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7003 Background: FLT3 Internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations are seen in 30% of AML patients (pts) and are associated with poor survival. Secondary FLT3-TKD mu...

Tumor lysis syndrome (TLS) is defined by metabolic derangements occurring in the setting of rapid tumor destruction. In acute myelogenous leukemia (AML), TLS frequency, risk stratification, monitoring, and management strategies are based largely on case series and data from other malignancies. A single-center, retrospective cohort study was conducted to estimate TLS incidence and identify TLS predictive factors in a patient population undergoing myeloid leukemia induction chemotherapy. This study included 194 patients, aged 18-86 years, with AML or advanced myelodysplastic syndrome undergoing primary myeloid leukemia induction chemotherapy. Nineteen patients (9.8%) developed TLS. In univariate analysis, elevated pre-chemotherapy values for uric acid (P < 0.0001), creatinine (P = 0.0025), lactate dehydrogenase (LDH) (P = 0.0001), white blood cell (P = 0.0058), gender (P = 0.0064) and chronic myelomonocytic leukemia history (P = 0.0292) were significant predictors. In multivariate analysis, LDH (P = 0.0042), uric acid (P < 0.0001) and gender (P = 0.0073) remained significant TLS predictors. A predictive model was then designed using a scoring system based on these factors. This analysis may lay the groundwork for the development of the first evidence-based guidelines for TLS monitoring and management in this patient population.

Nonmyeloablative stem cell transplantation (NST) harnesses the graft-versus-tumor effect while minimizing regimen-related toxicity, and can result in donor chimerism and remission. Acute graft-versus-host disease (GVHD) and infections are major complications after sibling NST. Toxicity of unrelated-donor (UD) NST and the most appropriate GVHD prophylaxis in this setting remain poorly defined. We describe 25 patients who received UD-NST conditioned with fludarabine and cyclophosphamide. The first six patients received cyclosporine (Cs) and mycophenolate mofetil (MMF) (n=5) or methotrexate (MTX) (n=1) as GVHD prophylaxis (group 1) and all developed grade III-IV acute GVHD. The next 19 patients received the same conditioning regimen with the addition of alemtuzumab, and all received Cs/MTX post-transplant. Engraftment and donor chimerism were achieved in all but one evaluable patient. In all, 15 patients died: five of six deaths in group 1 were attributable to acute GVHD, while deaths in group 2 were due to infection or progressive disease (P=0.05). The combination of Cs/MMF is inadequate GVHD prophylaxis for UD-NST. The use of Cs, MTX, and alemtuzumab eliminated severe acute GVHD; its impact on response merits further study.

Abstract 673 FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) in acute myeloid leukemia (AML) are associated with early relapse after standard chemotherapy and poor survival. Quizartinib (AC220) is an oral FLT3 receptor tyrosine kinase inhibitor that is active against both ITD mutant and wild type FLT3, and has shown promising activity in a Phase 1 study of patients (pts) with AML. This Phase 2 study was conducted to assess the efficacy and safety of quizartinib monotherapy in FLT3-ITD positive (+) and FLT3-ITD negative (−) pts with a total of 333 pts included in two cohorts. Cohort 2 included pts aged ≥ 18 yrs with AML relapsed or refractory to 2nd-line, salvage chemotherapy or relapsed after hematopoietic stem cell transplantation (HSCT). A total of 137 pts were included in this cohort and constitute the basis for this analysis. Data through 31 January 2012 from 137 pts in this cohort were analyzed. These pts included 99 (72%) who were FLT3-ITD(+) and 38 (28%) who were FLT3-ITD(-). A total of 50% of the 99 FLT3-ITD(+) pts and 61% of the 38 FLT3-ITD(-) pts were male. The FLT3-ITD(+) pts had a median age of 50 yrs (range 19 to 77 yrs), and the FLT3-ITD(-) pts had a median age of 55 yrs (range 30 to 73 yrs). Pts received quizartinib at a starting dose of 90 mg/day (females) or 135 mg/day (males), and were treated continuously during 28-day cycles. The composite complete remission (CRc) rate included complete remission (CR), complete remission with incomplete platelet recovery (CRp), and complete remission with incomplete hematologic recovery (CRi). For FLT3-ITD(+) pts the CRc rate was 44% (4% CR, 0 CRp, and 40% CRi), with a median duration of response of 11.3 weeks and median overall survival of 23.1 weeks. Of those refractory to their last AML therapy, 47% achieved a CRc with quizartinib. For FLT3-ITD(-) pts the CRc rate was 34% (3% CR, 3% CRp, and 29% CRi), with a median duration of response of 5.0 weeks and median overall survival of 25.6 weeks. Of those refractory to their last AML therapy, 31% achieved a CRc with quizartinib. Efficacy Results in AML Pts Relapsed/Refractory to 2nd-line Therapy or HSCT FLT3-ITD(+) N = 99 FLT3-ITD(−) N = 38 Cumulative CRc, n (%) 44 (44) 13 (34) CRc + PR, n (%) 67 (68) 18 (47) Prior nonresponders achieving CRc to quizartinib, n (%) 27/57 (47) 9/29 (31) Subjects discontinuing quizartinib because of HSCT, n (%) 34 (34) 13 (34) Median CRc duration, weeks 11.3 5.0 Median overall survival, weeks 23.1 25.6 CRc = composite complete remission; HSCT = hematopoietic stem cell transplantation; NR = not reached; PR = partial remission. The most common (≥20%) treatment-related adverse events (AEs) were nausea (38%), anemia (29%), QT interval prolongation (26%), vomiting (26%), febrile neutropenia (25%), diarrhea (20%), and fatigue (20%). The most common (≥10%) Grade 3 or 4 treatment-related AEs were anemia (26%), febrile neutropenia (25%), thrombocytopenia (15%), neutropenia (12%), and QT interval prolongation (10%). An AE of QT interval prolongation occurred in 36/137 pts (26%) and was Grade 3 in 13 pts (10%). No Grade 4 QT interval prolongation occurred. A total of 14 pts (10%) experienced a treatment-related AE resulting in discontinuation of quizartinib. The final data from this Phase 2 study confirm the high degree of activity of quizartinib monotherapy in FLT3-ITD(+) and FLT3-ITD(-) AML pts relapsed/refractory to 2nd-line treatment or HSCT. These data represent the highest level of single agent activity observed to date for FLT3-targeted therapy in FLT3-ITD(+) relapsed/refractory AML. Of clinical significance in this heavily pretreated population, approximately 1/3 of pts were successfully bridged to potentially curative HSCT, and many pts who were refractory to prior therapy responded to quizartinib. Safety findings were manageable, and were primarily myelosuppression and QT prolongation that was mitigated with dose modifications. Further Phase 1 and 2 studies with lower doses of quizartinib as monotherapy and in combination with other agents in FLT3-ITD(+) and FLT3-ITD(-) AML are being planned/ongoing. Disclosures: Levis: Ambit Biosciences: Consultancy. Perl:Ambit Biosciences: Consultancy. Dombret:Celgene: Consultancy. Döhner:Celgene, Amgen, Lilly, Genzyme: Consultancy. Steffen:Novartis: Travel/accommodations/meeting expenses Other. Rousselot:Ambit Biosciences: Consultancy. Martinelli:BMS, Novartis; Pfizer, Roche: Consultancy. Estey:Ambit Biosciences: Consultancy. Burnett:Ambit Biosciences: Consultancy. Gammon:Ambit Biosciences: Employment. Trone:Ambit Biosciences: Employment. Leo:Ambit Biosciences: Employment. Cortes:Novartis: Consultancy.

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase and is the most commonly mutated gene in acute myeloid leukemia (AML). Second generation FLT3 inhibitors such as quizartinib (AC220) are clinically active in relapsed FLT3-ITD+ patients. However, not all patients respond and to date primary resistance has not been characterized. A previous study proposed that AML cells from patients with relapsed FLT3 mutant AML and samples with high allelic burden for FLT3 ITD are more sensitive to FLT3 inhibitor cytotoxicity (Pratz KW, et al Blood 2010). We performed studies to test this hypothesis and determine if cells with homodimeric FLT3 ITD are more heavily dependent on FLT3 ITD for growth and survival than cells expressing heterodimeric FLT3 WT:FLT3 ITD. 16 primary AML samples that contain FLT3 ITD mutations were incubated in increasing concentrations of the second generation FLT3 inhibitor crenolanib and assayed for survival in short term liquid culture assays. Only 6 samples demonstrated greater than 30% inhibition of survival in this culture system whereas 10 samples showed little or no cytotoxic response. Consistent with previous results (Pratz, et. al, Blood 2010), responding samples tended to be from relapsed patients and to have higher FLT3 ITD allelic ratios. We then analyzed FLT3 expression and phosphorylation levels as well as inhibition of and crenolanib inhibition of FLT3 phosphorylation, as well as the canonical downstream signal transduction pathways STAT5, ras/MAPK, and PI3K/AKT/mTOR in 13 FLT3-ITD+ primary AML samples. For 11/13 samples, crenolanib strongly inhibited phosphorylation of FLT3 kinase. However, neither FLT3 protein expression nor baseline phosphorylation level correlated with cytotoxic response in liquid culture assays. Crenolanib inhibited phosphorylation of STAT5, ribosomal S6 and ERK to varying degrees and inhibition of none of these pathways consistently correlated with cytotoxicity. Overall, these results are consistent with the hypothesis that FLT3 ITD mutant AML with high allele burden or relapsed samples are more addicted to FLT3 ITD. To further examine this topic, we studied AML cell lines that express only wild type FLT3 (THP1), both FLT3-ITD and WT FLT3 (MOLM14) or FLT3-ITD but not WT-FLT3 (Mv4;11, TF1-ITD). Growth of all three cell lines expressing FLT3-ITD but not THP1 cells was inhibited by crenolanib. Crenolanib inhibited tyrosine phosphorylation and activation of downstream signaling pathways in all three FLT3-ITD+ cell lines. Importantly, crenolanib was active at 10 nM in TF1-ITD cells but higher concentrations were required to inhibit signaling in Mv4;11 cells. This suggests that allele ratio alone does not determine sensitivity to FLT3 inhibitors. Interestingly, FLT3 ligand (FL) impairs inhibition of FLT3 by kinase inhibitor and, again, mutant cell lines are similarly responsive to FL in the presence of kinase inhibitor. In summary, these data demonstrate that AML cells have variable dependence on mutant FLT3 for survival. Samples with high allele ratio and relapsed samples behave in a manner consistent with oncogene addiction and are likely to show cytotoxicity to FLT3 inhibition. However, the basis for oncogene addiction is unclear and does not depend on activation of the canonical signal transduction pathways known to be downstream of FLT3. Interestingly, a recent unbiased screen of phosphorylated proteins in FLT3-ITD+ AML to predict clinical response to AC220 did not identify tyrosine phosphorylation of canonical FLT3 targets, but correlated clinical response with serine phosphorylation on EEPD1-S160, BCL11A-S630, and RANBP3-S333 (Schaab C, et al. Leukemia 2014). Analysis of the effects of FLT3 inhibitors upon these proteins in FLT3 mutant primary samples and cell lines is ongoing. Disclosures Perl: Arog pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy.

Background: Additional sex combs like transcription factor 1 (ASXL1) is a member of the polycomb group protein. ASXL1 mutation has been implicated in myeloid malignancy transformation. It is hypothesized that mutated ASXL1 leads to the loss of polycomb repressive complex 2 (PRC2) mediated gene repression and subsequent transforming events. Recent studies identify ASXL1 mutation as a poor prognostic marker in patients (pts) with de novo acute myeloid leukemia (AML) who present with intermediate–risk cytogenetic lesions (Patel, NEJM 2012; Schnittger, Leukemia2013). To study the impact of ASXL1 mutations in an unselected AML population, we analyzed clinical and molecular characteristics of patients with untreated AML who express ASXL1 mutation at presentation. Methods: Using next generation sequencing, 254 adult patients with AML seen at the Hospital of the University of Pennsylvania were analyzed for mutations, including ASXL1, using a 33-gene hematologic malignancy panel. Clinical characteristics were obtained from retrospective chart review. Kaplan-Meier estimates were used to calculate overall survival (OS) from time of diagnosis. Living patients were censored at date last seen. Results: ASXL1 mutations were detected in 36/254 (14%) AML pts. There were 29 known pathologic mutations, 1 benign, 1 probable pathologic, and 9 variants of unknown clinical significance (VUS). In 6/36 (16.7%) pts, ASXL1 was the sole mutation identified. Of the 30 pts with additional mutations (Figure 1), 6/30 (20%) pts harbored 2 independent ASXL1 mutations. When the 27 patients with pathologic ASCL mutations were analyzed for co-mutations, TET2 (13/27, 48%) was the most frequent ASXL1 co-mutation. FLT3 (0/27, 0%) and NPM1 (1/27, 3.7%) were notable for their absence. Median age of pts at diagnosis was 69 years (range 23-80). Prior myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN) was noted in 9/36 (25%) and 11/36 (30.6%) pts, respectively. Four pts (11.1%) had received chemotherapy and/or radiation therapy for a prior non-myeloid neoplasm. Karyotype was normal in 18/36 (50%) pts, and 7 additional pts had intermediate cytogenetic lesions. There were 7 pts (19.4%) with unfavorable cytogenetics (complex karyotype (3 pts), 7q- (3 pts), and 5q- (1 pt)). Four pts (11.1%) had a favorable karyotype, with t(8;21) in 3 pts and t(15;17) in 1 pt. At presentation, median white blood cell count (WBC) was 6.4x103/uL (1.0 x -103). In pts whose AML transformed from prior MPN, median WBC was 50 X103/uL (3.3-140). Standard induction chemotherapy with an anthracycline and cytarabine was given to 17/36 (47%) pts. An additional 3/36 (8.3%) pts underwent induction therapy with clofarabine. Complete remission (CR) was documented in 14/20 (70%) evaluable pts. Of the remaining pts, 11 received a hypomethylating agent, and 5 received other therapies. Thirty-day treatment mortality for all 36 pts and for 27 pts with known ASXL1 pathologic mutation was 13.4% and 18.5% respectively. Kaplan-Meier estimate showed a median overall survival of 349 days (median follow up of 107 days (range 15-1570)). For the 27 pts with a pathologic ASXL1 mutation, the OS was 276 days (Figure 2, median follow up of 145 days (range 18-1570)). Conclusion: ASXL1 mutations in de novo AML with intermediate-risk cytogenetics is associated with poor clinical outcome in cooperative group trials. Strikingly we demonstrate in a single institution, retrospective analysis that 66.7% of pts who present with ASXL1 mutations in the setting of previously untreated AML had documented MDS, MPN and/or prior chemotherapy/radiation. Further studies are necessary to evaluate if ASXL1 mutation has independent prognostic significance in AML or if it is primarily a marker for secondary leukemia. Figure 1: ASXL1 and co-mutations Figure 1:. ASXL1 and co-mutations Figure 2: Overall survival for AML patients with ASXL1 pathologic mutation Figure 2:. Overall survival for AML patients with ASXL1 pathologic mutation Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Effective and well tolerated treatment options for patients with relapsed acute myelogenous leukemia (AML) are limited. Birinapant is a small molecule, peptidomimetic of second mitochondrial-derived activator of caspases (SMAC) that selectively targets Inhibitor of Apotosis proteins (IAPs) resulting in tumor cell apoptosis and inactivation of NF-kB. SMAC mimetics represent a novel class of anti-tumor agents and birinapant has been explored as a single agent and in combination with chemotherapy in trials in solid tumors. Based on pre-clinical response observed in a mouse model with AML, we developed an investigator initiated Phase I clinical trial using single agent birinapant in pts with relapsed AML and high risk myelodysplastic syndrome (MDS). METHODS: Eligible pts were >18 years old with non-M3 relapsed or refractory AML or high risk MDS refractory to a hypomethylating agent. A standard 3+3 dose escalation was planned using single agent birinapant at increasing dose levels and frequency. Subjects who did not complete at least one cycle of therapy (4 wks) or experience a dose limiting toxicity (DLT) were replaced. The primary endpoint was safety and determination a maximum tolerated dose (MTD). Secondary endpoints included pharmacokinetic (PK) and pharmacodynamics (PD) analysis as well as disease response. RESULTS: From 12/2011 to 05/2014, 20 subjects were enrolled at the Hospital of the University of Pennsylvania and received at least one dose of study drug, 1 had MDS, 19 had AML (9 with antecedent MDS). The median age was 75 (range 36 to 80). The median number of prior treatments was 2 (range 1 to 5) and 11 patients required hydroxyurea during study treatment. No other concurrent chemotherapy was permitted. Several dose levels were tested varying the dose (17mg/m2, 22mg/m2 and 26mg/m2) and frequency (weekly, twice weekly (BIW) and 3 times weekly (TIW)) for 3 out of 4 wk cycles. Evaluable subjects have stayed on study drug for PK and PD analysis were performed at specified time points. Feasibility of measuring cIAP1 and cIAP2 inhibition as well as NFkB activity as PD response parameters for birinapant was explored. cIAP1 and cIAP2 levels were evaluated via Western blot analysis of mononuclear cells from pts; NFkB activity was assessed by imaging flow cytometry prior to and during initiation of birinapant treatment. cIAP1/cIAP2 suppression as well as inhibition of NFkB were evident. PK levels in plasma and blasts were also assessed at the same specified time points. Based on analysis to date, birinapant has excellent drug exposure and target suppression of cIAP1/cIAP2 and inhibition of NF-kB activity during the BIW schedule of 17mg/m2 for 3 out of 4 wks. CONCLUSIONS: This is the first study evaluating the safety and role of birinapant in pts with MDS and AML and first study in humans evaluating administration of this drug on a BIW dosing schedule. Observed adverse events related to study drug in this pt population include exacerbation of underlying autoimmune disease, Bell’s palsy and reversible and clinically silent elevation of amylase and lipase. Feasibility of measuring NFkB and cIAP1/2 expression as pharmacodynamics markers is established. Our study supports the safety and on tumor targeting of BIW dosing of birinapant which is currently under evaluation in combination with other agents in MDS. Disclosures Frey: Tetralogic Pharmaceuticals: Research Funding. Minderman:Tetralogic Pharmaceuticals: Research Funding. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. Carroll:Tetralogic: Research Funding.

