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Abstract Introduction: High-dose cytarabine consolidation chemotherapy for acute myeloid leukemia (AML) induces profound transient myelosuppression. Thrombocytopenia is a limiting factor in chemotherapy administration for maintaining dose intensity and schedule as thrombocytopenic-related hemorrhage may contribute to significant morbidity and mortality. Therapeutic and prophylactic platelet (plt) transfusions remain the treatment of choice but the benefits typically last only 3-7 days, are expensive, time consuming, and may induce alloimmunization and transmit infection. PrE0901 was a phase I multi-center study to evaluate the safety and toxicity of eltrombopag, an orally bioavailable thrombopoietin (TPO) receptor agonist, for plt recovery in AML patients (pts) receiving consolidation therapy. Methods: The primary objectives were to determine the safety, tolerability, and optimal dosing of eltrombopag while describing the kinetics of plt recovery in AML pts receiving intensive consolidation. Plt recovery was defined as a sustained plt count of ≥ 20 x 109/L following the nadir with no plt transfusions in prior 7 days or a continued upward trend in plt count for at least 2 successive measurements despite no plt transfusions in 48 hours. We used a standard 3+3 design utilizing a novel dose escalation/de-escalation strategy for pts in either first or second complete remission, ≥18 and ≤70 years of age, who had an ECOG performance status of 0-2. Cytarabine was administered at 3 g/m2 (1.5 g/m2 for pts >60 years) IV over 3 hours twice daily on days 1,3,5. Eltrombopag at assigned dose was administered with a first cycle of cytarabine only. Planned eltrombopag dose levels -2, -1, 1, 2, 3, 4, and 5 were 50, 100, 150, 150, 150, 200, and 300 mg, respectively (Table 1). Eltrombopag was to start on days X, X, 3, -1, -5, -5, and -5 relative to day of cytarabine start and levels -2, -1, 1, 2, 3, 4, and 5, respectively, with day X being variable depending on timing of observed dose limiting toxicities (DLTs). The eltrombopag was continued until plt recovery or for up to 35 consecutive days, whichever occurred first. An exploratory objective was to determine if eltrombopag had an effect on TPO and EPO blood concentrations in this setting. Results: Accrual began on July 31, 2012 and the trial closed on January 8, 2016 upon recommendation after an independent Data and Monitoring Committee reviewed a separate trial of eltrombopag therapy in MDS which demonstrated futility. 104 pts were screened. 54 declined participation and 35 were deemed medically ineligible. A total of 15 eligible pts were enrolled; 14 were treated on study with one pt being withdrawn prior to starting treatment due to infection. Three pts were treated at each of dose levels 1-4 and two were treated on dose level 5. No DLTs were observed. Median time to plt recovery of all pts treated on study was 22.5 (range 16-43) days. Pts in cohort 1 dosed with eltrombopag 150 mg daily starting on day 3 of consolidation demonstrated the fastest plt recovery (median =19 days; range 19, 19, 19) compared to cohort 2 who received 150 mg daily starting on day -1 of consolidation and recovered the slowest (median 23 days; range 16, 23, 43). Dose escalation in terms of starting eltrombopag further in advance of chemotherapy exposure as within cohort 3 (150 mg starting day -5) was associated with a median plt recovery of 27 (range 22, 27, 27) days. Eltrombopag dose intensification with cohort 4 at 200 mg daily and cohort 5 at 300 mg daily was associated with median plt recoveries of 24 (range 18, 24, 26) and 23 (range 19, 27) days, respectively. Kinetics of plt recovery for all cohorts are represented in Figure 1 and duration of eltrombopag exposure for each individual patient is represented in Table 2. Conclusions: Endogenous cytokine (TPO and EPO) levels varied inversely with plt and hematocrit values and did not appear to be affected by eltrombopag dose / schedule (details to be provided at ASH Annual Mtg). Unusually high screen fail numbers warrant examination to guide future trial design in the post-remission setting. The addition of eltrombopag to high-dose cytarabine consolidation therapy was well-tolerated and no DLTs were observed. The results for cohort 1 [eltrombopag 150 mg daily starting on day 3 of consolidation] demonstrated the fastest plt recovery. Further investigation is needed to define the optimal role, dose, and schedule of eltrombopag in the treatment of chemotherapy associated myelosuppression. Disclosures Strickland: Alexion Pharmaceuticals: Consultancy; Ambit: Consultancy; Baxalta: Consultancy; Boehringer Ingelheim: Consultancy, Research Funding; CTI Biopharma: Consultancy; Daiichi Sankyo: Consultancy; Sunesis Pharmaceuticals: Consultancy, Research Funding; Abbvie: Research Funding; Astellas Pharma: Research Funding; Celator: Research Funding; Cyclacel: Research Funding; GlaxoSmithKline: Research Funding; Karyopharm Therapeutica: Research Funding; Sanofi: Research Funding.

