IL-1RA levels, there was no correlation between IL-1 beta and IL-1RA levels in our CML patients.(ABSTRACT TRUNCATED AT 400 WORDS)

The associations of mutations in the enhancer of trithorax and polycomb family gene ASXL1 with pretreatment patient characteristics, outcomes, and gene-/microRNA-expression profiles in primary cytogenetically normal acute myeloid leukemia (CN-AML) are unknown. We analyzed 423 adult patients for ASXL1 mutations, other prognostic gene mutations, and gene-/microRNA-expression profiles. ASXL1 mutations were 5 times more common in older (≥ 60 years) patients (16.2%) than those younger than 60 years (3.2%; P < .001). Among older patients, ASXL1 mutations associated with wild-type NPM1 (P < .001), absence of FLT3-internal tandem duplications (P = .002), mutated CEBPA (P = .01), and with inferior complete remission (CR) rate (P = .04), disease-free survival (DFS; P = .03), overall survival (OS; P = .006), and event-free survival (EFS; P = .002). Within the European LeukemiaNet (ELN) genetic categories of older CN-AML, ASXL1 mutations associated with inferior CR rate (P = .02), OS (P < .001), and EFS (P < .001) among ELN Favorable, but not among ELN Intermediate-I patients. Multivariable analyses confirmed associations of ASXL1 mutations with unfavorable CR rate (P = .03), DFS (P < .001), OS (P < .001), and EFS (P < .001) among ELN Favorable patients. We identified an ASXL1 mutation-associated gene-expression signature, but no microRNA-expression signature. This first study of ASXL1 mutations in primary CN-AML demonstrates that ASXL1mutated older patients, particularly within the ELN Favorable group, have unfavorable outcomes and may be candidates for experimental treatment approaches.

In this issue of Blood , Fernandez et al demonstrate that human leukocyte antigen ( HLA ) DRB1 alleles confer high-affinity binding to asparaginase epitopes, leading to higher frequency of allergic reactions.[1][1] The authors initially examined HLA data from European ancestry patients enrolled onto

We previously reported the adverse prognostic impact of Wilms tumor 1 gene (WT1) mutations in younger adult cytogenetically normal acute myeloid leukemia (CN-AML). Here, we investigated 243 older (≥ 60 years) primary CN-AML patients. WT1 mutated (WT1mut) patients (7%) had FLT3-ITD more frequently (P < .001), lower hemoglobin (P = .01), higher white blood cell count (P = .03) and percentage blood blasts (P = .03), and a shorter overall survival (P = .08) than WT1 wild-type (WT1wt) patients. Comparing older and younger WT1mut patients, they had similar pretreatment characteristics and outcome. By contrast, among WT1wt CN-AML, younger patients had a significantly better outcome. A WT1 mutation-associated gene-expression signature, reported here for the first time, included CD96, a leukemia stem cell-specific marker, and genes involved in gene regulation (eg, MLL, PML, and SNRPN) and in proliferative and metabolic processes (eg, INSR, IRS2, and PRKAA1), supporting the role of mutated WT1 in deregulating multiple homeostatic processes. Our results indicate that WT1mut CN-AML represents a distinct entity with poor treatment response across age groups. This study has been registered at www.clinicaltrials.gov as #NCT00900224.

Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients with leukemia; however, many will ultimately die because of disease relapse and development of drug resistance. Leukemias are cancers of the blood cells that result from alteration of the normal physiological constraints that regulate hematopoietic stem cells (HSCs). General characteristics of leukemia stem cells (LSCs) such as self-renewal, self-protection and proliferative quiescence represent inherent mechanisms that at least partially explain drug resistance and recurrence in post-therapy leukemia patients. Acute myeloid leukemia (AML) is a heterogeneous disease, both biologically and clinically, in which a number of distinct genetic abnormalities have been described. Several recent studies suggest that this heterogeneity extends to LSCs and can vary between patient subgroups, and even within individual patients. Moreover, the complexity of AML is further complicated by the existence of functionally diverse leukemic and preleukemic clones. Accordingly, the hierarchical organization of AML suggests that this may be relevant to current therapies that primarily target proliferating progenitors/blast cells, which lack self-renewal capacity, and not LSCs. In the current study, we rationalized that understanding how LSCs differ from normal HSCs at the molecular level, is an essential first step towards developing novel targeted therapies and achieving permanent disease remission. Despite the identification of novel LSC-specific markers, there is considerable heterogeneity in expression of these markers amongst AML patients. However, in addition to marker-enrichment strategies, LSCs can be identified by virtue of their quiescent and slow-cycling properties. For example, label-retaining cells can be isolated and used in functional assays but significant technical limitations impede broad utility of this approach. To this end, we describe the development and use of novel multi-fluorescent protein markers and DNA bar codes integrated into the cellular genomes by lentivirus, as single-cell tracking devices for monitoring LSCs in vivo. We demonstrate how LSCs can transition between a "proliferation phase" and a "quiescence phase" in vivo. Furthermore, using high-throughput quantitative transcriptome sequencing (Q-RNA-Seq) and RNAi genetic perturbation's focusing on well-defined self-renewal signaling pathways, we develop a differential network-based model to identify LSC-specific genes and subsequently prioritize/rank candidates as potential drug targets. In the current study, we identify several molecular targets deregulated in quiescent versus proliferating LSCs and a mutual set of signaling pathways that facilitate leukemic transformation downstream of diverse initiating mutations/lesions. Remarkably, both quiescent and dividing LSCs but not HSCs, were 'addicted' to SSRP1 - an essential component of the ubiquitous FACT chromatin remodeling complex. Two orally available quinacrine-related DNA-intercalating compounds inhibiting function of FACT (CBL0100 and CBL0175, respectively) suppressed LSC proliferation in vitro and in vivo, as demonstrated by production of leukemic clonogenic cells (CFU) and long-term engraftment of immunodeficient NSG mice, by simultaneous inhibition of NF-kB (stimulated and basal forms) and activation of p53. Furthermore, in a secondary transplantation experiment, leukemic cells obtained from CBL0175 treated mice (primary) failed to engraft into secondary NSG mice in a serial transplantation model by selectively targeting the LSC compartment. Collectively, we present a novel network-based polypharmacology approach that provides unique opportunities to preferentially ablate LSCs (quiescent and dividing types), with potentially profound clinical implications. Disclosures Frangou: Cellecta: Employment. Portwood:ImmunoGen: Research Funding. Wang:ImmunoGen: Research Funding.

Signal transducer and activator of transcription (STAT) proteins are involved in hematopoietic cytokine receptor signaling pathways that regulate cell proliferation, differentiation, and survival. STATs are dysregulated in acute myeloid leukemia (AML); mechanisms of dysregulation include constitutive activation and truncation of the C-terminal transactivation domain; the latter results in a beta isoform that has a trans-dominant negative effect on gene induction mediated by the full-length STAT alpha form. It was hypothesized that constitutive STAT activity might correlate with unfavorable treatment outcome in AML. Pretreatment bone marrow samples from 63 adult patients with AML were analyzed by electrophoretic mobility shift assay for the presence of STAT DNA-binding activity. Isoforms and relative levels of STAT proteins were determined by immunoblotting. Constitutive STAT3 activity was detected in samples from 28 (44%) patients. Pretreatment clinical characteristics, expression of STAT alpha/beta isoforms, and treatment regimens did not differ significantly between patients with and without constitutive STAT3 activity. Disease-free survival (DFS) was significantly shorter in patients with than in patients without constitutive STAT3 activity (median 8.7 vs 20.6 months; P =.01). Overall survival did not differ significantly. The subgroup of patients with constitutive STAT3 activity and the STAT3 beta isoform had the shortest DFS (P =.006) and shorter overall survival (P =.049) than all other patients. Whether adverse treatment outcome is attributable to constitutive STAT activity itself or to a process that leads to constitutive STAT activity remains to be determined. This is the first demonstration of a prognostic significance for STAT proteins in a malignancy.

In AML, t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22) and corresponding molecular rearrangements RUNX1/RUNX1T1 and CBFB/MYH11 (each involving a gene encoding a subunit of CBF) predict for favorable outcome in patients (pts) receiving consolidation with high-dose cytarabine (HiDAC) after achievement of complete remission (CR). However, 40-45% of these pts relapse. The subset of CBF AML pts harboring gain-of function mutations in the KIT gene (25%) and others overexpressing wild type (wt) KIT have inferior outcomes. Therefore, inhibiting KIT with DAS is a rational therapeutic strategy in CBF AML. We provide updated results (median follow-up 21.3 months (mo); range: 1.8-32.3 mo) for CALGB 10801, a phase II trial that combined DAS with standard chemotherapy for newly diagnosed with CBF AML pts. Enrollment required confirmation of CBF AML by the Alliance Molecular Pathology central lab using RT-PCR and Sanger sequencing-based assays for RUNX1/RUNX1T1 or CBFB/MYH11. Positive pts received induction chemotherapy with cytarabine (C) 200 mg/m2/day continuous intravenous (IV) infusion d (d) 1-7, daunorubicin (DNR) 60 mg/m2/d IV bolus d 1-3 and DAS 100 mg/d PO d 8-21. Pts with residual disease (>5% blasts) on d 21 were re-induced with same doses of C d1-5, DNR d1-3 and DAS d6-19. Pts who achieved CR received consolidation with HiDAC 3000 mg/m2 over 3 hours (if Between April 2011 and January 2013, we screened 779 pts and 61 adult CBF AML pts were enrolled. Two pts were excluded from these analyses – one had no follow-up information and one was not molecularly eligible. Median age was 51 yr (range: 19-85 yrs); 14 pts (24%) were older (>60 yr). Fifty-two% of patients were male; 76% were Caucasian. Of the 59 pts, 65% were CBFB/MYH11-positive and 35% were RUNX1/RUNX1T1-positive; 18% of the 56 who were molecularly evaluable harbored KIT-mutated (mut). Treatment (tx) began on average 4 d from molecular diagnosis (range: 0-11 d). Seven pts are still undergoing tx; 4 pts died on tx (2 older), 9 (4 older) had an adverse event (AE) requiring tx interruption, and 8 refused to complete the tx (mainly DAS maintenance). Observed AEs were those expected with C and DNR (hematologic and non-hematologic) and with DAS (nausea, liver toxicity, pleural effusions). Only 2 and 3 pts had gr4 pleural effusions or increased liver function tests, respectively. Gr 5 AEs included respiratory failure (1) and sepsis (2). Three pts died during induction (2 older CBFB/MYH11 and 1 younger RUNX1/RUNX1T1). One pt died from sepsis during consolidation in CR (CBFB/MYH11, 48 yr). The 30-d survival rate was 97% overall (98% in younger and 93% in older pts). The outcomes of all pts and molecular subsets are summarized in the Table. Table Clinical outcomes All (n=59) Younger (n= 45) Older (n= 14) RUNX1/RUNX1T1 (n= 20) CBFB/MYH11 (n= 38) KIT-wt (n=46 ) KIT-mut(n=10 ) CR* (%) 90 93 79 90 89 89 90 Relapseº (%) 17 13 29 20 16 17 20 2-yr DFS# (%) 72 74 65 79 67 72 70 2-yr OS¶ (%) 87 96 61 85 89 86 90 *CR, complete remission; º Relapse rate at any time #DFS, disease-free survival, ¶OS, overall survival These updated results suggest that 1) rapid molecular screening and treatment-protocol allocation for CBF AML are feasible within a cooperative group, 2) DAS+chemotherapy is tolerable in CBF AML pts of all ages, 3) clinical outcomes for CBF pts receiving DAS+chemotherapy remain at least comparable to those historically observed in CBF pts who received chemotherapy alone; 4) older CBF AML pts seem to benefit from this intensive approach; 5) among pts treated with DAS+chemotherapy, outcomes of KIT-mut pts seem similar to those of KIT-wt pts (see Figure), although we recognize the limitations with relatively small numbers. Pts on CALGB 10801 continue to be followed for survival endpoints. Overall, these results support the continued evaluation of KIT inhibitors in CBF AML through prospective randomized trials but emphasize that further outcome enhancements in this disease subset could be achieved via the use of additional rationally targeted agents. Figure 1 Figure 1. Disclosures Stock: Sigma-Tau: Membership on an entity's Board of Directors or advisory committees, Research Funding. Larson:Ariad: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy.

Introduction: Mutations in IDH1 and IDH2 occur in 15-20% of AML cases, inducing the production of the oncometabolite, 2-hydroxyglutarate, which results in aberrant hypermethylation of DNA in leukemic cells. DNA methyltransferase inhibitors (DNMTI), which induce DNA hypomethylation, are used off-label for the treatment of AML and have been shown to result in remission in 20-40% of patients. We hypothesized that DNMTI treatment would produce superior clinical responses in AML patients with IDH mutations compared to those AML patients without IDH mutations. Method: The medical records of 42 patients with AML treated with decitabine or azacitidine as induction therapy between 2008 and 2013 were reviewed. No patient who received a DNMTI for treatment of AML was excluded. Patient and disease characteristics, including cytogenetics, were collected. Treatment response and outcome to DNMTIs were identified according to International Working Group criteria. The presence of IDH mutation (R132 in IDH1 and R140 or R172 in IDH2) was identified by DNA sequencing by an independent research group. Analysis of the genomic data was performed by individuals who were blinded to the clinical data. The protocol was reviewed and approved by the institutional review board (IRB). Results: Themean age of patients in this study was 76.0±7.4 years. Thirty-three (78.6%) were male. Thirty six (86%) were treated with decitabine and six (14%) with azacitidine. Of the 42 patient samples analyzed, seven (16.7%) had IDH mutations (three IDH1, four IDH2). The clinical characteristics of patients with or without IDH mutation are presented in the table below. Twenty patients had normal karyotype, and five of these (25%) had an IDH mutation. Thirteen patients (31%) achieved remission (Complete response/Complete response with incomplete count recovery/Partial Response) after treatment with DNMTIs; 5 of 7 (71.4%) patients with an IDH mutation and 8 of 35 (22.9%) patients without an IDH mutation (p=0.01). When adjusted for age at diagnosis, sex, bone marrow blast percentage and cytogenetic profile, the odds of attaining remission after administration of a DNMTI among patients with an IDH mutation was 14.2 when compared to patients without an IDH mutation (95%CI: 1.3-150.4). Two patients with abnormal cytogenetics and mutations of IDH were identified, both of these patients achieved remission. Conclusion: The presence of IDH1 or IDH2 mutations may predict a favorable response to DNMTIs in patients with AML. These findings, although novel, are based on a small number of individuals in a retrospective study. Prospective assessment of this association with response prediction is necessary to confirm these results. | | Wild type IDH (n=35) | Mutant IDH1/2 (n=7) | P value | | -------------------------- | -------------------- | ------------------- | ------- | | Age | 76.6±1.3 | 72.7±2.2 | 0.2 | | WBC (k/µL) | 19.5±3.6 | 10.5±3.3 | 0.3 | | Hemoglobin (g/dL) | 9.2±0.3 | 9.6±0.6 | 0.9 | | Platelet (k/µL) | 71.2±10.5 | 175.9±65.0 | 0.006 | | Bone marrow blast (%) | 54.8±3.99 | 54.4±10.1 | 0.9 | | Peripheral blood blast (%) | 25.6±4.1 | 21.7±7.5 | 0.6 | Table 1. Disclosures Griffiths: Astex Pharmaceuticals, Inc.: Research Funding. Off Label Use: Use of decitabine in treatment of AML.