FTL3 mutations are found in about 30% of AML patients, conferring a leukemic blast growth advantage, drug therapy resistance in the bone marrow (BM) and poor outcome. Mesenchymal stem/stromal cells (MSCs) are essential components of the bone marrow microenvironment, and growing evidence suggest that MSCs play a critical role in AML chemo-resistance, although the molecular mechanisms involved are poorly understood. The purpose of the study was to (1) establish an novel in vitro co-culture system between primary AML blasts and healthy donor BM-MSCs (HD-MSCs) or AML patient-derived MSCs (AML-MSCs), (2) evaluate the impact of culture with BM-MSCs on the sensitivity of AML cells to AC220 using patients samples with FLT3-ITD (n=4) or FLT3-WT (n=3). We first cultured HD-MSCs (n=5) and AML-MSC (n=3) and observed no phenotypical differences (CD14- CD34- CD45- CD73+ CD90+ CD105+), although HD-MSCs grew faster. We evaluated the effect of co-culturing AML samples (n=6) with HD-MSCs or AML-MSCs for 5 and 12 days on leukemic cell growth and found that both types of MSCs significantly and equally enhanced AML cell proliferation while maintaining blast phenotype. Using clonogenic assays on 4 AML specimens cultured alone or with either HD- or AML-MSCs for 5 and 12 days, we found that co-culture with either source of BM-MSCs drastically increased colony-forming cells number at day 5 and day 12 while CFC number decreased in the absence on BM-MSCs (no colonies at day 12 for the 4 samples), indicating that AML co-culture with HD/AML-MSCs supports the survival and/or proliferation of AML stem/progenitor cells. We next assessed the effect of increasing doses of AC220 (1, 10, 50, 100 and 500nM) on the apoptosis of FLT3-ITD (n=3) and FLT3-WT (n=4) AML cells cultured alone or with HD-MSCs. Exposure to AC220 for 72 hours significantly, and in a dose-dependent manner, increased the apoptosis of AML FLT3-ITD cells in monoculture (n=3, 21±1% of Annexin V positive cells for control, AC220 1nM 29±3.7%, 10nM 31±2.5%, 50nM 32±1.5%, 100nM 34±1.7% and 500nM 38±3.6%). In contrast, AML FLT3-ITD cells co-cultured with HD-MSCs were resistant to the drug (n=3, 21±2.6% of Annexin V positive cells for control, AC220 1nM 23±3%, 10nM 22±3%, 50nM 25±5.7%, 100nM 30±8.3% and 500nM 33±9.5%). Interestingly, we found that AML FLT3-WT are much less sensitive to increasing doses of AC220 compared to ITD samples (n=4, 27±3.9% of Annexin V positive cells for control, AC220 1nM 30±6.5%, 10nM 35±14%, 50nM 37±11%, 100nM 39±13% and 500nM 43±11%), and co-culture with BM-MSCs further decreased the sensitivity of AML FLT3-WT cells to AC220-induced apoptosis (n=4, 19±3.2% of Annexin V positive cells for control, AC220 1nM 17±3.9%, 10nM 20±3.4%, 50nM 19±3.7%, 100nM 21±4.5% and 500nM 26±1%). AC220 treatment for 3 days significantly, and in a dose-dependent manner, inhibited CFCs in AML FLT3-ITD (n=4, with 26±8%, 46±6%, 60±9%, 69±10% and 86±3% inhibition with 1, 10, 50, 100 and 500nM of AC220 respectively) while AML FLT3-ITD co-culture with HD-MSCs were less sensitive (n=4, with 9±10%, 30±6%, 42±9%, 57±11% and 72±7% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Similarly to the AC220-induced apoptosis, we observed that AML FLT3-WT CFCs are less sensitive to AC220-induced growth inhibition compared to ITD samples, although a 3 days exposure to AC220 significantly, and in a dose-dependent manner, inhibited AML FLT3-WT CFCs (n=3, with 38±16%, 44±14%, 58±12%, 70±21% and 81±19% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Interestingly, we observed that co-culture of AML FLT3-WT with stromal cells were significantly more resistant to increasing doses of AC220 (n=3, with 22±7%, 36±5%, 43±8%, 46±8% and 57±6% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Altogether, these results suggest that AML FLT3-ITD cells in monoculture are more sensitive to AC220 treatment compared to AML FLT3-WT primary cells, but more importantly, upon interaction with primary HD-MSCs, both WT and FLT3-ITD primary samples are protected from apoptosis and growth inhibition induced by AC220, indicating a critical role for the BM microenvironment in AC220 resistance. We are currently testing the impact of BM-MSCs co-culture on leukemic stem cell sensitivity to AC220 using transplantation in NSG mice. We will also evaluate if this co-culture model can be predictive of the response to in vivo treatment with AC220 in a patient-derived xenograft model. Disclosures Dos Santos: Janssen R&D: Research Funding. Danet-Desnoyers:Janssen R&D: Research Funding.

Background:Leukemia cutis (LC) occurs in 10-30% of AML cases and may be a marker of poor prognosis. However, outside of monocytic AML (FAB M4/M5), no clinical or genetic predictors of LC are known. Recently, a number of somatic molecular mutations have been described in AML. Using amplicon-based next-generation sequencing (NGS) of a panel of recurrent, hematologic malignancy-associated mutations, we sought to determine potential molecular markers associated with the development of LC. Methods: A cohort of non-M3 AML patients treated at the University of Pennsylvania was identified in which NGS had been performed on either leukemic blasts obtained during clinical care or from the institutional tissue bank.Average read depth for 33 hematologic malignancy-associated genes was approximately 3000X, minimal depth was 250x, and reporting frequency cutoff for variants was 5%. Mutations were reported as pathogenic or variants of uncertain significance (VUS, further sub-classified internally as likely disease associated, VUS, or likely benign) based on the University’s Center for Personalized Diagnostics (CPD) review of publically available data; only pathogenic or likely disease-associated mutations were included in this analysis. A database maintained by dermatopathology was reviewed to identify cases of leukemia cutis at any time during the disease course. Independent dermatopathology review was obtained for indeterminate cases. Association between presence of each of the 3 most common molecular mutations (FLT3-ITD, DNMT3A, and NPM1) and development of LC was assessed by logistic regression, with adjustment for FAB M4/M5, as appropriate. The association between presence of a molecular mutation in different functional classes (tumor suppressors, activated signaling, chromatin modifiers, transcription factors, splicing machinery) and the development of LC was also assessed. Results:279 adult patients with AML with known molecular genotype were identified. Molecular profile was determined from AML diagnosis in (243, 88%) with the remainder undergoing assessment after prior therapy (relapsed or refractory). 56% were male with median age of 60 years (range 18-87) and median WBC count at diagnosis of 22 K/uL (range 0.4 -388 K/uL; 17% ≥100K/uL). The majority of patients had intermediate cytogenetic risk (12% favorable, 59% intermediate, 23% unfavorable, 6% unknown) and 41% of patients had FAB M4/M5 AML (9% unknown). The three most common mutations were NPM1 (29%), DNMT3A (25%), and FLT3-ITD (23%). NPM1mutations were enriched in patients with FAB M4/M5 AML (41% vs 23%, p=0.003). Leukemia cutis was present in 26 (9%) of patients. NPM1 mutant status was present in 14 of 26 cases of leukemia cutis (OR 3.17, 95% CI 1.40-7.20, p=0.006). No association was detected for LC and the presence of mutant FLT3-ITD (OR 1.27, p=0.613), mutant DNMT3A (OR 1.7, p=0.224), or a mutation in any functional class of AML mutations (all p-values NS). The impact of NPM1 mutant status remained significant after adjustment for association with M4/M5 AML (OR 3.91, p=0.005). As the histologic subtype of AML might modify the association between NPM1 mutations and leukemia cutis, we next examined the impact of NPM1 mutant status on patients with FAB M4/M5 AML and non-M4/M5 AML. Among patients with M4/M5 AML, 10/12 (80%) patients with LC were NPM1 mutant compared to 32/91 (35%) without LC suggesting that the presence of mutated NPM1 was significantly associated with the development of LC (OR 9.22, p=0.006). Among patients with non-M4/M5 AML, 3/9 (33%) of patients with leukemia cutis were NPM1 mutant compared to 32/142 (22.5%) without LC indicating no association in the non-M4/M5 subgroup (OR 1.72, p=0.461). Interestingly, M4/M5 AML was not associated with LC in the NPM1 WT cohort (OR 0.65, p=0.6). Conclusion: Using NGS, we identify a novel association between NPM1 mutation status and the presence of leukemia cutis, particularly within monocytic AML. Confirmation of these observations in a larger dataset is planned. Our data suggest potential cellular effects of NPM1 mutation affecting homing of leukemic blasts to skin and support the World Health Organization’s provisional classification of NPM1-mutated AML as a distinct biologic entity. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia (AML) results from a combination of oncogenic events that can involve multiple signal transduction pathways including mutation-induced activation of tyrosine kinases. Kinase inhibitors are increasingly studied as promising targeted approaches either alone or in combination with other agents. However, only subsets of patients respond to respective targeted therapies. Internal tandem duplication (ITD) of FLT3 is one of the most common mutations in AML. It causes constitutive activation of FLT3. Quizartinib (AC220) is an example of a potent FLT3 inhibitor that was studied in a recent phase II open-label study in patients with relapsed/refractory AML. However, the presence of activating mutations within FLT3 can predict response to a certain extent only. Here, we investigated whether large-scale analyses of phosphorylation-based signalling events allows identification of more accurate markers based on the hypothesis that the read-out is closer to the mode of action of FLT3 inhibitors. Therefore, we applied quantitative mass-spectrometry to globally profile the phosphoproteome of 12 pre-treatment bone marrow aspirates obtained from AML patients treated with the quizartinib. A signature derived from this analysis consists of five phospho-sites within the proteins EEPD1, BCL11A, RANBP3, RP3, and LMN1 and it accurately predicted response to treatment with AC220 as revealed by validation in additional independent nine AML patients. Although the combined signature of five phospho-sites showed the highest prediction accuracy, we could demonstrate that in particular phosphorylation of S640 on BCL11A and S333 on RANBP3 lead to almost equally good predictions if used as individual markers. Furthermore, we could show that in case of BCL11A, EEPD1, and LMN1 the expression of the total protein correlates with its phosphorylation and thus with response. The phosphorylation markers were identified and validated in bone marrow aspirates. Although, it is clinical standard procedure to use bone marrow aspirates for diagnosis of AML patients, a predictive test that can be applied to peripheral blood samples would have many advantages. Indeed, we could show that the phosphorylation of the marker proteins strongly correlate between bone marrow and peripheral blood samples from the same patients, suggesting that the phosphorylation or protein markers can be measured and are predictive in both, bone marrow and peripheral blood samples. Citation Format: Christoph Schaab, Felix Oppermann, Martin Klammer, Heike Pfeifer, Andreas Tebbe, Thomas Oellerich, Juergen Krauter, Mark Levis, Alexander Perl, Henrik Daub, Bjoern Steffen, Klaus Godl, Hubert Serve. Global analysis of the phosphoproteome of human blasts reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-325. doi:10.1158/1538-7445.AM2014-LB-325

7027 Background: Development of D835 is a common mechanism of resistance to most FLT3 inhibitors. Crenolanib, a type I FLT3 tyrosine kinase inhibitor, has in vitro activity against FLT3 ITD and FLT3 D835. Crenolanib trials in relapsed/refractory FLT3 mutant AML (FLT3 ITD, FLT3 D835, FLT3 ITD/D835) are ongoing (NCT01522469, NCT01657682). Methods: Clinical data on the first 19 patients (7M, 12F) with a median age of 47 years (21-81) are currently available. 6 patients were refractory and 7 had relapsed within 6 months of induction therapy. 4 had undergone prior allogeneic transplants. 11/19 had progressed after exposure to ≥1 prior FLT3 TKI (8 Sorafenib, 7 AC220, 2 PLX3397, 1 PKC412,). 3 had received 2 or more prior FLT3 TKIs. Initially, crenolanib was given at a fixed dose of 100 mg PO TID but the dose was subsequently individualized to 200 mg/m2/d given in 3 divided doses. Results: Crenolanib had a Tmax of 1.5-2 hours and a T1/2 of 8-9 hours. Median day 15 trough levels (from 11 patients) ranged from 136 ...

7043 Background: Relapse is the most common reason for failure of allogeneic stem cell transplant (SCT). Identifying patients at increased relapse risk is crucial for improving outcomes. Prognostic somatic mutations have been identified in AML but their prognostic value after SCT is unknown. We hypothesize that next generation sequencing (NGS) can predict SCT outcome in AML. Methods: We performed NGS for 33 hematologic malignancy associated genes in 62 adult patients treated with SCT for de novo AML (2003-2013). Relapse free survival (RFS) was compared by Cox regression with adjustment for cytogenetic risk, conditioning intensity, donor type and graft source. Results: At least 1 mutation was identified in 56 (90%) patients (median 2, range 0-6) including 35 of 36 (97%) patients with intermediate cytogenetics (median 3, range 0-6). Among patients with intermediate cytogenetics transplanted in CR, poor prognostic mutations were found in 9 of 15 FLT3ITD negative patients (2 DNMT3A; 3 DNMT3A + IDH; 1 DNMT3A +...

7093^ Background: For FLT3-ITD(+) AML patients (pts) who are relapsed or refractory to chemotherapy, allogeneic HSCT offers the best prospect for long-term survival. Pts are unlikely to undergo HSC...

7100 Background: The presence of FLT3-internal tandem duplication (ITD) in patients (pts) with acute myeloid leukemia (AML) is associated with early relapse and poor survival. Quizartinib (AC220) i...

FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) in acute myeloid leukemia (AML) are associated with early relapse after standard chemotherapy and poor survival. Quizartinib (AC220) is an oral FLT3 receptor tyrosine kinase inhibitor that has shown the highest level of single agent activity seen with a FLT3 targeted agent in FLT3+ relapsed AML to date. This Phase 2 study was conducted to assess the efficacy and safety of two lower doses of quizartinib monotherapy in FLT3-ITD positive (+) pts aged 18 yrs or older with AML relapsed or refractory to 2nd-line, salvage chemotherapy or relapsed after hematopoietic stem cell transplantation (HSCT) to identify the optimal dose for future studies. The co-primary endpoints of the study were the composite complete remission (CRc) rate, which included complete remission (CR), complete remission with incomplete platelet recovery (CRp), and complete remission with incomplete hematologic recovery (CRi) and the rate of grade ³2 QTc prolongation. The Phase 2 study enrolled a total of 76 pts who were randomized to either 30 mg or 60 mg daily. Preliminary data from the entire study of 76 pts are included here; updated information will be provided for presentation. Thirty eight patients each were randomized to each arm and treated continuously during 28-day cycles. 58% of the randomized patients were male. The pts had a median age of 53 yrs (range 19 to 77). For 30 mg pts the derived CRc rate was 50% (5% CR, and 45% CRi). For 60 mg pts the CRc rate was 50 % (3% CR, 3% CRp, and 45% CRi). 29% of pts at 30mg and 37% of pts at 60mg were successfully bridged to HSCT. Efficacy Results in AML Pts Relapsed/Refractory to 2nd-line Therapy or HSCT30 mg Arm N = 3860 mg Arm N = 38*Cumulative CRc, n (%)19 (50)19 (50)*CRc + PR, n (%)22 (63)26 (68)*Subjects receiving HSCT after discontinuing quizartinib, n (%)11 (29)14 (37)*Median overall survival, days146197*Two subjects randomized did not receive drug due to ineligibility but are included in the ITT analysis.CRc = composite complete remission; HSCT = hematopoietic stem cell transplantation; PR = partial remission. The rate of grade 2 or higher QTc prolongation assessed by central reading was 11% and 17% in 30 mg and 60 mg arms respectively, grade 3 QT prolongation was 3% in both arms and there was no grade 4. The mean maximum prolongation from baseline was 31.5 ms and 39.7 ms in 30 mg and 60 mg arms respectively. A majority of patients (78%) had a treatment related adverse event. The most common (10%) treatment-related adverse events (AEs) were anemia (18%), nausea (15%), fatigue (12%) and diarrhea (11%). Sixteen patients (22%) had a serious adverse event (SAE) possibly related to study drug. The most common (5%) treatment-related SAE was febrile neutropenia (5%). A total of 4 pts (5%) experienced a treatment-related AE resulting in discontinuation of quizartinib. The data from this Phase 2 study confirm the high degree of activity of quizartinib monotherapy in FLT3-ITD(+) AML pts relapsed/refractory to 2nd-line treatment or HSCT, previously shown in the larger Phase 2 ACE study of 333 patients treated at higher doses (90mg, 135mg or 200mg). Importantly, 25 of 76 pts (33%) were successfully bridged to a potentially curative HSCT, an almost identical rate to that seen with higher doses. The safety profile is improved specifically decreasing QT prolongation rate at lower doses. Other safety findings were manageable, and were primarily gastrointestinal adverse events and myelosuppression. The data from this Phase 2 study confirms an improved risk:benefit profile with lower doses of quizartinib and multiple randomized Phase 3 studies with quizartinib in both relapsed and newly diagnosed AML patients are planned. Disclosures: Cortes: Ambit: Research Funding. Schiller:Astellas: Consultancy. Trone:Ambit: Employment. Gammon:Ambit: Employment. Goldberg:Astellas: Research Funding. Marie:Astellas: Travel expenses and medical writing services provided by Astellas Other. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau. Levis:Ambit Biosciences: Consultancy, Research Funding.

