ALBERT

Description: Abstract 325 Background: Acute Myeloid Leukemia (AML) is the most common myeloid malignancy in adults and represents an aggressive disease with significant biological and clinical heterogeneity. Currently, cytogenetics and molecular changes are used to inform treatment strategies. However a wide range of clinical responses are observed in these patient subgroups necessitating alternative methodologies to provide information that could inform clinical decisions for AML disease management. Since the net result of the cytogenetic and molecular changes is necessarily a functional alteration of proteins within signal transduction networks the current study was undertaken to understand the diversity of signaling responses in AML patient samples. Critically, in these studies treatment of samples with a variety of input stimuli allowed intracellular phospho-signaling and apoptosis network properties to be revealed that would otherwise remain unseen in resting cells. Objectives: Single cell network profiling (SCNP) using muliparameter flow cytometry was used to identify intracellular pathway responses to treatment with myeloid cytokines and growth factors in addition to apoptosis-inducing agents in individual AML patients. Identification of unique signaling profiles in patient sample sub-groups may inform the choice of specific therapeutic regimens. Methods: JAK/STAT, PI3K/S6 and apoptosis signaling pathways were measured after in vitro exposure of 34 diagnostic non-M3 AML samples to a panel of myeloid growth factors (e.g FLT3L, SCF), cytokines (e.g G-CSF, GM-CSF) interleukins (e.g IL-6, IL-27) and apoptosis-inducing agents (etoposide, staurosporine). Samples were processed for cytometry by paraformaldehyde/methanol fixation and permeabilization followed by incubation with fluorochrome-conjugated antibody cocktails that recognize both cell surface proteins to delineate cell subsets in addition to intracellular signaling molecules. Results: JAK/STAT and PI3K/S6 signaling responses to individual modulators revealed a range of activity in these pathways within individual patients. Analysis of JAK/STAT and PI3K/S6 pathways across individual patient samples identified subgroups of samples with distinct pathway profiles: A) high JAK/STAT activity, B) high PI3K/S6 activity, C) high activity in both pathways, and D) low activity in both pathways. In vitro exposure of samples to staurosporine and etoposide revealed three distinct “apoptosis” profiles: 1) Staurosporine Refractory 2) Etoposide Refractory and samples that were 3) Apoptosis Competent in response to both these agents. In this study, elevated PI3K/S6 pathway activity and elevated IL-27/IL-6 induced JAK/STAT pathway activity was associated with in vitro refractoriness to apoptosis inducing agents. Analysis of JAK/STAT, PI3K/S6 and Apoptosis pathway activities characterized biologically distinct patient-specific signatures, even within cytogenetically and phenotypically uniform patient subgroups. Notable were differences in signaling profiles between samples from different patients and in cell subpopulations within the same sample. Conclusions: SCNP revealed a range of signaling responses within AML samples consistent with the heterogeneity of the disease. The data suggest the importance of characterizing and tracking signaling profiles within the subpopulations of an AML sample over time and in response to therapeutic pressure with the aim of guiding the choice of a targeted agent to be used alone or in combination with chemotherapy to improve patient response rates. Disclosures: Rosen: Nodality, Inc.: Employment, Equity Ownership. Putta:Nodality, Inc.: Employment, Equity Ownership. Covey:Nodality, Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership.

