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A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.

Background Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. Methods Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. Results Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. Conclusions SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.

In this paper, we discuss the computation of first-passage moments of a regular time-homogeneous semi-Markov process (SMP) with a finite state space to certain of its states that possess the property of universal accessibility (UA). A UA state is one which is accessible from any other state of the SMP, but which may or may not connect back to one or more other states. An important characteristic of UA is that it is the state-level version of the oft-invoked process-level property of irreducibility. We adapt existing results for irreducible SMPs to the derivation of an analytical matrix expression for the first passage moments to a single UA state of the SMP. In addition, consistent point estimators for these first passage moments, together with relevant R code, are provided.

While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily management of patients. B-cell receptor signaling is a driving event in the progression of B-cell chronic lymphocytic leukemia and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometry-based assay, allows functional signaling analysis at the level of the single cell. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant associations of single cell network profiling data with clinical outcome (i.e. time to first treatment), as assessed by Cox regression models, were then confirmed in patients' samples in two other sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patients' clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of patients with Binet stage A disease (n=21), increased anti-IgM-modulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in two test cohorts from distinct populations of patients. In conclusion, these findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.

Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1) derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ(1) resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1). Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.

Abstract 2834 Background: B-cell chronic lymphocytic leukemia (B-CLL) follows a highly variable clinical course, with some patients having indolent disease and not requiring treatment, whereas others experience rapid disease progression, treatment resistance, and death within 2 to 3 years. Widely accepted staging systems, biological parameters, and prognostic indices help stratify patients into risk groups, yet there remains substantial intragroup heterogeneity. Single cell network profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, quantitatively 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 BCR signaling in 21 cryopreserved samples from Binet Stage A B-CLL patients. In that study, a panel of signaling nodes (a node is defined as the combination of extracellular modulator and corresponding intracellular readout and is abbreviated node→readout) was examined and increased anti-IgM-induced phospho (p)-Erk signaling (anti-IgM→p-Erk) was associated with shorter time from diagnosis to first treatment (TTFT) (Scupoli et al. EHA 2010). Objectives: The present study was undertaken to independently verify the association between BCR responsiveness and shorter TTFT, to assess repeatability of SCNP measurements in B-CLL cells, and to explore additional signaling nodes relevant to B-CLL biology. Methods: SCNP was performed as previously described on cryopreserved peripheral blood mononuclear cells (PBMC) collected between 2004 and 2009 from 32 patients with untreated Binet Stage A B-CLL at various points during their clinical follow up; at the time of the analysis, 9 (28%) progressed to active disease, requiring treatment. Median follow-up was 47 months (range 4 to 179 months). SCNP analysis of 21 signaling nodes investigating B cell receptor, survival and NFkB signaling in B-CLL cells was performed in replicate, on separate plates run on the same day. Repeatability was assessed by regressing paired induced signaling measurements from experimental replicates. Associations between SCNP measurements and TTFT were determined using Cox Proportional Hazards regression. Results: Excellent repeatability was observed for SCNP signaling measurements in B-CLL cells (e.g., anti-IgM→p-Erk: R2=0.97, slope=1.01). Consistent with the previous study, an association between BCR responsiveness, measured by anti-IgM→p-Erk, and shorter TTFT was also observed in this study, although the pre-specified significance criteria were not met (likelihood ratio (LR) Chi square (χ2) test p=0.07). A post hoc analysis excluding two samples with low viability (low % aqua negative cells) was also performed (Figure 1a) showing a statistically significant association between anti-IgM→p-Erk signaling and TTFT (p=0.03, Figure 1b). Among the new nodes investigated in this study, an additional significant association between increased anti-IgD→p-NFkB and shorter TTFT was identified (p=0.02). Conclusions: This study confirms, in an independent data set, the association between increased anti-IgM→p-Erk signaling and shorter TTFT in B-CLL that was observed previously in a separate study. In addition, a newly identified association between anti-IgD→p-NFkB and TTFT was observed, thus supporting the role of BCR signaling in the pathogenesis of the disease and the utility of SCNP assay in elucidating these signaling deregulations with the potential for the development of prognostic and predictive tests. Disclosures: Cesano: Nodality: Employment, Equity Ownership. Evensen:Nodality: Employment, Equity Ownership. Ptacek:Nodality Inc.: Employment, Equity Ownership. Cordeiro:Nodality Inc.: Employment, Equity Ownership. Hawtin:Nodality Inc.: Employment, Equity Ownership. Ware:Nodality Inc.: Employment, Equity Ownership.