Background Activating mutations [primarily internal tandem duplication (ITD) events] in FLT3 are detected in 30% of acute myeloid leukemia (AML). The clinically active FLT3 tyrosine kinase inhibitor (TKI) AC220 (quizartinib) has achieved complete remissions in relapsed/refractory FLT3-ITD+ AML patients in a phase II study (Cortes, et al and Levis et al, ASH 2012, abstracts 48 and 673) but is vulnerable to resistance-conferring mutations in the FLT3 kinase domain (KD). The F691L “gatekeeper” substitution was the most commonly detected mutation in an in vitro mutagenesis screen for AC220 resistance (Smith et al, Nature 2012). This mutation, and substitutions at the activation loop residue D835, have been associated with acquired clinical resistance to AC220 (Smith et al, Nature 2012; Alber et al, Leukemia 2013). Mutations at gatekeeper residues such as F691 have repeatedly surfaced as mediators of clinical resistance to TKIs. Identifying TKIs that retain activity against these substitutions has consistently proven challenging. PLX3397 is a potent and selective inhibitor of FMS, KIT and FLT3-ITD with a half-life of 20 hours in humans, resulting in µM steady-state plasma concentrations at the recommended phase II dose for AML patients. PLX3397 retains activity against the AC220-resistant FLT3-ITD/F691L mutant, but not against several D835 mutants (Smith et al., ASH 2011, abstract 764). In this study, we conducted a mutagenesis screen of FLT3-ITD and FLT3-ITD/F691L to identify single and compound mutations that confer resistance to PLX3397 and may cause acquired resistance in patients. Results PLX3397 inhibited the proliferation of BaF3/ FLT3-ITD cells at a concentration well below that achieved in patients (IC50 0.14 µM) and retained activity against cells expressing the FLT3-ITD/F691L mutant (IC50 0.350 µM). Other AC220-resistant mutants (D835V/Y/F and Y842C/H) conferred substantial cross-resistance to PLX3397 (∼50 to 400-fold shift in IC50 of FLT3-ITD; ranging from 7.2 to >10 µM). An in vitro mutagenesis screen of FLT3-ITD identified several mutations conferring resistance to PLX3397, including novel substitutions in 3 residues which conferred ≥10X resistance relative to FLT3-ITD: D835E/G/N, D839A/G and R845G (IC50s 1.4 to 4.1 µM). Given the in vitro activity of PLX3397 against the AC220-resistant F691L mutant, it is anticipated that PLX3397 will be administered to patients who acquire resistance to AC220 or sorafenib due to this mutation; a mutagenesis screen of FLT3-ITD/F691L was therefore conducted. We identified multiple KD mutations in FLT3-ITD/F691L conferring ≥10X resistance to PLX3397 (compared to FLT3-ITD) including several mutations in the FLT3 activation loop: D835H/G/E/N, D839A/G/N, N841K, Y842S, R845G (IC50s 1.6 to >10 µM), and 2 mutations in residues located in the tyrosine kinase domain 1 (TK1) domain: N676S, a residue previously implicated in clinical resistance to the FLT3 inhibitor PKC412 (IC50 2.8 µM), and M664I, a residue not previously linked to FLT3 inhibitor resistance (IC50 2.0 µM). While all identified mutants conferred some degree of resistance to PLX3397 in the absence of an F691L mutation, most conferred a higher degree of resistance in the setting of F691L, suggesting a cooperation between the gatekeeper residue and residues in the activation loop and TK1 domain that impacts PLX3397 binding. Finally, we conducted a preliminary analysis of samples from AML patients who relapsed after an initial response to PLX3397. Using Pacific Biosciences Single Molecule Real-Time Sequencing, we identified evolution of polyclonal FLT3 KD mutations at the D835 residue at the time of relapse in 2 patients, including, in one patient, novel PLX3397-resistant D835E/H mutations identified in our mutagenesis screen. Analysis of additional patient samples for single and compound resistant mutations is ongoing and will be presented. Conclusions PLX3397 harbors promise for the treatment of FLT3-ITD+ AML, particularly for patients who have developed resistance to FLT3 TKIs due to the gatekeeper F691L mutation. However, a mutagenesis screen reveals PLX3397 is vulnerable to mutations in the FLT3 activation loop and TK1 domain. Patients acquire secondary FLT3 KD mutations at the time of resistance to PLX3397, confirming the mechanism of action of this clinically active FLT3 inhibitor. A multi-site phase I/II study of PLX3397 in FLT3-ITD+ AML is ongoing. Disclosures: Smith: Plexxikon Inc: Research Funding. Off Label Use: Unapproved drugs for AML: AC220 and PLX3397. Le:Plexxikon Inc: Employment. Zhang:Plexxikon Inc: Employment. West:Plexxikon Inc: Employment. Shah:Ariad Pharmaceuticals: Consultancy, Research Funding; Plexxikon Inc: Research Funding; Ambit Biosciences: Research Funding.

7012 Background: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) in acute myeloid leukemia (AML) is associated with early relapse after chemotherapy and poor survival. Relapse after HSCT or failure of salvage chemotherapy in FLT3-ITD AML is rarely associated with subsequent HSCT or durable survival. Quizartinib, an oral FLT3 inhibitor, shows promising activity; a Ph 2 study (n=333) of quizartinib monotherapy (Levis et al, ASH 2012) reported that quizartinib often bridged patients (pts) to HSCT. Methods: 136 FLT3-ITD(+) and 40 FLT3-ITD(-) pts, relapsed/refractory after HSCT or 1-second line regimen, were included. The response category of composite complete remission (CRc) comprised complete remission [CR] + complete remission with incomplete platelet recovery [CRp] + complete remission with incomplete hematologic recovery [CRi]. Results: Quizartinib was discontinued for HSCT in 47/136 FLT3-ITD(+) pts (35%), with 44/47 having at least a PR (2 CRp, 24 CRi, 18 PR). Median overall survival (OS) was 41.5 wks for pts with CRc prior to HSCT and 29 wks for pts with PR. The 1y survival rate was 39% for both response groups. Pts with a CRc (n=36) or PR (n=20) but no HSCT, respectively, had a median OS of 24.5 and 20.9 wks and 1y survival rates of 25% and 5%. Of 27 pts with OS >52 wks, 17 (63%) had HSCT. Quizartinib was discontinued for HSCT in 14/40 FLT3-ITD(-) pts (35%), with 13/14 having at least a PR (1 CR, 1 CRp, 7 CRi, 4 PR). Median OS was not yet reached for pts with CRc, and was 40.7 wks for pts with PR. The 1y survival rate was 78% for pts with CRc and 50% for pts with PR. 8 of these 14 pts had detectable ITD mutation but the level was below the prespecified 10% cutoff. Conclusions: Of clinical significance in these heavily pretreated pts who had failed salvage chemotherapy or HSCT, approximately 1/3 were successfully bridged to potentially curative HSCT, with encouraging 1y survival rates. To extend the potential benefits of FLT3 inhibitor therapy in combination with allogeneic HSCT, studies of maintenance quizartinib to prevent relapse post HSCT are ongoing. Clinical trial information: NCT00989261.

7023 Background: Advanced age and FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) in AML are each associated with early relapse after standard chemotherapy and poor survival. Quizartinib, an oral FLT3 inhibitor active against ITD mutant and wild type FLT3, has shown promising activity in Ph 1 and 2 studies. Methods: We provide detailed analysis from a Ph 2 study (N = 333) of quizartinib monotherapy, focusing on patients (pts) aged ≥ 70 y with AML that relapsed and/or was refractory to prior therapy. Results: A total of 83 pts age ≥70 y included 60 (72%) FLT3-ITD(+) and 23 (28%) FLT3-ITD(-). Median duration of treatment was 14.6 wks for FLT3-ITD(+) pts and 5.9 wks for FLT3-ITD(-) pts. Composite complete remission (CRc) rate included complete remission (CR), complete remission with incomplete platelet recovery (CRp), and complete remission with incomplete hematologic recovery (CRi). Of 60 FLT3-ITD(+) pts, 32 (53%) had a CRc (1 CR, 3 CRp, 28 CRi). Of 23 FLT3-ITD(-) pts, 10 (43%) had a CRc (2 CR, 1 CRp, 7 CRi). 12/27 FLT3-ITD(+) pts (44%) and 5/10 FLT3-ITD(-) pts (50%) refractory to prior therapy responded to quizartinib, and 12/83 (14%) survived >1 y. The most common (≥10%) Grade 3 or 4 treatment-related adverse events (TRAEs) were febrile neutropenia (22%), anemia (20%), transient QT interval prolongation (17%; no Grade 4), and thrombocytopenia (12%). 15 pts (18%) had TRAEs resulting in discontinuation. Conclusions: Because AML in the elderly, particularly in those aged ≥70 y, is genetically heterogeneous and often follows a myelodysplastic syndrome, there is a wide assumption that it may be less amenable to FLT3-targeted therapy than AML in younger pts. Our data argue against these conclusions and show that pts aged ≥70 y with chemotherapy-resistant AML have preserved high response rates, and promising survival to quizartinib. Clinical trial information: NCT00989261. [Table: see text]

7021 Background: Advanced age and FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) in acute myeloid leukemia (AML) are associated with early relapse after standard chemotherapy and poor survival. Quizartinib (AC220), an oral FLT3 inhibitor active against ITD mutant and wild type FLT3, has shown promising activity in Ph 1 and 2 studies. Methods: Patients (pts) in a Ph 2 open label study (N = 333) of quizartinib monotherapy included 154 aged ≥60 y with known FLT3-ITD status and AML relapsed in 52 wks. The median age of these pts surviving >52 wks was 69.5 y (range 66–80 y) and median OS was 76.3 wks (range 56.9–96.0 wks). All of these pts responded to quizartinib (2 CR, 2 CRp, 8 CRi, 4 partial remission [PR]). 2 pts were still alive >1 ½ y (OS 93.0 and 96.0 wks). Median OS in FLT3-ITD(-) pts was 19.1 wks and 6/44 FLT3-ITD(-) pts (14%) survived >52 wks. The median age of these pts was 70.0 y (range 65–77 y) and their median survival was 76.6 wks (range 54.9–98.4 wks). 5 of these pts responded to quizartinib (1 CR, 3 CRi, 1 PR). Conclusions: These data for an FLT3-targeted agent show encouraging survival in a subset of elderly pts with relapsed/refractory FLT3-ITD(+) AML. Clinical trial information: NCT00989261. [Table: see text]

Abstract 674 Background. Activating mutations in FLT3 occur in ∼30% of adult acute myeloid leukemia (AML) cases, including internal tandem duplication (ITD) mutations (∼25%) and point mutations in the tyrosine kinase domain (KD), primarily at the activation loop (AL) residue D835 (∼5%). FLT3-ITD and FLT3-AL mutations can co-occur in individual patients. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity in FLT3-ITD+ AML, as evidenced by a composite complete remission rate of 45% in 53 chemotherapy refractory/relapsed patients in an interim analysis of a phase II study (Cortes et al, ASH 2011 abstract #614). While some patients without FLT3 mutations in the AC220 phase I dose escalation study responded, no objective response was observed in a small number of FLT3-AL mutant patients (Cortes et al, ASH 2008 abstract #767). We recently reported that secondary mutations at F691, D835 and Y842 in FLT3-ITD confer in vitro resistance to AC220, and that relapse in 8/8 AML patients who initially responded to AC220 was associated with evolution of secondary KD mutations in the FLT3-ITD+ allele (Smith et al., Nature, 2012). In 6 of 8 patients, relapse was associated with D835 substitutions, making this amino acid position the most commonly substituted residue in FLT3-ITD+ patients at the time of disease relapse. Given that D835 mutations in FLT3-ITD appear to confer a high degree of pre-clinical and clinical resistance to AC220, we hypothesized that D835 mutations in the absence of the ITD might also confer resistance to AC220 and could be associated with clinical relapse, especially in patients harboring a mixture of FLT3-WT, FLT3-ITD+, and FLT3-AL mutant blasts. Results. In vitro binding studies revealed that FLT3-D835V and FLT3-D835Y have decreased affinity for AC220 (Kd=4.8 and 7nM, respectively) compared to native FLT3 (Kd=1.8nM). In proliferation assays of BaF/3 cells, FLT3-D835V/Y mutants demonstrated increased relative AC220 resistance (IC50 24 and 6.8nM) compared to FLT3-ITD (IC50 0.13nM). To sensitively and precisely determine the frequency and phase (ITD+ vs ITD-) of mutations in clinical isolates, we utilized single molecule real-time (SMRT; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the KD of FLT3 ITD+ and ITD- alleles. We analyzed ITD- sequences from pretreatment and relapse samples obtained from the 8 FLT3-ITD-positive AML patients with acquired AC220 resistance reported in our previous analysis. Interestingly, all 6 patients with secondary mutations at D835 in ITD+ alleles at relapse also harbored D835 mutant ITD- alleles. All D835 mutations detected in ITD- alleles were also observed in ITD+ alleles from the same patient sample. In ITD+ alleles, we detected D835Y (n=6), D835V (n=3), and D835F (n=2) (range 2.7–50.6% of ITD+ alleles). In ITD- alleles, we found D835Y (n=6), D835V (n=2), and D835F (n=1) (range 3.8–50% of ITD- alleles). One patient had 2 D835 mutations (D835Y/F) in ITD- alleles at relapse. Although 2 of 8 patients evolved the F691L mutation in ITD+ alleles at relapse, this substitution was not detectable in ITD- sequences in any patient. In 7/8 patients, samples obtained immediately prior to AC220 administration were available for analysis. We were unable to document the presence of AC220-resistant mutations in either ITD+ or ITD- sequences in these pretreatment samples, suggesting that these mutants evolved under the selective pressure of AC220 treatment. We are currently performing single cell RT-PCR to determine if D835 mutations in ITD+ and ITD- alleles occur in distinct leukemic cells, which would suggest a polyclonal blast population at relapse. Conclusions. FLT3-AL mutations at D835 confer resistance to AC220 in vitro. The evolution of D835 substitutions in ITD- alleles in the majority of patients who respond and relapse on AC220 suggests that constitutively activating FLT3-AL mutations at residue D835 can confer acquired clinical resistance to AC220. AML patients with FLT3-D835 mutations may exhibit de novo resistance to AC220 and other FLT3 inhibitors. Our findings predict that the clinical activity of potent FLT3 inhibitors that are tolerant of D835 substitutions will mechanistically involve inhibition of D835 mutants in both the presence and absence of an ITD mutation. Disclosures: Chin: Pacific Biosciences: Employment. Levis:Astellas Pharma: Consultancy; Plexxikon: Consultancy; Symphogen: Consultancy; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Perl:Astellas Pharmaceuticals: Consultancy. Travers:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Equity Ownership. Radich:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Shah:Ariad: Consultancy, Research Funding.

