ALBERT

Description: 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.

Description: Key Points Serum 2HG analysis by LC-MS can accurately identify patients with AML with and without IDH mutations. Oncometabolite testing of serum 2HG is indicated as a diagnostic, prognostic, and therapeutic monitoring tool in AML.

Description: 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

Description: 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

Description: 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.

Description: 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.

Description: Abstract 2600 Background: Older patients diagnosed with acute lymphoblastic leukemia (ALL) receiving intensive induction therapy often suffer from poor outcomes due to therapy-related toxicity and high rates of relapse. We previously reported that patients age ≥60 diagnosed with ALL and treated at our institution with either hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone alternating with high-dose methotrexate and cytarabine (hyperCVAD) or daunorubicin, vincristine and prednisone (DVP) induction therapies achieved comparable rates of survival. Now with longer follow-up and further analysis, we describe additional outcomes and factors predictive of survival in this patient population. Methods: Thirty-seven patients diagnosed with ALL at age ≥60 and treated at the University of Pennsylvania between July 2003 and June 2011 who received induction therapy with either hyperCVAD (≥1 A+B cycle) or DVP (phase I+II) were analyzed. HyperCVAD was administered as first described at the MD Anderson Cancer Center and DVP per the ECOG 2993/UKALL XII protocol. Therapy adjustments and bone marrow biopsy to confirm remission were performed at the discretion of the treating physician. Almost all Philadelphia chromosome (Ph) positive patients received a tyrosine kinase inhibitor. Event-free survival (EFS) was defined as the time from diagnosis to either relapse or death from any other cause. Results: Table 1 describes baseline characteristics. Table 2 describes outcomes. If achieved, morphologic remission was recognized upon the first bone marrow assessment performed while on therapy, which occurred after a median of 4 (2 A+B) cycles of hyperCVAD and by completion of phase II induction of DVP. EFS and overall survival (OS) trended in favor of DVP. HyperCVAD patients were more likely to complete intensive therapy but less likely to receive maintenance therapy, and more likely to relapse with the majority of relapses occurring off active treatment. Primary reasons for not starting or stopping maintenance therapy were infections and cytopenias. Relapsed disease was the most frequent cause of death. Table 3 describes univariate Cox regression analysis. Receipt of maintenance therapy demonstrated the strongest association with survival (p=0.0001, hazard ratio 0.06 for EFS; p=0.0002, hazard ratio 0.05 for OS). Valid multivariate analysis could not be performed due to small sample size. Conclusions: In older ALL patients treated with aggressive induction therapies and achieving remission, receipt of maintenance therapy appears to be most predictive of EFS and OS. Outcomes in the DVP and hyperCVAD groups were similar although a trend towards prolonged survival in the DVP group was seen, which may be explained by a lower rate of relapse due to a higher likelihood of remaining on therapy over time. Our findings suggest that these patients may benefit from attenuated courses of intensive initial therapy in order to avoid developing toxicities that may prohibit tolerance of prolonged maintenance therapy. Disclosures: No relevant conflicts of interest to declare.

Description: 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

Description: 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