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EP3189161A1 - Procédés de diagnostic, de pronostic et procédés de traitement - Google Patents

Procédés de diagnostic, de pronostic et procédés de traitement

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Publication number
EP3189161A1
EP3189161A1 EP15838003.0A EP15838003A EP3189161A1 EP 3189161 A1 EP3189161 A1 EP 3189161A1 EP 15838003 A EP15838003 A EP 15838003A EP 3189161 A1 EP3189161 A1 EP 3189161A1
Authority
EP
European Patent Office
Prior art keywords
cells
cell
apoptosis
individual
aml
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15838003.0A
Other languages
German (de)
English (en)
Other versions
EP3189161A4 (fr
Inventor
Alessandra Cesano
Rachael HAWTIN
Santosh Putta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nodality Inc
Original Assignee
Nodality Inc
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Filing date
Publication date
Application filed by Nodality Inc filed Critical Nodality Inc
Publication of EP3189161A1 publication Critical patent/EP3189161A1/fr
Publication of EP3189161A4 publication Critical patent/EP3189161A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • AML Acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • myeloproliferative neoplasms are examples of disorders that arise from defects of hematopoietic cells of myeloid origin. These hematopoietic disorders are recognized as clonal diseases, which are initiated by somatic and/or inherited mutations that cause dysregulated signaling in a progenitor cell. The wide range of possible mutations and accompanying signaling defects accounts for the diversity of disease phenotypes and response to therapy observed within this group of disorders. For example, some leukemia patients respond well to treatment and survive for prolonged periods, while others die rapidly despite aggressive treatment. Some patients with myelodysplastic syndrome suffer only from anemia while others transform to an acute myeloid leukemia that is difficult to treat.
  • CR complete remission
  • Ara-C standard-dose cytarabine
  • TRM treatment-related mortality
  • One embodiment disclosed herein is a method of diagnosing, prognosing, determining progression, predicting a response to a treatment or choosing a treatment for pediatric acute myeloid leukemia (AML) in an individual.
  • the method comprises (1) classifying one or more AML cells in an individual younger than 21 years old by: a) subjecting a cell population comprising said one or more AML cells from said individual to a modulator selected from the group consisting of Etoposide, Thapsigargin, or FLT3L; b) determining an activation level of p-Erk or p-S6 in one or more cells from said individual, c) determining the level of cleaved PARP in the one or more cells; and c) classifying said one or more hematopoietic cells based on said activation levels of p-Erk and S6 and the level of cleaved PARP; and (2) making a decision regarding a diagnosis, prognosis, progression, response to a treatment, selection of treatment or
  • One embodiment shown herein is a method for prognosing, predicting, or monitoring disease states in single cells, comprising: determining the level of an activatable element in cells in a sample on a single cell basis; classifying the cells in the sample as mature or immature; excluding cells from further analysis that are classified as mature and limiting further analysis to only those cells that are classified as immature; and correlating the activation level of the activatable elements to levels of activatable elements for disease profiles.
  • the method can use extracellular markers such as CD1 lb, CD117, CD45 and CD34 to determine maturity of the cells.
  • the method can also limit the analysis to those cells that are not in active apoptosis.
  • the method can also use a FAB classification to determine maturity of the cells, such as M0, Ml, M2 and M6.
  • the analysis can be performed on bone marrow cells, single cells, and pathways, such as apoptosis and DNA damage repair.
  • One embodiment can determine response to therapy, non- response, or risk of relapse.
  • One embodiment discloses multiple methods to analyze the data received from the above method.
  • the invention provides a method of treating an individual suffering from AML, wherein the individual is greater than 55 years old, comprising
  • araC administering araC to the individual based on a decision to treat the individual, wherein the decision to treat the individual is based at least in part on the results of a test comprising (i) contacting cells from a sample from the individual with one or more agents that induce apoptosis; (ii) determining the level of apoptosis in the cells by a process that comprises determining, in single cells, the level of a marker of apoptosis.