48 FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) in acute myeloid leukemia (AML) are associated with early relapse after standard chemotherapy and poor survival. Quizartinib (AC220) is an oral FLT3 receptor tyrosine kinase inhibitor that is active against both ITD mutant and wild type FLT3, and has shown promising activity in a Phase 1 study of patients (pts) with AML. This Phase 2 study was conducted to assess the efficacy and safety of quizartinib monotherapy in FLT3-ITD positive (+) and FLT3-ITD negative (-) pts with a total of 333 pts included in two cohorts. Cohort 1 included pts aged ≥ 60 yrs with AML relapsed in

Abstract 2481 Purpose: Cancer-associated IDH mutations produce the metabolite 2-hydroxyglutarate (2HG), but the clinical utility of serum 2HG measurements has not been previously established. We studied whether 2HG measurements in AML patients correlate with the presence of IDH mutations and whether diagnostic or remission 2HG measurements predict survival. Patients and Methods: Serum samples from 223 previously untreated adults (≤ 60 years of age) with de novo AML from the Eastern Cooperative Oncology Group E1900 clinical trial (62 IDH mutated, 161 IDH wild-type) were analyzed for 2HG concentration by reverse-phase liquid chromatography coupled to mass spectrometry (GC-MS). Results: Pretreatment 2HG levels ranged from 10 to 30000 ng/ml and were significantly elevated in IDH-mutant samples (median 3004.1 ng/ml), as compared to the wild-type cohort (median 61.2 ng/ml) (p < 0.0005). 2HG levels did not differ among the specific IDH1 or IDH2 allelic variants. In ROC analysis, a discriminatory level of 700 ng/ml segregated patients with and without IDH mutations with 86.9% sensitivity and 90.7% specificity. On repeat mutational analysis of 13 IDH wild-type samples with 2HG levels >700 ng/ml, IDH mutations were identified in nine samples, most often at low allele burden. IDH mutant patients with 2HG levels ≤ 200 ng/ml at complete remission experienced improved overall survival compared to those with higher 2HG levels (HR 3.5, p = 0.02) (Figure 1). Conclusion: We establish a firm association between IDH mutations and elevated serum 2HG concentration in AML. These data confirm that peripheral blood measurement of an oncometabolite provides useful diagnostic and prognostic information for cancer therapy, and furthermore can inform patient selection of IDH mutant targeted therapies. Disclosures: Levine: Agios Pharmaceuticals: Research Funding. Straley:Agios Pharmaceuticals: Employment. Yen:Agios Pharmaceuticals: Employment. Agresta:Agios Pharmaceuticals: Employment. Carroll:Agios Pharmaceuticals: Research Funding.

Abstract 885 Although high clinical response rates to the FLT3 inhibitor quizartinib (AC220) among subjects with relapsed/refractory AML suggest a potential clinical breakthrough, there remains concern that few subjects achieved CR or PR by strict IWG response criteria. Prior reports describe terminal neutrophil differentiation of AML blasts during clinical response to AC220 (Sexuaer, et al. ASH 2011, #943), suggesting that traditional chemotherapy metrics may not prove relevant to measure FLT3 inhibitors' clinical activity. Here we characterize responses to AC220 from a recent Phase II clinical trial, based on histomorphologic, molecular, and cytogenetic analysis and independent of that study's modified IWG metrics. We describe at least two distinct types of morphologic responses to AC220 among the 21 subjects treated at our site on this single-arm trial. All subjects had AML that relapsed or was refractory to prior chemotherapy. Subjects received single agent AC220 daily at 90–200 mg and were evaluated by bone marrow aspiration and biopsy on treatment days 15 and 29 and then monthly until blasts were cleared from blood and marrow. 15 of 21 enrolled subjects were FLT3-ITD+; 6 were FLT3 wild type (WT). 14/16 subjects with circulating or extramedullary blasts at study entry showed complete morphologic elimination of these populations. 18/20 subjects with marrow blasts >20% at study entry reduced marrow blasts to Disclosures: Carroll: GlaxoSmithKlein: Research Funding. Perl:Astellas Pharmaceuticals: Consultancy.

Abstract 2453 Background: Quizartinib (AC220) is a potent, highly selective inhibitor of FLT3, KIT, and FMS tyrosine kinases with promising clinical activity in AML patients, particularly those with FLT3 internal tandem duplication (FLT3-ITD) mutations. However, limited biochemical FLT3 inhibition in leukemic blasts in vivo by AC220 has been previously described (Perl, et al ASH 2012 #3502). As a biomarker for FLT3 inhibition, we assessed monitoring of phosphorylated ribosomal protein S6 (pS6) at serines 235/236 by flow cytometry. S6 is a downstream target of the PI3 kinase/mammalian target of rapamycin (mTOR) pathway, is constitutively phosphorylated in nearly all FLT3-ITD+ samples, and its phosphorylation shows dynamic changes in response to FLT3 ligand (FL) or FLT3 inhibitors in vitro. We hypothesized that S6 phosphorylation would be a biomarker for FLT3 inhibition and here provide final analysis of AC220 phamacodynamic monitoring at our center. Methods: Serial peripheral blood samples were collected during a phase 2 AC220 clinical trial (Cortes, et al. ASH 2011, #2576). Blood was aliquoted within four hours of collection and a subset was exposed to signaling inhibitors (ex vivo AC220, rapamycin × 30 min.) or activators (phorbol ester/PMA or FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed and red cells lysed with the permeabilizing agent triton X-100. Samples were stored at −20C in glycerol-containing medium. After collecting all time points, samples were simultaneously thawed, denatured with ice-cold methanol, and stained for flow cytometry. Blasts were identified by using CD45 and side scatter and confirmed by expression of multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Constitutive S6 phosphorylation was defined by comparing unstained (fluorescence minus one, FMO) and PMA and/or cytokine-stimulated cells. Biochemical sensitivity to AC220 was defined as a reduction in the percentage of positive phospho-S6 blasts by >50% as compared to baseline. Results: 21 subjects provided whole blood samples (15 FLT3-ITD+, 6 FLT3-WT), 15 had evaluable peripheral blasts (>100 blasts/microliter) for p-S6 monitoring. Only one subject with FLT3-WT had sufficient circulating blasts for analysis. By contrast, 13/15 FLT3-ITD+ subjects' blasts were evaluable. As previously described by our group and others using flow cytometry, pS6 is heterogeneous in primary AML samples and, at basal state, frequently only demonstrable in a subset of blasts. The mean percentage of blasts demonstrating S6 phosphorylation (pS6+) prior to AC220 therapy was 15% (median 4%, range Conclusions: We demonstrate the feasibility and utility of flow cytometry for phospho-S6 to monitor the biochemical efficacy of FLT3 inhibitors. The potential of these methods to predict clinical response/resistance paired with the rapid turnaround time of flow cytometry suggests potential future application of this technology in the screening evaluation of patients being considered for therapy with novel signal transduction inhibitors. Disclosures: Perl: Astellas Pharmaceuticals: Consultancy. Carroll:GlaxoSmithKlein: Research Funding.

Abstract 141 Background: The clinically active FLT3 inhibitor AC220 is vulnerable in vitro to resistance-conferring mutations at 3 residues in the FLT3-ITD kinase domain: the “gatekeeper” residue F691, and activation loop (AL) residues D835 and Y842. Mutations at 2 of these residues, F691 and D835, were identified in 8/8 FLT3-ITD+ acute myeloid leukemia (AML) patients who relapsed on AC220, including mutations at residue D835 in 6/8 patients. A molecular docking analysis suggests that mutations at D835 favor the active “DFG-in” kinase conformation and thereby impair binding of AC220, which presumably requires an inactive conformation for binding (Smith, et al, Nature 2012). Therefore, we predict that a type I inhibitor capable of binding the “DFG-in” active conformation of FLT3 will be required to inhibit AC220-resistant FLT3-ITD AL mutants. Crenolanib (CP-868,596) is a potent, selective ATP-competitive inhibitor of the FLT3-related receptor tyrosine kinases PDGFR-a and -b. Notably, crenolanib retains activity against the imatinib-resistant PDGFR-a D842V mutation, which is analogous to the AC220-resistant FLT3-ITD/D835V mutation. Low micromolar concentrations of crenolanib have been safely achieved in a phase I study of solid tumor patients with a half-life of ∼14 hours (Lewis et al, JCO 2009). We hypothesized that crenolanib may be a Type I inhibitor of FLT3 that retains activity against FLT3 mutant isoforms, including AC220-resistant FLT3 AL mutants, which are highly cross-resistant to multiple FLT3 TKIs. Results: In vitro binding studies demonstrate that crenolanib binds preferentially to the phosphorylated form of ABL (Kd =140nM vs Kd=440nM for non-phosphorylated ABL), confirming crenolanib is a Type I inhibitor. Additionally, crenolanib potently binds native FLT3 in vitro (Kd=0.26nM) and retains affinity for FLT3 harboring substitutions at D835 (H/V/Y; Kd= 0.24, 0.048 and 0.26nM respectively). Crenolanib demonstrates substantially more potent in vitro binding affinity for the compound FLT3-ITD/D835V mutant than AC220 (Kd=0.05nM vs Kd=210nM). In cellular assays, crenolanib induces apoptosis and inhibits the proliferation of the patient-derived FLT3-ITD+ cell lines MV4;11 and Molm14 with an inhibitory concentration 50 (IC50) of 5.2 and 9nM, respectively. FLT3 autophosphorylation and downstream signaling in MV4;11 and Molm14 cells were inhibited at similar concentrations. Treatment with crenolanib prolonged survival in a murine bone marrow transplant model of FLT3-ITD+ leukemia. Crenolanib inhibits the proliferation of BaF/3 cells transformed with FLT3-ITD (IC50 7.8 nM), and retains activity in BaF/3 cells harboring highly AC220-resistant FLT3-ITD/D835V/Y/F and FLT3-ITD/Y842C/H mutants (IC50 15–19nM). Crenolanib also potently suppresses the growth of BaF/3 cell lines containing the FLT3-activating point mutations D835V and D835Y in the absence of ITD (IC50 3.1nM), which we have recently found to be associated with AC220 resistance (Smith et al, ASH 2012, submitted). The FLT3-ITD/F691L mutation confers modest resistance to crenolanib (IC50 49.7nM). Western blot analysis reveals dose-dependent decrease in FLT3 autophosphorylation and downstream signaling. Crenolanib potently inhibits the proliferation of an AC220-resistant Molm14 subclone that harbors a D835Y mutation (IC50 15.4nM). In these cells and native Molm14 cells, crenolanib appears to retain maximal biochemical inhibition of FLT3 autophosphorylation and downstream signaling at nanomolar concentrations in human plasma, indicating relatively low plasma protein binding. Finally, treatment with crenolanib inhibited FLT3 autophosphorylation in human primary FLT3-ITD+ AML cells, including those from a patient who developed resistance to AC220 associated with a D835 mutation. Conclusions: Crenolanib is a Type I inhibitor of FLT3 that retains activity in the low nanomolar range against native and AC220-resistant FLT3-ITD mutant isoforms in in vitro binding studies, cell line and murine leukemia models, as well as in primary human AML cells. Crenolanib therefore has the potential to be clinically active in AML patients with activating FLT3-ITD or AL mutations, and to recapture clinical response in patients with acquired AC220-resistant kinase domain mutations. Clinical trials of crenolanib in TKI-naïve and TKI-pretreated FLT3-mutant AML are currently being planned or have recently been initiated. Disclosures: Perl: Astellas Pharmaceuticals: Consultancy. Carroll:GlaxoSmithKlein: Research Funding. Shah:Ariad: Consultancy, Research Funding.

Abstract 786 Acute Myeloid Leukemia (AML) results from a combination of oncogenic events that can involve multiple signal transduction pathways including mutation-induced activation of tyrosine kinases. Kinase inhibitors are increasingly studied as promising targeted approaches either alone or in combination with other agents. However, only subsets of patients respond to respective targeted therapies. We hypothesize that the phosphorylation status of certain sets of proteins (phosphosignatures) can predict the clinical response. In recent years, advances in sample processing, mass spectrometry, and computer algorithms for the analyses of proteomics data have enabled the application of mass spectrometry-based proteomics to monitor phosphorylation events in a global and unbiased manner. These methods have become sufficiently sensitive and robust to identify and quantify thousands of phosphorylation sites in a single experiment. Furthermore, spiking-in a SILAC- (stable-isotope labelling by amino acids in cell culture) reference sample (Super-SILAC) allows for the precise quantification of phosphorylation events also in in-vivo samples. Internal tandem duplication (ITD) of FLT3 is one of the most common mutations in AML. It causes constitutive activation of FLT3. AC220 (Quizartinib®, Ambit) is a selective inhibitor of the receptor-type tyrosine-protein kinase FLT3 that is currently under development for the treatment of AML. In a recent phase II open-label study, patients with relapsed AML were treated with AC220. The results of this trial will be reported elsewhere. Here, we sought to identify phosphorylation events that predict clinical response with high accuracy, especially in the group of FLT3-ITD-positive patients. To this end, we established a global, quantitative phosphoproteome analysis of AML cells derived from patient bone marrow. We analyzed samples from 15 patients before treatment with AC220 and employed the obtained phosphoproteomics data to discover a predictive signature of phosphorylation markers. In total, we confidently identified and quantified roughly 10,000 phosphorylation sites. Unsupervised clustering of the data revealed two large clusters that are both characterized by stronger phosphorylation of associated protein kinases. Next, we correlated these clusters with the reported clinical response of the respective patients. Although the clustering was unsupervised regarding the response information, the two clusters almost perfectly separated responders (complete or partial remission) from non-responders (stable or progressive disease). Furthermore, we identified a signature comprising four phosphorylation sites that accurately predict the outcome of therapy based on the corresponding phosphorylations obtained before treatment. We evaluated the performance of this signature in a cross-validation set-up and in additional test samples that have not been used for training. Moreover, we validated the robustness of the selected predictive features. Our study supports that the phosphorylation of four proteins are candidate biomarkers for predicting response to AC220 in AML. Notably, the phosphorylation markers are more predictive than the FTL3-ITD status alone and will thus allow a more precise stratification of AML patients for treatment with AC220. In addition, our results demonstrate for the first time that identifying predictive phosphorylation signatures directly from clinical patient samples is possible. Disclosures: Schaab: Kinaxo, Evotec: Employment, Research Funding. Oppermann:Kinaxo, Evotec: Employment, Research Funding. Pfeifer:Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other; Kinaxo, Evotec: Research Funding. Klammer:Kinaxo, Evotec: Employment, Research Funding. Tebbe:Kinaxo, Evotec: Employment, Research Funding. Oellerich:Kinaxo, Evotec: Research Funding; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Krauter:Ambit: Joined performance of clinical trial Other. Levis:Astellas Pharma: Consultancy; Plexxikon: Consultancy; Symphogen: Consultancy; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Perl:Ambit: Joined performance of clinical trial Other. Daub:Kinaxo, Evotec: Employment, Research Funding. Steffen:Kinaxo, Evotec: Research Funding; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Godl:Kinaxo, Evotec: Employment, Research Funding. Serve:Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other; Kinaxo, Evotec: Research Funding.

Abstract 143 Background: Mammalian target of rapamycin (mTOR) inhibitors enhance cytotoxic chemotherapy effects in primary acute leukemia cells in preclinical assays. This prompted a multi-center evaluation of a combination of mTOR inhibitor plus induction chemotherapy in AML. As mTOR is frequently but not uniformly activated in primary AML samples, it is unclear which patients benefit from this targeted approach. Thus, we sought to monitor mTOR kinase activity during therapy to determine whether target activation and/or inhibition predicted clinical response. We previously reported our preliminary experience monitoring pS6 in AML blasts by flow during clinical trials combining sirolimus and AML induction chemotherapy (Kasner et al, ASH 2011, #230). Here we provide the final clinical and pharmacodynamic results from this cohort of subjects. Methods: Subjects had relapsed/refractory AML or untreated AML with unfavorable risk factors (e.g. therapy-related, prior MDS or MPN, or age >60 without favorable karyotype) with a median age of 60.5 years (range 32–77). Subjects received oral sirolimus (12 mg on day 1, then 4 mg daily on days 2–9) plus MEC (mitoxantrone 8 mg/m2/day, etoposide 100 mg/m2/day, cytarabine 1 gm/m2/d on days 4–8) on one of two successive clinical trials. Clinical response was assessed at hematologic recovery or day 42 using IWG criteria (CR, CRp, PR vs. non-response). Pharmacodynamic samples were collected from blood or marrow at baseline, 2 hours post-sirolimus dose on days 1 and 4, and at trough on day 4 (prior to chemotherapy administration). Concurrent blood rapamycin concentration was measured by immunoassay or HPLC. Whole blood/marrow fixation was performed using published methods (Perl, et al. Clin. Cancer Res. 2012). Positive gates for pS6 were created by comparing blasts in ex vivo stimulated (phorbol ester/PMA) and inhibited (rapamycin) conditions and/or autofluorescence (FMO) controls. Results: We enrolled 52 subjects in 2 consecutive trials; 51 were evaluable for clinical response. Toxicity was similar to published MEC data. 3 infectious deaths occurred (6%). Prolonged aplasia was not observed. 24/51 (47%) subjects responded, with 18 CR (35%), 1 CRp, and 5 PR's observed. Mean peak and trough rapamycin concentrations on day 4 were 22.0 and 8.9 ng/ml, respectively, and did not differ among clinically responding or non-responding subjects. Median survival time for the whole group was 243 days (longest follow up 1584 days). Among the 24 subjects achieving CR or PR, median duration of time to the first event (relapse or death) was 261 days. 20 subjects were able to proceed to a stem cell transplant following therapy. Serial flow cytometric analysis was performed in 46 subjects, of which 37 provided paired day 1 and day 4 flow samples and were evaluable for clinical response at count recovery. The overall response rate (ORR) among subjects with baseline constitutive pS6 was 14/27 (52%, 9 CR, 1 CRp, 4 PR). The ORR for subjects without constitutive pS6 was 4/10 (40%, 3 CR, 1 PR). Subjects with >50% reduction in pS6 positive blasts on day 4 were considered to be biochemically sensitive to rapamycin, while subjects with Conclusions: Sirolimus plus MEC is a tolerable and active regimen for patients with high risk AML. The addition of an mTOR inhibitor augmented chemotherapy response particularly among those with demonstrable baseline mTOR activation and target inhibition during therapy. These results demonstrate the diversity of AML with reference to the activation of ribosomal S6 and suggest that phospho-flow monitoring may be an effective tool for patient selection for use of signaling inhibitors in AML. Future trials of this regimen may benefit from enrichment for subjects with mTOR activation and/or rapamycin sensitivity assessment. Disclosures: Off Label Use: Rapamycin. FDA approved for solid organ transplant. Investigational use for treatment of leukemia. Weiss:Celgene: Consultancy.