Abstract Background: Persistence of minimal residual disease (MRD) after induction, consolidation and salvage therapies is an independent poor prognostic marker in AML. The bone marrow (BM) microenvironment is a protective niche that promotes chemotherapy resistance of leukemia cells. This residual subset of chemotherapy-resistant cells present during remission eventually contributes to the leukemia relapse/persistence. Adhesion of AML cells to BM stroma is dependent on the CXCR4 receptor binding to its ligand CXCL12. High levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis will dislodge AML cells from the protective microenvironment and, when combined with chemotherapy, may improve the response to therapy. BL-8040 (BKT140) is a short peptide which inhibits the binding of CXCR4 to CXCL12, resulting in mobilization of leukemic blasts from the BM to the peripheral blood (PB). BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy and as a single agent induces differentiation and apoptosis of leukemia cells in preclinical models. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of relapsed/refractory AML patients was recently completed (NCT01838395). Here we report results from correlative studies done on samples from patients who participated in this trial. Objective: To assess BL-8040 single agent anti leukemia effects in relapsed/refractory AML. Method: Patients received a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5g/m2 for patients ≥60; 3g/m2 for patients <60) per day on days 3-7. Six dose levels of BL-8040 (0.5 - 2.0 mg/kg) were tested. PD parameters such as mobilization of leukemic blasts and induction of differentiation and apoptosis were assessed after monotherapy with BL-8040 using frequent PB sampling and BM aspirates at baseline and on day 3 prior to Ara-C administration. Results: 45 patients (including 3 receiving compassionate use BL-8040) with a median age of 61 yrs were treated. The majority of patients had poor risk disease with 24% secondary AML and 17% with prior allogeneic stem-cell transplantation. Most patients were heavily pretreated with 19% relapsing after a short first remission (CR1 ≤12 months), 17% had ≥2 relapses and 45% refractory to 1-2 inductions. The CR+CRi rate was 38% in subjects receiving BL-8040 dose ≥1.0 mg/kg (n=39). Ongoing follow-up (FU) of responding patients (expansion phase; N=19) shows median EFS of 9.3 months (range 4.3-12.8 months). At the time of this analysis 10/19 patients are alive with a FU range of 0.96 - 12.8 months. FACS analysis revealed that BL-8040 monotherapy triggered an average 4.3-fold increase of immature AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+) in PB (i.e., mobilization). Response to treatment was associated with more robust mobilization of AML blasts following BL-8040 monotherapy (responders 9.5 vs non-responders 1.7 fold median). Preferential mobilization of AML blasts over normal cells (4.7 fold vs. 1.4 fold, respectively) was confirmed by FISH analysis in a subset of patients with informative cytogenetic abnormalities. Induction of granulocytic differentiation of immature leukemia progenitors (2.5 fold) in the BM following BL-8040 monotherapy was noted. Finally, induction of AML blasts apoptosis (40% increase) by BL-8040 alone was evident by FACS in day 3 (pre-araC) bone marrow biopsies. Conclusions: The data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 is safe and well tolerated and when given in combination with Ara-C improves the response rate achieved historically with Ara-C alone. In addition, treatment with BL-8040 as a single agent rapidly and efficiently induces mobilization, differentiation and cell death of AML blasts. This selective effect on chemotherapy-resistant cells may be translated into reduction of residual disease arguing for incorporation into front-line trials and such studies are ongoing. Disclosures Foran: Millennium Pharmaceuticals, Inc.: Research Funding; medscape: Honoraria; karyopharm: Honoraria; pfizer: Honoraria; novartis: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding. DiPersio:Incyte Corporation: Research Funding. Peled:Biokine Therapeutics: Employment. Abraham:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Vainstein:BioLineRx Ltd.: Employment. Oberkovitz:BioLineRx Ltd.: Employment. Aharon:BioLineRx Ltd.: Employment. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Abstract Abstract 524 The International UKALLXII/E2993 trial enrolled 2091 pts with newly diagnosed adult ALL between 1993 and 2006. Patients received 2 months of combination induction chemotherapy followed by an intensification course of high dose methotrexate and L-asparaginase after which pts were randomized to autologous hematopoietic stem cell transplant (HSCT) or consolidation and maintenance chemotherapy. Patients <55 years of age with a matched sibling donor were assigned myeloablative allogeneic HSCT (Goldstone, et al, Blood 2008; 111:1827). Here we report a detailed analysis of 1,229 pts from this trial with B-ALL, excluding Philadelphia-chromosome-positive patients. The median age was 30 years (range 14–65) and 59% were male. The median WBC was 9.2×109/L (range 0.1–930) and 73% of pts had a WBC <30 × 109/L. CNS involvement was present in 4% of pts at diagnosis. Comprehensive centralized immunophenotyping was performed