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

Description: Abstract 1588 Poster Board I-614 Background Mutations in the receptor tyrosine kinase (RTK) Fms-like tyrosine kinase 3 (FLT3) gene are among the most common somatic mutations in AML with FLT3 internal tandem duplications (ITDs) occurring in 20-35% of adult and 5-15% of pediatric AML. While the presence of FLT3 ITD mutation does not appear to influence outcome to induction chemotherapy, this mutation has been shown to confer a poor prognosis with significantly shorter disease free and relapse free survival. For patients with intermediate risk cytogenetically normal AML, molecular testing for FLT3 ITD has recently been incorporated into the National Comprehensive Cancer Network (NCCN) guidelines for clinical practice. However, while molecular testing can identify a subset of patients at high risk for relapse, there remains clinical heterogeneity likely due to differences in activation of signal transduction networks. Objectives This study tested the ability to use single cell network profiling (SCNP), in which cells are perturbed with extracellular modulators and their response ascertained by multiparametric flow cytometry, to identify a more clinically predictive functional readout of activation state, intracellular signaling capabilities and pathway dysregulation in the context of FLT3 mutational status. Methods Modulated SCNP was performed sequentially on two independent sets of patient samples (n=32 peripheral blood and n=85 bone marrow samples respectively). 304 and 201 “node-metric” i.e. modulated read outs of dynamic elements on individual proteins in signaling pathways were measured in the two sets respectively. These were derived from pathways known to be relevant to Flt3 WT and Flt3-ITD signaling (e.g. Ras-Raf-Erk-S6, PI3K-Akt-S6, STATs), as well as in-vitro chemotherapeutic induction of apoptosis (cleaved PARP, cleaved caspases), phosphatases, drug transporters (e.g. MDR-1, ABCG2) and expression of growth factor RTKs (e.g. Flt3R, c-Kit). Results In the first study, univariate analysis revealed 76 nodes out of 304 tested that distinguished FLT3 ITD from FLT3 WT patient samples (i.e. AUC of ROC 〉0.7; p

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

Description: Abstract 4768 Introduction: As aberrant JAK/STAT signaling in hematopoietic cells is associated with certain hematologic and immune diseases, understanding the regulation of JAK/STAT signaling is critical. Signaling pathway- and cell type-specific responses to various cytokines in immune system signaling networks can elicit a wide range of biological outcomes due to the combinatorial use of a limited set of kinases and STAT proteins. Although advances have been made in uncovering the intracellular mechanisms relating to cytokine signaling, the biological outcome may vary depending on composition and activation state of the cellular network. Single Cell Network Profiling (SCNP) by flow cytometry allows the interrogation of intracellular signaling networks within a heterogeneous cellular network, such as in unfractionated whole blood. We applied SCNP to investigate cytokine induced JAK/STAT signaling in whole blood across healthy human donors (n=11) to 1) measure the relative contribution of distinct cytokines to induce signaling across multiple cell subsets; 2) measure the kinetics of signaling activation across cytokines and cell subsets; 3) measure the variation among donors in their overall signaling characteristics. Our aim was to better characterize “normal” cytokine responses across healthy individuals as a basis to eventually describe abnormal states. Methods: Whole blood from 11 healthy donors (20-65 yrs, 7 males, 4 females, 8 Caucasians, 2 Hispanics, 1 East Asian) was stimulated at 37°C in 96-well plates with a low, medium, and high dose of GM-CSF, IFN-α, IL-27 and IL-6, each added separately. For each dose, a stimulation time course was run with 6 time points between 3 and 45 minutes. Each well had a final concentration of 90% whole blood. SCNP assay was performed using a fluorophore-labeled antibody cocktail to simultaneously measure signaling in six distinct cell populations, including: neutrophils, CD20+ B cells, CD3+CD4+T cells, CD3+CD4- T cells (CD8 enriched), CD3-CD20- lymphocytes (NK cell enriched), and CD14+ monocytes. The median fluorescent intensity of phospho (p)-STAT1(Y701), p-STAT3(Y705), and p-STAT5(Y694) were measured in each defined cell population for each experimental condition. Results: This SCNP assay was relatively high-throughput and provided high-content data, that equates to 19,000 gel lanes if attempted by Western analysis (11 donors × 4 cytokines × 4 concentrations × 6 time points × 6 cell subsets × 3 p-readouts). In general, each cytokine demonstrated unique dose-dependent signaling characteristics (e.g. activation/termination kinetics, magnitude of response) for each cell type analyzed, and in some cases, the kinetics differed between p-STAT readouts within the same cell subset for the same cytokine. For instance, IL-6 induced signaling was only observed in CD4+ T cells and monocytes with peak p-STAT3 levels at 3 minutes followed by p-STAT1 and p-STAT5 at 10–15 minutes. In addition, signal resolution fell to baseline levels at 45 minutes in monocytes, while the CD4+ T cells showed sustained elevated signaling, suggesting a cell-type specific regulation. In contrast to IL-6, IFN-α stimulation activated all 3 STAT proteins, peaking at 10 minutes with similar kinetics in all cell subsets. However, termination of IFN-α signaling was faster and almost complete at 45 minutes in monocytes, while in the all other subsets the signal was sustained. This efficient signal termination in monocytes was also observed with GM-CSF→p-STAT5, while neutrophils maintained persistent p-STAT5 levels. IL-27 induced p-STAT1 and p-STAT3 in T cell subsets, B cells, and monocytes with peak activation at 30 minutes. In general, signaling characteristics were remarkably uniform across the healthy donors. IL-6→p-STAT3 was particularly consistent across time points and ligand concentrations, while p-STAT1 and p-STAT5 showed more variation. Conclusions: Approaching cell signaling from the perspective of the cellular network under physiological conditions (whole blood) allows for a more comprehensive and clinically relevant view of the signaling state of complex tissues. As many small molecule compounds targeting JAK/STAT pathways enter the clinic, this study provides an important reference point for comparison of healthy signaling networks with networks altered either by pathological disease state or therapy. Disclosures: Covey: Nodality Inc.: Employment, Equity Ownership. Gulrajani:Nodality Inc.: Employment, Equity Ownership. Marimpietri:Nodality Inc.: Employment, Equity Ownership. Chiang:Nodality Inc.: Employment, Equity Ownership. Panganiban-Lustan:Nodality Inc.: Employment, Equity Ownership. Cordeiro:Nodality Inc.: Employment, Equity Ownership. Longo:Nodality Inc.: Employment, Equity Ownership. Rosen:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership.