The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and to avoid over- or under- treatment. Minimal residual disease (MRD) is a very effective method for predicting response to treatment and potential relapse in patients with ALL. The most validated method used to assess MRD in ALL is flow cytometric (FCM) analysis of leukemia-associated immunophenotypes which can detect up to 1 leukemic cell per 10,000 normal cells (0.01%). There is no standard method for detecting MRD by FCM in ALL patients. Our institutional approach has been to evaluate 20,000- 50,000 events using a specific immunophenotypic panel and to analyze the data using WinList software with a concurrent comparison with the diagnostic immunophenotype. GemStone™ is a new data analysis program that uses a Probability State Modeling (PSM) approach to analyze FCM data. The incorporated TriCOM technology numerically correlates all the immunophenotype combinations which can be used to detect MRD and other rare populations. In this study, we compared the GemStone and WinList analysis methods on the same patient data files. We identified 33 patients suitable for comparison by performing a retrospective review of the medical records from 2004-2010 in our pre-B cell ALL database. Patients were newly diagnosed, untreated, age ≥17 years and had FCM data available at diagnosis, week 4 (range 3-8) and week 16 (range 12-20) post therapy. We analyzed the FCM files to detect MRD using both GemStone and WinList methods separately, we subsequently reviewed the clinical course of the patients and identified those who sustained complete remission or relapsed. The clinical and FCM results were statistically correlated in Chi Square 2x2 table to identify the predictive value of each analysis method. WinList: we used CD10, CD19, CD34 and CD38 as a backbone marker set and tracked the same abnormal immunophenotype upon diagnosis in the subsequent follow up samples. GemStone: We used the same marker set and incorporate the TriCOM technology identify the MRD numerically. Figure 1 compares the outcome between GemStone and WinList methods at 16 weeks follow up. Table 1 reviews Chi Square with both method of analysis which gives comparable results. In summary, our retrospective review showed that GemStone method in detecting the MRD had comparable clinical and statistical significance to the WinList method. However, we found that the limited number of events collected (20,000) reduced our ability to completely automate the analysis process in GemStone due to intensity differences in the data. We are now conducting a prospective analysis by GemStone on ALL patients with higher number of collected events (5 x 10⁵- 2x 10⁶) Further, in our review, the MRD at 4 months post diagnosis (average 12-20 weeks) was more clinically significant than the one at 4 weeks follow up (range 3-8 weeks). Figure 1: A. Correlation between the clinical and FCM result using WinList method at 16 weeks follow up B. Correlation between the clinical and FCM result using GemStone method at 16 weeks follow up Figure 1:. A. Correlation between the clinical and FCM result using WinList method at 16 weeks follow up B. Correlation between the clinical and FCM result using GemStone method at 16 weeks follow up Abstract 5348 Table 1: comparison between WinList and GemStone method. Sensitivity Specificity PPV NPV P value Statistically significant WinList at 4 week F/U 33% 58% 23% 70% 0.49 no GemStone at 4 week F/U 44% 79% 44% 79% 0.17 no WinList at 16 week F/U 78% 58% 41% 88% 0.0323 yes GemStone at 16 week F/U 89% 67% 50% 94% 0.0022 yes Disclosures Hill: Verity Software house: Employment. Griffiths:Astex Pharmaceuticals: Research Funding; Celgene, Incyte and Alexion: Honoraria.

Background: Treatment of acute promyelocytic leukemia (APL) with arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) is highly effective as first-line therapy, although approximately 10% of patients relapse after treatment. Several mechanisms of ATRA resistance have been proposed including accelerated clearance of ATRA and increased levels of cellular retinoic acid-binding protein (CRABP), which induces retinoic acid metabolism. Tamibarotene is a synthetic retinoid that does not significantly bind CRABP, suggesting that it might be effective in APL patients with ATRA resistance. Tamibarotene has shown efficacy in APL patients with relapse after chemotherapy and ATRA, but has not been studied in relapse after treatment with ATO and ATRA Methods: We conducted a multicenter phase II clinical trial of tamibarotene in adult patients with relapsed or refractory APL after treatment with ATRA and ATO (concomitant or sequential). Participants were treated with single agent tamibarotene at a daily dose of 6 mg/m2 for up to 56 days during induction. Patients achieving a complete response were eligible to continue on consolidation treatment with tamibarotene at the same dose for a maximum of six 28 day cycles. The primary outcome for this trial was to determine the rate of durable complete response (CR) using tamibarotene as a single agent. Secondary outcomes included the rate of morphologic complete remission, partial response, cytogenetic complete response, molecular complete response as well as the safety profile and tolerability of this medication. Results: We enrolled 14 patients from March 2008 to October 2011 at 8 centers. The median age of the participants was 56 years (range 20-76). Twelve patients had relapsed APL and 2 had primary refractory disease. Patients had a median of 2 remissions (range 1-5) prior to enrollment with a median time from the most recent remission to study screening of 23 months (range 2-102). Twelve patients (86%) had received other treatments including stem cell transplant (n=4) in addition to ATO and ATRA prior to enrollment. Eight patients achieved a morphologic remission during treatment with tamibarotene and 2 had a partial response (>50% reduction in BM blasts). Three (21%) patients with complete morphologic remission had a CR, whereas 5 (36%) had a complete remission with incomplete hematologic recovery (CRi), without meeting pre-specified recovery of neutrophil (>1,000/μL) and platelet counts (>100,000/μL). Seven out of 8 patients who achieved CR or CRi relapsed after treatment. The median duration of response for patients achieving CR was 203 days (range 183-212). The median overall survival for the entire group was 233 days (95% CI 196-526 days). Thirteen patients reported treatment-emergent adverse events, although the majority were mild (Grade 1-2). The most frequently reported adverse events included rash (n=6, grade 1-3), headache (n=4, grade 1) and neutropenia (n=3, grade 3-4). Two patients experienced APL differentiation syndrome (grade 2). Two patients discontinued the study due to adverse events: one patient developed progressive multifocal leukoencephalopathy and the other developed pneumonia, although neither of these events was thought to be related to tamibarotene. Discussion: These results suggest that tamibarotene has activity in patients with relapsed APL after treatment with ATO and ATRA. Although the CR rate of 21% is lower than that reported in a previous trial using tamibarotene (58%; Tobita, 1997), the 24 patients in that study had received ATRA alone or in combination with chemotherapy, but not ATO. Thus, tamibarotene has significant clinical activity in this heavily pre-treated population with acceptable toxicity. Further studies using tamibarotene as initial therapy and in combination with ATO are warranted. Disclosures LoCoco: Lundbeck: Consultancy, Speakers Bureau; Teva: Consultancy, Speakers Bureau. Cortes:CytRx: Research Funding.

Background: Relapsed/refractory (r/r) Acute Myeloid Leukemia (AML) remains a therapeutic challenge. Although cytarabine arabinoside (AraC) is the most active drug, constituting the backbone of a majority of r/r regimens, the benchmark response to therapy remains a dismal 17 to 20% (Burnett, Wetzler et al. JCO, 2011.). The immunomodulatory drug lenalidomide (Len), is approved by the Food and Drug Administration for multiple myeloma and myelodysplasia and has demonstrated activity as a single agent in AML at doses as high as 50 mg for 21 days (d) of a 28 d cycle (Blum et al, JCO, 2010.). Based upon this activity profile we developed a phase I study to evaluate the safety and tolerability of Len in combination with AraC in patients with r/r AML. Methods: Eligible patients were older than 18 years(y), had r/r AML with an Eastern Cooperative Oncology Group performance status better than 2 and adequate renal and hepatic function. Patients were excluded for active CNS disease, uncontrolled infections, congestive heart failure, adrenal insufficiency, anti-cancer therapy within 14 d of enrollment, or prior exposure to Len. All enrolled patients had to practice appropriate contraception. Patients received AraC 1.5 g/m2/d over 3 hours on d 1-5 of a 28 day cycle, with a plan for standard 3+3 Len dose escalation. Initial patients received Len 25 mg on d 6-10 (n= 3), subsequent patients received doses between 25 and 10 mg (dose de-escalation) on d 6-26 with 2 d of rest prior to the next cycle. Following induction, patients who had residual AML (>5%) could receive a second identical course of therapy, provided they demonstrated an improvement in blast percentage relative to baseline. Patients who achieved CR received maintenance with Len 10 mg/d continuously. A 12 patient expanded cohort was enrolled at the maximum tolerated dose (MTD) to assess efficacy. Responses were assessed by International Working Group Criteria for AML (Cheson B et al. JCO, 2003.). Patient Characteristics: Fifty-one patients were consented and 45 were treated on study, 32 of these were evaluable for response, all patients were evaluated for toxicity. Approximately half the patients were female (20/45). The median age was 66 y (range 33-82) and median WBC 2.42x109/L (range 0.18-63.15). Four patients (8%) had an antecedent hematological disorder. By European LeukemiaNet criteria 2 patients (4%) had favorable risk disease, 8 (18%) were Int-1, 12(27%) were Int-2 and 11 (24%) were adverse risk; 12(27%) patients were not evaluable by ELN due to lack of karyotype or molecular data from diagnosis. Twelve patients had primary refractory AML. Results: The MTD for Len given on d 6-26 in combination with AraC at 1.5 g/m2/d x 5 d was 10 mg. Dose de-escalation from the starting dose of 25 mg on this schedule was required due to excess toxicity. The most commonly observed non-hematologic drug related adverse events seen on the study (all < grade 2 unless indicated) were nausea, increased liver function tests (>grade 3), rash (grade >3), hypokalemia (> grade 3) and fatigue. At the 25 mg dose level the dose limiting toxicity was rash, while patients enrolled at the 15 mg dose level experienced dose limiting elevation in LFTs, fatigue and bleeding. Five patients achieved a CR (16%), 5 demonstrated CRi (16%) and there were 3 hematological improvements (HI) for an overall response rate (CR+Cri+HI) of 41% (13/32). The median overall survival (OS) (95% confidence interval) for patients treated on study was 5.8 (2.5, 10.6) months and disease free survival was 3.4 (2.3, 6.2) months. Conclusions: Although prior interesting data support the activity of single agent high dose Len in r/r AML, our single institute phase I study of intermediate dose AraC followed by Len was associated with marked skin and other toxicities at the Len 25 mg dose level, precluding dose escalation to the historically more active 50 mg dose. The CR rate in this study was not dissimilar to previously reported responses with single agent or combination AraC based regimens. Issues of dose and schedule for this combination may have had a significant impact on the potential benefit for these two drugs in combination. Nevertheless, the overall low CR rate from this study does not suggest any superiority for this combination in comparison with the historical single agent response rate for intermediate dose AraC in r/r AML. Disclosures Griffiths: Celgene, Incyte and Alexion: Honoraria; Astex Pharmaceuticals: Research Funding. Wang:Incyte: Speakers Bureau; Immunogen: Other. Wetzler:MedPace: Consultancy; Bristol Myers Squibb: Research Funding; Jazz Pharmaceuticals: Consultancy; Sigma Tau: Consultancy; Amgen: Honoraria; Novartis: Honoraria; Teva: Honoraria; Plexus: Consultancy; Celgene: Research Funding.