in 372 of the 409 (91%) ECOG pts enrolled and initial analysis demonstrated pro-B/pre-pre-B in 17%, early pre-B in 52%, pre-B in 14%, either early pre-B or pre-B in 6%, mature B in 2% and B-lineage, not otherwise specified, in 9%. Outcomes were similar between patients with immunophenotyping data and those without. The complete remission (CR) rate was 93% and there were 4% induction deaths. The overall survival (OS), event-free survival (EFS), relapse risk (RR) and non-relapse mortality (NRM) at 5 years for the overall group is shown in the table. There were no significant differences in CR rate, OS, EFS, RR or NRM for the immunophenotypic subgroups. There was better OS and EFS with less relapse and similar risk of NRM for pts randomized to chemotherapy as compared with autologous HSCT (Figure 1). In the matched sibling donor-no donor comparison, OS and EFS were improved, RR was less and NRM was higher in the donor group (Figure 2). Cytogenetic analysis confirmed that patients with t(4;11) (n=77, 8% total), t(8;14) (n=19, 2% total), a complex karyotype (≥5 abnormalities in the absence of an established chromosomal abnormality) (n=38, 5% total), or low hypoploidy/near triploidy (n=41, 5% total) had an inferior OS and patients with high hyperdiploidy had a superior OS when compared to other patients. In conclusion, this is the largest series of B-ALL in uniformly treated adults ever reported and confirms the superior outcome of patients randomized to chemotherapy as compared with autologous HSCT and shows that patients with a matched sibling donor have an improved survival over patients without a donor. No difference in CR rates or outcome was noted for patients with different immunophenotypic subtypes of B-ALL. Future improvements in outcome of pts with B-ALL will rely on the addition of new agents such as B cell monoclonal antibodies and improvements in allogeneic HCT.Table.Outcomes at 5 years of follow-upnCR rate (%)OSEFSRRNRMOverall12299342375021Pro-B/pre-pre-B639236345421Early pre-B/pre-B2689340345421Age ≤ 35 years746 (61%)9653454516Chi square p<0.0001<0.0001<0.0001<0.0001<0.0001Age >35 years483 (39%)8932265930Randomized to autologous HSCT15735316610Log rank p0.030.010.01NSRandomized to chemotherapy1694742558Matched sib donor34452483032Chi square p0.050.03<0.001<0.001No Donor47445415510Values are % except n and p values. CR=complete remission, OS=overall survival, EFS=event-free survival, RR=relapse risk, NRM=non-relapse mortality NS=not significant. The p values represent the difference between age groups for the first comparison, randomized to autologous HSCT versus chemotherapy for the second comparison and between matched sibling donor and no donor for the third comparison. Overall survival by randomized allocation at 10 years Overall survival by donor availability at 10 years Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 579 Background: In AML, age and cytogenetics remain among the major prognostic factors. Age above 55 years and a complex karyotype (CK; ≥ 3 abnormal clones) or the presence of monosomy 7 (M7) are universally associated with unfavorable prognosis. Recent data from HOVON-SAKK (J Clin Onc 26: 4791) suggest that in patients under the age of 60 years, subgroups with poor risk versus very poor risk can be delineated based on the presence of a MK. MK was defined as either the presence of at least two autosomal monosomies or one monosomy plus at least one structural abnormality. Limited data exists regarding the impact of MK in AML patients over the age of 55 years. Methods: Data from 935 AML patients above age 55 enrolled on three prospective ECOG therapeutic clinical trials (E1490, E3993, E3999) between 1990 and 2005 were retrospectively reviewed to identify those with MK with single and/or multiple monosomies, MK in the presence or absence of a CK, CK with/without MK, MK with/without M7, and normal karyotype (CN). CK was defined as ≥ 3 clonal abnormalities for the purposes of this study. Complete remission (CR), disease-free survival (DFS), and overall survival (OS) were assessed. Results: The median age of evaluable patients was 68 years (range 56 – 93) with a male/female ratio of 391/299. Cytogenetic data were evaluable in 690 (74%) subjects. Clonal cytogenetic abnormalities occurred in 399 (58%). Core-binding factor (CBF) abnormalities accounted for 16/399 (4%) whereas CK was detected in 181/399 (45%). MK was present in 172 (25%) evaluable cases, predominantly in the setting of CK (140/172, 81%). Overall CR was 44%. CR was achieved in 49% MK−, 29% MK+, 31% CK+, and 49% of CK−. Data for the combinations of MK and CK are presented in Table 1. Median follow-up for survivors was 5.0 years. OS at 4-years was 9% for all evaluable patients and 4y-DFS was 12% in those achieving CR. Median OS of MK+/CK+ (0.3y) was significantly worse (p<0.001) than MK+/CK−(0.6y), MK−/CK+ (0.8y), and MK−/CK−(0.9y). Median DFS of MK+/CK+ (0.3y) was also significantly poorer (p<0.001) than MK+/CK− (0.9y), MK−/CK+ (0.8y), and MK−/CK− (0.9y). M7 was present in 82 (13%) enrolled patients with 98% (80/82) of M7 occurring within a MK. CR was achieved in 31% of MK+/M7+ and 27% of MK+/M7−. Neither median OS nor DFS were significantly impacted by M7 status as noted in Table 2. Conclusions: AML patients above the age of 55 years have an unfavorable prognosis with the majority of patients harboring adverse clonal cytogenetic abnormalities and failing to achieve a CR. Fewer than 10% survive 4-years from diagnosis. Results from the ECOG experience demonstrate the prognostic predictive value of MK in older AML patients. In contrast to the data presented by the HOVAN-SAKK group, the prognostic value of MK in patients over 55 years is limited to those with MK in combination with CK. These results also demonstrate the failure of monosomy 7 to provide prognostic value in the setting of MK. