The proposed study will be an open-label prospective investigation using fresh bone marrow and paired peripheral blood samples obtained from patients with AML starting at the time of diagnosis and including samplings at times of response assessment (CR and NR) continuing through relapse for responders and for other subsequent treatments including salvage therapy after resistance/relapse. Approximately 100 AML patients are expected to be enrolled. There is no extra marrow and blood sampling taken from patients who enroll into this study because we only study their marrow and blood when they undergo such sampling for routine medical needs. The SCNP results will not be used to guide or adjust current, or future, treatment for patients enrolled in this study and they will not be contacted regarding the study results.

Fresh whole bone marrow (5-10ml) and paired peripheral blood (5-10ml) will be collected from AML patients at participating institutions when these patients need marrow/blood tests prior to initiation of therapy, post each induction, post consolidation, at relapse and/or refractory, which is according to their standard care. When marrow cannot be obtained it is acceptable to collect only peripheral blood from the patient. Patient's demographic information, diagnosis, treatment option, outcome will be de-identified by the study PI. Samples must be collected in a standard 10 ml green top heparinized vacutainer labeled with sample de-identified ID, time, and date of collection. Samples will then be shipped to Nodality Inc. via FedEx in ambient temperature shipping kits on the same day. Nodality Inc. personnel will process the sample for study within 36 hours from collection. Leukemic blast SCNP will be conducted under the supervision of the researchers at Nodality Inc. and at facilities owned by Nodality Inc. Samples will be fractionated into bone marrow mononuclear cells (BMMC) or peripheral blood mononuclear cells (PBMC) and then aliquoted. All but one of these fractionated aliquots will be cryopreserved. The fresh, fractionated aliquot will be incubated with cytokines (e.g. interleukins, Flt3L), growth factors (e.g. SCF, GM-CSF and G-CSF), chemotherapeutic agents (e.g. cytarabine, ara- C, etoposide), and other modulators. Cells will then be fixed, permeabilized, and stained with antibodies that recognize extracellular markers (i.e. surface phenotypic markers such as clusters of differentiation, drug transporters, and receptors) in conjunction with intracellular activation-state specific epitopes (readouts) of designated signaling molecules. Subsequently, cells will be processed by multiparametric flow cytometry for SCNP. Blast populations will be defined by using combination of surface markers (CD33, CD34, CD38, CD45, CD11b) and approximately 30 different signaling nodes (paired modulator/readout e.g. Flt3L → phospho-Erk), depending on sample cell number (ideally 5-6 million; minimum 3 million). Signaling readouts will be evaluated in the total blast population, as well as within individual subpopulations, defined by CD33, CD34, CD 38, CD45, CD11b, and Side Scatter (SSC). Signaling readouts will be analyzed for each signaling node (univariate analysis) as well as a combination of nodes when the data set allows for multivariate analyses. In the "bridging" assay, cell surface markers and signaling readouts will be compared between fresh and cryopreserved AML samples including BMMC and PBMC when available in addition to BM samples.