Abstract 3576 Eltrombopag, a non-peptide, small molecule, thrombopoietin receptor agonist, has been shown to increase platelet counts in patients with idiopathic thrombocytopenic purpura (ITP), hepatitis C associated thrombocytopenia and severe aplastic anemia. Pre-clinical studies show that eltrombopag stimulates megakaryopoiesis in bone marrow samples from patients with acute myelogenous leukemia (AML) and inhibits leukemic cell growth. The clinical use of eltrombopag in the AML patient population has not been previously explored. We report the results of an investigator-initiated, phase I dose escalation clinical trial to evaluate the safety and efficacy of eltrombopag monotherapy in older patients with AML. Eligible patients were ≥ 60 years old with non-M3 AML and a baseline platelet count ≤75,000/ul. A standard 3+3 design was used employing dose escalation in 4 sequential cohorts of 50mg, 100mg, 200mg and 300mg of eltrombopag daily. Subjects who did not complete at least one cycle of therapy (4 weeks) or experience a dose-limiting toxicity (DLT) were replaced. The primary endpoint was safety. Secondary endpoints included platelet and disease response. Bone marrow biopsies to evaluate for disease response and fibrosis were planned at one month, 3 months and 6 months. Twenty-three subjects were enrolled from June 2010 to February 2012 at the Hospital of the University of Pennsylvania and received at least one dose of study drug. The median patient age was 73 (range 60–86). Twenty-one of 23 subjects were relapsed or refractory after prior therapy. Two of 23 subjects (ages 85 and 86) were previously untreated. Nineteen subjects were platelet transfusion-dependent. Thirteen subjects had a history of antecedent myelodysplastic syndrome (MDS) and 6 were requiring hydroxyurea at time of study entry. Fourteen of 23 subjects completed the first cycle of therapy. Three remained on study for the entire 6-month study period. At the time of this report one subject remains on eltrombopag in an extension phase (8+ months of therapy). Indications for drug cessation included: possible DLT=1; progressive disease=5; death due to sepsis=5; lack of response=6, loss of response=1; and pursuit of hospice or alternative therapy=4. Overall eltrombopag was well tolerated at all doses studied and a maximally tolerated dose (MTD) was not reached. One subject at the 300mg dose level developed Grade 4 hepatic laboratory abnormalities possibly related to study drug, which met criteria for a DLT. The subject had concurrent polymicrobial sepsis and subsequently died. No other grade 3 or greater liver function abnormalities were observed. Asymptomatic skin discoloration with associated abnormal serum pigmentation was noted in subjects at the 200mg and 300mg dose levels. There were no platelet responses in the 50 mg or 100 mg cohorts (7 subjects total). Two of 7 subjects treated at the 200mg dose level and 2 of 9 subjects treated at the 300mg dose level achieved a platelet response (25% platelet response rate at these higher dose levels). All 4 of these subjects became platelet transfusion independent. The duration of platelet responses were 5 weeks, 12 weeks, 6 months and 8+ months (ongoing at time of this report). One subject with primary refractory AML, associated with monosomy 7, obtained a complete morphologic and cytogenetic response by 3 months of therapy at the 300mg dose level. This response was sustained at most recent bone marrow biopsy at 6 months of therapy and the patient remains on study drug. Increased reticulin fibrosis was also observed in this subject over time. No other subjects experienced a PR or CR. 6 patients had stable disease (SD) at one month, which was maintained at 3 months for 4 patients. Our study shows that eltrombopag is well tolerated in patients with AML at doses well above those typically used to treat ITP. At higher doses, we observed clinically meaningful platelet and antileukemic responses. Further exploration of eltrombopag as both a platelet agonist and disease-modifying agent in the treatment of AML is justified. Disclosures: Frey: GSK: Consultancy, Research Funding. Porter:Novatis: Patents & Royalties; Celgene: Honoraria; Genentech: Employment; Pfizer: Research Funding. Perl:Astellas: Consultancy. Carroll:GlaxoSmithKlein: Research Funding.

Abstract 4245 Background. Therapies for adults with acute lymphocytic leukemia (ALL) fail to cure the majority of patients. The mTOR inhibitor, rapamycin, is a potent chemosensitizer in ALL cells and combination rapamycin- methotrexate is curative in an ALL xenotransplantation model. We therefore explored the feasibility of adding rapamycin to a multi-drug ALL regimen in subjects with de novo or relapsed Philadelphia chromosome negative ALL or other aggressive lymphoid malignancies. Additionally we performed pharmacodynamic analysis of peripheral blood blasts to estimate mTOR activation at baseline and with rapamycin. Methods. Subjects were treated with rapamycin 12 mg on day 1, followed by 4 mg daily on days 2–7. To allow steady state inhibition of mTOR, systemic therapy with hyper-CVAD (A cycles) alternating with high dose methotrexate and high dose cytarabine (B cycles) was given following the day 3 rapamycin dose of each cycle, with rituximab if CD20 positive and intrathecal prophylaxis if indicated. Peripheral blood specimens were collected serially during the first cycle of chemotherapy and were aliquoted, incubated with signal modulators as controls for 30 minutes then fixed with 4% formaldehyde and permeabilized. No ficoll separation was performed. Thereafter, cell samples from all time points for a subject were thawed, denatured with ice-cold methanol, and stained for cytometer analysis using a uniform antibody cocktail including alexa fluor-488 conjugated to phosphorylated S6 kinase. Results. We report results on the first 7 subjects, median age 45, (range 26–65) of which 3 had relapsed B-precursor ALL and the remainder were newly diagnosed T lymphoblastic lymphoma, adult T- cell lymphoma (ATLL), mantle cell (MCL) and Burkitt’s lymphoma (BL). Feasibility. Thus far 16 ‘A’ and 15 ‘B’ cycles have been administered. The median time to next rapamycin was 23 days (range 19–57) and to chemotherapy was 25 days (range 21–59). Median time to recovery of absolute neutrophil count of > 500 and platelet count of >50 was 16 (range 0–27) and 17 (range 0–46) days, respectively. Two subjects never had count recovery in the setting of persistent disease. The following > grade 3 non- hematologic toxicities were observed: 5 neutropenic fevers, 5 infections, 4 non-neutropenic fevers, 1 ataxia (cytarabine related), psychosis, and hypophosphatemia. No fungal infections were noted. No treatment-related mortality has been observed. Responses. Of 7 subjects, 4 achieved complete responses (CR) after cycle 2B (MCL, BL, ATLL and T- lymphoblastic lymphoma). The ATLL and MCL patients completed 5 and 4 cycles respectively and remain in CR after allogeneic BMT. The T-lymphoblastic lymphoma and BL patients completed 8 and 7 cycles respectively and remain in CR. All three subjects with relapsed ALL were taken off study with persistent disease after 1 to 4 cycles, and have subsequently died. Pharmacokinetics. Rapamycin levels fell within the range typically targeted for transplant immunosuppression -mean (SD) 8.57 (3.33), range 4.7–14.7. Pharmacodynamics. Two of 3 subjects with pre B- ALL had sufficient peripheral blast count of >200/μ l to perform intracellular phosphoflow. Both subjects’ blasts displayed constitutive phosphorylation of the ribosomal S6 protein (25% & 31%) at baseline with maximal inhibition at 72 hours of in vivo rapamycin in the 1st sample and submaximal inhibition in the 2nd sample (2.9% & 12.4%). Rapamycin trough levels were 9.5 and 8.7 respectively. The addition of 1000nM ex vivo rapamycin produced further reduction (0.68% & 2.07%). Despite exhibiting varying degrees of rapamycin sensitivity, both subjects had persistent disease after only 1 and 2 cycles of therapy respectively. Conclusion. We show that the addition of rapamycin to Hyper-CVAD in adults with ALL and other aggressive lymphoid malignancies is feasible and results in similar toxicities to Hyper-CVAD alone without increased myelosuppression or treatment related mortality. At basal state, pS6, measured by flow cytometry, is heterogeneous in primary ALL samples and likely only demonstrable in a subset of blasts. Whole blood intracellular flow cytometry is a novel, feasible and potentially powerful technique to monitor pharmacodynamic response to novel therapeutics that inhibit mTOR signaling in ALL. Expansion of this trial to better characterize toxicity, response rates and the feasibility of performing PD/PK correlation is planned. Disclosures: Off Label Use: Rapamycin. This is FDA approved for rejection prevention in solid organ transplant. It has been used investigationaly in this study for treating ALL. Carroll:Glaxo Smith Kline, Inc.: Research Funding; Sanofi Aventis Corporation: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.

Abstract 764 Background: Activating mutations in FLT3 are detected in approximately 30% of adult acute myeloid leukemia (AML) cases, most commonly involving internal tandem duplication (ITD) events. The clinically-active FLT3 inhibitor AC220 (quizartinib) was recently reported to be associated with a composite complete remission (CR) rate of 43% in relapsed/refractory FLT3-ITD+ AML patients in an interim analysis of a phase II study (Cortes, et al, EHA 2011, abstract #1019). AC220 is vulnerable in vitro to a limited number of resistance-conferring mutations in the FLT3 kinase domain (Smith et al, AACR 2011, abstract #4737). Mutations at two of these residues, F691 and D835, have been detected in 9/9 patients with AML with loss of response to AC220 analyzed to date (Smith et al, ASH 2011, submitted). Mutations at “gatekeeper” residues such as F691 have repeatedly surfaced as important mediators of clinical resistance to inhibitors of BCR-ABL, EGFR, ALK and KIT kinases. Identifying tyrosine kinase inhibitors that retain activity against these substitutions has proven challenging. PLX3397 is a potent and selective inhibitor of FMS, KIT and oncogenic FLT3 with an elimination half-life of 20 hours in humans. Steady-state plasma concentrations of 10–20 uM have been achieved in solid tumor patients enrolled in a phase I study, where minimal myelosuppression has been observed (Anthony et al, J Clin Oncol 29: 2011, suppl abstr 3093). Results: PLX3397 selectively inhibited the proliferation of the human FLT3-ITD+ AML cell lines MV4-11 and MOLM-14 with a 50% inhibitory concentration (IC50) in the submicromolar range (0.09–0.2 uM). PLX3397 inhibited phosphorylation of FLT3-ITD in cells with a dose response similar to the growth inhibition range. We next evaluated the ability of PLX3397 to inhibit the proliferation of Ba/F3 cells transformed with FLT3-ITD and AC220-resistant FLT3-ITD mutant isoforms F691L, D835V/Y, and Y842C/H. PLX3397 inhibited the proliferation of Ba/F3/FLT3-ITD cells (IC50 0.07 uM) at a concentration comparable to that needed to inhibit the growth of MV4-11. Encouragingly, PLX3397 retained activity against Ba/F3 cells expressing the clinically-relevant F691L gatekeeper mutation at a similar concentration (IC50 0.37 uM). Other AC220-resistant mutations evaluated conferred substantial cross-resistance to PLX3397 (IC50 >200x IC50 of FLT3-ITD alone for all mutations; range 14.3–130,000 uM). Western blot analysis of FLT3 autophosphorylation demonstrated dose responsiveness in the same concentration range as observed in cellular proliferation results for each FLT3-ITD mutant isoform. Based upon molecular docking studies, PLX3397 forms non-specific hydrophobic interactions with the gatekeeper side-chain, and thus is less sensitive to the F691L mutation than AC220, for which the combined effects of steric hindrance and polarity mismatch impede its binding to the FLT3-ITD/F691L. To further assess if PLX3397 warrants clinical evaluation as a FLT3 inhibitor in AML, we performed a modified plasma inhibitory assay by incubating MOLM-14 cells in either normal donor or AML patient plasma spiked with increasing concentrations of PLX3397 as well as unmanipulated, steady-state plasma samples from the solid tumor PLX3397 phase I trial. Using phospho-specific flow cytometry to evaluate FLT3 signaling through the downstream protein ribosomal S6, we observed near-maximal reductions in phospho-S6 in both normal and AML patient plasma containing >10 uM PLX3397 as well as plasma samples obtained from the solid tumor trial. Conclusions: PLX3397 harbors substantial promise for the treatment of FLT3-ITD+ AML. Our data demonstrate that potent FLT3 inhibitory concentrations are achieved in humans. Moreover, while mutations in the FLT3-ITD kinase domain represent an important cause of loss of response to clinically-active FLT3 inhibitors, PLX3397 retains activity against the FLT3-ITD/F691L “gatekeeper” mutation, which we have found to be a common cause of preclinical and acquired clinical resistance to AC220. A multi-site phase I/II study of PLX3397 in FLT3-ITD+ AML has been initiated. Disclosures: Off Label Use: Investigational agent PLX3397 will be discussed. Zhang:Plexxikon Inc.: Employment. Carroll:Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding. Shah:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.

Abstract 3502 Background: AC220 (quizartinib) is a potent, highly selective inhibitor of FLT3, c-KIT, and c-FMS tyrosine kinases with promising clinical activity in AML, particularly among patients with FLT3 internal tandem duplication mutations (FLT3-ITD). However, direct confirmation of biochemical FLT3 inhibition in AML blasts in vivo by AC220 has not been previously described. We report separately that phospho-ribosomal protein S6 (pS6) by flow cytometry provides a sensitive and dynamic readout of FLT3 kinase activity during FLT3 inhibitor therapy. S6 is a downstream target of the PI3 kinase/mammalian target of rapamycin (mTOR) pathway, is constitutively phosphorylated in nearly all FLT3-ITD+ samples, and its phosphorylation shows dynamic changes in response to FLT3 ligand (FL) or FLT3 inhibitors. We hypothesized that reductions in S6 phosphorylation by flow could serve as a biomarker for FLT3 inhibition and predict response and/or resistance to AC220. Methods: Serial peripheral blood samples were collected during a phase II AC220 clinical trial (Cortes, et al. EHA 2011, abstract #1019). Samples were aliquoted into FACS tubes within four hours of collection and a subset was exposed to signaling inhibitors (ex vivo AC220, rapamycin × 30 min.) or activators (phorbol ester/PMA or FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed and red cells were lysed with the permeabilizing agent triton X-100. Samples were stored at −20C in glycerol medium. Samples from all time points were simultaneously thawed, denatured with ice-cold methanol, and stained with a single antibody cocktail. Blasts were identified by using CD45 and side scatter (SSC) and confirmed by expression of multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Positive and negative pS6 gates were created by comparing blasts in stimulated and unstimulated conditions and/or autofluorescence (FMO) controls. Results: 6 subjects provided blood samples (5 FLT3-ITD, 1 FLT3-WT) and had evaluable peripheral blasts (>100/microliter) for pS6 monitoring. As previously described by our group and others using intracellular flow, pS6 is heterogeneous in primary AML samples and, at a basal state, frequently only demonstrable in a subset of blasts. The mean percentage of blasts demonstrating S6 phosphorylation prior to AC220 therapy was 29% (median 9.8%, range 2–97%). To quantify FLT3 kinase inhibition in vivo, we monitored pS6 prior to and following subjects' initial dose of AC220. Samples obtained two hours following the initial dose consistently reduced the percentage of blasts with pS6 to a mean of 2.8% (median 1, range 0.02 to 11). Consistent with its long half-life, pS6 following a single dose of AC220 remained consistently and potently suppressed at trough concentration on day 2. Comparing clinical response and biochemical inhibition by AC220, all 5 subjects with marked reduction in pS6 to AC220 cleared peripheral blasts by day 29 and 3 subjects achieved marrow blasts Conclusions: We demonstrate the feasibility and utility of intracellular flow cytometry for phospho-S6 to monitor the biochemical efficacy of FLT3 inhibitors. The potential of these methods to predict clinical response/resistance paired with the rapid turnaround time of flow cytometry suggests potential future application of this technology in the screening evaluation of patients offered novel signal transduction inhibitors. Studies correlating the magnitude of signal inhibition with IWG responses are ongoing in a larger cohort treated with AC220 and will be reported once clinical data mature. Disclosures: Carroll: TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding; Agios Pharmaceuticals: Research Funding.