Introduction Subcutaneous (SC) omacetaxine mepesuccinate (OMA) is indicated for the treatment of CP and AP CML in adults with resistance/intolerance (R/I) to ≥2 tyrosine kinase inhibitors (TKIs). Unlike TKIs, OMA inhibits protein synthesis and is not a direct inhibitor of Bcr-Abl kinase activity. The clinical activity of OMA was demonstrated in a combined cohort of patients from 2 single-arm trials. The cohort consisted of patients who had received ≥2 approved TKIs and, at a minimum, documented evidence of R/I to dasatinib and/or nilotinib. This is the final ≥24-month follow-up analysis in a cohort subset from the 2 studies as originally approved by the FDA. Methods Patients received OMA 1.25 mg/m2 bid SC in 28-day cycles: 14 days for induction and 7 days as maintenance, adjusted for tolerability. Primary endpoints were major cytogenetic response (MCyR) for CP and major hematologic response (MaHR) for AP. Secondary objectives included time to onset and duration of response, progression-free survival (PFS), overall survival (OS), and adverse events (AEs). Survival data were collected after study discontinuation. Efficacy was analyzed by a data monitoring committee (DMC) on-study and investigators during follow-up for the cohort used for approval of OMA, which excluded patients with a best response at baseline or from a site without verified protocol compliance. Safety was analyzed for all CP and AP patients receiving SC OMA. Results As of October 12, 2012, the efficacy analysis cohort included 76 CP and 35 AP patients. The safety group included 163 patients (108 CP and 51 AP, plus 4 AP patients from a prior study). The majority of the 76 CP (median age, 59 y; range, 26-83 y) and 35 AP patients (median age, 64 y; range, 23-83 y) had received hydroxyurea (CP 41/76, AP 22/35) and 3 prior TKIs (CP 36/76, AP 22/35). At data cutoff, 5 CP patients and 1 AP patient continued OMA treatment (CP: 57, 43, 57, 52, and 48 months; AP: 52 months). Median treatment duration was 7.5 (range, 0-55.6) months for CP and 1.6 (0-49.7) months for AP patients. Median total months of follow-up for survival was 29.5 (95% CI, 17.6-36.4) for CP and 14.3 (95% CI, 4.7-18.7) for AP patients. Of patients receiving ≥2 cycles, 53/60 CP and 16/26 AP patients had a cycle delay; median number of cycles requiring delay per patient were 4 (CP) and 1 (AP). DMC-assessed responses were unchanged at 24 months; only investigator assessments were performed during follow-up. The MCyR rate for CP was 18% (14/76) with a mean time to onset of 3.5 months and median duration of 12.5 months. For AP, the MaHR rate was 14% (5/35) with a mean time to onset of 2.3 months and median duration of 4.7 months. No AP patients achieved MCyR. For ongoing patients, responses at data cutoff were complete cytogenetic response for all CP patients and hematologic improvement for the AP patient. Median PFS was 9.6 months for CP and 3.6 months for AP patients. At data cutoff, 16 CP and 1 AP patients had not progressed. Survival data were collected after therapy discontinuation; however, of the 6 patients in the efficacy cohort who remained on treatment at data cutoff, 2 (both CP) lost response as assessed by investigators. Based on the 24-month update, median OS was extended from 33.9 to 40.3 (95% CI, 23.8 to not reached) months for CP patients and was unchanged for AP patients at 14.3 (95% CI, 6.7-18.7) months (Figure). Safety and tolerability across the 24-month analysis were unchanged from the original analysis. The most common AEs (≥20%) were thrombocytopenia, anemia, neutropenia, diarrhea, nausea, fatigue, asthenia, and pyrexia for CP and AP patients; leukopenia and headache were ≥20% for CP only; febrile neutropenia was ≥20% for AP only. Patients with severe myelosuppression were at risk for infections and hemorrhage. Grade 3/4 hematologic laboratory toxicities for CP and AP patients were thrombocytopenia (CP 92/108, AP 44/50), neutropenia (CP 88/108, AP 35/50), leukopenia (CP 82/108, AP 30/50), and anemia (CP 65/108, AP 39/50). There were 53 deaths in the CP and 38 in the AP groups in the ≥24-month period; the most common causes were disease progression (25, 15), unknown (13, 17), sepsis (5, 0), and hemorrhage (cerebral [2, 3], pulmonary [1, 1]). Conclusions This 24-month update demonstrates that OMA induces clinically meaningful and durable responses in a subset of heavily pretreated patients with CP or AP CML who failed prior TKI therapies. Most grade 3/4 AEs were related to myelosuppression. Support: Teva BPP R&D, Inc. Disclosures: Cortes: BMS : Research Funding; Novartis: Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Ariad: Consultancy, Research Funding. Wetzler:Teva: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Lipton:Ariad: Consultancy, Equity Ownership, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria, Research Funding. Khoury:Teva: Honoraria. Michallet:Astellas: Honoraria; Merck: Honoraria; Genzyme: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Research Funding; Teva: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Honoraria; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Baccarani:BMS: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Teva: Consultancy; Novartis: Consultancy, Honoraria. Rea:BMS: Honoraria; Teva: Honoraria; Novartis: Honoraria. Chuah:BMS: Honoraria; Novartis: Honoraria. Parikh:Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Dr Reddys: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Glenmark: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Spectrum Pharmaceuticals: Equity Ownership; OncoRx: Equity Ownership; Biocon: Research Funding; GSK: Research Funding; Intas: Research Funding; Alkem: Research Funding. Li:PharmaStat, LLC: Consultancy. Munteanu:Teva: Employment, Equity Ownership. Brown:Teva: Employment, Equity Ownership. Nicolini:Novartis: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Teva: Honoraria; Ariad Pharmaceuticals: Honoraria; Pfizer: Honoraria.

Background Epigenetic therapy (Epi) with the hypomethylating agents, azacitidine (Aza) and decitabine (Dec), is increasingly being utilized for induction treatment of older AML patients (pts) based on studies demonstrating both tolerability and prolonged survival. By contrast, standard “7+3” intensive chemotherapy (IC) effectively induces remission in many individuals but is associated with significant toxicity and higher mortality. We compared our institute's experience with Epi vs. IC for the upfront treatment of newly diagnosed AML pts ≥60 years old. Methods We performed a retrospective chart review of 164 pts ≥ 60 yrs old with newly diagnosed AML who underwent initial therapy at our center between 3/2008-2/2013. Half (n=84; 51%) received IC with regimens containing cytarabine 100 mg/m2 IV for 7 days and daunorubicin 60 mg/m2 IV for 3 days. Half were treated with Epi (n=82; 49%) regimens containing either Dec (20 mg/m2 IV daily for 5 or 10 days) or Aza (75 mg/m2 sq daily for 7 days). Kaplan Meier method, log rank test, and univariate cox proportional hazard models were used to assess overall survival (OS) and correlation with covariates of interest. Results Baseline pt characteristics demonstrated a difference in the median age of pts in each group (IC 67 vs. Epi 75 yrs; p At our center, older AML pts receiving IC had superior complete response at any time point (CRatp)(43% vs. 21%; p< 0.01) and higher overall response rates (ORR=CR+CRp) (50% vs. 28%; p Conclusions Our results suggest that IC and Epi represent clinically equivalent approaches for the upfront treatment of older AML pts. In spite of significantly higher CR and ORR in the IC group, our finding of improved OS following IC vs. Epi was not substantiated in multivariate analysis, suggesting that this difference may be explained by the comparatively younger age of pts in the IC group. Leukemia-free survival and 30-day mortality were the same for IC vs. Epi-treated pts, as were all response and survival outcomes in the poor-risk cytogenetics subgroup. These data highlight the growing need for prospective clinical trials to conclusively determine the respective roles of IC vs. Epi therapy in older AML pts. Disclosures: No relevant conflicts of interest to declare.

Background Sapacitabine is an orally administered nucleoside analogue which causes single-strand DNA breaks and induces G2 cell cycle arrest. A multi-center, randomized phase 2 study was conducted to evaluate 3 dose regimens in older patients with MDS refractory to hypomethylating agents. The primary endpoint was 1-year survival. Secondary endpoints included the rate of CR, CRp, PR, major hematological improvement (HI) or stable disease and their corresponding durations. The study used a selection design to identify a dose regimen which produced a better 1-year survival rate in the event that all three dose regimens were active. The planned sample size was 60 patients (20 patients in each arm). Methods Eligible patients were ≥ 60 years with intermediate-2 or higher risk MDS and 6 - < 20% blasts in bone marrow after receiving hypomethylating agents, ECOG 0-2, adequate renal and hepatic functions. Patients were randomized 1:1:1 to receive sapacitabine every 4 weeks at 200 mg b.i.d. x 7 days (Arm G), 300 mg q.d. x 7 days (Arm H), or 100 mg q.d. x 5 days per week x 2 weeks (Arm I). Treatment was continued indefinitely until unacceptable toxicity or disease progression. Results Sixty three patients were randomized and treated (21 patients per arm). Median follow-up was 23.6 months. Median age was 73. Prior therapy also included lenalidomide (n=8), clofarabine (n=1) and fludarabine-based regimen for bone marrow transplant (n=2). Thirteen patients had high risk and all others had intermediate-2 risk. Baseline bone marrow had 10-19% blasts in 40 patients. Median number of cycles was 3 (range: 1- >21) and 30 patients received ≥ 4 cycles. To date, 9 patients have responded (2 CRs, 2 CRp, and 5 major HIs): 19% (Arm G), 10% (Arm H) and 14% (Arm I). Time to response is 1- 3 cycles. Additionally, 21 patients achieved stable disease lasting longer than 16 weeks. Median overall survival was 8.6 months: 9.7 months (Arm G), 9.7 months (Arm H) and 7.6 months (Arm I). One –year survival is 38% (Arm G), 24% (Arm H), and 33% (Arm I). Thirty-day death rate was 5% on each of the arms. Common adverse events regardless of causalities included fatigue, nausea, diarrhea, constipation, pneumonia, edema, pyrexia, epistaxis, febrile neutropenia, anemia, neutropenia and thrombocytopenia, most of which were mild to moderate. Conclusions Sapacitabine appears to be safe and active across all 3 dose regimens. The median survival in all three arms appears to exceed the median survival in published reports of the outcome of MDS patients after treatment failure of hypomethylating agents (Jabbour Cancer 2010, Prebet JCO 2011: median survival 4.3 – 5.6 months). The dose regimen of 200 mg b.i.d. x 7 days (Arm G) has better 1-year survival rate and the highest response rate. Disclosures: Garcia-Manero1: Cyclacel: Research Funding. Wetzler:Cyclacel: data safety monitoring of another study Other, Research Funding. Seiter:Cyclacel: Research Funding. Chiao:Cyclacel: Employment, Equity Ownership, Patents & Royalties. Kantarjian:Cyclacel: Research Funding.

Background The DNA methyltransferase inhibitors (DNMTi) 5-azacitidine (Aza) and decitabine (Dac) are a standard of care for patients with myelodysplatic syndrome (MDS) and acute myeloid leukemia (AML). Many hypotheses exist for the mechanism of these agents, including re-expression of epigenetically silenced tumor suppressor genes and direct cytotoxicity. We and others have shown hypermethylation of WNT/β-catenin inhibitory genes in primary MDS and AML samples. Activation of WNT/β-catenin signaling has furthermore been shown to play a role in the development and relapse of AML. Inhibition of WNT/β-catenin signaling is proposed to be clinically beneficial in AML. Based on published and preliminary data demonstrating that AML cell lines exposed to Aza and Dac re-express WNT/b-catenin inhibitors and down-regulate WNT/β-catenin signaling, we hypothesized that patients with AML receiving Dac would demonstrate suppression of WNT/β-catenin signaling in circulating blasts. Methods We obtained serial peripheral blood samples from 5 patients with AML during their first cycle of Dac therapy (20 mg/m2 per day for 10 days). These patients were ineligible for high-dose induction chemotherapy based on age, performance status or comorbid illness and were of intermediate or poor risk karyotype. All patients had circulating blasts at presentation, which remained stable or declined over time. Blast populations were identified using flow cytometric selection based upon the characteristics of the diagnostic sample. Global methylation was assessed (using LINE-1 as a surrogate) by bisulfite pyrosequencing of mononuclear cell DNA extracted from the peripheral blood. To determine whether reduced methylation was associated with decreases in WNT/β-catenin signaling, we performed imaging flow cytometry (ImageStream, Amnis Corporation) on viable blasts harvested pre-treatment and on days 5-9 of the first Dac cycle (at the methylation nadir). ImageStream was used to assess nuclear localization of β-catenin, a biochemical hallmark of active WNT signaling, in CD34+ blasts. Nuclear β-catenin was quantified using a similarity score: a log-transformed Pearson's correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). A resolution metric (Fishers discriminant ratio, Rd) was calculated to determine shifts in the population distributions of this similarity score between CD34+ blasts at diagnosis versus after Dac therapy; a negative Rd value indicates decreased similarity scores between β-catenin and DAPI in treated versus diagnostic samples and thus decreased nuclear β-catenin. To determine whether changes in nuclear β-catenin were associated with changes in gene transcription, we performed whole genome analysis of RNA extracted from diagnostic and follow up samples (n = 5 paired samples, Illumina Human-HT12v4 Expression BeadChip). Results As expected, Dac treatment significantly reduced global DNA methylation (LINE-1 methylation) from 77.0 ± 1.6% pre-treatment to a nadir of 60.9 ± 6.8% (p< 0.01, n = 6). During the methylation nadir (days 5 to 9), the cellular distribution of β-catenin in CD34+ blasts shifted from the nucleus to the cytoplasm as indicated by an average Rd value of -0.22 (n = 5). This Rd value is similar to that observed after treating U937 cells with 0.5 µM Dac. The total amount of β-catenin in CD34+ blasts from treated patients did not change compared to their pre-treatment levels (0.98 ± 0.40 fold, n = 5) indicating that the shift in the cellular distribution of β-catenin from the nucleus towards the cytoplasm was not accompanied by decreased protein level. Comparison of samples from day 5-9 of Dac treatment to pre-treatment samples revealed 128 genes with a ≥ 1.2-fold change (75 up, 53 down, p< 0.05) in mRNA levels. We did not observe changes in expression of WNT regulatory genes, suggesting that alternative mechanisms may explain the reduction in nuclear β-catenin following Dac therapy. Of the 10 genes up-regulated more than 1.5-fold, 4 (FOS, EGR1, CLEC12A, RNASE2) are associated with myeloid differentiation. Of these, FOS and EGR1 are negatively regulated by WNT/β-catenin signaling. Conclusions Given that activation of WNT/β-catenin signaling can suppress hematopoietic differentiation, these data suggest a model in which Dac exposure reduces nuclear β-catenin, leading to increased expression of pro-differentiation genes. Disclosures: Wetzler: Teva: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Griffiths:Astex Pharmaceuticals: Research Funding; Celgene, Inc.: Honoraria; Alexion Pharmaceuticals: Honoraria.