Biomarkers of immune function from viable cells would be of high value for many clinical applications. The immune system responds rapidly to a variety of stimuli, so identifying immune measures that are stable, robust, and relevant to clinical features/outcomes can be challenging. SCNP is a multiparametric flow cytometry assay measuring induced changes in intracellular signaling, providing a functional measure of pathway capacity in multiple cell subsets without physical separation. To assess the longitudinal stability of signaling capacity, PBMCs from 11 healthy donors (7 female, age 29 - 60) were collected at 2 time points and characterized by SCNP of 32 nodes (combinations of a modulator and an intracellular signaling readout) in 21 cell subsets. Signaling stability was assessed for each node:subset combination, calculating the ratio of the signaling variability between time points to the mean signaling level. Of 258 node:subset combinations measured: 160 (62%) had a ratio

Abstract 1783 Background CLL follows a highly variable clinical course. Evidence suggests that BCR signaling is a driving event in disease onset and progression. SCNP is a multiparametric flow cytometry-based assay that measures changes in intracellular signaling proteins in response to modulators, providing a functional measure of pathway activity. In prior studies using samples collected from patients with Binet Stage A CLL but at different time points in the natural history of their disease, an association between elevated BCR signaling and shorter TTFT was observed (Cesano et al. ASH 2011 Abstract 2834). In order to assess the correlation of CLL biology (measured by SCNP) and clinical course in a clinically more homogeneous population, samples collected as part of a clinical trial from elderly patients with previously untreated CLL prior to therapy initiation were assessed. Objectives 1) To confirm the association between αIgM-induced (→) p-ERK signaling and TTFT in an independent CLL patient cohort, and 2) to identify additional associations between signaling and prognostic, molecularly defined clinical subgroups (i.e. IGHV and p53 mutational status). Methods: 29 evaluable cryopreserved CLL peripheral blood patient samples were collected between November 2008 and January 2010 as part of a Phase II trial. These studies were not part of the trial. Of the 29 samples, 20 were IGHV unmutated and 12 had unfavorable cytogenetics (del11q22.3 and/or del17p13) with 2 carrying p53 mutations. SCNP analysis was performed to quantitatively measure 18 intracellular signaling proteins within CD19+CD5+ CLL cells using a panel of 14 disease-relevant modulators. Cox Proportional Hazards regression was performed and Kaplan–Meier curves were used to assess signaling associations with TTFT in the 24 Binet A/B samples, as the prior TTFT association with BCR signaling was identified in Binet A samples. Wilcoxon signed-rank test and logistic regression were used to identify signaling associations with p53 and IGHV mutational status. Results: Consistent with prior studies, αIgM→p-ERK signaling was associated with TTFT (p=0.05, likelihood ratio (LR) test). Notably, the combination of SDF1α and αIgM modulation induced greater p-ERK signaling than observed with either agent alone and displayed a stronger association with TTFT (p=0.02) (Figure 1A). For this cohort, only the IGHV mutational status (p=0.01) (Figure 1B) and not cytogenetic risk categories, CD38, or ZAP-70 showed a significant association with TTFT. In addition, combining IGHV with αIgM→p-ERK did not improve predictive power. Significant associations to IGHV unmutated status (Figure 2A) included multiple nodes modulated by αIgM (p-ERK, p-PLCγ2, p-SYK). The strength of this relationship was greater using concurrent stimulation with αIgM+SDF1α. R848 (TLR7/8 agonist) and thapsigargin (Ca2+ influx) signaling were also increased in the unmutated samples. Finally, since the induction of p21 is in part regulated by p53 we tested the hypothesis that the lack of p21 induction by the DNA damaging agent, bendamustine, will be associated with p53 mutations. Samples with high spontaneous apoptosis in the absence of drug were removed prior to unblinding. Of the 13 evaluable samples, there was a significant association to p53 mutational status and p21 induction by bendamustine (p=0.0125, LR test, Figure 2B). Conclusions: These data confirm the association of BCR and BCR+SDF1α signaling with disease progression in CLL, and the potential for SCNP to identify patients more likely to require early treatment. These data support the potential utility of SCNP to: (1) identify in one assay those patients with a more aggressive form of CLL, including both unmutated IGHV and p53 pathway alterations, and (2) identify patients with signaling profiles who may be more likely to respond to targeted therapies. Disclosures: Ptacek: Nodality, Inc.: Employment, Equity Ownership. Evensen:Nodality, Inc.: Employment, Equity Ownership. Hawtin:Nodality, Inc.: Employment, Equity Ownership. Friedland:Nodality, Inc.: Employment, Equity Ownership. Ware:Nodality, Inc.: Employment, Equity Ownership. Cordeiro:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc: Employment, Equity Ownership.