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: The bone marrow (BM) niche protects acute myeloid leukemia (AML) cells from chemotherapy. BM homing of AML cells is dependent on CXCR4 and its ligand CXCL12; high levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis with a potent CXCR4 antagonist will disrupt the interaction of the leukemic blasts with the BM and augment the anti-leukemic effect of chemotherapy. BL-8040 (BKT140) is a short synthetic peptide which inhibits the binding of CXCR4 to CXCL12, resulting in the mobilization of leukemic blasts from the bone marrow to the peripheral blood. BL-8040 also inhibits CXCR4/CXCL12 mediated pro-survival ERK/Akt signaling and alters balance of pro/anti-apoptotic Bcl-2 proteins. BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy, and, in contrast to other CXCR4 inhibitors, as a single agent induces apoptosis of leukemia cells ex-vivo in clinical samples. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of adult relapsed/refractory AML patients is currently ongoing (NCT01838395). Objective: To assess the safety, efficacy and pharmacodynamics/pharmacokinetic parameters of BL-8040 in combination with Ara-C in first or second relapse and refractory AML patients. Method: The study includes a dose escalation phase (3+3 design) followed by an expansion phase. Each patient receives a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5 g/m2 for patients ≥60; 3 g/m2 for patients <60) on days 3-7. Five dose levels of BL-8040 (0.5, 0.75, 1, 1.25 and 1.5 mg/kg) have been tested. Extensive pharmacodynamic parameters such as CXCR4 expression on the leukemic cells, receptor occupancy, mobilization of leukemic cells and induction of apoptosis are being assessed after monotherapy with BL-8040 using peripheral blood sampling (PB) and BM aspirates at baseline and on day 3 prior to Ara-C administration. Clinical response to treatment is evaluated by BM biopsy on day 30 (-/+ 2). Results: Twenty two patients (median age, 61 y; range, 43-74 y) with relapsed/refractory AML were treated during the escalation phase. BL-8040 was escalated up to 1.5 mg/kg (recommended phase 2 dose) without reaching the MTD. Combination of BL-8040 with Ara-C was safe and well tolerated at all doses tested. Only one SAE (Sweet Syndrome) was reported as possibly related to study drug. The primary treatment related AEs were transient injection site and systemic reactions. The composite complete remission including complete remission (CR) and complete remission with incomplete blood count recovery (CRi) rate was 38% in subjects receiving BL-8040 doses of 1 mg/kg and higher (n=16). Two days of BL-8040 monotherapy triggered an average 40.2-fold (range 1.0-350.9) mobilization of immature AML progenitors (CD45+Low/CD34+/CD117+/HLA-DR+) from the BM to the PB. Moreover, a significant apoptotic effect on the immature leukemia progenitors in the BM following 2 days of BL-8040 monotherapy was noted. Flow cytometric evidence of apoptosis by BL-8040 was corroborated by an increase in cleaved caspase 3 positive blasts in day 3 bone marrow biopsies. In addition, BL-8040 treatment resulted in a median decrease of 57.7% (range, -10.4% to -96.2%) in the number of BM Leukemia progenitor cells (CD45+Low/CD34+/CD117+/HLA-DR+ out of total CD45+/CD34+ normal/progenitor cells) compared with the baseline biopsy. This decrease was accompanied with a 3.1 fold increase in granulocytes in the day 3 BM biopsy supporting differentiation effect. Finally, long receptor occupancy (up to 24 hours post dosing) was confirmed by FACS analysis measuring surface CXCR4 staining. Pharmacokinetics analysis confirmed dose incremental BL-8040 exposure with a short plasma half-life. Conclusions: The current data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 has potent anti leukemic activity and in combination with Ara-C may improve the response typically achieved in this advanced AML population. Mechanistically, BL-8040 was able to induce apoptosis of leukemic progenitor cells, overcoming the stroma protection, inducing mobilization and differentiation. Disclosures Rowe: BioLineRx Ltd.: Consultancy; Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:Novartis: Research Funding. Altman:Seattle Genetics: Consultancy; Novartis: Consultancy; Spectrum: Consultancy; Astellas: Consultancy; Ariad: Consultancy; BMS: Consultancy. Peled:Biokine Ltd.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BiolineRX: Consultancy, Research Funding. Pereg:BioLineRx: Employment. Vainstein:BioLineRx: Employment. Aharon:BioLineRx: Employment. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Cortes:Teva: Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; BerGenBio AS: Research Funding; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