  • the marker of apoptosis comprises a marker selected from the group consisting of pChk2, p-H2AX, Bcl-2, cytochrome c, c-caspase 3, c-caspase 8, and cPARP, for example, cPARP.
  • the test can further include determining the level of a second marker in the single cells, such as a marker of cell maturity, e.g., CD34.
  • the level of the marker of apoptosis can be adjusted by a first factor and the level of the second marker can be adjusted by a second factor, where the first factor is at least 1.5 times greater than the second factor.
  • the level of the marker or markers can be determined by a process comprising (a) contacting the single cells with a detectable binding element specific for the marker; (b) detecting the detectable binding element in the single cells with a detector.
  • the detectable element can be an antibody or antibody fragment.
  • the detector comprises can be a flow cytometer.
  • the detector can be a mass cytometer.
  • the test can comprise gating the cells from the sample so that results from a subset of cells are used in the decision. The gating can be by one or more of side scatter (SSC) and forward scatter (FSC), Amine Aqua or other indicator of cell death and SSC, SSC and CD45.
  • SSC side scatter
  • FSC forward scatter
  • the gating is by at least two of side scatter (SSC) and forward scatter (FSC), Amine Aqua or other indicator of cell death and SSC, SSC and CD45. In certain embodiments, the gating is by three of side scatter (SSC) and forward scatter (FSC), Amine Aqua or other indicator of cell death and SSC, SSC and CD45. In certain embodiments, cells are first gated by side scatter and forward scatter (SSC and FSC) to eliminate cell debris, then by Amine Aqua or other indicator of cell death and SSC to eliminate dead cells, then by SSC and CD45 to select for blasts, and finally measures of the marker of apoptosis are taken.
  • SSC side scatter
  • FSC forward scatter
  • the test can further comprise contacting cells from a sample from the individual with a modulator that is not an agent that induces apoptosis and determining, in single cells, the levels of an intracellular activatable element.
  • the modulator can be selected from the group consisting of FLT3L, PMA, SCF, IL-27, G-CSF, etoposide, and thapsigargin, such as selected from the group consisting of FT3L and PMA.
  • the intracellular activatable element can be selected from the group consisting of pAKT, pCREB, p- EPvK, p-S6, p-STATl, p-STAT3, p-STAT5, such as selected from the group consisting of pAKT and pCPvEB.
  • the agents that induce apoptosis comprise etoposide, araC, or daunorubicin, or a combination thereof. In certain embodiments at least two agents are used, such as araC and daunorubicin.
  • the individual is further treated with an agent selected from the group consisting of daunorubicin, G-CSF, GM-CSF, cyclosporine, idarubicin, mitoxantrone, and combinations thereof.
  • the decision to treat the individual is further based one or more of age, sex, race, absolute blast count, percent of blasts, monocytes, neutrophils, FLT3 ITD status, NPM1 status, hemoglobin, platelet count, or a combination thereof.
  • the sample is a bone marrow (BM) sample or a peripheral blood (PB) sample.
  • PB peripheral blood
  • the sample is a BM sample.
  • the individual is suffering from de novo AML or secondary AML. In certain embodiments, the individual is suffering from de novo AML.
  • the test further comprises determining the viability of the cells and proceeding with the test only if the viability exceeds a certain threshold. In certain embodiments, determining the viability of the cells comprises measuring, in single cells, the levels of one or more markers of apoptosis and comparing the level to the threshold level.
  • the marker of apoptosis can comprise cPARP.
  • the invention provides kit for determining whether or not to treat an individual greater than 55 years of age suffering from AML with a treatment comprising administering araC to the individual, comprising (i) at least two agents that induce apoptosis, selected from the group consisting of etoposide, araC, and daunorubicin; (ii) a detectable binding element for detecting a marker of apoptosis selected from the group consisting of pChk2, p- H2AX,Bcl-2, cytochrome c, c-caspase 3, c-caspase 8, and cPARP; (iii) at least two detectable binding elements that bind to cell surface markers; (iv) instructions for use, wherein the instructions for use may be physically included with the other elements of the kit or may be supplied separately for use with the kit by electronic or physical delivery to an end user of the kit.