Background Marrow angiogenesis plays an important role in the pathogenesis of acute myeloid leukemia (AML). Among the most prominent pro-angiogenic factors produced by AML cells are the angiopoietins (Ang-1/2), ligands of the Tie2 endothelial cell receptor. Elevated expression of Ang-2 is an independent prognostic factor for overall survival in AML. Trebananib (AMG 386) is a first-in-class neutralizing peptibody that provides potent and selective inhibition of angiopoietins by sequestering Ang1 and Ang2 and preventing their interaction with the Tie2 receptor. Purpose Here we evaluated the safety, tolerability, pharmacokinetic profile, and biologic effects of trebananib in de novo and relapsed/refractory AML patients considered to be poor candidates for standard induction chemotherapy. Methods Thirteen AML patients were enrolled in single agent arm A of this open-label, non-randomized phase Ib trial. (ClinicalTrials.gov ID: NCT01555268, NCI-2011-02979). Drug was administered at two dose levels (15 and 30 mg/kg) via weekly intravenous infusion over 1 hour. Circulating angiogenic factor levels (Ang-2, VEGF-A/C/D, HGF, IL-8, FGF-1/2, G-CSF, PlGF) in the plasma were measured by multiplex flow cytometry prior to first dose, on cycle 1 day 29 (following 4 weekly doses of trebananib), and at the end of study. Results Median age was 76 years (range 53-82). Nine patients were male (69%). Two patients had de novo, 7 had refractory, and 4 had relapsed AML. One patient had undergone prior allogeneic stem cell transplantation. Aside from one possibly drug-related incident of grade 3 mucositis on the 15 mg/kg cohort, no other dose-limiting toxicities were reported. The most common toxicities were abdominal bloating, diarrhea, edema, and weight gain. Preliminary pharmacokinetic analysis of trebananib was dose-proportional. At the 15 and 30 mg/kg dose levels, median Cmaxand AUC on Day 1 were 193 and 473 ug/mL and 11,670 and 35,217 ug-hr/mL, respectively. No neutralizing antibodies to trebananib were detected. Among seven AML patients with consecutive plasma samples, therapy was associated with marked elevations of Ang-2 levels (from 15 to 336-fold higher than baseline) after 1-4 weeks of therapy (Table 1). Induction of Ang-2 did not differ significantly between the 15 mg/kg and 30 mg/kg cohorts. Ang-2 levels measured in bone marrow or plasma samples at the same time point were comparable. In contrast, levels of other angiogenic growth factors (VEGF-A/C/D, IL-8, G-CSF, HGF, FGF-1/2, PlGF) were not consistently altered by trebananib therapy. One AML patient (on the 15 mg/kg cohort) had a partial response with >50% decrease in marrow blasts. Two patients (15 mg/kg, 30 mg/kg) had stable disease over >1 cycle. Conclusions We report here the preliminary results of phase 1b study evaluating the safety, tolerability, pharmacokinetic profile, and biologic effects of trebananib in adult AML patients. Weekly infusions of trebananib administered at 15 and 30 mg/kg were well tolerated. Drug pharmacokinetics were similar to those previously reported in solid tumor patients. Trebananib resulted in substantial elevations in measurable circulating and marrow Ang-2 levels in AML patients, presumably due to a negative compensatory feedback loop resulting from potent in vivo Ang-1/2 inhibition. Arm B of this study (trebananib plus low dose cytarabine) is currently accruing. Further in depth studies examining the biological effects of trebananib on marrow angiogenesis and clinical response in AML patients are ongoing. Disclosures: Becker: Millenium: Research Funding.

Acute Myelogenous Leukemia (AML) is a disease that clinically evolves over time as many patients who are responsive to therapy upfront acquire resistance to the same agents when applied in the relapse setting. The stem cell model for AML has been invoked to explain primary resistance to standard therapy; the leukemia stem cell (LSC) population representing a therapy-refractory reservoir for relapse. There have been no prospective efforts to formally assess the evolution of the LSC population during patients’ clinical course. We performed a prospective characterization of specimens from a well-defined cohort of patients with AML at diagnosis and relapse to assess the frequency and phenotype of functionally defined LSCs. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Pre-therapy and post-relapse LSC frequencies were assessed using xenotransplantation limiting dilution analyses (LDA). We assessed the frequencies of CD45RA, CD32, TIM-3, CD96, CD47, and CD97 expressing populations that have been previously published to possess LSC activity. Functionally validated pre-therapy and post-relapse LSC populations were identified using fluorescent labeled cell sorting and NSG xenotransplantation. LSC activity was confirmed for each population using secondary xenotransplantation. Gene expression analysis of highly enriched LSC populations from pre-therapy and post-relapse samples was performed using ABI TILDA qPCR analyses following pre-amplification. Results We demonstrated by LDA an 8 to 42-fold increase in LSC frequency between diagnosis and relapse in paired primary patient samples. The increase in LSC activity was not associated with an increase in frequency for phenotypically-defined populations previously reported to possess LSC activity. Rather, we found that LSC activity expanded at relapse to immunophenotypic populations of leukemic cells that did not possess LSC activity prior to treatment. Moreover, in all patients, the number of phenotypically distinct LSC populations (as defined by CD34 and CD38 or CD32 and CD38) detectable at relapse was dramatically expanded. Further, while the majority of the LSC populations’ gene expression profile remained stable between diagnosis and relapse, a subset of genes were enriched in defined LSC populations at relapse including IL3-receptor alpha and IL1-RAP, both previously demonstrated to play a role in LSC biology. Conclusions This study is the first to characterize the natural evolution of LSCs in vivo following treatment and relapse. We demonstrate an increase in LSC activity and greatly increased phenotypic diversity of the LSC population, suggesting a loss of hierarchical organization following relapse. These findings demonstrate that treatment of AML patients with conventional chemotherapy regimens can promote quantitative and qualitative expansion of the LSC compartment. Further, the data indicate that surface antigen immune-phenotype is not predictive of function in relapse and suggest a major limitation to efforts targeting specific surface antigens in the relapse setting. Understanding the mechanisms by which LSC expansion occurs and how to target it will likely improve our currently poor treatment options for patients who relapse. Disclosures: Becker: Millenium: Research Funding.

Among the prognostic cytogenetic and molecular aberrations in AML, t(8;21)(q22;q22) and inv(16)(p13q22) and their corresponding molecular rearrangements RUNX1/RUNX1T1 and CBFB/MYH11 (each involving a gene encoding a protein chain of the key transctiption factor CBF), predict for a favorable outcome in pts receiving consolidation with high-dose cytarabine (HiDAC) after achievement of complete remission (CR). However, approximately 40% of these pts eventually relapse. Approximately 25% of CBF AML pts carry gain-of function mutations in the KIT gene. These mutations result in a constitutively active tyrosine kinase (TK) that contributes to aggressive leukemia growth, and is associated with unfavorable outcome. In addition, CBF AML pts with wild type KIT overexpress this protein, and this is also associated with an inferior outcome. Therefore, inhibiting KIT with DAS is a rational therapeutic strategy in CBF AML. We report here on a phase II trial that combined DAS with standard chemotherapy for CBF AML. Enrollment required molecular confirmation of CBF AML by the Alliance Molecular Pathology central lab using RT-PCR and Sanger sequencing-based assays. Overall, 779 patients were screened for CBF; 69 were found to be CBF-positive and 61 were subsequently enrolled. Newly diagnosed RUNX1/RUNX1T1 or CBFB/MYH11-positive pts received induction chemotherapy with cytarabine (C) 200 mg/m2/day continuous intravenous (IV) infusion on days 1-7, daunorubicin (DNR) 60 mg/m2/d IV bolus on days 1-3 and DAS 100 mg/d PO on days 8-21. Pts with residual disease (>5% blasts) on day 21 after first induction received a re-induction treatment with same doses of C on days 1-5, DNR on days 1-3 and DAS on days 6-19. Pts who achieved CR received consolidation therapy with HiDAC 3000 mg/m2 over 3 hours (if Between April 2011 and January 2013, we completed the planned accrual of 61 adult CBF AML pts. Median age was 51 years (yrs; range: 19.6 to 85 yrs), and 15 pts (24%) were older (>60 yrs). Half of pts were male (51%) and a majority were Caucasian (75%). Of all 61 pts, 65% were CBFB/MYH11-positive and 35% were RUNX1/RUNX1T1-positive. Treatment was started on average 4 days from molecular diagnosis (range: 0 to 11 days). To date, 51% of pts are still undergoing treatment; 4 pts died on treatment (2 older), 7 (4 older) had an adverse event requiring treatment interruption, and 6 refused to complete the treatment (mainly the continuation component). Observed toxicities were those expected with C and DNR (hematologic and non-hematologic) and with DAS (nausea, liver toxicity). 55 pts are currently evaluable for treatment-related toxicity. The most common grade 4 toxicities were sepsis (5), acute kidney injury (3), and respiratory failure (3). Grade 5 toxicities included respiratory failure (1) and sepsis (2). Two of these pts died during induction (respiratory failure, sepsis); both were older and CBFB/MYH11. One pt died from sepsis during consolidation in CR (CBFB/MYH11, 48 yrs). The 30-day survival rate was 97% (95% CI: 89% to 99.6%) overall (98% in younger and 93% in older pts). Of 59 pts currently evaluable for response, 54 (92% of all pts; 96% younger and 80% older) achieved CR. Of the 5 patients who failed to achieve CR, 2 had RUNX1/RUNX1T1 and 3 had CBFB/MYH11. Among the 54 CR pts, no younger pt has relapsed, while 2 older pts with CBFB/MYH11 have relapsed. The median follow-up (f/u) was 11.2 months (range: 1.2 to 23.2 mos.). The 1-yr DFS and OS rates were respectively 90% and 87% for all pts; 97% and 95% for younger pts, and 63% and 62% for older pts, respectively. Early results from this study show that 1) rapid screening for CBF AML is feasible within a cooperative group, 2) DAS plus chemotherapy in CBF AML pts is tolerable including in older pts, and 3) the initial clinical outcomes are at least comparable to those historically observed in this patient population. Patients continue to be followed for survival endpoints. Molecular characterization for KIT mutations and expression levels of marrow and blood blasts is ongoing and will be correlated with toxicity and clinical outcome. Disclosures: No relevant conflicts of interest to declare.

Abstract 816 We hypothesized that imatinib plus sequential chemotherapy would result in significant leukemia cell cytoreduction (molecular remission) in patients with Ph+ acute lymphoblastic leukemia (ALL), allowing collection of normal hematopoietic stem cells uncontaminated by residual BCR/ABL+ lymphoblasts and thus reduce the likelihood of relapse after autologous (auto) stem cell transplant (SCT) for patients 120 days had CMR; 2 additional patients achieved MMR, and 1 patient had residual MRD >0.001 (less than MMR); however, the sample size was too small to analyze the effect of MRD on outcome. After a median follow up time of 5.1 years, 9 (47%) of 19 auto-SCT patients and 7 of 15 (47%) of the allo-SCT patients remain alive in continuous CR. Ten of the transplanted patients have relapsed (8 following auto-SCT and 2 following allo-SCT); relapses occurred at a median of 5.9 months following auto-SCT. The DFS (median, 5.5 vs 4.1 years; P=0.84) and OS (median, 6.0 years vs. not reached at >6 years; P=0.90) were similar between those who underwent auto- or allo-SCT (Panels C and D). We conclude that patients who have MRD at levels lower than or equal to MMR following auto-SCT have prolonged survival and that auto-SCT represents a safe and effective alternative to allo-SCT for Ph+ ALL patients without sibling donors. The intergroup trial CALGB 10701 (Alliance) is now testing this strategy in Ph+ ALL patients >50 year old, using dasatinib as the BCR/ABL inhibitor. Figure: Disease-free (A, C) and overall (B, D) survival, stratified by minimal residual disease (MRD) at Day +120 and by transplant type. (A, B) MRD ≤0.001 (major molecular response) vs. >0.001 at day +120 for patients undergoing autologous-stem cell transplant (SCT); (C, D) allogeneic (allo) vs. autologous (auto) SCT. Figure:. Disease-free (A, C) and overall (B, D) survival, stratified by minimal residual disease (MRD) at Day +120 and by transplant type. (A, B) MRD ≤0.001 (major molecular response) vs. >0.001 at day +120 for patients undergoing autologous-stem cell transplant (SCT); (C, D) allogeneic (allo) vs. autologous (auto) SCT. Disclosures: Wetzler: BMS: Research Funding; Novartis: Research Funding. Off Label Use: Dasatinib is off label but in a clinical trial. 615A Oral Session 1 (3 abstracts) Acute promyelocytic leukemia 622A Oral Session 1 (3 abstracts) Autologous and Allogeneic Transplantatin CLL - Biology and Pathophysiology, excluding Therapy: Cell Signaling Leukemias - Biology, Cytogenetics and Molecular Markers in Diagnosis and Prognosis: Chronic Lymphoid and Myeloid Leukemias 111. Hemoglobinopathies, excluding Thalassemia IV

Abstract 44 CALGB (now part of the Alliance for Clinical Trials in Oncology) performed a non-randomized phase II study testing one year (yr) of investigational maintenance therapy with decitabine for newly diagnosed adult AML patients (pts) 1 × 109/L, platelets >75 × 109/L, and be within 90 days after day 1 of the final consolidation. Decitabine 20mg/m2 was given IV over 1 hour for 4–5 days, every 6 weeks, for 8 cycles. 546 pts enrolled with a median age of 48 yrs (range, 17–60) and median presenting white blood count (WBC) of 12.6 × 109/L (range, 0.3–380 × 109/L). 75% achieved CR (412/546). Reasons for CR pts not receiving maintenance were mainly early relapse, alloHCT in 1st CR, inadequate count recovery, and patient refusal (Blum et al. ASH 2011). 33% of CR pts (134/412) received investigational maintenance. Of these, 46 (34%) were favorable risk; among the remaining 88 pts, 73 were consolidated with autoHCT, and 15 received HiDAC-based consolidation. The median time from initial study registration to initiation of maintenance therapy was 6.3 months. Pts receiving decitabine had a median age of 45 yrs (range, 18–60) and presenting WBC of 13.5 × 109/L (range, 0.4–221 × 109/L). Treatment with decitabine maintenance was feasible and reasonably well tolerated; the primary toxicity was myelosuppression. Preplanned dose reduction criteria for neutropenia were met after the first 20 pts had been treated. After this early timepoint, all subsequent pts who had been consolidated with autoHCT received only 4 days of decitabine per cycle (HiDAC consolidated pts continued to receive 5 days per cycle). The median number of cycles of decitabine received was 7 (range, 1–8); 46% of pts (62/134) received all 8 cycles, and 75% completed at least 4 cycles. Reasons for discontinuation of decitabine before completing 8 cycles included relapse (28%, 38/134), pt refusal (13%), adverse events (4%), and others. In this selected cohort of pts who received post-CR maintenance with decitabine, 1-yr and 3-yr overall survival (OS) were 96% and 66%; 1-yr and 3-yr disease-free survival (DFS) were 80% and 53%. 1-yr “failure-free survival” calculated from the time of registration to decitabine was 68% (70% for favorable risk, 68% for others). These results are similar to the outcomes for comparable pts enrolled on our prior CALGB study 19808 who received identical chemotherapy for induction and consolidation and then were randomized to observation or maintenance with interleukin-2 (3-yr OS 61% and 68%, 3-yr DFS 45% and 56%, for observation and IL-2 respectively). Post-consolidation maintenance with decitabine appears to provide only modest, if any, benefit overall. Correlative studies for CALGB 10503 are ongoing, including investigations into the impact of decitabine in unique molecular and cytogenetic subsets of disease, the prognostic/predictive value of aberrant methylation and other molecular markers, and assessment of minimal residual disease. Supported by CA33601 and CA128377. Disclosures: No relevant conflicts of interest to declare.