Abstract Background: Midostaurin (M) is a multi-targeted small molecule FLT3 inhibitor which has single agent activity in both internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutant FLT3 AML. The objective of this global rand phase III trial was to determine if the addition of M to ind and consol therapy followed by one year of maint would improve overall survival (OS) compared to standard chemotherapy in younger adults with activating FLT3 muts. Methods: Between May 2008 and October 2011, 3279 previously untreated AML pts age 18-60 (exclusive of acute promyelocytic leukemia) in 225 sites/17 countries were screened for FLT3 muts at one of 7 academic labs (subject to extensive assay cross-validation). Hydroxyurea was allowed for up to 5 d prior to beginning ind therapy while awaiting results of mut testing. Pts were rand for the duration of therapy to M or P stratified by FLT3 mut subtype (TKD v ITD high allelic mut fraction (>0.7) vs low mut fraction (0.05-0.7). Ind therapy consisted of D 60 mg/m2 IV d1-3 and C 200 mg/m2 d1-7 CIV plus M or P (50 mg po bid, d 8-22). Re-treatment with a second blinded course was allowed if residual AML was noted on a d 21 marrow exam. Pts achieving complete remission (CR) received 4 cycles of C 3g/m2 over 3h q 12h on days 1, 3, and 5 plus M or P (50 mg po bid, d 8-22) followed by a year of maint therapy with M or P (50 mg po bid). Transplantation (SCT) was allowed. With a sample size of 717 pts, the trial was powered to detect an improvement from 16.3 (P) to 20.9 (M) months in median OS (HR = 0.78) using a one-sided alpha of 0.025 and power of 84%. The final analysis was to occur after 509 deaths, but given the slow rate of events (359 deaths by April 2015), the trial was amended to change the timing of the OS analysis, and promote event free survival (EFS, defined as the earliest of death, relapse, or no CR within 61 d of the start of ind) as a key secondary endpoint. The critical value for this primary analysis is set at 0.02286 (1-sided) accounting for the alpha spent at the interim analysis (0.5%). Support: U10CA180821, U10CA180882, CA31946, Novartis Results: 717 pts (341 FLT3 ITD-Low, 214 FLT3 ITD-High; 162 FLT3 TKD) were rand to either M (n=360) or P (n=357). There were no significant differences between the arms in age (median, 48y), race, FLT3 subtype, or baseline CBC except for gender (M, 48.2% male; P, 40.6% male; p=.04). All pts are off active treatment, with a median follow-up of 57 months for surviving pts. No statistically significant differences were observed in the overall rate of grade 3 or higher hematologic or non-hematologic adverse events (AEs) between M and P (regardless of attribution). A total of 37 grade 5 AEs were reported (M, 5.3%; P, 5.0%; p=1.0). No differences in treatment-related grade 5 AEs were observed (M, 3.1%; P, 2.5%; p=0.82). CR rate is 59% (M) and 54% (P) (p=0.18). The HRs comparing M to P for OS is 0.77 (one-sided p = 0.007; Figure 1), and for EFS is 0.80 (one-sided p = 0.004; Figure 2). 402/717 (57%) pts received an allogeneic SCT (M, 58%; P, 54%) at any time; 177/717 (25%) in CR1 (M, 27%; P, 22%). Median time to allogeneic SCT was similar on each arm (M, 5.0 months; P, 4.6; p=0.23). Secondary analyses for OS and EFS censoring at the time of SCT provided similar results (Table). The benefit of M was consistent across all FLT3 subgroups for both EFS and OS (Figure 3). Conclusions: The C10603 trial demonstrated that a prospective trial in a pre-therapy genetically defined subgroup of AML pts was feasible and that the addition of the multi-kinase inhibitor M to standard chemotherapy and for one year of maint therapy significantly improved EFS and OS (in both uncensored and censored for transplant analyses) in pts whose blasts had a TKD or ITD (low or high FLT3 mut burden). These findings may lead to improved outcomes through the use of M as a component of therapy in younger adults with mutant FLT3 AML. Table.ArmMedian, mos (95% CI)p-value 15-year Event rate% (95% CI)HR2(95% CI)OSM74.7 (31.5, * )0.00750.8 (45.4-55.9)0.77 (0.63, 0.95)P26.0 (18.5, 46.5)43.1 (37.6-48.4)OS, SCT censoredM* (*,*)0.04762.6 (54.6-69.7)0.77 (0.56,1.05)P* (36.9, *)54.9 (46.2-62.8)EFSM8.0 (5.3, 10.6)0.004426.7 (22.2-31.5)0.80 (0.67, 0.95)P3.0 (1.9, 5.8)19.1 (15.1-23.6)EFS, SCT censoredM8.2 (5.5, 10.7)0.02524.2 (18.9-29.8)0.84 (0.70, 1.0020)P3.0 (1.9, 5.8)21.8 (16.8-27.3)1Stratified on FLT3 subtype; one-sided, log-rank p-value.2Cox model stratified on FLT3 subtype.*= not attained Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Stone: Celgene: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Novartis: Research Funding; Amgen: Consultancy; Agios: Consultancy; Roche/Genetech: Consultancy; Merck: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Celator: Consultancy; Juno: Consultancy; Abbvie: Consultancy; Karyopharm: Consultancy. Off Label Use: midostaurin- FLT 3 inhibitor. Thiede:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AgenDix GmBH: Equity Ownership. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Medeiros:Celgene: Honoraria, Research Funding; Agios Pharmaceuticals: Honoraria. Schlenk:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Teva: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding. Larson:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy, Research Funding; Pfizer: Consultancy.