Abstract 2876 Background: B-CLL follows a variable clinical course, with a subset of patients progressing quickly. The reasons for this outcome disparity are not fully understood; however, evidence suggests that B-cell receptor (BCR) signaling is a driving event in disease onset and progression. B-CLL cells also receive survival signals through additional receptors. SCNP is a multiparametric flow cytometry-based assay that measures, quantitatively at the single cell level, changes in intracellular signaling proteins in response to extracellular modulators. This provides a functional measure of pathway activity and intraclonal signaling differences within the larger B-CLL cell population. In prior studies, we observed higher αIgM-induced (→) p-ERK signaling in B-CLL samples from patients who had a shorter time to first treatment (TTFT) (Cesano et al. ASH 2011 Abstract 2834). Herein we examined the feasibility for SCNP to further define patient risk stratification. Objectives: The current study was designed to 1) map signaling profiles in early-stage B-CLL and to 2) identify signaling associations with clinical subgroups defining B-CLL prognosis (IGHV mutational status, cytogenetic risk, CD38 / ZAP-70 expression). Methods: Between 2006 and 2007, peripheral blood mononuclear cells (PBMCs) were collected and cryopreserved from a cohort of 39 untreated B-CLL patients, Rai stage 0 - II, at different time points during their clinical course. Of the 39 samples evaluated; 15 and 20 expressed CD38 (≥30% of cells) and ZAP-70 (≥20% of cells), respectively; 19 were IGHV unmutated (98% cutoff); and cytogenetic risk groups were evenly represented. SCNP analysis quantitatively measured 22 intracellular signaling proteins within CD19+CD5+ B-CLL cells, using a panel of 14 disease-relevant modulators. The Wilcoxon rank-sum test was used to identify signaling associations with CD38 and ZAP-70 expression, cytogenetic risk categories, del17p (includes p53 gene deletion) and IGHV mutational status. Results: Significant associations between patient risk categories and signaling are summarized in Table 1. IGHV unmutated and ZAP-70+ samples showed (1) elevated α-IgM or α-IgD-induced BCR signaling, either alone or in combination, (2) decreased CpGβ → p-ERK induction and (3) increased thapsigargin-induced (Ca2+ signaling) signaling. Of note, CD38+ samples did not show the same associations, but shared with unmutated IGHV samples a higher responsiveness to IFNα and weaker induction of p21 in response to bendamustine. The unfavorable cytogenetic group samples showed increased αIgM→p-ERK and had higher basal p-S6 that further increased with IgD crosslinking. Lack of p21 induction was also associated with unfavorable cytogenetics, which includes deletion of p53 (del17p), a regulator of p21 expression. Conclusions: This is the second, independent SCNP analysis of B-CLL signaling showing decreased bendamustine→p21 and increased αIgM→p-ERK signaling in samples with unfavorable cytogenetics. Further associations with IGHV unmutated status included increased BCR signaling in multiple nodes, altered TLR9 responsiveness, and decreased drug-induced p21. These data support the potential utility of SCNP in: (1) identifying in one assay those patients with a more aggressive form of B-CLL, including both unmutated IGHV and p53 pathway alteration, and (2) identification of patients with signaling profiles that may be more likely to respond to targeted therapies. Disclosures: Ptacek: Nodality, Inc.: Employment, Equity Ownership. Evensen:Nodality, Inc.: Employment, Equity Ownership. Friedland:Nodality, Inc.: Employment, Equity Ownership. Cordeiro:Nodality, Inc.: Employment, Equity Ownership. Ware:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc: Employment, Equity Ownership. Hawtin:Nodality, Inc.: Employment, Equity Ownership.