Abstract 3753 Background Subcutaneous omacetaxine mepesuccinate (“omacetaxine”) is a first-in-class cephalotaxine. Omacetaxine is a protein synthesis inhibitor that does not depend on direct binding of Bcr-Abl. Omacetaxine reduces levels of multiple oncoproteins, including Bcr-Abl, and induces apoptosis in leukemic stem cells. Omacetaxine has shown clinical activity and adequate tolerability in two phase 2, open-label, international, multicenter studies in patients with chronic myeloid leukemia (CML): the first in patients with history of T315I mutation who had failed prior imatinib and the second in patients with resistance or intolerance to ≥2 tyrosine kinase inhibitors (TKIs). This is a pooled analysis of the efficacy and safety data of omacetaxine in patients with CML in the blast phase (BP). Methods Data from patients with CML-BP were included from two phase 2 studies. All patients received omacetaxine 1.25 mg/m2 subcutaneously twice daily for up to 14 consecutive days every 28 days for induction and the same dosage for up to 7 days every 28 days as maintenance. The number of consecutive days of dosing could be adjusted, as clinically indicated. Recombinant growth factor support was allowed in the event of febrile neutropenia. The primary outcome measures were major hematologic response (MaHR) and major cytogenetic response (MCyR). MaHR was defined as complete hematologic response (CHR) lasting ≥4 weeks, no evidence of leukemia, or return to chronic phase. MCyR included confirmed or unconfirmed complete or partial response. Secondary endpoints included duration of response, time to disease progression, overall survival and safety. Results Forty-four patients with CML-BP (median age 53.5 years [range, 19–68]) received treatment with omacetaxine. Forty-two patients (96%) had ECOG status ≤2. All but 1 patient had prior treatment with imatinib; 5 (11%) patients received imatinib only, 20 (45%) received 2 approved TKIs (imatinib, dasatinib, or nilotinib), and 19 (43%) were treated with all 3 approved TKIs. Most common non-TKI agents included hydroxyurea in 18/44 (41%), anthracyclines and related agents in 17 (39%), cytarabine in 14 (32%), and interferons in 11 (25%). Mutation analysis was not performed per protocol; however, point mutations were detected in 23 patients, and 17 (74%) of these had the T315I mutation. No mutations were identified in 7 patients and 14 were not assessed for mutations. The median number of cycles of omacetaxine administered was 2 (range 1–12) with a median duration of exposure of 1.5 months (range 0–13.8). Four patients (9%) had a MaHR (3 with CHR and 1 returned to chronic phase); additional 2 patients had hematologic improvement as a best response. No patient achieved MCyR; 3 patients (7%) achieved a minimal cytogenetic response. Median duration of MaHR was 1.7 months (range 1.7–4.9 months). Median survival was 3.5 months (95% CI 2.2–4.5); in 4 patients with MaHR, median survival was not reached as of >1 year of follow-up by Kaplan-Meier analysis (95% CI 2.4 to not reached) versus 3.5 months (95% CI 2.2–3.9) in patients without MaHR (Figure). Median time to disease progression was 2.2 months (95% CI 1.5–2.9). Grade 3/4 laboratory hematologic toxicities included thrombocytopenia (43/44 patients), anemia (36/44), neutropenia (36/44), and leukopenia (29/44), with most of the thrombocytopenia and neutropenia toxicities occurring in earlier cycles and attenuating in later cycles. The most common (≥5%) grade 3/4 nonhematologic AEs were hypercalcemia and bone pain (4/44 each), followed by confusional state (3/44). The most common reasons for discontinuation were progressive disease (21 patients) and death (14 patients); 2 patients discontinued due to AEs. One patient discontinued due to transfer for stem cell transplantation (7 cycles received on study over 6.7 months). Nineteen deaths occurred on study and 19 during follow-up; disease progression was the most common cause (25/38). One death was deemed related to the study drug (sepsis). Conclusions Among heavily pretreated patients with CML-BP who had failed prior TKI therapy, omacetaxine demonstrated limited activity, although 13% of patients had hematologic improvement, 2 patients had responses with duration longer than one year. Most grade 3/4 events were hematologic; grade 3/4 nonhematologic AEs were less common. Support: Teva Pharmaceutical Industries Ltd. Disclosures: Off Label Use: Subcutaneous omacetaxine mepesuccinate (“omacetaxine”) is a protein synthesis inhibitor that does not depend on direct binding of Bcr-Abl. Omacetaxine has shown clinical activity in 2 studies of chronic myeloid leukemia (CML), one in patients with a history of the T315I Bcr-Abl mutation and the other in patients failing at least 2 tyrosine kinase inhibitors. Cortes:Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Chemgenex (Teva): Consultancy, Research Funding; Deciphera: Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding. Wetzler:BMS: Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees. Baccarani:Teva: Research Funding. Douer:Teva: Consultancy. Craig:Teva: Consultancy. Kantarjian:ChemGenex (Teva): Research Funding. Akard:Celgene: Research Funding, Speakers Bureau; Millenium: Speakers Bureau; Eisai: Speakers Bureau; Pfizer: Research Funding; Merck: Research Funding; Ariad: Research Funding; Teva: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

Abstract 2787 Background Subcutaneous omacetaxine mepesuccinate (“omacetaxine”) is an investigational, first-in-class cephalotaxine, a protein synthesis inhibitor that does not depend on direct binding of Bcr-Abl. Omacetaxine reduces levels of multiple oncoproteins, including Bcr-Abl, and induces apoptosis in leukemic stem cells. It has shown clinical activity and adequate tolerability in two phase 2, open-label studies of chronic myeloid leukemia (CML), the first in patients with a history of the T315I Bcr-Abl mutation (enrolled September 2006 through March 2010) and the second in patients with resistance or intolerance to ≥2 approved tyrosine kinase inhibitors (TKIs; enrolled March 2007 through June 2009). This ad hoc analysis presents the efficacy and safety results of patients in the phase 2 studies who remained on omacetaxine as of March 31, 2012. Methods Efficacy and safety data were pooled from patients with CML chronic phase (CML-CP) and CML accelerated phase (CML-AP) who received omacetaxine in either of the phase 2 studies and had not discontinued omacetaxine as of March 31, 2012. Omacetaxine 1.25 mg/m2was given subcutaneously twice daily up to 14 consecutive days per 28-day cycle for induction and at the same dosage for up to 7 days/cycle as maintenance. Results Of the 203 patients enrolled in the 2 studies (108 CML-CP, 51 CML-AP, and 44 CML blast phase), 9 CML-CP patients (median 35 cycles [range 26–53]) and 2 CML-AP patients (median 20.5 cycles [range 19–22]) continue to receive omacetaxine. Median age of ongoing CML-CP patients at baseline was 61 years (range 45–73); 7 were male. Median age of ongoing CML-AP patients was 58 years (range 48–67); all were male. For CML-CP patients, the median number of dosing days was 137 (range 68–342) over a median of 35 cycles (range 26–53); the 2 CML-AP patients had 189 and 277 dosing days during 19 and 22 cycles, respectively. Other baseline clinical characteristics are presented in the Table. Seven of the CML-CP patients achieved MCyR, including 6 complete responses. One CML-CP patient achieved a minimal cytogenetic response and 1 had no response. No CML-AP patient achieved MCyR. Time to onset of MCyR for the CML-CP patients were Six CML-CP patients were in CHR at baseline and 3 achieved CHR within 3 months. One CML-AP patient achieved CHR at All patients had at least 1 AE and 1 serious AE. The most common AEs were anemia (11/11), thrombocytopenia (9/11), neutropenia (6/11), nausea (7/11), diarrhea or fatigue (5/11 each), and headache (4/11). Grade 3/4 AEs occurred in 10 (91%) patients. The most common were hematologic: thrombocytopenia (79% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(79\) \end{document} CML-CP, 100% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(22\) \end{document} CML-AP), anemia (67% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(49\) \end{document} CML-CP, 100% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(22\) \end{document} CML-AP), and neutropenia (56% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(59\) \end{document} CML-CP, 0% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(02\) \end{document} CML-AP). Leukopenia and pancytopenia were reported in 1 patient each in the CML-AP group. Blast crisis, which was deemed treatment-related, was reported in 1 patient in the CML-AP group; this patient was discontinued. No deaths were reported. Conclusions A subset of heavily pretreated patients with CP or AP CML who participated in phase 2 studies of omacetaxine were able to continue omacetaxine for up to 53 cycles and experience durable major cytogenetic and hematologic responses. Grade 3/4 AEs were primarily hematologic and consistent with the known safety profile of omacetaxine. Disclosures: Kantarjian: ChemGenex (Teva): Research Funding. Off Label Use: Subcutaneous omacetaxine mepesuccinate (“omacetaxine”) is a protein synthesis inhibitor that does not depend on direct binding of Bcr-Abl. Omacetaxine has shown clinical activity in 2 studies of chronic myeloid leukemia (CML), one in patients with a history of the T315I Bcr-Abl mutation and the other in patients failing at least 2 tyrosine kinase inhibitors. Baccarani:Teva: Research Funding. Nicolini:BMS: Research Funding, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Teva: Speakers Bureau. Wetzler:Teva: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Akard:Merck: Research Funding; Pfizer: Research Funding; Eisai: Speakers Bureau; Millenium: Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Ariad: Research Funding; Teva: Research Funding. Legros:Celgene: Speakers Bureau; BMS: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Craig:Teva: Consultancy. Cortes:Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Chemgenex (Teva): Consultancy, Research Funding; Deciphera: Research Funding.

Abstract 4433 Background Subcutaneous omacetaxine mepesuccinate (“omacetaxine”) is an investigational, first-in-class cephalotaxine and is a protein synthesis inhibitor that does not depend on direct binding of Bcr-Abl. Omacetaxine reduces levels of multiple oncoproteins, including Bcr-Abl, and induces apoptosis in leukemic stem cells by blocking the initial step of protein translation at the level of the ribosome. Omacetaxine has shown clinical activity and adequate tolerability in two phase 2, open-label, international, multicenter studies in patients with chronic myeloid leukemia (CML). Patients with a history of the T315I mutation who failed prior imatinib were enrolled in the first study (CML202). Patients were enrolled in the second study (CML203) if they had resistance or intolerance to ≥2 tyrosine kinase inhibitors (TKIs). Point mutations occur frequently in CML patients who develop resistance to imatinib. This is a post hoc analysis of responses to omacetaxine in patients with any phase CML by mutational status. Methods Data from patients with CML in chronic phase (CP), accelerated phase (AP), or blast phase (BP) were included from the two phase 2 studies. Omacetaxine 1.25 mg/m2was administered subcutaneously twice daily for 14 consecutive days every 28 days for induction and the same dosage for 7 days every 28 days as maintenance. An exploratory analysis was conducted to elucidate associations between mutation status and complete hematologic response (CHR) or major cytogenetic response (MCyR) following omacetaxine treatment. Results Among CP, AP, and BP patients, grade 3/4 hematologic toxicities were thrombocytopenia (in 89/105, 43/49, and 43/44 patients, respectively), neutropenia (86/106, 35/49, 36/44), leukopenia (76/106, 30/49, 29/44), and anemia (66/107, 39/49, 36/44). Common grade 3/4 nonhematologic adverse events included fatigue (5/108, 5/51, and 2/44 patients, respectively) and pneumonia (3/108, 4/51, and 2/44). Seventy-six deaths occurred during long-term follow-up (33, 24, 19); 8 were treatment-related (5, 2, 1), most commonly due to sepsis (n=3). The pooled baseline mutational analysis included 203 CML patients (108 CP, 51 AP, and 44 BP). Unknown mutations were noted in 24%, 25%, and 32% of CP, AP and BP patients, respectively. Mutations were identified in 52%, 51%, and 52% while no mutation status was recorded for 24%, 25%, and 16% of CP, AP, and BP patients, respectively. T315I was the most frequently identified mutation (Table), occurring in 36%, 24%, and 39% of patients, respectively. Multiple mutations, which include individual mutations listed separately, were detected in 12% CP, 14% AP, and 7% BP patients. Among patients with CP or AP CML, CHR rate was high in the presence of the T315I mutation. MCyR rates for CP patients were similar for patients with or without mutations but appeared to be relatively lower among patients with multiple mutations. Among AP patients, MCyR only occurred in those without mutations. Conclusions Point mutations, particularly T315I, were common in this heavily treated population of CML patients although T315I may be overrepresented due to CML202 inclusion criteria. Efficacy of omacetaxine, as measured by rates of CHR and MCyR, appeared to be largely independent of the presence or absence of point mutations. However, interpretation of these results is limited by small sample sizes in many of the groups. Support: Teva Pharmaceutical Industries Ltd. Disclosures: Off Label Use: Subcutaneous omacetaxine mepesuccinate (“omacetaxine”) is a protein synthesis inhibitor that does not depend on direct binding of Bcr-Abl. Omacetaxine has shown clinical activity in 2 studies of chronic myeloid leukemia (CML), one in patients with a history of the T315I Bcr-Abl mutation and the other in patients failing at least 2 tyrosine kinase inhibitors. Nicolini:BMS: Research Funding, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Teva: Speakers Bureau. Wetzler:BMS: Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees. Akard:Celgene: Research Funding, Speakers Bureau; Millenium: Speakers Bureau; Eisai: Speakers Bureau; Pfizer: Research Funding; Merck: Research Funding; Ariad: Research Funding; Teva: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Baccarani:Teva: Research Funding. Kantarjian:ChemGenex (Teva): Research Funding. Craig:Teva: Consultancy. Cortes:Chemgenex (Teva): Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Deciphera: Research Funding.