Abstract Abstract 2739 Background: Internal tandem duplication mutations of the FMS-like tyrosine kinase 3 receptor (FLT3-ITD) are among the most frequent mutations in de novo acute myeloblastic leukemia (AML) and result in constitutive activation of the receptor tyrosine kinase. Based on data showing that these mutations negatively affect outcome in AML, FLT3 receptor kinase activity is currently being targeted in the clinic. To date, only FLT3 receptor mutant AML patients have been selected for trials involving FLT3 receptor kinase inhibitors, with surprisingly variable clinical responses. Downstream targets of FLT3 receptor activation, whether by receptor mutation or FLT3 ligand binding, involve Stat5, PI3-kinase/Akt and the Ras/Raf/Erk kinase signal transduction pathways which ultimately affect cell survival and proliferation. Functional characterization of those signaling pathways in mutated versus non-mutated FLT3 receptor (FLT3 -WT) myeloblasts has significant potential to reveal heterogeneity among these genetically defined subgroups and to predict for response to kinase inhibition, independent of FLT3 receptor mutation status. Methods: Modulated single cell network profiling (SCNP) was used to evaluate the activation state of intracellular signaling molecules (i.e. nodes), including phosphorylated (p)-Akt, p-Erk, p-S6, p-Stat5 and cleaved-PARP, at baseline and after treatment with specific modulators [including cytokines (such as IL-27) growth factors (such as FLT3 ligand) and drugs (such as cytosine arabinoside)] in 7 healthy bone marrow mononuclear blasts (BMMb) and leukemic myeloblasts, characterized for FLT3 receptor mutation status, from 44 AML patients (38 FLT-WT and 6 FLT3-ITD), aged >60 years (ECOG trial E3999). A total of 64 node-metrics were analyzed. Results: Signaling profiles differed significantly in FLT3-ITD vs. FLT3-WT AML, and in FLT3-WT vs. BMMb (shown in Figure 1 for a representative node, FLT3 ligand induced p-S6). Specifically, compared to BMMb, FLT3-ITD blasts uniformly showed increased basal p-Stat5 levels, decreased FLT3 ligand-induced activation of P13K and Raf/Ras/Erk pathways, minimal IL-27 induced activation of the Jak/Stat pathway, and higher apoptotic responses to DNA-damaging agents. Two AML samples harboring a low FLT3-ITD mutant burden, however, exhibited a signaling pattern similar to FLT3-WT AML samples. By contrast, FLT3-WT samples displayed heterogeneous signaling profiles, overlapping both with those of FLT3-ITD and BMMb samples, suggesting that a fraction of FLT3-WT AML samples exhibit FLT3 receptor pathway deregulation even without FLT3-ITD. Conclusions This study showed that SCNP, which provides a detailed view of intracellular signaling networks at the single-cell level, subclassified patients with AML beyond their molecularly determined FLT3 mutation status. In particular, a fraction of FLT3-WT AML samples signaled as if containing a FLT3 receptor length mutation while FLT3-ITD samples with low mutational load signaled like FLT3-WT AML samples. The clinical relevance of this observation, both for disease prognosis and response to kinase inhibitors, will be revealed only if AML patients are accrued to kinase inhibition trials irrespective of FLT3 receptor mutation status. The wide range of signaling responses observed in FLT3-WT AML suggests that disease across FLT3-WT patients is heterogeneous, likely promoted through distinct mutations and alterations, giving rise to distinct signaling profiles in individual patients Our data also provide evidence for the co-existence of differentially signaling blast populations in individual patients. The potential impact of signaling heterogeneity on clinical response needs to be assessed and may require an individualized combination of treatment modalities. Disclosure: Rosen: Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership.