  • the agents can comprise araC and daunorubicin.
  • the detectable binding element can comprises an antibody or antibody fragment.
  • the cell surface markers can comprise CD45 and CD34.
  • the marker of apoptosis can be cPARP.
  • the kit can further comprise suitable packaging.
  • the kit can further comprise at least one, or at least two, or at least three, control cell lines.
  • the kit can further comprise a set of rainbow control particles (RCPs).
  • Figure 1 shows some examples of cellular pathways.
  • cytokines such as G-CSF or growth factors such as FLT-3 Ligand (FLT3L) will activate their receptors resulting in activation of intracellular signaling pathways.
  • chemotherapeutics such as AraC can be transported inside the cell to cause effects, such as DNA damage, caspase activation, PARP cleavage, etc.
  • Figure 2(a) shows the use of four metrics used to analyze data from cells that may be subject to a disease, such as AML.
  • the median (mean can be used as well) fluorescence intensity (MFI) was computed for the cells in one of the gated populations measured under various conditions of staining and stimulation.
  • the "basal" metric is calculated by subtracting the MFI of cells in the absence of a stimulant and stain
  • the "total phospho” metric is calculated by measuring the fluorescence of a cell that has been stimulated with a modulator and stained with a labeled antibody and then subtracting the value for
  • the "fold change” metric is the measurement of the fluorescence of a cell that has been stimulated with a modulator and stained with a labeled antibody and then subtracting the value for unstimulated stained cells (log 2 (MFI Stimuiated stained ) - log 2 (MFI Unstimulated stained ).
  • the "quadrant frequency” metric is the percentage of cells in each quadrant of the contour plot.
  • Figure 2(b) shows that additional metrics can also be derived directly from the distribution of cell for a protein in a gated population for various conditions.
  • NewlyPos % of newly positive cells by modulator, based on a positive gate for a stain.
  • AUC unstim Area under the curve of frequency of un-modulated cells and modulated cells for a stain.
  • Figure 2B measures the frequency of cells with a described property such as cells positive for cleaved PARP (% PARP+), or cells positive for p-S6 and p-Akt. Similarly, measurements examining the changes in the frequencies of cells may be applied such as the Change in % PARP + which would measure the % PARP+ Stimuiated stained - % PARP+ Unstimuiated Stained .
  • the AUC unstim metric also measures changes in population frequencies measuring the frequency of cells to become positive compared to an unstimulated condition.
  • Figure 3 shows a diagram of apoptosis pathways.
  • Figure 4 shows the use of signaling nodes to select patients for specific targeted therapies.
  • Figure 5(a) depicts a gating analysis to define leukemic blast population.
  • Figure 5(b) shows that cell surface markers did not identify resistance-associated myeloblasts subpopulations.
  • Figure 6 shows that an examination of signaling profiles revealed differences in relapse and diagnosis samples for SCF and FLT3L.
  • Figure 7 shows that c-kit expression is not predictive of SCF responsiveness.
  • Figure 8 shows univariate analysis for first study. Univariate analysis of modulated signaling and functional apoptosis nodes stratify NR and CR patient groups.
  • A Stratification of NR and CR patient groups with SCF modulated, but not Basal, p-Akt signaling.
  • B Stratification of NR and CR patient groups with SCF modulated, but not Basal, p-Akt signaling.
  • Figure 9 shows combinations of independent nodes from distinct pathways improve stratification for first study. Examples demonstrate how corners and thresholds for the classifiers are set.
  • O CR
  • X NR
  • A Doublet combination of nodes i.e. SCF induced p-Erk and IL-27 induced p-Stat3.
  • B Triplet combinations of nodes i.e. SDF-a induced p-Akt, IL-27 induced p- Stat3, and Etoposide induced p-CHK2-, c PARP+ cells.
  • C Comparison of AUCs of ROCs of raw data vs. AUCs of ROCs on bootstrapped data to illustrate robustness of individual combinations. Combinations with AUCs of ROCs above 0.95 on the raw data are shown.