Abstract 937 Background . Activating in-tandem duplication (ITD) mutations within the FLT3 juxtamembrane region are detected in ∼25% of adult acute myeloid leukemia (AML) cases and portend a poor prognosis. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity, as evidenced by a composite complete remission rate of 43% in 53 chemotherapy refractory/relapsed AML patients evaluable for an interim analysis of the ongoing phase II study in FLT3-ITD+ relapsed/refractory AML (Cortes et al, EHA 2011 abstract 1019). Many patients who initially respond ultimately suffer disease progression. We sought to utilize the clinical efficacy of AC220 to determine if FLT3-ITD is a valid therapeutic target in human AML. A saturation mutagenesis strategy identified amino acid substitutions at three amino acid residues (F691, D835, Y842) in FLT3-ITD that confer a high degree of resistance to AC220 in vitro. Of these, substitutions at F691 and D835 conferred the greatest degree of relative resistance. Results . To assess the validity of FLT3-ITD as a therapeutic target in AML, we analyzed paired pretreatment and relapse samples obtained from a cohort of 9 FLT3-ITD-positive AML patients enrolled in the exploratory part of the ongoing phase II study of AC220 in relapsed/refractory AML (clinicaltrials.gov NCT00989261) who relapsed after initially achieving morphologic clearance of bone marrow blasts to In an expanded analysis of genomic DNA samples from 30 patients enrolled in the exploratory part of the Phase II study who discontinued study drug for any reason, we observed the occurrence of acquired mutations in the kinase domain (D835 and F691) in a total of 10 of 30 (33%) patients at the off study timepoint. One patient had a D835Y mutation prior to going on AC220. Molecular docking studies were undertaken to provide mechanistic insights into the structural basis of resistance conferred by AC220-resistant mutations. These studies revealed that AC220 likely binds strongly to the DFG-out inactive FLT3 kinase domain. AC220 directly interacts with the gatekeeper residue F691, explaining the drug-resistance associated with the F691L mutation. Mutations at D835 and Y842 may potentially de-stabilize the inactive conformation, and result in a more active, AC220 binding-deficient FLT3 kinase. Indeed, substitutions at these residues are known to activate FLT3 in the absence of an ITD mutation. To more precisely assess the frequency and identity of resistance-conferring mutations at relapse, we analyzed a subset of samples using single molecule real-time (SMRT™; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the kinase domain of FLT3-ITD alleles. With this assay, more than 350 reads of >1000 nucleotides were reliably obtained. Analyses of pretreatment and relapse samples from four patients confirmed the presence of resistance-conferring FLT3-ITD kinase domain mutations at F691 or D835 at relapse in 36–60% of FLT3-ITD sequence reads. Additionally, this method detected polyclonal resistance in two of the four samples assessed. Conclusions . Our studies validate FLT3-ITD as a therapeutic target in a proportion of AML cases, and demonstrate that the clinical activity of AC220 is mediated by FLT3-ITD inhibition. AC220-resistant FLT3-ITD gatekeeper and activation loop mutations identified in clinical samples from relapsing patients represent high-value therapeutic targets for next-generation FLT3 inhibitors. Disclosures: Off Label Use: AC220 is an investigational agent and has no approved drug indication in AML. Chin:Pacific Biosciences: Employment. Hunt:Ambit Biosciences: Employment. Levis:Ambit Biosciences, Inc: Consultancy. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.

Abstract 1557 Introduction: The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway controls key cellular processes and is frequently activated in both acute and chronic myeloid leukemia (AML, CML) as well as high-risk MDS, thus representing an attractive therapeutic target. UCN-01 inhibits a number of serine-threonine kinases, including phosphatidylinositide-dependent kinase 1, which phosphorylates and activates Akt. Perifosine, an alkylphospholipid, targets the pleckstrin homology domain of Akt, thereby inhibiting its plasma membrane translocation and activation. Synergistic inhibition of Akt by UCN-01 and perifosine has been demonstrated in prostate and lung cancer cell lines (Clin Cancer Res 10: 5242, 2004). Methods: We conducted a Phase I study to determine the maximum tolerated dose (MTD) and recommended Phase II dose of UCN-01 and perifosine, using a standard 3+3 dose escalation design. Patients (pts) >18 years with poor-risk, or relapsed/ refractory AML or acute lymphoblastic leukemia (ALL), CML in accelerated/blastic phase or MDS failing standard therapy, and peripheral blast count Results: Thirteen pts with AML (11), ALL (1), and MDS (1) were treated (DL1, n=6; DL2, n=4; DL3, n=3). Median age was 69 years (range, 20–85), 77% pts were male, and median number of prior treatments was 3 (range, 1–6). Nine of 11 AML patients had secondary (7)/therapy-related (2) disease, 4 had adverse and 5 intermediate karyotypes, and all were either primary refractory (4) or had first remission duration < 12 months (7). Pts received median 1 (range, 1–3) cycle of therapy. Five pts did not complete 1st cycle due to disease progression (n=3; DL1), cholecystitis/sepsis (n=1; DL3), or drug-related toxicity (n=1; DL3), and 4 received hydroxyurea temporarily to control increasing blast counts. The most frequent drug-related toxicities (NCI CTC v3) were grade (gr) 1/2 nausea and diarrhea (62% each), vomiting (54%), fatigue and hyperglycemia (31% each) anorexia, hypocalcemia and constipation (23% each), increased AST/ALT (15%), hypokalemia and hypotension (8% each). Few ≥gr 3 toxicities were seen, all at DL3: fatigue (gr 3), hyperglycemia (gr 4), hypokalemia (gr 3), pericarditis/tamponade/hypotension (gr 4) and respiratory distress (gr 4). MTD was therefore defined as 65 mg/m2 based on 2 pts with dose limiting toxicities at DL3. One AML pt (DL1) had disease stabilization for 3 cycles and one MDS pt (DL2) had improvement in neutrophil count. Given lack of sufficient evidence of clinical efficacy, the DL2 cohort was not expanded to 6 pts as planned. However, no gr 3/4 drug-related toxicities were observed in 10 pts treated at DL1/2. Pharmacodynamics: Western blot analysis did not consistently demonstrate appreciable direct Akt inhibition in ficolled blood/marrow mononuclear cells from 5 pts (DL1/2) on d4 (perifosine alone) or d21-28, however, a 38–74% reduction in phosphorylated ribosomal protein S6 in leukemic blasts from 3 pts on d4 was noted by intracellular flow cytometry, consistent with inhibition of signaling through the Akt downstream target p70S6 kinase. Pharmacokinetics: The average steady state concentration (Css) of perifosine was 4.75±2.53 μg/mL, consistent with published data (Eur J Cancer 38: 1615, 2002), with 1 pt having an unusually high Css of 9.25 μg/mL. The end-of-infusion concentration for UCN-01 was also consistent with published data (Clin Cancer Res 13: 2667, 2007), with average Cmax 13.2±0.424 μg/mL and 20.7±3.08 μg/mL at doses of 40 and 65 mg/m2, respectively. Conclusion: UCN-01 (40 or 65 mg/m2)/perifosine can be safely administered, but this regimen lacked clinical efficacy. This approach may have failed because of insufficient Akt inhibition in vivo and/or need to inhibit more than one signaling pathway to produce clinical response. Future studies with more potent Akt inhibitors should examine the validity of this approach in conjunction with pharmacodynamic monitoring. Disclosures: Carroll: Glaxo Smith Kline, Inc.: Research Funding; Sanofi Aventis Corporation: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding. Gojo:Keryx Inc.: Research Funding. Off Label Use: Neither UCN-01 nor perifosine have an approved indication.

Abstract 230 Background: Mammalian target of rapamycin (mTOR) inhibitors enhance cytotoxic chemotherapy effects in primary acute leukemia samples in preclinical assays, prompting multi-center evaluation of regimens combining mTOR inhibitors plus induction chemotherapy in AML. As mTOR is frequently but not uniformly activated in primary AML samples, it is unclear which patients benefit from this targeted approach. Thus, we sought to monitor mTOR kinase activity during therapy to determine whether target activation and/or inhibition predicted clinical response. We previously reported the feasibility of real-time, pharmacodynamic monitoring of ribosomal S6 phosphorylation (pS6) in leukemic blasts using flow cytometry of fixed whole blood (Perl et al, ASH 2009, #413). mTOR directly regulates the p70S6 kinase and its phosphorylation of S6 at serines 235/6 is inhibited by rapamycin. Thus pS6 provides a surrogate marker of mTOR kinase activity. Fixing whole blood and/or marrow preserves phosphorylation states in the presence of administered signal transduction inhibitors, thus avoiding cell-processing effects. Here we update our experience monitoring pS6 in AML blasts by flow during clinical trials combining sirolimus and AML induction chemotherapy. Methods: Subjects had relapsed/refractory AML or untreated AML with unfavorable risk factors (e.g. therapy-related, prior MDS or MPN, or age >60 with non-favorable karyotype) and received oral sirolimus (12 mg on day 1, then 4 mg daily on days 2–9) plus MEC (mitoxantrone 8 mg/m2/day, etoposide 100 mg/m2/day, cytarabine 1 gm/m2/d on days 4–8) on one of two successive clinical trials. Clinical response was assessed at hematologic recovery or day 42 using IWG criteria (CR, CRp, PR vs. non-response). Pharmacodynamic samples were collected from blood or marrow at baseline, 2 hours post-sirolimus dose on days 1 and 4, and at trough on day 4 (prior to chemotherapy administration). Concurrent blood rapamycin concentration was measured by immunoassay or HPLC. Whole blood/marrow fixation was performed using published methods (Chow & Hedley, Cytometry A, 2005). Positive gates for pS6 were created by comparing blasts in ex vivo stimulated (phorbol ester/PMA) and inhibited (rapamycin) conditions and/or autofluorescence (FMO) controls. Results: 27 subjects provided paired day 1 and day 4 flow samples and were evaluable for clinical response at the time of submission. Mean peak and trough rapamycin levels were 22.8 and 9.2 ng/ml, respectively, and did not differ among clinically responding and non-responding subjects. 17/27 (63%) subjects' blasts had constitutive S6 phosphorylation at baseline. Consistent with prior reports, pS6 was heterogeneous and typically present in a subset of blasts. In these 17 subjects, we observed a median of 13% pS6+ blasts (mean 14, range 2–33). 14/17 showed a therapy-induced reduction in pS6+ blasts to a mean of 4.5% (median 2.5, range 0.4–22) on day 4. The remainder had either no change or increased pS6+ blasts. Comparing the percentage of pS6+ cells on day 4 to baseline, the median reduction in pS6 differed among clinically responding and non-responding subjects (72% and 43%, respectively). The clinical response rate was 9/17 (53%, 6 CR 3 PR) among subjects with baseline S6 phosphorylation and 4/10 (40%, 3 CR, 1 PR) in patients with no baseline S6 phosphorylation. Subjects with >50% reduction in pS6 blasts on day 4 were considered to be biochemically sensitive to rapamycin, while subjects with Conclusions: Data from these ongoing trials suggest that sirolimus plus MEC preferentially benefits the subset of patients with demonstrable baseline mTOR activation. The greatest response is seen when mTOR is activated in leukemic cells at baseline and its function is potently inhibited during therapy. Future trials may benefit from enrichment for subjects with mTOR activation and/or rapamycin sensitivity assessment. These data provide in vivo evidence that mTOR inhibitors augment cytotoxic chemotherapy effect in AML and demonstrate the utility of fixed whole blood flow cytometry for real-time pharmacodynamic evaluation of novel signal transduction inhibitors. Disclosures: Off Label Use: Rapamycin. FDA approved for solid organ transplant. Investigational use for treatment of leukemia. Carroll:Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

Abstract 2576 FLT3-ITD mutations in Acute Myeloid Leukemia (AML) are associated with early relapse after standard chemotherapy and poor survival. AC220 is a potent, selective, oral FLT3 tyrosine kinase inhibitor that showed promising activity in FLT3-ITD+ patients (pts) in a Phase I study. This Phase II monotherapy trial was conducted to examine the safety and efficacy of AC220 monotherapy in pts with relapsed/refractory FLT3-ITD+ AML. Patients were enrolled into two cohorts: Cohort 1 enrolled pts ≥60 yrs and relapsed/refractory to 1st-line chemotherapy and Cohort 2 pts ≥18 yrs and relapsed/refractory to 2nd-line chemotherapy or hematopoietic stem cell transplantation (HSCT). A planned analysis was performed in February 2011 on the first 62 pts which comprised the exploratory group with the subsequent ∼240 pts being the confirmatory group for which the data will remain sequestered until study completion. The exploratory group was enrolled between 19 November 2009 and 25 August 2010. 53/62 (85%) pts were evaluable for efficacy (FLT3-ITD+ by central laboratory, received at least 1 dose of AC220, 1 post-tx response assessment and no major efficacy-related protocol deviations). The composite CR (CRc=CR+CRp+CRi) rate was 45% (24/53: 2 CRp, 22 CRi) and PR rate was 24% (13/53). Importantly, of the pts refractory to any prior therapy, 62% (16/26) had CRc and 19% (5/26) had PR in response to AC220. Median duration of CRc has not yet been reached in Cohort 1 and was 10.6 wks in Cohort 2. Overall, 8% (2/25) of pts in Cohort 1 and 30% (11/37) of pts in Cohort 2 underwent HSCT and were censored for duration of CRc. Median overall survival was 24.7 weeks for efficacy evaluable pts, 24.1 wks for Cohort 1 and not yet reached in Cohort 2 (pts were not censored at HSCT). At the time of the analysis 51/62 patients were off study (most commonly due to disease progression (19), HSCT (13) or adverse event (7). 55% of pts were still alive. The most common (>19%) drug-related AEs were nausea, QTc prolongation, vomiting, fatigue, dysgeusia, anorexia, febrile neutropenia, diarrhea, and dyspepsia. QTc prolongation occurred in 21 (34%) pts (Grade 3 in 11 pts, 18%). The incidence of QTc prolongation was decreased by reducing AC220 starting dose from 200 (35%) to 135 mg/day (8.3%) (males) and 90 mg/day (5.9%) (females). 15 pts (24%) experienced fatal treatment-emergent AEs; none were considered drug-related. An additional analysis will be conducted when all pts have > 1 yr follow up which will be available at the time of the meeting. These preliminary data suggest that AC220 achieves clinically meaningful reductions in marrow blasts in a substantial proportion of pts with both refractory and relapsed FLT3-ITD+ AML, and many of these pts were successfully bridged to HSCT. These encouraging efficacy results and an acceptable safety profile in this high risk population support continued clinical evaluation in mono- and combination therapy. Disclosures: Cortes: Ambit: Research Funding; Novartis: Research Funding.

Abstract 3508 Although first-generation FLT3 inhibitors may have had limited anti-leukemic effects due to suboptimal target inhibition, newer drugs such as AC220 and KW-2449 have substantially greater in vitro potency and bioavailability. Ex vivo assays such as the plasma inhibition assay (PIA) are useful to estimate free drug bioavailability, but direct confirmation of biochemical FLT3 inhibition in leukemic blasts in vivo has proven more challenging to employ systematically for drug development. Here we report the development of a fixed whole blood intracellular flow cytometry platform to measure real-time signal inhibition during a clinical trial of the second-generation FLT3 inhibitor KW-2449. Methods: Anticoagulated blood samples were aliquoted into FACS tubes within four hours of collection; a subset was exposed to signaling inhibitors (KW-2449, rapamycin × 30 min.) or activators (phorbol ester/PMA or FLT3 ligand/FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed, red cells were lysed with the permeabilizing agent triton X-100, and specimens were stored at −20C in glycerol medium. Subjects' samples from all time points were simultaneously thawed, denatured with ice-cold methanol, and stained with a single cocktail of antibodies. Blasts were identified by CD45 and side scatter (SSC) and confirmed by multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Positive gates for phospho-proteins were created by comparing blasts in stimulated and unstimulated conditions and/or autofluorescence (FMO) controls. Results: Despite adequate controls, flow demonstrated limited changes in FLT3-ITD+ blasts' pSTAT5 signal following either FL stimulation or ex vivo KW-2449 treatment of these peripheral blood primary samples. This contrasted with the FLT3-ITD+ cell line Molm14, in which FLT3 inhibition reduced pSTAT5. However, the PI3K/AKT/mTOR downstream target ribosomal protein S6 (S6) was consistently observed to be constitutively phosphorylated in both Molm14 cells and peripheral blood FLT3-ITD+ AML blasts. pS6 in all FLT3-ITD+ samples markedly augmented with ex vivo FL, and decreased following ex vivo KW-2449 treatment. We therefore serially monitored S6 phosphorylation during therapy on a phase 1/2 trial of KW-2449. In this clinical trial, subjects were treated with KW-2449 every 6–8 hours, due to the drug's relatively short half life. 10 subjects (9 FLT3-ITD+, 1 FLT3-WT) provided serial blood samples for analysis. All FLT3-ITD+ subjects had blasts identifiable by morphology and immunophenotype. Samples with as few as 500 blasts/uL were informative for pS6. In all cases, blasts showed dynamic changes in pS6 in response to ex vivo FL. As previously described using intracellular flow cytometry, pS6 in primary AML samples was heterogeneous, and, at basal state, frequently only demonstrable in a subset of blasts. We observed constitutive S6 phosphorylation in 8/9 subjects' leukemic cells. The mean percentage of blasts with constitutive pS6 was 21% (median 7%, range 5–70%). To directly quantify FLT3 kinase inhibition in vivo, we serially monitored pS6 in blasts by flow prior to and following their initial oral KW-2449 dose. In 8/8 patients with baseline constitutive S6 phosphorylation, blood obtained two hours following the initial dose showed marked reduction in the percentage of pS6+ blasts to a mean of 3.8% (median 1.3% range 0.1 to 20%). This reflected an 83% mean reduction in the percentage of pS6+ blasts. PIA was performed in 8/9 of FLT3-ITD+ subjects and confirmed that potent FLT3-inhibitory concentrations were present 2 hours after a single dose of KW-2449 (mean reduction from baseline of 79% for pFLT3 and 88% for pSTAT5). Two subjects' samples were followed serially by flow cytometry throughout the dosing interval. One showed sustained inhibition (consistent with concurrent PIA), while in the other, pS6 returned to baseline within 4–6 hours of the initial dose (concurrent PIA not done). Summary: We confirm that PI3K/AKT/mTOR is a major downstream pathway of FLT3 signaling in primary AML samples. We further demonstrate the feasibility of intracellular flow cytometry for S6 phosphorylation to monitor the biochemical efficacy of FLT3 inhibitors in patients. Studies are underway to correlate biochemical FLT3 inhibition by flow cytometry with clinical response/resistance to KW-2449 and other FLT3 inhibitors. Disclosures: Sato: Kyowa Hakko Kirin Co., LTD: Employment. Akinaga:Kyowa Hakko Kirin Co., LTD: Employment. Rao:Kyowa Hakko Kirin Co., LTD: Employment. Levis:Kyowa Hakko Kirin Co., LTD: Research Funding; Ambit Biosciences: Consultancy. Carroll:Kyowa Hakko Kirin Co., LTD: Research Funding.