Description: Abstract 2745 Poster Board II-721 Background: Gemtuzumab Ozogamicin (GO, Mylotarg), a humanized CD33 monoclonal antibody linked to calicheamicin was approved by the US FDA for use as a monotherapy in patients older than 60 years with relapsed acute myeloid leukemia (AML) unfit to tolerate standard salvage therapy. GO is internalized rapidly after infusion, and calicheamicin, a potent enediyene, is subsequently released and acts as a cytotoxic agent by causing double strand DNA breaks. Currently GO is in multiple clinical trials as a single agent or in combination with other therapies for both induction and consolidation treatment of various clinical subgroups of AML. However, the mechanisms of action and resistance of GO are incompletely understood and it is unclear which patient subgroups benefit from GO-based therapy. Single cell network profiling (SCNP) has shown promise as a methodology wherein multiple signaling networks are measured after treatment with an exogenous modulator such as a growth factor, cytokine or therapeutic agent and the identified signaling profiles can be used as clinical and therapeutic enablement tools. Objectives: SCNP using multiparameter flow cytometry was used to identify intracellular pathways that were associated with responsiveness or refractoriness to in vitro GO exposure in both cancer cell lines and primary AML samples. Methods: Signaling pathways emphasizing DNA damage response, cell cycle, apoptosis and drug transporter activity were measured by SCNP after in vitro exposure of cell lines and AML primary samples to clinically relevant concentrations of GO. Samples were processed for cytometry by paraformaldehyde /methanol fixation and permeabilzation followed by incubation with fluorochrome-conjugated antibody cocktails that recognize cell surface proteins to delineate cell subsets and intracellular signaling molecules. Results: In cell lines, responsiveness to in vitro GO exposure was defined as a) induction of DNA Damage as measured by increased p-ATM, p-Chk2 and p-H2AX, b) cell cycle arrest at G2/M as measured by increased cyclin B1 and DNA content & c) induction of apoptosis as measured by cleaved PARP and viability dyes. Of note, inhibition of drug transporter activity in 2 MDR-1+ cell lines did not restore GO responsiveness, suggesting the presence of additional relevant resistance mechanisms in these cell lines. In primary AML diagnostic samples, DNA damage and apoptosis pathway readouts were able to identify responsiveness or refractoriness to GO exposure. In the GO responsive profile, induction of both DNA damage responses and apoptosis were seen. Within the refractory samples, two distinct profiles were observed: a) robust and early induction of DNA damage response without apoptosis and 2) delayed and attenuated DNA damage response without apoptosis. Conclusions: Characterization of intracellular Cell Cycle, DNA Damage, and Apoptosis networks in single cells after GO exposure distinguishes GO responsive from refractory AML cells. Further, these pathway signatures provide information about mechanisms of refractoriness. (e.g. a block between a successful DNA damage response and initiation of apoptosis versus a block in the initial induction of DNA damage after GO exposure). The ability of the same profiles to predict clinical responses to the drug will be tested in future studies. Disclosures: Rosen: Nodality, Inc.: Employment, Equity Ownership. Cordeiro:Nodality Inc.: Employment, Equity Ownership. Soper:Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership. Fantl:Nodality Inc.: Employment, Equity Ownership.