Abstract Abstract 3405 Poster Board III-293 Background: Autologous hematopoietic cell transplantation (AutoHCT) as consolidation has been an alternative to standard intensive chemotherapy and/or allogeneic HCT for younger patients with acute myeloid leukemia (AML) in first complete remission (CR). However concerns about toxicity and true benefits in long-term outcome have hampered its use. Methods: We present the follow-up of younger patients with de novo AML treated on ECOG study E1900, on an intention-to-treat (ITT) and per protocol (PP) analysis. DFS and OS were measured from the time of registration to consolidation therapy. Results: 657 patients younger than 61 years were initially randomized to standard-dose cytarabine and either standard- or high-dose daunorubicin in a 7+3 approach. Of the 352 patients who entered consolidation phase, 307 patients, 68 with favorable-risk cytogenetics (FRC), 128 with intermediate-risk cytogenetics (IRC), 90 with indeterminate cytogenetics (IDT) and 21 with unfavorable-risk cytogenetics (URC), were allocated to AutoHCT. Patients in these cohorts were treated with high-dose cytarabine 3g/m2 x 6 doses in 5 days, for two cycles. Peripheral blood cells were collected after the second cycle. Initially patients were randomized to receive gemtuzumab ozogamicin (GO) or not; however, no benefit was derived from GO therapy and the randomization was closed. 156 patients did not receive the planned AutoHCT due to disease progression, alternative therapy, failure to collect, insurance denial, toxicity and other reasons. 151 patients (33 FRC, 64 IRC, 47 IDT and 7 URC) were conditioned with IV busulfan 0.8 mg/kg daily x 16 doses (without dose adjustment) and cyclophosphamide 60 mg/kg x 2 days. Autologous cells were infused on day 0 and patients were supported with sargramostim until ANC recovery > 500/μL. Veno-occlusive disease occurred in three patients. Treatment-related mortality (TRM) of AutoHCT was 1.3% (2/151) at 30 days. Median follow-up was 31.8 months. In the ITT analysis, median disease-free survival (DFS) was 21.1 months and overall survival (OS) was 31.8 months. For patients treated PP, median DFS and OS were 29.8 months and 39.7 months, respectively. Median DFS by cytogenetics was, 20.5 months for IRC and 22.4 months for IDT; not reached for the FRC group (p=0.10) and median OS was 32.8 months for IRC and 34.4 months for IDT; not reached for the FRC group (p=0.06). For FRC patients treated with high dose daunorubicin induction and AutoHCT the DFS and OS was ≥ 80% at 4 years based on K-M estimates. Patients who were FLT3-positive (n=27) demonstrated a trend towards worse DFS (16.2 vs. 30.3 months) and OS (20.0 vs.39.7 months) than those who were FLT3-negative (n=113) (p=0.12 and 0.10, respectively). Conclusions: Auto HCT is a safe consolidation approach with a low TRM in younger patients with de novo AML in first remission. Results are excellent in respect to DFS and OS particularly for the FRC patients on the high-dose daunorubicin arm. Due to small numbers, we could not demonstrate a negative outcome in FLT3-positive patients receiving AutoHCT. Further study, however, with a larger number of patients is warranted. Disclosures: Fernandez: Otsuka: Honoraria. Off Label Use: IV busulfan in the treatment of acute myeliod leukemia .

Abstract Abstract 2695 Background: Age and cytogenetics are major determinants of outcome in older patients with AML. Complete response (CR) to induction chemotherapy is observed in less than half of de novo AML patients (excluding APL) older than 60 years. Because unfavorable treatment results are often obtained despite intensive chemotherapy, which carries the potential for significant treatment-induced morbidity and mortality, it would be valuable to be able to predict with high accuracy at the individual patient level who will achieve a CR. Prospective identification of patients unlikely to benefit from anthracycline/cytarabine-based chemotherapy could spare patients from unnecessary treatment-related toxicities and allow consideration for alternative therapeutic strategies. Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on signaling cascades at the single cell level. Using SCNP technology we have developed a set of classifiers that predict for likelihood of response to anthracycline/cytarabine-based induction therapy in older patients with AML. Methods: SCNP assays were performed on paired, bone marrow (BM) and peripheral blood (PB), samples from 44 AML patients (de novo, evolved from an antecedent MDS or MPN or treatment related), >60 years old, enrolled on ECOG trial E3999. Based on two previous training studies, 38 combinations of modulators and intra-cellular proteins (signaling nodes along the phosphoinositide 3-kinase (P13K ), the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) and the DNA damage response and apoptosis pathways) were investigated. Basal and modulated protein levels and the effect of modulation on proteins levels in the leukemic blast cells were expressed using a variety of metrics. A total of 64 node/metric combinations (dimensions) were used to build multi-parametric classifiers (ranging from 2 to 10 nodes/metrics) using different modeling methodologies (including random forest, boosting, lasso and a bootstrapped best subsets logistic modeling approach that shrinks regression coefficients (BBLRS)) able to predict the likelihood of response to induction therapy. The performance characteristics of the classifiers built on the BM samples were then evaluated independently on the paired PB samples. Results: Several promising models with high area under the operator/receiver curve (AUROC) values (indicating strong agreement between actual clinical responses and responses as predicted by the model) were developed based on SCNP proteomic read outs for BM samples. The observed and predicted values from the current best BBLRS model are shown in Figure 1. The unadjusted AUROC of this model is 0.98 and the expected AUROC for the model when applied to an independent (validation) sample is 0.84. Five signaling nodes are represented in this model; they include nodes belonging to growth factor- induced survival pathways (PI3K, RAS/MAPK) as well as DNA damage response and apoptosis pathways. When the predictive accuracy of the lead SCNP classifier was compared to that of a model based on traditional clinical/molecular predictors (i.e. the combination of age, therapy-related AML, and karyotype) the adjusted AUROC of the SCNP classifier far surpassed that of the clinical predictors (adjusted AUROC=0.61 for clinical/molecular predictors vs. adjusted AUROC= 0.84 for the SCNP classifier). Finally, when the nodes in the best BBLRS model developed on data from BM samples were used to model read outs from the paired PB samples, the adjusted AUROC of the resulting BBLRS model was comparable to that of the model fit to BM samples. Conclusion: This training set data show the value of performing quantitative SCNP under modulated conditions as the basis for developing highly predictive tests for response to induction chemotherapy. Most importantly, the predictions made by the SCNP classifier are independent of established prognostic factors, such as age and cytogenetics The ability of one set of nodes to accurately predict response in paired BM or PB samples from individual patients suggests that the predictive power of the SCNP assay is independent of sample source, further improving the practicality of the test. Independent validation studies are ongoing. Disclosures: Cesano: Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Hackett:Nodality Inc.: Consultancy. Rosen:Nodality Inc.: Employment, Equity Ownership.