Abstract 943 FLT3/ITD mutations are present in approximately 23% of adult AML cases. FLT3/ITD therefore presents an attractive therapeutic target for tyrosine kinase inhibitors. To date, the clinical responses to FLT3 inhibitors have been primarily limited to clearance of peripheral blood blasts, while bone marrow blasts remain largely unaffected. AC220 (quizartinib), a novel FLT3 inhibitor with 10–50 times more potency in vivo than other FLT3 inhibitors, has had a high response rate in an exploratory subset of patients in an ongoing clinical trial for relapsed and refractory FLT3/ITD AML. A high proportion of FLT3/ITD AML patients treated with AC220 displayed the typical rapid clearance of peripheral blasts seen with other FLT3 inhibitors. Strikingly, however, (as reported at last year's ASH meeting), the bone marrow blasts underwent terminal differentiation in response to AC220 treatment over the course of 4–8 weeks, as established by the presence of the same FLT3/ITD mutation in mature circulating neutrophils isolated after treatment with AC220. This differentiation was frequently accompanied by a syndrome of fever, skin nodules resembling Sweet's syndrome, and pulmonary nodules. To study the mechanisms associated with this phenomenon, we developed an in vitro model of FLT3 inhibitor-induced differentiation. In order to more closely reproduce the bone marrow microenvironment in vitro, we prepared human stromal cell layers derived from healthy bone marrow donors and co-cultured them with Molm14 cells (FLT3/ITD cell line), as well as with primary AML blasts from FLT3/ITD AML patients in the presence and absence of AC220. In the absence of co-culture with stroma, both the Molm14 cell line and the primary samples underwent rapid apoptosis in the presence of increasing concentrations of AC220, similar to the response of peripheral blood blasts in AC220 -treated patients. However, when grown in co-culture with stroma, Molm14 cells underwent relatively rapid (24–48 hours) morphologic differentiation, as evidenced by the development of characteristic multi-lobed nuclei, and by reduction of nitroblue tetrazolium (NBT) after stimulation with endotoxin (respiratory burst activity). Similar data were observed in Molm14 cells treated with the FLT3 inhibitor sorafenib, indicating that this differentiation is likely a class effect of FLT3 inhibitors. We have also used our model system to study differentiation in primary AML patient blasts. Pre-treatment blasts were obtained from a patient enrolled on the AC220 trial (who exhibited terminal differentiation of marrow blasts in response to AC220), and differentiation was induced in vitro by co-culturing the blasts with stroma in the presence of AC220. After five days of drug exposure during stromal co-culture, the blasts remained viable (assessed by trypan blue exclusion), but morphologically began to resemble myelocytes, and demonstrated reduction of NBT in response to endotoxin. Flow cytometry analysis of these blasts after eight days of AC220 exposure demonstrated increased side scatter, expression of CD15, and loss of CD117 and CD34, all indicative of maturation. Blasts cultured in the absence of stroma displayed decreased P-FLT3, P-STAT5, and P-ERK in response to AC220. In contrast, blasts exposed to AC220 on stroma demonstrated continued activity of P-ERK despite loss of P-FLT3 and P-STAT5. CEBPalpha protein levels were significantly higher in blasts on stroma, but decreased rapidly over the first 48 hours of culture. These findings confirm that a FLT3/ITD mutation can be sufficient to induce a differentiation block, and, in the bone marrow microenvironment, FLT3 inhibition does not induce immediate apotosis, but rather differentiation. The continued activity of P-ERK (presumably by parallel signaling pathways activated by stromal cells) may be responsible for the survival of the blasts in the setting of FLT3 inhibition, with CEBPalpha inducing a differentiation program in this context. We conclude, therefore, that the response of a FLT3/ITD AML patient to potent FLT3 inhibition consists of rapid apoptosis of blasts in the peripheral blood, and, in most cases a slower process of terminal differentiation in bone marrow blasts. Furthermore, our in vitro model will allow us to explore the precise mechanisms whereby differentiation is blocked in FLT3/ITD AML, and how this can be overcome with FLT3 inhibition and other targeted therapies. Disclosures: Borowitz: BD Biosciences: Research Funding. Levis:Ambit Biosciences, Inc: Consultancy.

Abstract 2366 Background: Allogeneic hematopoietic stem cell transplant (HSCT) has been shown to be of some benefit for patients with acute myelogenous leukemia (AML) with untreated early relapse. However, outcomes for patients who have morphologic evidence of relapsed leukemia are not well defined. We describe our experience with HSCT for patients with active AML at the time of transplant, to determine prognostic factors for outcome after transplant. Patients and Methods: We analyzed recipients of myeloablative HSCT from 1997 to 2009 at our institution with morphologically active AML at the time of transplant. Patients were identified through our transplant database, and disease status was verified through medical record review. Forty patients were included based on the presence of circulating blasts at the time of admission for transplant, and/or a positive bone marrow biopsy (>5% bone marrow blasts) immediately prior to transplant. There were 6 patients coded as “relapsed/refractory” in our database whose disease status at the time of transplant was unable to be confirmed and thus were excluded from analysis. Results: Baseline patient and transplant characteristics are shown in Table 1. 30% of patients were transplanted for primary induction failure, 22% for first untreated relapse (no treatment between first documented relapse and HSCT), 35% for first refractory relapse (did not attain CR with treatment administered at time of first relapse), and 13% had second or greater refractory relapse. The overall survival for all patients was 82% at 30 days; 55% at 100 days; 28% at one year; and 18% at two years post-HSCT. Of 31 deaths, 71% were attributable to disease, 19% to regimen-related toxicity and infection, and 10% to graft-versus-host-disease (GVHD). Seven patients remain alive at the time of analysis, with 6 patients (15%) alive and free of disease more than 3 years post-HSCT. The median follow-up of the disease-free survivors is 9 years; Table 1 shows characteristics for this subgroup. We identified several patterns of interest. Four disease-free survivors were transplanted in first untreated relapse, yielding 44% (4/9 patients) with first untreated relapse that are long-term disease-free survivors. In addition, while most patients were transplanted with circulating blasts, 5 of 6 disease-free survivors did not have circulating disease. Although 40% of transplants were from unrelated donors, 5 of 6 disease-free survivors received sibling transplants. Conclusions: Our review of patients with morphologically apparent relapsed or refractory AML confirmed that a subset of patients can achieve a durable remission from HSCT. The majority of these long-term survivors shared important characteristics, including (1) lack of circulating blasts at transplant, (2) sibling donors, and (3) first untreated relapse disease status. This information may serve a prognostic purpose, and may assist in identifying appropriate candidates for transplant or for alternative therapies. However, it was not possible based on our analysis to predict reliably those who would not experience long-term survival. Except for second or greater refractory relapse, there was no factor that identified patients who had no benefit from HSCT. As such, HSCT remains a viable option with the potential for long-term disease-free survival in this population. Disclosures: No relevant conflicts of interest to declare.

Abstract 4643 Systemic steroids are the mainstay of therapy for GVHD, but treatment failure is common. Inflammatory cytokines, including IL-2 and TNF-a, are important mediators of GVHD, and may be critical targets for therapy. Given the ineffective treatment options for patients (pts) with steroid-refractory GVHD (SR-GVHD) and the almost universally poor outcomes with available therapies, between June 2001-May 2008, we treated 22 patients with SR-GVHD with a combination of anti-cytokine therapy that included daclizumab and infliximab. Seventeen of these patients were evaluable for a retrospective review of detailed outcomes. The median age was 48 years (range 35-63) and grafts were from HLA matched siblings (5), matched unrelated donors (9), 1 antigen mismatched unrelated donors (2), or cord blood (1). Indications for stem cell transplant (SCT) were acute leukemia (9), chronic myelogenous leukemia (1), lymphoma (5) chronic lymphocytic leukemia (1) and multiple myeloma (1). Most patients received methotrexate with either tacrolimus (14) or cyclosporine (CSA) (2) and 1 patient received CSA with steroids as initial GVHD prophylaxis. High dose steroids were started for acute GVHD a median of 39 days after transplant in 12 pts (range 25-119). Five additional patients developed SR-GVHD a median of 46 days (range 25-119) after donor lymphocyte infusion. All patients had persistent or progressive acute GVHD despite 1mg/kg/day (4 pts) or 2 mg/kg/day (13 pts) of corticosteroids for a median of 8 days (range 5-26) and were treated for acute GVHD severity index B (3), C (10) or D (4). GVHD involved the skin in 9 pts, liver in 9 pts, and gut in 15 pts. Daclizumab was given at 1.5 mg/kg day 1 and 1 mg/kg day 4, 8, 15, and 22. Infliximab was given at 10 mg/kg day 1,8,15, and 22. Overall, 47% of patients responded to therapy. Four patients (24%) had complete resolution of symptoms and 4 (24%) had partial responses. The remaining 9 patients failed to respond, progressed or had a mixed response. Additional therapies were started in 7 patients. Similar to other available reports on SR-GVHD, survival was limited and all pts died at a median of 6.7 months (range 1.6-26) from transplant and 37 days from initiation of daclizumab/ infliximab (8 from infection, 5 from GVHD, 2 from relapsed disease, and 2 from other causes). Data from additional patients will be presented. Although this is a retrospective analysis, these results suggest that combination anti-cytokine therapy with daclizumab/infliximab has significant activity in SR-GVHD, but outcomes remain poor. New methods to prevent and treat GVHD are urgently needed. Disclosures: Off Label Use: Daclizumab and Infliximab were used to treat acute Graft versus Host Disease. Porter:Genentec: Spouse employed by Genentech, owned by Roche, manufacturer of daclizumab.

Abstract 413 The mammalian target of rapamycin (mTOR) is an emerging molecular target in cancer therapy, however the relationship between target activation in individual tumors as well as inhibition of target and clinical response is poorly defined. This is in large part due to the difficulty of real-time monitoring of mTOR activation in patient samples. mTOR is activated in acute myelogneous leukemia (AML) cells and the mTOR inhibitor rapamycin enhances cytotoxic effects of chemotherapy in vitro and in vivo. We therefore developed a new application of a recently-developed, whole blood fixation/permeabilization technique for intracellular flow cytometry (Chow, et al. Cytometry A 2005). Using this approach, we sought to serially monitor S6 ribosomal protein (S6) phosphorylation in peripheral blood leukemic blasts during clinical trials of mTOR inhibitors. S6 is a known target of mTOR and its phosphorylation is a surrogate marker for mTOR kinase activation. We applied this methodology during a recent pilot trial in which an oral mTOR inhibitor, sirolimus (rapamycin), was administered in sequence with intensive combination chemotherapy (mitoxantrone, etoposide, and cytarabine, or MEC) in patients with relapsed, refractory, or secondary AML. The whole blood fixation process sufficiently preserved surface epitopes and light scatter properties for immunophenotyping, allowing specific signaling analysis of leukemic blasts as well as non-malignant cell populations. Importantly, even leukopenic trial samples containing as few as 20% blasts provided robust signaling data in malignant cells. S6 phosphorylation was readily apparent in leukemic blasts prior to therapy and, consistent with prior reports, occurred only in a subset of blasts. Exposing aliquots of pre-treatment whole blood samples to increasing concentrations of rapamycin ex vivo determined that leukemic blasts from most samples showed inhibition of S6 phosphorylation at clinically achievable concentrations (between 10-20 nM). Notably, some subjects' leukemic blasts showed no inhibition to >50 nM rapamycin, which far exceeded trough concentrations measured on our studies. To examine rapamycin's in vivo biochemical effects, we performed a paired analysis of clinical samples drawn at study entry and after 72 hours of oral sirolimus. 10 subjects provided paired samples, of which 2 did not show baseline S6 phosphorylation, 6 showed baseline S6 phosphorylation that inhibited during therapy, and 2 showed baseline S6 phosphorylation but no inhibition. Trough rapamycin levels were similar among rapamycin responsive and resistant subjects. Considering the 6 subjects with in vivo mTOR inhibition, 3 subjects achieved complete or partial remissions from the regimen. Neither subject with in vivo rapamycin resistance had a clinical response. Overall, we conclude that effective inhibition of mTOR signaling in AML blasts occurs in the majority of subjects during sirolimus treatment at the dose studied. However, cell-intrinsic rapamycin resistance occurs in a minority of patients and requires further study to clarify its mechanism and effects upon concurrent chemotherapy response. These data demonstrate the feasibility of real-time, intra-tumoral pharmacodynamic monitoring of S6 phosphorylation by flow cytometry during clinical trials combining intensive chemotherapy and signal transduction inhibitors for leukemia. Our approach greatly clarifies pharmacokinetic/pharmacodynamic relationships and has broad application to pre-clinical and clinical testing of drugs whose direct or downstream effects disrupt PI3K/AKT/mTOR signaling. Such compounds include inhibitors of FLT3, c-KIT, BCR-ABL, JAK2, and ras/raf/MAPK. Multicenter/cooperative group phase II testing of sirolimus plus MEC in AML has been initiated to establish the regimen's response rate and test the extent to which our pharmacodynamic studies predict clinical response. Disclosures: Off Label Use: The use of sirolimus in the therapy of AML is investigational and off-label. Carroll:Cephalon consultancy: Consultancy; Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceutical: Research Funding.