Description: Abstract 2693 Background: Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on signaling cascades at the single cell level. Using this technology, ECOG in collaboration with Nodality is developing several novel biomarker assays with the aim to find blast functional signaling profiles predictive of response to induction therapy and risk of relapse in AML patients. To date, such assays utilized patient bone marrow (BM) as the sample source of blasts. However, in about 65% of patients with AML, circulating peripheral blasts are detected and peripheral blood (PB) sampling is easier and less invasive for patients than BM sampling. Objectives: The objective of this study was to compare by SCNP the functional effects of a panel of compounds simultaneously on different signaling pathways (such as the phosphoinositide 3-kinase (P13K )and the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) pathway) relevant both to the biology of the disease and the development of new therapeutics, in paired, diagnostic, cryopreserved PB mononuclear cells (PBMC) and BMMC samples from 44 AML patients. A paired sample was defined as a BMMC and PBMC specimen collected from the same patient on the same day. Methods: Modulated SCNP using a multiparametric flow cytometry platform was used to evaluate the activation state of intracellular signaling molecules in leukemic blasts under basal conditions and after treatment with specific modulators (Table 1). The SCNP phosphoflow assay was performed on 88 BMMC/PBMC pairs from ECOG trial, E3999. The relationship between readouts of modulated intracellular proteins (“nodes”) between BMMC and PBMC was assessed using linear regression, Bland-Altman method or Lin's concordance correlation coefficient. Results: Table 1 shows the goodness of fit (R2) values from the linear regression analysis for both the basal levels and the modulated levels of intracellular signaling proteins. Most of the signaling nodes show strong correlations (R2 〉0.64) with several of the exceptions belonging to nodes with weak response to modulation (e.g. SCF -〉 p-Akt) or antibodies with dim fluorphores (i.e. Alexa 647). The lack of response is however, consistent between the tissue types for the weak response nodes. Using a rank based metric that is less sensitive to the absolute intensity levels seem to perform better for the antibodies with dim fluorophores. Results from other methods; Bland Altman and Lin's Concordance also show good concordance between the tissue types. Conclusions: The data presented here demonstrate: 1) Specimen source (BM or PB) does not affect proteomic signaling in patients with AML and circulating blasts. 2) PB myeloblasts can be used as a sample source for Nodality SCNP assays to identify functionally distinct leukemic blats cell populations with distinct sensitivities to therapy. 3) The ability to use PB as a sample source will greatly improve the utility of these assays. In particular, our results will facilitate the monitoring of cellular signaling effects following the administration of targeted therapies, e.g., kinase inhibitors, at time-points when BM aspirates are not clinically justifiable. Disclosures: Cesano: Nodality Inc.: Employment, Equity Ownership. Rosen:Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership.