Abstract 2541 CALGB 10001 tested the feasibility of performing allogeneic or autologous stem cell transplantation (SCT) following combination chemotherapy and tyrosine kinase inhibition with imatinib in patients Disclosures: No relevant conflicts of interest to declare.

Abstract 3630 Background: Sapacitabine is a novel nucleoside analogue with a unique ability to induce single-strand DNA breaks after incorporation into DNA, leading to production of double strand DNA breaks and/or G2 cell cycle arrest. It is orally administered and has demonstrated promising activity in patients with acute myeloid leukemia (AML). In AML cell lines, the active metabolite of sapacitabine, CNDAC, is synergistic with hypomethylating agents with the synergy being more apparent if cells are treated with hypomethylating agents first. We are conducting a phase 1/ 2 study to evaluate the safety and efficacy of administering sapacitabine in alternating cycles with decitabine in elderly patients with newly diagnosed AML. Methods: Decitabine 20 mg/m2 was administered intravenously daily × 5 days of a 4-week cycle (odd cycles) alternating with sapacitabine 300 mg po b.i.d. × 3 days/week × 2 weeks of a 4-week cycle (even cycles). After these doses were shown to be safe in the phase 1 part, patients were treated in the Phase 2 part to reach a planned sample size of approximately 24 patients. The primary efficacy endpoint is response rate (CR, CRp, PR, or major HI). The regimen will be considered active if response rate is ≥30%. Eligible patients must be ≥70 years with untreated AML unsuitable for or unwilling to receive standard induction chemotherapy; patients who received hypomethylating agents for prior MDS or MPD are excluded. Results: As of August 2011, 25 patients were treated with the above regimen and 23 had ≥ 60 days of follow-up. Median age is 76 years (range, 72–90). No DLTs were observed. Four patients achieved CR, 3 PR and 2 major HIs in platelets. Median time to response is 2 cycles (range 2– 6). Fifteen patients have received ≥4 cycles of treatment. Three patients died within 60-days and the deaths were unrelated to study drugs by investigator assessment. Common adverse events (regardless of causality) included weakness, anorexia, nausea, constipation, diarrhea, dyspnea, edema, pneumonia, febrile neutropenia, neutropenia, thrombocytopenia, anemia, and hypocalcemia, mostly moderate in intensity. Conclusion: The sequential combination of decitabine and sapacitabine is safe and active in elderly AML. Disclosures: Off Label Use: Decitabine for the treatment of elderly AML. Wetzler:Cyclacel Limited: Research Funding. Chiao:Cyclacel Limited: Employment, Equity Ownership.

Abstract 3454 RUNX1 encodes the α subunit of core binding factor, a heterodimeric transcription factor required for normal hematopoiesis. Acquired RUNX1 mutations (muts) have been associated with poor clinical outcome in AML; however, prior studies analyzed pts heterogeneous for cytogenetics, age, AML type (primary or secondary), and treatment received [including allogeneic stem cell transplant (alloSCT) in 1st complete remission (CR1)] and contained limited data regarding the potential molecular drivers of the worse outcome. We report a relatively large study testing the prognostic impact of RUNX1 muts in primary CN-AML pts (n=392) treated similarly with intensive cytarabine/anthracycline-based 1st-line therapy and without alloSCT in CR1. This cohort comprised both younger [1 corresponds to a higher risk for higher values of continuous variables and the 1st level listed of a dichotomous variable. To gain biological insight, RUNX1 mut-associated gene and miR expression signatures were derived in CN-AML for the first time. Older, NPM1-wt pts were analyzed since RUNX1 muts are more common in this age group and are nearly exclusive from NPM1 muts, which have their own characteristic gene-expression signature. This yielded 484 probe sets representing 278 named genes differentially expressed between RUNX1-mut (n=31) and RUNX1-wt (n=45) pts (P In summary, RUNX1 muts are twice as common in older CN-AML pts than younger. They negatively impact on outcome in both younger and older pts not receiving alloSCT in CR1. RUNX1-mut blasts have molecular features of normal/malignant stem cells and B cells, which may explain their chemoresistance and guide novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Abstract 2521 We asked whether a modified European LeukemiaNet (ELN) karyotype-based classification without molecular markers has prognostic significance in AML pts aged 80 to 89 years (y). We queried the German-Austrian AML Study Group (AMLSG; 7 pts), the Acute Leukemia French Association Group (ALFA; 17 pts), the German AML Cooperative Group (AMLCG; 35 pts) and the Cancer and Leukemia Group B (CALGB; 81 pts) for pts treated with intensive induction (7+3 or similar) on whom conventional karyotype analyses were completed and reviewed centrally. Of the 140 pts, including 82 (59%) men and 58 (41%) women, with a median age of 82 y (range, 80–89), 117 pts had de novo and 23 had secondary or therapy-related AML (s-AML/t-AML). There were 2 pts in the modified Favorable Group [t(8;21) and inv(16)], 67 pts in the modified Intermediate I Group [cytogenetically normal (CN-AML) pts], 44 pts in the ELN Intermediate II Group [t(9;11) and abnormalities (abn) classified as neither favorable nor adverse] and 27 pts in the ELN Adverse Group [inv(3) or t(3;3), t(6;9), t(v;11)(v;q23), -5 or del(5q), -7, abn(17p) and complex karyotype (≥3 abn)]. In order to assess the impact of karyotype on outcome, we eliminated early deaths. The complete remission (CR) rate for all 92 (66%) pts surviving beyond 30 days (d) was 46% and their median disease-free survival (DFS) was 6 months (mo); 35% were disease-free at 1 y and 12% at 3 y. Similarly, the median overall survival (OS) for pts surviving beyond 30 d was 6 mo, with 35% alive at 1 y and 11% at 3 y. There was no difference in DFS or OS based on AML type (de novo v s-AML/t-AML; Table 1). As shown in Table 2, pts in the modified Intermediate I Group had better OS than pts in the Adverse Group (P=0.03). Among CN-AML pts with molecular testing completed, 9/21 (43%) were NPM1-mutated (mut), 8/25 (32%) had FLT3-internal tandem duplication (ITD) and 1/12 (8%) was CEBPA-mut. Interestingly, FLT3-ITD did not have prognostic significance in the CN-AML cohort alive beyond 30 d (data not shown), but NPM1 mutation resulted in trends for higher CR rates (67% v 25%, P=.09) and longer OS (91 v 10 mo, P=0.07) in the CN-AML cohort alive beyond 30 d (Figure). Of note, there was no difference between the 2 larger cohorts (AMLCG and CALGB) in regards to pt characteristics or outcome in de novo pts; there were insufficient numbers of pts with s-AML/t-AML to compare the 2 cohorts. We conclude that there is a difference in accrual to clinical trials of octogenarian AML pts among the different cooperative groups. Further, CN-AML pts had better OS in octogenarian AML, and NPM1 mutation may be of prognostic significance among the CN-AML pts.Table 1:Treatment outcome by disease presentation for 92 pts alive beyond 30 dEnd Pointde novo AML (n = 78)s-AML/t-AML (n = 14)All pts (n = 92)P de novo v s-AML/t-AMLCR, no. (%)35 (45)7 (50)42 (46).78DFS.76 Median, y0.50.60.5.27 % Disease-free at 1 y36 (26–51)29 (4–61)35 (21–49) % Disease-free at 3 y14 (5–29)012 (4–24)OS Median, y0.50.60.5 % Alive at 1 y35 (24–46)36 (13–59)35 (25–45) % Alive at 3 y14 (6–23)011 (5–19)Table 2:Disease outcome by ELN karyotype-based classification without molecular markers for 92 pts alive beyond 30 dEnd PointIntermediate I (n = 43)Intermediate II (n = 29)Adverse (n = 20)PCR, no. (%)23 (53)13 (45)6 (30).23DFS.17* Median, y0.60.40.3 % Disease-free at 1 y36 (17–56)40 (16–63)17 (1–52) % Disease-free at 3 y23 (8–41)00OS.03† Median, y0.90.40.3 % Alive at 1 y43 (28–57)33 (16–50)21 (7–41) % Alive at 3 y17 (8–31)5 (0–20)5 (0–22)*Only Intermediate I compared to Intermediate II (too few pts in the Adverse Group)†This is overall P-value. Adjusted P-values were not significant for the differences between Intermediate-I and Intermediate-II Groups (P=.26) and between Intermediate-II and Adverse Groups (P=.87). There was a statistically significant difference between Intermediate-I and Adverse Groups (P=.03)Figure.Overall survival by NPM1 status in CN-AML ptsFigure. Overall survival by NPM1 status in CN-AML pts Disclosures: Krug: MedA Pharma: Honoraria; Novartis: Honoraria; Alexion: Honoraria; Boehringer Ingelheim: Research Funding; Sunesis: Honoraria.

Abstract 3761 Background Omacetaxine is a first-in-class, reversible, transient inhibitor of protein elongation that does not depend on BCR-ABL binding. By blocking ribosomal function, the drug decreases intracellular levels of several antiapoptotic regulatory proteins, inducing antitumor activity via apoptosis [Pérez-Galán P et al. Blood 2007;109:4441–9]. Omacetaxine had clinical activity and was well tolerated in two phase 2, open-label, multicenter studies, including 1 in chronic myeloid leukemia (CML) patients with T315I mutation who had failed prior imatinib [Cortes et al, EHA 2011 Abstract 1012] and the second in CML patients with resistance or intolerance to ≥2 tyrosine kinase inhibitors (TKIs) [Cortes et al, ASH 2009 Abstract 861]. Methods This analysis included patients with CML-chronic phase (CML-CP) and CML-accelerated phase (CML-AP) from either of the two phase 2 studies mentioned above. All patients had previously been treated with ≥2 TKIs (including imatinib), to which they had shown resistance (eg, via point mutations) or intolerance. All patients received omacetaxine 1.25 mg/m2 subcutaneously twice daily for 14 consecutive days every 28 days for induction and for 7 days every 28 days as maintenance. The number of consecutive days of dosing could be adjusted, as clinically indicated. Recombinant growth factors could be administered if necessary. The primary endpoints for CML-CP patients were major cytogenetic response (MCyR) or complete hematologic response (CHR), and for CML-AP patients they were major hematologic response (MHR) or no evidence of leukemia (NEL). Patients were followed for survival after omacetaxine discontinuation. Results Of 122 patients, 81 had CML-CP (median age 59 years; range 26–83) and 41 had CML-AP (median age 63 years; range 23–83). All patients had previously received imatinib; 85% of CML-CP patients and 93% of CML-AP patients had received dasatinib, 59% and 63% nilotinib, and 14% and 17% other antineoplastic agents other than TKIs, respectively. Sixty-nine patients had shown resistance to ≥2 TKIs, 7 showed intolerance, and 5 resistance to 1 and intolerance to another. Previous treatments ended a median 1.3 (range 0.2–28) months prior to the study in CML-CP patients and a median 2.1 (range -4 to 33) months in CML-AP patients. Median on-study exposure to omacetaxine was 7.4 (range 0–39) months among CML-CP and 1.9 (0–30) months among CML-AP patients. In the CML-CP group, 16 patients (20%) achieved a MCyR (8 [10%] complete, 8 [10%] partial); the median duration of MCyR was 18 months (range 4 to ongoing). In addition, 4 (5%) had a minor cytogenetic response. Fifty-six (69%) achieved CHR with a median response duration of 12.2 months (range 8–26); median onset time was 0.7 (95% CI, 0.5–1.2) months. The median overall survival was 34 months. In the CML-AP group, 11 patients (27%) had a MHR, including 10 (24%) with CHR and 1 (2%) with NEL; the median duration of MHR was 9 months (range 4–14). In addition, 2 (5%) achieved a return to chronic phase and 3 (7%) had hematologic improvement. Three (7%) patients had a minor and 3 (7%) had a minimal cytogenetic response. The median overall survival was 16 months. The most common cause for discontinuation was disease progression: 5 (6%) of CML-CP and 5 (12%) of CML-AP patients. Among CML-CP and CML-AP patients, 12% and 24%, respectively, discontinued treatment due to adverse events (AEs) of all types, excluding disease progression. The most common serious AEs (>5% in either group) for CML-CP and CML-AP patients, respectively, were febrile neutropenia (7.4%, 12.2%), bone marrow failure (11.1%, 0), and thrombocytopenia (11.1%, 7.3%), excluding disease progression. The most common (>15% in both groups) grade 3/4 hematologic toxicities for CML-CP and CML-AP patients, respectively, were anemia (37%, 34%), neutropenia (47%, 22%), and thrombocytopenia (67%, 46%). The most frequent (≥5% in either group) nonhematologic grade 3/4 AEs in CML-CP and CML-AP patients, respectively, were general disorders (eg, aplasia, fatigue, generalized edema) (9%, 5%), infections (5%, 2%), metabolism and nutrition (0, 10%), and respiratory (1%, 5%). Conclusions Patients with CML who had failed previous treatment with ≥2 TKIs showed clinically meaningful response to omacetaxine therapy. Omacetaxine was well tolerated in this population. Disclosures: Cortes: ChemGenex: ChemGenex is now Cephalon, Inc., Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Deciphera: Research Funding. Off Label Use: Omacetaxine is an investigational drug. Nicolini:Bristol Myers Squibb France: Consultancy, Speakers Bureau; Norvartis Pharma France: Consultancy, Research Funding, Speakers Bureau. Wetzler:Novartis Pharmaceuticals Corporation: Consultancy; ChemGenex (Cephalon): Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding. Akard:Novartis: Speakers Bureau; Millenium: Speakers Bureau; Eisai: Speakers Bureau; Celgene: Speakers Bureau; BMS: Speakers Bureau. Craig:Cephalon, Inc.: Employment. Nanda:Cephalon, Inc.: Employment. Dial:Cephalon, Inc.: Employment. Benichou:Cephalon, Inc.: Employment. Cairati:Cephalon, Inc.: Employment. Baccarani:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer Oncology: Consultancy, Honoraria. Kennealey:Cephalon, Inc.: Employment. Kantarjian:ChemGenex: ChemGenex is now Cephalon, Inc., Employment.