Abstract 788 FLT3 mutations are common in AML and are associated with poor prognosis. Lestaurtinib is a multi-targeted kinase inhibitor with potent activity against FLT3. In an attempt to definitively establish whether or not there is clinical benefit for FLT3 inhibition in the relapse setting, we conducted a randomized trial of chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse. Patients were randomized to receive either chemotherapy alone, consisting of mitoxantrone, etoposide, and cytarabine (MEC) or high-dose cytarabine (HiDAc) depending on the duration of CR1) or chemotherapy followed by 80 mg lestaurtinib orally BID. Patients on the chemotherapy only arm were eligible for crossover if they were refractory to therapy. Patients on the lestaurtinib arm received the drug for up to 112 days on study (they could continue to receive the drug through a separate extension protocol). The primary objective was complete remission (CR/CRp). Secondary objectives included overall survival (OS), safety, and tolerability. From January 2004 through December 2008, 224 patients at 52 different centers were enrolled and randomized, with 220 patients actually receiving therapy. Blood samples from patients on the lestaurtinib arm were obtained at baseline, on Day 15, and on Day 42 for pK studies and for determination of in vivo FLT3 inhibition and measurement of plasma FLT3 ligand (FL) and alpha-1 acid glycoprotein (AGP) levels. The median age for all patients enrolled was 55 years (53.5 in the control arm, 58.5 in the lestaurtinib arm), with 88% having FLT3/ITD mutations, 8% having D835 mutations, and 4% having both. The duration of CR1 was less than 6 months in 47% of patients in each arm. On the lestaurtinib arm, 107 patients received at least 1 week of lestaurtinib (mean 60.1 days, range 8-418), with 17 of these patients receiving drug on the extension protocol. On the chemotherapy only arm, 7 patients crossed over to receive lestaurtinib, and 31 patients went on to receive lestaurtinib on the extension protocol after outcome evaluation. Lestaurtinib was generally well tolerated following chemotherapy, with 104 Grade 3/4 adverse events in the lestaurtinib arm versus 99 in the control arm. There were 61 serious adverse events in the lestaurtinib arm versus 49 in the control arm. Early death (by Day 42) occurred in 22 lestaurtinib patients (2 progressive leukemia, 14 infectious, 6 organ failure) and16 control patients (6 progressive leukemia, 5 infectious, 5 organ failure). By intention to treat analysis, there were 29 CR/CRp in the lestaurtinib arm and 23 in the control arm (26% versus 21%; p = 0.35), with no difference in OS (4.73 months versus 4.57 months) between the two arms. Blood samples from 79 of the 107 lestaurtinib-treated patients obtained on Day 15 were analyzed for FLT3 inhibitory activity (plasma inhibitory activity [PIA] assay), as well as FLT3 ligand (FL), lestaurtinib, and AGP concentrations. Mean lestaurtinib plasma concentration was 12 uM at Day 15 and fell to 7.5 uM on Day 42. Mean lestaurtinib concentration at Day 15 was 13.3 uM in patients achieving CR/CRp and 11.5uM in non-responders. A target of >85% inhibition of FLT3 maintained at trough (pre-dose) was defined from previous studies. Of the 79 patients tested, 46 (58%) achieved this degree of FLT3 inhibition on Day 15. FL concentrations rose from baseline (15.6 pg/mL) to Day 15 (1148 pg/mL), and AGP concentrations rose by an average of 52% over the same period, both of which may have reduced the degree of FLT3 inhibition. Of the 46 patients with target FLT3 inhibition on Day 15, 18 (39%) achieved CR/CRp, while only 3 (9%) of the 32 patients with below target FLT3 inhibition achieved CR/CRp. Thus, in patients with FLT3 mutant AML at first relapse, pharmacokinetic factors and possible physiologic factors (FL and AGP) limit lestaurtinib's ability to effectively inhibit FLT3. FLT3 inhibition by lestaurtinib, when achieved, correlates with better CR rates, but in this trial that benefit was negated by a poor CR rate in those patients for whom the drug did not reach the target level of FLT3 inhibition. Overall, lestaurtinib treatment following re-induction chemotherapy failed to increase response rates or prolong survival of patients with FLT3 mutant AML in first relapse. Disclosures: Levis: Cephalon: Clinical Advisory Board member. Ravandi:Cephalon: Honoraria, Member, clinical advisory board, Research Funding. Erba:Cephalon: Research Funding, Speakers Bureau. Baccarani:Novartis Pharma, Bristol Myers Squibb, Merck Sharp & Dome, Pfizer: Consultancy, Speakers Bureau. Stone:Cephalon: ad hoc consultancy; Novartis: Research Funding, ad hoc consultancy. Advani:Cephalon: Research Funding. Douer:Cephalon: Honoraria, Research Funding, Speakers Bureau. Litzow:Cephalon: Research Funding. Tremmel:Cephalon: Employment, Equity Ownership. Bensen-Kennedy:cephalon: Employment. Smith:Cephalon: Research Funding.

In vitro, bexarotene inhibits the proliferation of non-M3 AML cell lines and induces differentiation of leukemic blasts. Our previous phase I study in non-M3 AML showed evidence of leukemic response as manifested by reduction in bone marrow blast counts (15% response rate), improved platelet counts (41%) and improved neutrophil counts (26%). Based on these results, a phase II trial in non-M3 AML was initiated at the phase I MTD. In the current phase II trial, bexarotene (300mg/m2) was administered daily as monotherapy until disease progression or unacceptable side effects occurred. Fourteen patients have been enrolled: 8M/6F, median age 74 (range 20–83), 9 secondary AML (MDS or prior chemotherapy), 9 primary refractory or relapsed < 1 year after induction, 5 no prior induction chemotherapy, 5 requiring hydroxyurea at the time of enrollment for leukemic blast control, 4 prior allogeneic stem cell transplant, 12 blood transfusion dependent, 11 platelet transfusion dependent, and 8 neutropenic. Overall, no significant adverse events were noted. All patients received prophylactic antihyperlipidemic agents and achieved good lipid control. Two patients developed mild hypothyroidism related to bexarotene. Five patients were evaluable with bone marrow biopsy at 2 months: 1 50% reduction in absolute blasts, 1 SD and 3 PD. Similar to data from our prior phase I study, evidence of clinical activity was manifested as platelet count response in 1 patient and neutrophil increases attributable to bexarotene in 2 patients. When combining the results of our phase I experience (27 patients) with our phase II data (14 patients), there is a suggestion of increased activity in patients with 5q minus abnormalities with 4/7 (57%) benefiting (2 BM response, 4 neutrophil improvements and 1 platelet response). Conversely rates of clinical benefit were lower in patients with multiple (>3) cytogenetic abnormalities (2/13), relapse after stem cell transplant (1/9) or requiring hydroxyurea for peripheral blast control at the time of study enrollment (0/10). Bexarotene is very well tolerated at the dose level studied. Early evidence for clinical activity has been seen as exemplified by improvement in platelet count, increased neutrophil counts and decreased bone marrow blasts. In summary, we conclude that bexarotene is an active agent in a subgroup of patients with AML. Study enrollment continues with amended inclusion criteria to focus on patients more likely to benefit from treatment.

BACKGROUND: Conventional treatment options for patients with relapsed hematologic malignancies are both limited and highly toxic driving the pursuit of more tumor specific and less toxic therapies. RNA targeted oligonucleotides are potentially powerful drugs with the ability to silence genes required for malignant hematopoietic cell growth at the post transcriptional level. Studies from our laboratory have validated the c-myb proto-oncogene, which regulates important hematopoietic cell functions and is overexpressed in many hematologic malignancies, as a target for this technology. A prior Phase I trial using a 24 nucleotide phosphorothioated antisense oligodeoxynucleotide targeted to c-Myb mRNA (C-MYB AS ODN) did not identify a maximum tolerated dose (MTD) of the drug. Here we report initial results of a follow up Phase I dose escalation trial using C-MYB AS ODN at higher dose levels than previously studied in subjects with refractory hematologic malignancies. METHODS: C-MYB AS ODN is administered as a 7 day continuous infusion. 5 dose levels ranging from 3mg/kg/day to 12mg/kg/day are planned. Subjects are enrolled using an accelerated dose escalation scheme in which one subject is enrolled on each dose level (DL) with plans to revert to the standard 3+3 design in the event of significant attributable toxcity. C-MYB AS ODN concentrations are measured in peripheral blood (PB) and in mononuclear cells (MNC) by slot blotting at baseline, days 3 and 7 of infusion and two weeks after cessation of infusion. C-myb expression is assessed at these timepoints though QRT-PCR for c-myb RNA. Disease specific assessments of response are measured at predefined timepoints after therapy. RESULTS: 6 subjects, all with refractory acute myelogenous leukemia (AML) have enrolled to date. Escalation through the first 3 DLs occurred without any toxicities. At DL4 (10mg/kg/day) abnormalities have been noted in coagulation assays. The first subject enrolled on DL 4 developed a grade 3 prolongation of the activated partial thromboplastin time (PTT) attributable to drug which returned to normal within 48 hours of drug cessation. Factor levels, DIC parameters and reptilase time were normal. The PTT abnormality was consistent with a “lupus like” inhibitor effect (DRVVT was abnormal and the PTT corrected with the addition of phospholipid in two independent tests.) The 2nd subject treated at DL 4 developed a milder but similar PTT prolongation. The 3rd subject enrolled on DL 4 had a normal PTT throughout therapy. No subjects developed bleeding complications. Plasma and intracellular drug concentrations were dose related (320–640pg/l and 2–80 ng/5×10e6 cells respectively). Peak drug concentrations were found on Days 3–7. By 14 days after infusion, most ODN was cleared from plasma, but remained measurable in MNC at concentrations 30–50% of the maximum value detected. QRT-PCR for c-myb mRNA was performed in 3 subjects. Subject 1’s (DL 1) c-myb mRNA levels gradually decreased from baseline during infusion and nadired two weeks after cessation of infusion. Subjects 3 (DL 3) & 5 (DL 4) had a decrease in c-myb mRNA levels midway through infusion but c-myb RNA levels increased back to baseline by cessation of infusion. To date no subject has had a clinically important response to therapy. Accrual continues for DL 5. CONCLUSIONS: C-MYB AS ODN is detectable in plasma and MNCs of subjects during continuous drug infusion with a steady state reached by day 3 of infusion. Plasma drug levels were markedly reduced 14 days after cessation of infusion but MNC drug levels remained elevated. C-MYB AS ODN at DL 4 (10mg/kg/day) is associated with a PTT prolongation consistent with a “lupus inhibitor” like effect. While encouraging biological activity was identified optimal dose and delivery remain to be established before clinically significant effects can be reasonably expected.

Recent results have demonstrated that multiple signal transduction pathways are activated in acute myeloid leukemia (AML) cells, however, the tyrosine kinase(s) that phosphorylates these signaling proteins is not identified. We have analyzed AML cells using a phosphoproteomics screen and demonstrate that the Src family kinases, Lyn, Lck and Fgr, are phosphorylated on their activation sites in AML samples. Expression and activation of Lyn has been previously confirmed. Evaluation of Lck demonstrated that Lck is expressed to a variable degree but consistently in AML samples (n=20). Lck kinase assays show activation of Lck in 17/20 samples tested at levels above the level of activation detectable in normal CD34+ progenitor cells. Lyn and Lck both contribute to AML cell growth as siRNA depletion of either kinase leads to decreased leukemia colony forming activity. Interestingly, both Lyn and Lck contribute to phosphorylation of STAT5 as STAT5 phosphorylation is decreased but not abrogated by siRNA modification of either kinase alone. Consistent with the necessity for this signaling pathway for optimal AML cell growth, siRNA knockdown of STAT5 leads to decreased expression of both STAT5A and STAT5B, decreased expression of the STAT5 target protein, Bclxl and decreased AML colony forming ability. Based on this data, we have studied the FDA approved compound, Dasatinib, and demonstrate that Dasatinib decreases AML colony formation in 4 out of 5 samples tested. Overall, these results demonstrate that SFK’s act redundantly to regulate STAT5 phosphorylation and AML cell growth in primary cells and that phosphoprotein analysis is a robust approach to identify new targets for therapy of malignancy. Src family kinase inhibitors may be valuable in the therapy of AML.

Xenotransplantation of human acute myeloid leukemia (AML) in immunocompromised animals has been critical for the definition of leukemic stem cells. However, existing immunodeficient strains such as NOD/SCID and NOD/SCID/b2mnull have short life spans, age dependent leakiness of humoral immunity and low levels of AML cell engraftment making long-term evaluation of primary human AML biology difficult. A recent study suggested that the nonobese diabetic/severe combined immondeficient/IL2Rgnull (NOG) mouse has enhanced ability to engraft AML cells but this study relied on neonatal injections that are technically challenging. We performed an extensive analysis of AML engraftment in adult NOG mice using intravenous tail vein injection. Thirty-six different AML samples were analyzed including 2 samples of acute promyelocytic leukemia (APML). We used a threshold for AML engraftment of >0.5% human CD45+33+ cells in the murine bone marrow. Based on this threshold, 22 samples (61%) showed engraftment in NOG mice. Of these samples, 14 (64%) showed high levels of engraftment (greater than 10% of murine marrow replaced with human CD45+CD33+ cells). Engraftment did not correlate with FAB subtype or cytogenetic abnormalities to a statistically significant degree, however we noted that one sample with an 11q23 translocation and several samples with Flt3 ITD mutations showed consistent high level engraftment. Several samples demonstrated engraftment as high as 95% of the murine marrow with total AML cell expansion of 2-30 fold. Evaluation of AML stem cell frequency and expansion is ongoing. Engraftment in spleen was variable and in general significantly lower than in bone marrow. For most samples, peripheral blood engraftment was barely detectable. In contrast to NOD/SCID mice, both APML samples engrafted well in the NOG mouse with high levels of peripheral blood involvement. Some samples occasionally showed engraftment of a population of cells expressing CD2 and other T cell associated markers by flow cytometry, however this observation was inconsistent even between mice injected with the same sample. All samples tested (n=5) showed consistent engraftment in secondary and tertiary recipients with most samples tested showing further expansion of total AML cells in subsequent transplants. Importantly, a number of animals developed organomegaly and a wasting illness consistent with advanced leukemic disease. Several such animals showed extramedullary leukemic infiltration into non-hematopoietic tissues. Etoposide monotherapy (40 mg/kg in divided doses) of heavily engrafted mice did not induce a significant response in terms of leukemia regression. Studies of other chemotherapeutic agents are ongoing. We conclude that the NOG xenotransplantation model is a robust model for studying human AML cell engraftment which will allow for better characterization of AML biology and testing of new therapies

In vitro, bexarotene has been shown to inhibit the proliferation of non-M3 AML cell lines and induce differentiation of leukemic blasts. This phase I study was designed to evaluate the safety of escalating doses of bexarotene in patients with non-M3 AML. Bexarotene was administered orally daily until disease progression occurred. Prophylactic antihyperlipidemic agents were used in all patients. Six dose levels ranging from 100 to 400mg/m2 are planned. Dose escalation occurred in cohorts of 3–6 patients based on dose-limiting toxicity. Twenty patients have been enrolled in 5 dose cohorts (100–300mg/m2) with enrollment demographics: 13M/7F, median age 69 (range 51–82), 11 prior MDS, 8 primary refractory, median number of induction attempts 2, no prior induction chemotherapy 2, prior autologous stem cell transplant 4, 19 blood transfusion dependent, 13 platelet transfusion dependent, and 16 neutropenic. Consistent with reported toxicity, 2 patients developed hypothyroidism, 7 patients developed grade 2–4 hypertriglyceridemia and 1 patient developed grade 2 pancreatitis. Two patients developed a syndrome reminiscent of retinoic acid syndrome, consisting of dyspnea/hypoxia, pleural/pericardial effusions, weight gain/edema and dry cough in the setting of a rapidly rising neutrophil count. This syndrome resolved within 48 hours of stopping bexarotene and initiating steroids. No CR’s were noted, however significant evidence of drug activity were seen. Six patients showed evidence of neutrophil response (pretreatment median ANC 286/μL, range 28–1,037/μL, posttreatment ANC 3,150/μL, range 1,100–27,207/μL). Flow sorted peripheral blood neutrophils were collected from three of these patients and examined by FISH. Between 92–100% of purified neutrophils contained the patient’s leukemic cytogenetic abnormality suggesting differentiation of the leukemic blasts. Bone marrow blasts decreased to 30,000/μL (peak range 40–292x103/μL). Four of these patients with platelet counts

In vitro studies have suggested that AML cells are sensitive to treatment with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. In particular, mTOR inhibition is known to enhance the sensitivity of primary AML cells and AML stem cells to etoposide based chemotherapy leading to inhibition of leukemic SRC activity in NOD/SCID mice. To determine the feasibility of applying this approach in vivo, we performed a Phase I dose escalation study of the mTOR inhibitor sirolimus (rapamycin) with a combination chemotherapy induction regimen in adults with relapsed or refractory non-M3 AML. The purpose of this trial was to determine the safety and dose limiting toxicities of sirolimus and chemotherapy in this patient population. Patients received a loading dose of oral sirolimus on day 1 followed 24 hours later by daily doses of oral sirolimus on days 2–7 plus MEC (mitoxantrone 8 mg/m2/day IV, etoposide 100 mg/m2/day IV, and cytarabine 1000 mg/m2/day IV) on days 1–5. Five sirolimus dose levels were explored by a standard 3+3 design. Sirolimus was studied at loading doses from 3–15 mg and daily doses from 1–5 mg/d. Clinical response was assessed by bone marrow biopsy upon hematologic recovery or day 42, whichever occurred first. 23 adults (14 women, 9 men) of median age 58 (range 22 to 65) with relapsed, refractory, or secondary AML were treated with sirolimus and MEC. Five subjects had antecedent hematologic disorders or prior leukemogenic chemotherapy, 18 had relapsed or refractory disease. Sirolimus was well tolerated and did not increase non-hematologic toxicity of MEC chemotherapy. Asymptomatic, reversible liver transaminase or bilirubin elevations occurred in 4 patients, two of which were > grade 2. One patient with a history of prior cytarabine cerebellar toxicity (unknown at the time of study entry) developed reversible cerebellar ataxia. Three patients died of complications related to bacterial infections during chemotherapy-induced aplasia. Dose limiting toxicity was prolonged myelosuppression at the highest planned dose level and was responsible for one treatment-related death due to infectious complications from unresolved aplasia on study day 119. For the first four dose levels the median time to ANC recovery >500/uL among evaluable patients was 27 days (range16–38). Pharmacokinetic data showed that doses of 3 mg and higher consistently achieved rapamycin levels considered therapeutic in solid organ transplantation (4–9.2 ug/L). Bone marrow studies in 2/2 evaluable patients on dose level 4 (12 mg loading dose and 4 mg per day sirolimus) showed inhibition of p70S6 kinase phosphorylation consistent with effective inhibition of mTOR at this dose level. Complete remissions occurred in four patients, all treated for first relapse. Two patients subsequently proceeded to allogeneic transplantation. These results indicate that the combination of mTOR inhibition and chemotherapy is feasible in human AML and establish an appropriate dose for phase II studies to be 12mg loading dose followed by 4 mg daily. Patient recruitment at this dose is ongoing. Confirmation of the efficacy of this regimen, which targets signal transduction in leukemic < stem cells, is planned in a randomized phase II trial at the cooperative group level.