Description: Abstract 2512 Background: FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations (FLT3 ITD+) result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients (pts) with AML; however, substantial heterogeneity in clinical outcomes still exists within both the FLT3 ITD+ and FLT3 ITD- AML subgroups, suggesting alternative mechanisms of disease relapse not accounted for by FLT3 mutational status. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Previously, we reported the use of this assay to functionally characterize FLT3 receptor signaling in healthy bone marrow and AML samples (Rosen et al. PLoS One 2010). By applying it to a separate cohort of samples collected from elderly non-M3 AML pts at diagnosis, a subclassification of AML samples beyond their “static” molecular FLT3 ITD status was generated (Rosen et al. ASH 2010 Abstr 2739). Specifically, FLT3 ITD- AML samples displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD+ AML samples. Conversely, FLT3 ITD+ AML samples displayed more homogeneous induced signaling, with the exception of those with low mutational load, which had profiles more analogous to FLT3 ITD- AML samples. Due to the small numbers of pts in that exploratory study (n=44 [38 FLT3 ITD- and 6 FLT3 ITD+ pts]), an independent study was undertaken to confirm the observations, as well as to evaluate their clinical relevance (i.e., association with disease free survival (DFS) following anthracycline/cytarabine-based induction therapy). Methods: SCNP was performed as previously described on cryopreserved bone marrow or peripheral blood samples collected prior to anthracycline/cytarabine-based induction therapy from 104 elderly (〉60y) non-M3 AML pts enrolled on ECOG trial 3999 or 3993 for whom ITD mutational status (including % mutational load), response and DFS data were available. Samples included 85 FLT3 ITD- and 19 FLT3 ITD+ AML, 30 and 8 of which, respectively, were collected from patients who achieved complete remission (CR). Objectives: The primary study objective was to confirm that levels of FLT3 ligand (FLT3L)-induced signaling (as measured by changes in intracellular phospho-S6 level) are more homogeneous in FLT3 ITD+ than in FLT3 ITD- myeloblasts. Four FLT3 ITD+ groups were pre-defined based on % mutation load (〉0, 30%, 40%, or 50%). In addition, FLT3 ITD mutational status and signaling data from the SCNP assay (FLT3L and stem cell factor-induced phospho-S6 signaling and cytarabine/daunorubicin-induced apoptosis [cleaved PARP]) were combined to mathematically model their association with DFS among pts who achieved CR. DFS was defined as time from date of confirmed CR to date of relapse or death. Results: As shown in Figure 1a, our previous observations that variance in FLT3L-induced signaling is higher in FLT3 ITD- AML samples than in FLT3 ITD+ ones and that variance is decreased with increasing mutational load were verified in this study (Levene Test for FLT3 ITD- vs FLT3 ITD+ 50 p value=0.023). Further, when the association of DFS with FLT3 ITD mutational status and signaling data from the SCNP assay was measured using a Cox Proportional-Hazards model, the SCNP data were shown to provide independent information from FLT3 ITD mutational status (p =0.0115 for FLT3L-induced phospho-S6 signaling, Figure 1b). Conclusions: These data add to the growing body of evidence that, even within currently accepted risk stratification groups, AML is a heterogeneous disease. Functional characterization of FLT3 receptor signaling deregulation using SCNP provides prognostic information independent from FLT3 ITD mutational status and allows for more accurate pt stratification by functionally defining DFS risk sub-groups. Characterization of FLT3 signaling deregulation by SCNP could ultimately aid in the improved clinical management of AML pts and help identify candidates for FLT3 receptor inhibitor studies. Disclosures: Cesano: Nodality: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Mathi:Nodality: Employment. Rosen:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Hawtin:Nodality: Employment, Equity Ownership.

Description: Abstract 1582 Poster Board I-608 Background Traditional AML prognostic markers are based on clinical characterization (e.g. age) or static measurements of leukemia biology present at diagnosis, such as cytogenetics and isolated molecular events (e.g. presence of FLT3 ITD mutation). No validated methods currently exist to predict the disease response to standard AML induction chemotherapy for individual patients. Objectives: Single Cell Network Profiling (SCNP) was used to measure intracellular signaling in response to extracellular modulators in order to develop a new proteomic tool to characterize and monitor AML biology in the context of therapeutic applications. Methods Modulated SCNP using a multiparametric flow cytometry platform was performed evaluating the phosphorylation of intracellular signaling molecules in their basal states and after treatment with modulators in specific cell populations (e.g. leukemic cells). Since multiple signaling pathways may be dysregulated in AML and contribute to the likelihood of response to a given therapy, pathways that affect proliferation, apoptosis, and DNA damage were analyzed. Analyses were aimed to assess assay reproducibility, identify a signaling profile associated with likelihood of response to standard induction chemotherapy (first training set, n=34), and test extrapolation of the identified profile to a fully independent set of AML samples (second training set; n=88). Results High assay reproducibility (Pearson correlation coefficients ≥ 0.8) was observed. In the first training study univariate analysis revealed multiple “nodes” (modulated read outs of proteins in signaling pathways) associated with disease response to conventional induction therapy (i.e. AUC of ROC 〉0.66; p