Abstract 1462 Despite progress made in understanding the biology and risk-adapted treatment of AML, prognosis of older pts [age ≥60 years (y)] remains poor. We have shown that high expression of the BAALC gene bestows worse outcome in older CN-AML pts. Yet, the mechanism(s) by which BAALC affects response to therapy remain unknown. MicroRNAs (miRs) are small noncoding RNAs that hybridize to target mRNAs and inhibit their translation or promote degradation, resulting in downregulation of the corresponding proteins. Recently, miR-3151 was discovered embedded in intron 1 of BAALC. We thus hypothesized that miR-3151 might be co-expressed with BAALC and contributes alone or in concert with its host to poor prognostic impact in older CN-AML. MiR-3151 and BAALC expression were measured by quantitative real time (RT) PCR in pretreatment blood of 179 older CN-AML pts enrolled on Cancer and Leukemia Group B (CALGB) cytarabine/daunorubicin-based protocols. The expression values of miR-3151 and BAALC were normalized to the internal controls SNORD44 and RN18S1, respectively. MiR-3151 expression correlated only weakly with the expression of BAALC (Spearman correlation r= 0.3). For clinical and prognostic analyses pts were dichotomized into high and low miR-3151 and BAALC expressers at the median miR and gene transcript expression values and analyzed for other molecular prognosticators (FLT3 -ITD, FLT3 -TKD, CEBPA, IDH1, IDH2, NPM1, TET2, WT1 mutations and ERG expression). Gene (GEP) and microRNA (MEP) expression profiles associated with miR-3151 expression were derived, using Affymetrix U133 plus 2.0 & OSU CCC v4.0 arrays, respectively. At diagnosis, higher miR-3151 expression was associated with lower % blood blasts (P =.02), FAB types M4 and M5 (P =.05), and wild-type NPM1 (P Disclosures: No relevant conflicts of interest to declare.

Abstract 3096 Relapsed or refractory AML is difficult to treat even with allogeneic BMT. In an attempt to reduce leukemia burden and limit toxicities from multiple re-induction attempts, we initiated a treatment strategy to offer allogeneic BMT prior to count recovery, within 3 to 4 weeks after re-induction. Among 156 consecutive AML patients referred to our BMT program between 01/2003 and 03/2008, 84 (54%) patients did not receive an allogeneic BMT due to: death (n=25, 30%), patient refusal (n=13, 16%), disease progression (n=13, 16%), autologous BMT (n=11, 13%) and other (n= 22, 26%). Seventy-two (46%) patients received an allogeneic BMT: 21 (29%) patients were prospectively identified with very high risk features (primary induction failure (PIF) or refractory relapse (RR)) and received re-induction with the intent-to-induce hypoplasia and transplant prior to count recovery. The remaining 51 patients received re-induction with the goal of inducing complete remission (CR) prior to BMT (the intent-to-induce remission group). Patient and BMT-related characteristics, as well as BMT outcomes are summarized in Table 1. Patients were prospectively accrued to myeloablative or reduced intensity BMT protocols depending on their age, KPS, co-morbidities, disease risk and the degree of HLA match. The intent-to-induce hypoplasia group had a lower 2-year PFS and OS (reflecting their very high risk disease characteristics). In subgroup analysis in patients with PIF or RR, there was no difference in 2-year PFS (15% vs. 22%, p=0.39) or 2-year OS (20% vs. 33%, p=0.32) between the intent-to-induce hypoplasia and the intent-to-induce remission groups. The overall and day 100 TRM was similar in both groups of patients with PIF or RR. Using this prospective strategy of identifying patients with very high risk disease with the intent to transplant them during hypoplasia after re-induction therapy, 46% of all the referred AML patients were able to undergo allogeneic BMT. This is higher than other published series where < 30% of patients proceeded to transplant where the goal of re-induction was to induce and achieve CR prior to transplant. Our data show that it is safe to transplant advanced AML patients early (within 3 to 4 weeks) after re-induction with acceptable mortality in patients with very high risk AML who otherwise might not be transplant candidates.Table.Patient Characteristics and OutcomesFactorIntent-to-induce remission (N=51)Intent-to-induce hypoplasia (N=21)PN(%)N(%)Patient and Disease CharacteristicsAge at BMT18-4016 (31)4 (19)NS41-6028 (55)13 (62)61+7 (14)4 (19)KPS at BMT≥9023 (45)1 (5)0.1 Disclosures: Hahn: Novartis: stock.

Abstract 3542 Background: Achieving major molecular response (MMR) is an important milestone in chronic myeloid leukemia (CML) therapy. MMR has been defined as a 3-log reduction in BCR-ABL transcript levels from a standardized baseline (BL) established in the IRIS trial (Hughes TP, N Engl J Med. 2003). Standardization has been achieved through the development of an IS, which defines MMR as BCR-ABLIS = 0.1%. In contrast, the NCCN defines MMR as a 3-log reduction in BCR-ABL transcript levels but is indefinite on the definition of BL. Here, using reconstructed samples emulating CML patient BCR-ABL levels, the pairwise concordance of MMR determination was examined within and between 3 labs using the IS-standardized GeneXpert® (GX) system and 3 labs using laboratory-developed tests (LDTs). For comparative purposes, this analysis assumes BL is established at the time of diagnosis. Methods: 100 virtual patients (VPs) were emulated based on data from the REVEAL BCR-ABL Methods Comparison Study, in which 8 discrete levels of blinded K562 cell–spiked blood corresponding to BCR-ABLIS ratios ranging from ∼10% to ∼0.01% were analyzed by 3 labs using the IS-standardized GX system and 3 labs using non-IS LDTs. VP emulations were guided by actual patient outcomes in landmark analyses of 7- treatment response (Hughes TP, Blood. 2010). Treatment response profiles over an 18-month time horizon were modeled by assigning one of the 8 BCR-ABL levels ranging from approximately 10%-0.01% IS sampled in the REVEAL study to each of 4 virtual time points (eg, 3, 6, 12, and 18 months). BL levels were selected from quartiles representing pretreatment BCR- ABL ratios between 50–150%; results based on BL levels observed in the IRIS clinical trial will also be presented. 600 VP transcript profiles (VTPs) were then reconstructed using data from each of the 6 laboratories for all 100 VPs. The final 18-month time point in each VTP provided the BCR-ABL level against which the IS or NCCN objective criterion was applied to make MMR determinations. MMR concordance was evaluated by inspecting all possible inter-lab pairwise comparisons among the 100 VPs. Results: Pairwise concordance in MMR as determined by NCCN criterion among all 6 labs is shown in Fig 1A. MMR determinations among the 3 GX labs were concordant in 88% to 93% of VPs. In contrast, MMR determinations among the LDTs were concordant in 43% to 80% of VPs, and MMR determinations were concordant in 53% to 91% of VPs when compared between GX labs and LDTs. When MMR determination based on IS criterion for GX was considered, MMR concordance improved to 93% to 96% among the GX labs in contrast to 51% to 92% concordance observed between the GX and LDT sites (Fig 1B). It is noteworthy that Lab D results more closely approximated the IS than results from the other LDTs examined in the REVEAL study (data not shown). Although Lab D does not report results per the IS, it does report results relative to a median diagnostic BL, similar to the approach used in the IRIS trial. A healthcare system based on LDTs without any attempted IS standardization resulted in MMR concordance of only 43%. Potential sources of discordance among tests will be discussed in detail. Conclusions: These results illustrate that the NCCN criterion for MMR determination is not adequate for inter-lab comparisons of BCR-ABL transcript levels near the clinically important level of MMR. In contrast, standardization to the IS improves inter-lab concordance in MMR determination. Taken together, these results highlight the discrepancies that may result when comparing molecular responses between labs not standardized to the IS. As attainment of MMR is a critical milestone of CML therapy, errors in MMR determination may have an adverse impact on CML disease management. Disclosures: Reddy: Novartis: Research Funding, as Presenting Author, sponsorship to attend ASH. Höfling:Novartis: Employment. Manning:Novartis: Employment. Mignault:Novartis: Employment. Mullaney:Novartis: Employment. Ossa:Novartis: Employment. Stein:Novartis: Employment. Wang:Novartis: Employment. Yang:Novartis: Employment.

Abstract 2441 Background: Cancer testis or cancer germline (CG) antigens are a group of genes whose usual expression is limited to embryonic or germ cell tissues and which can also be aberrantly expressed in a variety of malignancies (Fratta E et al. Mol Oncol. 2011;5:164). The CG antigen genes MAGE-A1, XAGE-1, NY-ESO-1, and PRAME (among others) are epigenetically regulated. Most adult non-germline tissues demonstrate dense CpG island promoter methylation, with no gene expression. Endogenous expression of CG genes in epithelial malignancy can be immunogenic and a number of vaccine strategies are currently underway, however studies in hematologic malignancy have shown limited baseline expression, and thus there has been limited interest in them as a target in AML. Recently, several groups have shown that treatment with the hypomethylating agents, azacitidine(Aza) and decitabine(Dac) are sufficient to re-induce gene expression in myeloid cell lines, prompting interest in these genes as a therapeutic target in myeloid malignancy (Almstedt M et al. Leuk Res 2010;34:899). Re-expression of these genes has also been observed in MDS/AML patients treated with Dac, albeit at higher doses (15–270mg/m2) than those currently in standard use (Sigalotti L et al. Blood 2003;101:4644). Hypothesis: We hypothesized that conventional therapy with Aza(75mg/m2) and Dac(20mg/m2) induces CG promoter hypomethylation and gene expression, and that this may be sufficient to induce anti-CG antigen directed antibody responses. Methods: We obtained samples every 2–4 days from 2 AML patients during the first cycle of Aza or Dac treatment, and analyzed baseline and post-treatment samples for methylation (by pyrosequencing) and CG gene expression (by RT-PCR) of the above genes. We further tested serum from 10 patients for the presence of baseline and post treatment (following 1–10 cycles of Aza or Dac) anti-CG antibodies using an ELISA-based method. Results: Both AML patients treated with hypomethylating agents demonstrated a time dependent decrease in global methylation (by LINE-1 pyrosequencing), and CG antigen promoter methylation which nadired at day 6 post treatment. Baseline methylation of MAGE-A1 (90%), XAGE-1 (85%), NY-ESO-1 (92%) and PRAME (50–60%) was similar and high in both patients pre-treatment. Both Dac and Aza induced hypomethylation of all the CG genes studies, and this hypomethylation correlated with changes in LINE-1 methylation. Low level expression of MAGE-A1, XAGE-1, and PRAME were induced in a time dependent fashion following treatment with both Aza and Dac, however only limited re-expression of NY-ESO-1 was observed. ELISA testing for antibodies against a panel of 25 CG antigens (including MAGE-A1, XAGE-1 and NY-ESO-1) was performed on 10 patients with either MDS or AML. Following hypomethylating agents there was evidence for increased antibody levels, most notably against MAGE proteins, compared to baseline. Conclusion: Conventional hypomethylating agent therapy can induce CG gene expression, which may be sufficient to induce anti-CG antigen antibody responses. Augmentation of anti-tumor immune responses may be possible in myeloid malignancies using a combination of hypomethylating agents and CG antigen-directed vaccines. Disclosures: Odunsi: Ludwig Institute for Cancer Reserach: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 1395 We have previously shown that constitutive activation of the signal transducer and activator of transcription (STAT) 3 protein is associated with worse outcome in patients with acute myeloid leukemia (AML). To clarify the role of STAT3 signaling in AML leukemogenesis, STAT3 shRNAmirs were introduced into the AML cell line HEL that carries the Janus kinase 2 with V617F mutation leading to constitutive tyrosine phosphorylation and activation of STAT3 and STAT5. Clonal line of HEL cells expressing STAT3 shRNAmir or control shRNA were generated and then transduced with a lentiviral virus harboring a constitutively expressed firefly luciferase gene. Engraftment of these cells was evaluated in a disseminated xenograft non-obese diabetic severe immunocompromised mouse model. When tested in vitro, HEL STAT3 knock-down (HEL-STAT3KD) cells showed normal STAT5 expression and activity but had lower proliferation rates than control cells, PKC-bII and Pim1 were down-regulated, and activation of ERK1/2 was increased. Trans-signaling by hyper-IL-6 enhanced STAT3 phosphorylation proportional to STAT3 level. In vivo imaging of luciferase activity demonstrated significant delay in engraftment of STAT3KD cells compared with controls (Figure). To the best of our knowledge, this is the first demonstration that STAT3 knockdown delays leukemia cell engraftment, that STAT3 signaling is crucial for AML leukemogenesis and should promote STAT3-tailored therapeutic targeting.Figure:Down-regulating STAT3 results in delayed HEL engraftment. Panel 1A, Optical CCD images of HEL (non-silenced control in the upper panel and shRNAmir in the lower panel) xenografts in NOD/SCID mice from day 28 and day 45. Panel 1B, Graphs showing correlation of luciferase gene expression in HEL xenografts (p=0.0079, Man-Whitney test). The graphs were generated from repeated scanning of the mice on days 14, 28, 35, 45, 53 and 72. Individual mean values (photons/s/cm2/sr) calculated from the regions of interest were plotted.Figure:. Down-regulating STAT3 results in delayed HEL engraftment. Panel 1A, Optical CCD images of HEL (non-silenced control in the upper panel and shRNAmir in the lower panel) xenografts in NOD/SCID mice from day 28 and day 45. Panel 1B, Graphs showing correlation of luciferase gene expression in HEL xenografts (p=0.0079, Man-Whitney test). The graphs were generated from repeated scanning of the mice on days 14, 28, 35, 45, 53 and 72. Individual mean values (photons/s/cm2/sr) calculated from the regions of interest were plotted. Disclosures: No relevant conflicts of interest to declare.

