Single Cell Network Profiles in Non-M3 AML Associated with Patient Response to Standard Induction Therapy.

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<0.05). Importantly combination of some of the independently predictive nodes improved disease response stratification (AUC of ROC up to 1.0; p<0.05). Extrapolation of the assay to a second independent set of samples revealed similar findings after accounting for clinical covariates. Specifically, for patients <60 years, the presence of intact apoptotic pathways was correlated with complete response (CR) while in samples from patients ≥60 years increased p-Akt and p-Erk levels in response to FLT3L stimulation correlated with non response (NR). Importantly, the predictive values of these nodes was independent from cytogenetic and FLT3 mutational status. Conclusions The two studies reported here show that AML biology characterization in individual patients using modulated SCNP can be performed with high technical accuracy and reproducibility to quantitatively characterize the biology of AML. This approach can be used to generate highly predictive tests for therapeutic response independently of classic prognostic factors. Disclosures Kornblau: Nodality, Inc.: Consultancy. Rosen:Nodality, Inc.: Employment, Equity Ownership. Putta:Nodality, Inc.: Employment, Equity Ownership. Cohen:Nodality, Inc.: Employment, Equity Ownership. Covey:Nodality, Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership. Gayko:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc.: Employment, Equity Ownership.

Distinct Patterns of DNA Damage Response and Apoptosis Correlate with Jak/Stat and PI3Kinase Response Profiles in Human Acute Myelogenous Leukemia

“…However, the sample set was unintentionally biased with samples predominantly from NR, female patients of younger age with intermediate cytogenetics ( Table S1 ). In spite of these limitations, univariate analysis of this sample set and an independent sample set (with 57 CR samples out of a total of 88 samples) from a separate institution revealed common nodes for CR and NR stratification suggesting that survival, DDR and apoptosis pathways may be relevant ways to characterize AML disease subtypes with further follow-up warranted in additional AML sample cohorts [28] .…”

“…Heat map of Jak/Stat signaling responses, where each row represents a specific readout and metric such as: p-Stat1 | Basal, IL-27 → p-Stat1 | Total. The samples have been ordered according to IL-27 → p-Stat3 | Total, since this node had the highest AUC and p-value for stratifying CRs from NRs [28] . Cytogenetic risk and FAB categories are noted for each sample.…”

“…It is more relevant for pathway measurements regulated by activity thresholds. For apoptosis assays, the percentage of cells that undergo cell death in response to an agent was measured with the amine aqua viability dye and with antibodies against cleaved PARP [28] .…”

“…In the AML study by Irish, although a subset of samples were identified in which potentiated p-Stat3/p-Stat5 signaling correlated with clinical refractoriness to chemotherapy, not all AML patients who were refractory to chemotherapy showed a potentiated p-Stat3/p-Stat5 signaling response, suggesting the role of alternate oncogenic pathways in their leukemia [5] . In a recent report, SCNP analysis of a separate cohort of AML samples taken at diagnosis was used to describe correlations between an expanded panel of intracellular signaling pathways with clinical response to induction therapy [28] .…”