Background: Acute myeloid leukemias (AML) non-responsive to induction chemotherapy generally have poor prognoses. Understanding the biological mechanisms of drug resistance, or sensitivity, specific to each individual AML could inform biologically-based treatment selection in the salvage setting. The current study was undertaken to identify resistance mechanisms to commonly used AML drugs. Methods: 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). Herein we use SCNP to investigate biological mechanisms of in vitro resistance to chemotherapeutics relevant to AML treatment (Ara-C/Daunorubicin, Gemtuzumab Ozogamicin, Decitabine, Azacitidine and Clofarabine) by assessing in a quantitative fashion the induction of DNA Damage Responses (as measured by phospho-Chk2 and phospho-H2A.X levels) and apoptosis (as measured by cleaved PARP levels and loss of membrane integrity) in 6 pediatric and 6 elderly AML samples. In addition, SCF and FLT3L induced survival signaling (as measured by phospho-Akt and phospho-Erk levels) were simultaneously assessed. Results: Treatment with Ara-C/Dauno and GO revealed cross-resistance among AML samples and was associated with elevated SCF induced PI3K/Erk survival signaling. In vitro treatment of AML samples with the different genotoxic agents (Ara-C/Dauno, GO or CLO), resulted in three distinct profiles of DNA Damage and apoptosis responses: A) Samples with induced DNA Damage Response and apoptosis, B) Samples with induced DNA Damage response but no detectable apoptosis and C) Samples lacking significant induced DNA Damage response and apoptosis. In the latter group the presence of drug efflux activity in some samples, as measured by sensitization with an MDR1 (MultiDrug Resistance) inhibitor, may explain the observation. Of note, for each sample the drug response varied with the specific agent tested. Differential sensitivity among individual patient samples was also observed between treatment with two hypomethylating agents (Decitabine and Azacitidine). Additionally, distinct kinetics of DNA Damage and apoptosis were observed among drugs: genotoxic agents (CLO, Ara/Dauno, GO) induced DNA Damage prior to induction of apoptosis while hypomethylating agents (Decitabine and Azaciditine) induced DNA Damage coincident with induction of apoptosis. Conclusions: Using SCNP to dissect in vitro response to clinically used AML drugs reveals: 1) heterogeneity of individual patient biology characteristic of AML, 2) distinct resistance mechanisms present in unique samples and 3) mechanistic differences between distinct classes of agents. Studies are ongoing to assess the clinical relevance of these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B72.

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.

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.

Background: AML represents a group of clonal hematopoietic stem cell disorders in which aberrantly regulated signaling pathways lead to oncogenic progression. In a prior study, a panel of 36 cytokine response nodes was measured in single cells by multi-parameter phosphoflow cytometry in newly diagnosed AML patient samples. Unique cancer network profiles were revealed that correlated with response to chemotherapy (Irish et al, Cell (2004) 118, p217). Objectives: Given the heterogeneity of AML, the current study was designed to expand the number of signaling nodes to 152 per patient sample. The signaling nodes were organized into 4 biological categories: Protein expression of receptors and drug transporters Response to cytokines and growth factors, Phosphatase activity, Apoptotic signaling pathways. Methods: Multi-parameter flow cytometric analysis was performed on peripheral blasts taken at diagnosis from 33 AML patients who attained a complete response (CR, n=9) or no-remission (NR, n=24) to one cycle of standard 7 + 3 induction therapy (100–200mg/m2 cytarabine and 60mg/m2 daunorubicin). Results: The data show that expression of the receptors for c-Kit and FLT3L and the drug transporter ABCG2 were increased in patients who attained an NR versus CR. Readouts from the cytokine-Stat response panels and the growth factor-Map kinase and PI3-Kinase response panels (see Table 1) revealed increased signaling in blasts taken from NR patients versus blasts taken from patients who clinically responded to therapy. To determine the role of phosphatases, a physiologic phosphatase inhibitor, peroxide, (H2O2) revealed increased phosphatase activity in CRs versus NRs. In the absence of treatment with H2O2, CRs had lower levels of phosphorylated PLCƒ×2 and SLP-76 versus NRs, and attained higher levels of phosphorylated PLCƒ×2 and SLP-76 upon H2O2 treatment. Lastly, interrogation of the apoptotic machinery using agents such as staurosporine and etoposide showed that NR patient blasts failed to undergo cell death, as determined by cleaved PARP and cleaved Caspase 8. Of note, in NR patient blasts, these agents did promote an increase in phosphorylated Chk2 suggesting a communication breakdown between the DNA damage response pathway and the apoptotic machinery. In contrast, blasts from CR patients showed significant populations of cells with cleaved PARP and caspase 8 consistent with their clinical response outcomes. Conclusions : In this study, 152 signaling nodes per patient sample were measured by multi-parameter flow cytometry and revealed distinct signaling profiles that correlate with patient response to ara-C based induction therapy. Alterations were seen in expression for the c-Kit and Flt-3L receptors, the ABCG2 drug transporter, cytokine and growth factor pathway response, phosphatase activity and apoptotic response, all of which could stratify the NR from the CR patient subsets. Whether there is a combination of hierarchical nodes to best predict response to therapy is currently under investigation.