Abstract 1857 This study is registered with ClinicalTrials.gov, number NCT00590187. Background: Sapacitabine is an orally administered nucleoside analogue with a unique ability to cause irreparable single-strand DNA breaks and induce G2 cell cycle arrest. We are conducting a multi-center, randomized phase 2 study to evaluate 3 dose regimens of this drug in older patients with MDS refractory to hypomethylating agents. The primary endpoint is 1-year survival. Secondary endpoints include the rate of CR, CRp, PR, CRi or hematological improvement (HI) and their corresponding durations. The study uses a selection design to identify a dose regimen which produces a better 1-year survival rate in the event that all three dose regimens are active. The planned sample size consists of 60 patients, 20 patients in each arm. Methods: Eligible patients must be ≥60 years with intermediate-2 or higher risk MDS previously treated with hypomethylating agents, ECOG 0–2, and adequate renal and hepatic functions. Patients were randomized 1:1:1 to receive sapacitabine every 3 –4 weeks at 200 mg b.i.d. × 7 days (Arm A), 300 mg b.i.d. × 7 days (Arm B), or 400 mg b.i.d. × 3 days per week × 2 weeks (Arm C). The number of cycles is not limited. Results: As of July 2010, 61 patients were treated with mean follow-up of 230 days. Median age was 73 years and 77% of patients were 70 or older. Baseline bone marrow contained < 5% blasts in 10 patients (16%), 5–10% blasts in 20 patients (33%), 11–20% blasts in 26 patients (43%) and 21–30% blasts in 5 patients (8%). The median number of cycles was 2 and 34% of patients received ≥ 4 cycles. To date, 14 patients have responded (2 CR and 12 major HIs) with response rates of: 24% (Arm A), 35% (Arm B) and 10% (Arm C). The two CRs occurred on Arm A. Median time to response is 2–3 cycles (range 1 to 6 cycles). Four deaths occurred within 30 days of randomization (7%), one of which was considered to be possibly related to sapacitabine. Common adverse events (all grades, regardless of causality) included fatigue, nausea, diarrhea, constipation, abdominal pain, anorexia, peripheral edema, dyspnea, alopecia, pneumonia, arthralgia, febrile neutropenia, anemia, neutropenia and thrombocytopenia, most of which were mild to moderate in intensity. Conclusions: Sapacitabine appears to be safe across all 3 dose regimens with the highest overall response rates observed on the 7-day dose regimens. Results for the primary endpoint of 1-year survival will be presented at the meeting. Disclosures: Wetzler: Cyclacel: Research Funding. Claxton:Cyclacel: Research Funding. Foran:Cyclacel: Research Funding. Goldberg:Cyclacel: Research Funding; Cyclacel: Research Funding. Chiao:Cyclacel: Employment, Equity Ownership. Kantarjian:Cyclacel: Research Funding.

Abstract 98 Mutations in the TET2 gene were recently identified in a variety of myeloid neoplasms including AML. However, the frequency and clinical relevance of TET2 mutations in CN-AML, the largest cytogenetic subgroup of adult AML, have not been well defined. We report here the frequency and spectrum of TET2 mutations, and their associations with clinical and molecular characteristics, treatment outcomes and genome-wide gene- and microRNA (miR)-expression signatures in a relatively large cohort of 427 patients (pts) with primary CN-AML. The pts, aged 18–83 years, were intensively treated on CALGB frontline protocols, and were analyzed centrally for TET2 mutations by PCR and direct sequencing, and for other prognostic gene mutations (FLT3 internal tandem duplications [ITD] and tyrosine kinase domain mutations, MLL partial tandem duplications, and mutations in NPM1, CEBPA, WT1 and IDH1&IDH2). If available, buccal swabs or remission marrow samples were used to determine TET2 germline status. Gene- and miR-expression profiles were derived using microarrays (Affymetrix HG-U133 plus 2.0 and OSUCCC custom miR array v4.0). At least 1 sequence variation in TET2 was found in 104 pts. Frameshift (n=59) and nonsense (n=34) variations were distributed throughout all coding exons, while missense changes (n=37) clustered mainly (28/37) in 2 evolutionarily conserved domains of TET2. The remaining missense variations in 9 pts were located outside the conserved domains, and analysis of available buccal swabs or remission samples showed that these sequence changes were present in the germline. Since it is unclear whether they represent innocent polymorphisms or disease-relevant mutations, these 9 pts were excluded from further analyses. TET2-mutated (TET2-mut) pts were older (P Disclosures: No relevant conflicts of interest to declare.

Abstract 2176 Recent advances in frontline therapy for newly diagnosed AML include increased dose of anthracycline during induction, multi-agent regimens, high dose cytarabine (HiDAC) as consolidation for core binding factor (CBF) patients (pts), and allogeneic transplantation (alloHCT) during 1st remission for poor risk pts. Prolonged low-dose cytotoxic maintenance therapy does not appear to improve clinical outcomes, but investigation of novel maintenance strategies remains appealing, affording pts an opportunity to receive new therapies without increasing the considerable toxicities of induction/ consolidation. The CALGB performed a phase II study of induction and risk-adapted consolidation, followed by one year of maintenance therapy with decitabine, for newly diagnosed AML pts Correlative investigations for CALGB 10503 are ongoing, including identification of novel prognostic markers, targets for treatment, and markers of minimal residual disease. Clearly, clinical investigation of maintenance therapy as part of a comprehensive treatment approach for frontline therapy of AML (inclusive of homogeneous induction and consolidation therapy) requires considerable up-front enrollment in order to reach maintenance accrual goals. Randomized maintenance studies will likely need intergroup participation for timely completion. However, we conclude that the benefits of up-front enrollment (relative to a post-consolidation enrollment strategy) outweigh the accrual burdens. Benefits include uniform pre-maintenance therapy and the potential for novel discovery from correlative studies. Study designs for future maintenance trials for AML in 1st CR must balance expediency with the more comprehensive approach employed in CALGB 10503. Disclosures: Blum: Celgene: Research Funding. Off Label Use: decitabine in AML. DeAngelo: Deminimus: Consultancy.

Abstract 2290 Introduction: Multiple TKI failure is a growing problem in a subset of CML patients. Treatment with a third TKI after two have failed often yields poor results. New treatment options are needed for this patient population. OM is a first-in-class cetaxine with demonstrated activity as a single agent in CML. It inhibits the production of short-lived oncoproteins (such as Mcl-1) involved in cancer cell survival via a mechanism independent of Bcr-Abl binding. Several studies have suggested that OM has a favorable toxicity profile when given to patients with CML via the subcutaneous route. We explored OM efficacy and safety in a subset of patients who had received therapy with multiple prior approved TKI. Methods: We analyzed a subset of adult CML-CP patients who had received two or more TKI (imatinib, dasatinib, nilotinib), from a combined interim dataset of two prospective Phase 2 studies (CML-202, for patients with the T315I kinase domain mutation, and CML-203, for patients with failure to ≥2 TKI) utilizing OM in the treatment adult patients with all phases of CML who had failed TKI. TKI failure was defined as no complete hematologic response (CHR) by 12 weeks (wk), no cytogenetic response by 6 months (mo), no major cytogenetic response (MCyR) by 12 mo, loss of CHR or MCyR, or progressive leukocytosis. The focus of this analysis was to assess the CHR and MCyR response rates as well as the overall safety of OM in these patients. Adverse events presented are Grade 3/4 events that occurred in ≥ 5% of patients (regardless of causality). Results: A total of 73 of the 93 CML-CP patients from these two studies had received two or more TKI prior to OM treatment. Median time from initial CML diagnosis to first dose of OM was 74.4 months. Mutations of any kind were seen in 48% of the patients, whereas 29% had no identified mutation and 23% had no available data on mutation status. Sixty (82%) of these 73 patients achieved or maintained (twelve patients were in CHR at study entry) a CHR and 17 (23%) achieved a MCyR (9 complete and 8 partial). The median duration of MCyR was 4.4+ months (range 1.2–14.1+). Median overall survival for patients treated with OM after failure of 2 or more TKI has not yet been reached [95% Confidence Interval (CI) 22.9, NA months] (Figure 1). Eleven patients had a treatment—emergent adverse event leading to death, and two deaths were probably related to study drug. Median progression-free survival was 11.1 months (95% CI 6.5, 13.8 months). Median follow-up time was 7.5 months for all patients with twenty-five patients remaining on study at the time of this data cut. A total of 36 of the 93 CP patients from these two studies had been treated with three or more TKI; 27 (75.0%) achieved or maintained a CHR and 7 (19.4%) achieved a MCyR (4 complete and 3 partial) on OM treatment. The median duration of MCyR in this group was 4.0+ months (range 1.2–11.5+) at the time of data cut-off. The primary Grade 3/4 adverse events in patients who received OM after failure of 2 or more TKI were hematologic, including thrombocytopenia (64%), neutropenia (48%) and anemia (40%) most commonly, followed by febrile neutropenia (12%), bone marrow failure (12%), pancytopenia (7%) and febrile bone marrow aplasia (6%). These events were dosing schedule dependent. Clinical sequelae were uncommon and managed with transient treatment interruptions and dose adjustments. Grade 3/4 non-hematologic adverse events were infrequent, with only fatigue (6%) occurring ≥5% of patients. Conclusions: OM, through a mechanism of action independent of Bcr-Abl, may offer a clinically viable option for patients who have progressed on multiple TKI treatment. Disclosures: Off Label Use: The drug is currently in development and has an NDA submitted for use in TKI resistant CML. Cram: ChemGenex: Employment, Equity Ownership. Humphriss: ChemGenex: Employment, Equity Ownership. Benichou: ChemGenex: Consultancy, Equity Ownership. Craig: ChemGenex: Employment, Equity Ownership, Executive Management Level.

Abstract 1041 Several studies suggest that subnormal vitamin D levels may be prognostic in a number of malignancies, including non Hodgkin lymphoma, prostate, breast, colon and lung cancer. However, no studies have evaluated the impact of subnormal vitamin D levels on treatment outcome in AML. Vitamin D levels were evaluated in 97 consecutive newly diagnosed similarly treated patients with AML (excluding AML M3). There were 50 (52%) males and 47 (48%) females with a median age of 60 years (range 19–91). The median white blood cell (WBC) count at diagnosis was 18×109/L (range 0.57–555.23). Karyotype was favorable in 11%, intermediate in 50%, adverse in 23%, of unknown significance in 11% and not available in 5%. A total of 74 patients (76%) had de novo AML. Albumin levels were subnormal ( Consolidation therapy consisted of either high-dose cytarabine (47%), ADE (5+2+2) (15%), allogeneic (8%) transplantation, or other/no further treatment (30%). The median follow-up was 8.1(range, Disclosures: No relevant conflicts of interest to declare.

Interleukin-1 (IL-1) has recently been reported to play an important role in acute myelogenous leukemia (AML) blast proliferation. We therefore investigated the effect of soluble IL-1 receptors (sIL-1R) and IL-1 receptor antagonist (IL-1RA) on the growth of AML bone marrow blast progenitors from 25 patients. In the AML blast colony culture assay, sIL-1R and IL-1RA inhibited blast colony-forming cell replication in a dose-dependent fashion, at concentrations ranging from 10 to 500 ng/mL (sIL-1R) and 10 to 1,000 ng/mL (IL-1RA), and their inhibitory effect was partially reversed by IL-1 beta. A similar inhibitory effect was also noted with the use of anti-IL-1 beta neutralizing antibodies. When AML blast progenitors were grown either in the presence of fetal calf serum (FCS) alone or with one of the following: phytohemagglutinin leukocyte-conditioned medium (PHA-LCM), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, interleukin-3 (IL-3), or stem cell factor (SCF), addition of 100 ng/mL sIL-1R or IL-1RA inhibited blast colony formation by 3% to 96% and 2% to 97%, respectively. In sharp contrast, neither of these IL-1- inhibitory molecules significantly inhibited proliferation of normal marrow hematopoietic progenitors. Lysates of 2 x 10(7) low-density AML marrow cells were tested for intrinsic IL-1 beta content using an enzyme-linked immunoadsorbant assay (ELISA). Samples from five of six patients showed high concentrations (ranging from 501 to 2,041 pg), whereas 2 x 10(7) cells from two normal marrow aspirates yielded 54.6 pg of IL-1 beta. AML blast colony-forming cells from all six patients were inhibited by sIL-1R, IL-1RA, or both. Incubation of nine samples of AML low-density cells with either sIL-1R or IL-1RA reduced GM-CSF concentrations in cell lysates, and supernatants from nine (P less than .01) and six samples (P less than .037), respectively, and G-CSF concentration in lysates from six of nine samples (P less than .03), and in supernatants from five of six samples (P less than .06) when studied by ELISAs. Our data implicate IL-1 in AML blast proliferation and suggest the potential benefits of using IL-1-inhibitory molecules in future therapies for AML.