Abstract Abstract 2463 The treatment of relapsed/refractory B-Chronic Lymphocytic Leukemia (CLL) is difficult in terms of both inducing meaningful responses and being able to predict clinical outcome. In an attempt to address the latter aspect, we analyzed a battery of prognostic parameters obtained from a cohort of relapsed/refractory CLL patients entered onto ECOG trial E2903. This protocol included 2 steps: an induction phase (step 1) with pentostatin (4 mg/m2), cyclophosphamide (600 mg/m2), and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and a consolidation phase (step 2) where complete responders (CR) were given 4 weeks of subcutaneous (sc) alemtuzumab (30 mg/m2, 3 times a week) or patients attaining partial response (PR), stable or progressive disease were given 18 weeks of sc alemtuzumab. Sixty-five eligible patients were enrolled; median age was 63 yr; male to female ratio of 4:1; median (range) number of prior therapies was 3 (1–7); and there were 16, 7, and 40 patients in Rai 0–1, Rai 2; and Rai 3–4 stages respectively. The median serum β-2 microglobulin was 4 (1–28). The overall response rate for step 1 in this cohort was 46% with 1 complete and 29 partial responses and 38.5% judged as having stable disease. Strict CR criteria, including CT imaging were utilized, demonstrating the infrequency of total response using CT scans. For traditional prognostic parameters, we found that age over 70 yr and high Rai risk stage (3–4) did not preclude a clinical response, but there was a significant difference between responders and non-responders in serum β-2 microglobulin levels (p=0.03). For molecular prognostic parameters, we found that the immunoglobulin variable heavy chain region (IGVH) mutational status did not preclude a lack of clinical response. However, for patients who were ZAP70 negative (−) or CD38 negative (−) these factors were positively associated with clinical response (p=0.046, 0.0005, respectively). The sensitivity in predicting a clinical response was 82% in ZAP70- and 73% in CD38-. In multivariable logistic regression analysis, only CD38 was significantly associated with response (Odds Ratio: 41, p=0.002). When patients were segregated into low (13q- or normal), intermediate (Trisomy 12) and high risk FISH (17p-,) there was no association with clinical outcome. In addition, if patients' CLL cells were unmutated and high risk FISH, there was no reduced clinical response. Of 65 patients included in this analysis, the 2-year overall survival (OS) was 55% (±7%) and the 2-year progression-free survival (PFS) is 25% (± 6.6%). The median follow-up time amongst survivors was 23 (range 6–60) months. The univariable analysis for PFS revealed that the IGVH mutated (2-yr PFS 53% for mutated vs. 18% for unmutated, p=0.037, Figure) and low risk FISH (2-yr PFS 42% for low risk vs. 23% for high risk, p=0.04) were significant. However, only IGVH mutated was significant (HR 0.33, p=0.027) for PFS in multivariable Cox model. In univariable analysis for OS, the combination of age <70 yr and β-2 microglobulin<2 upper limits [UL] was significant (2-yr OS: 68% vs. 39%, p=0.048), whereas only β-2 microglobulin was significant (HR 3.45 for >=2UL vs. <2UL, p=0.02) in multivariable Cox model. A total of 24 patients entered onto step 2 of the consolidation phase, receiving alemtuzumab; 17 patients who were responders from step 1 entered onto the 4 weeks of step 2; and 7 of the non-responders went onto the 18 weeks of step 2. The 2-year OS of these 24 patients was 62% from the date of consolidation phase: 61% for 17 responding patients to PCR and 60% for non responders to PCR (p=0.87). Current conclusions of this study are: (1) heavily pretreated high risk CLL patients may still have a respectable survival with the PCR combination; (2) β-2 microglobulin, ZAP70, and CD38 were important factors associated with response to PCR; (3) both IGVH mutated and FISH were prognostic markers associated with PFS; (4) addition of alemtuzumab may confer comparable survival rates for PCR non responders to the responders; (5) β-2 microglobulin was the only prognostic factor significant for OS. In total this study shows that both traditional and molecular markers can be used to predict clinical outcome for high risk relapsed/ refractory CLL patients treated with chemoimmunotherapy. We anticipate further valuable prognostic information to come from this still accruing study. Progression Free Survival post PCR for IGVH patients as separated by Unmutated (UM) or Mutated cohorts Progression Free Survival post PCR for IGVH patients as separated by Unmutated (UM) or Mutated cohorts Disclosures: Rosen: Genentech: Speakers Bureau.