  • Figure 10 contains "box and whisker” plots and scatter plots that illustrate the different ranges of signaling observed in FLT3-WT and BMMC cells.
  • Figure 11 contains distribution plots that illustrate the different ranges of signaling observed in FLT3-WT and BMMC cells and distinct FLT3L responsive subpopulations in both sets of cells.
  • Figure 12 illustrates FLT3L signaling kinetics in FLT3-WT AML and healthy bone marrow myeloblast (BMMC).
  • Figure 13 depicts a table comparing FLT3 Receptor and FLT3L induced signaling between normal BM Myeloblast and FLT3-WT AML.
  • Figure 14 depicts the variance in signaling among different FLT3 subgroups.
  • Figure 15 contains "box and whisker” plots that demonstrate the range of values of both FLT3 receptor levels and FLT3L-induced S6 signaling.
  • Figure 16 contains "box and whisker" plots that demonstrate the observed differences between FLT3-WT and FLT3-ITD samples.
  • Figure 16(a) illustrates differences in FLT3L-induced Stat signaling.
  • Figure 16(b) illustrates differences in IL-27-induced Stat signaling.
  • Figure 16(c) illustrates differences in Etoposide-induced apoptosis.
  • Figure 17 graphically depicts stratifying nodes that distinguished FLT3-ITD from FLT3-WT samples.
  • Figure 18 tabulates the correlations between nodes that stratify FLT3-ITD from FLT3-WT samples.
  • Figure 19 provides a schematic overview of bivariate modeling.
  • Figure 20 contains scatter plots that illustrate the signaling profiles of clinical outliers relative to other study samples.
  • Figure 20(a) illustrates FLT3L-induced S6 signaling in the clinical outliers relative to FLT3-ITD and FLT3-WT samples.
  • Figure 20 (b) illustrates IL- 27-induced Stat signaling in the clinical outliers relative to FLT3-ITD and FLT3-WT samples.
  • Figure 20(c) illustrates IL-27-induced Stat signaling in the clinical outliers relative to FLT3-ITD and FLT3-WT samples.
  • Figure 21 tabulates the correlations between nodes that stratify FLT3-ITD from FLT3-WT samples.
  • Figure 22 tabulates results from a univariate analysis of differences between FLT3- ITD and FLT3-WT signaling.
  • Figure 23 depicts a summary table of common stratifying pathways between FLT3- WT and FLT3-ID signaling in AML samples.
  • Figure 24 depicts FLT3L-induced p-S6, p-Erk and p-Akt signaling in different FLT3 subgroups.
  • Figure 25 depicts IL-27-induced p-Statl, p-Stat3 and p-Stat5 signaling in different FLT3 subgroups.
  • Figure 26 tabulates results from a univariate analysis of differences between FLT3- ITD and FLT3-WT signaling.
  • Figure 27 list all combinations of nodes for which the bivariate model of the combination had an AUC greater than the best single node/metric within the combination
  • Figure 28 demonstrates the stratification that PCA achieves when applied to induced nodes in pathways and basal nodes in the same pathways.
  • Figure 29 illustrates three distinct responses to apoptosis and DNA damage repair (DNA) that were observed in AML blasts.
  • Figures 30(a) is a scatter plot comparing etoposide versus staurosporine-mediated apoptosis.
  • Figure 30(b) contains distribution plots that illustrate sample-specific differences in sensitivity to etoposide and staurosporine-mediated apoptosis.
  • Figure 31(a) illustrates the selection of staurosporine refractory and responsive cells.
  • Figure 31(b) contains scatter plots which illustrate IL-27-induced and G-CSF-induced Stat signaling responses in the staurosporine outliers.
  • Figure 31(c) contains scatter plots that compare a principle component representing Stat pathway activity (derived from PCA of the nodes associated Stat pathway).
  • Figure 31(d) tabulates the Pearson and Spearman correlations between staurosporine response and individual nodes.
  • Figure 32(a) illustrates the selection of etoposide and staurosporine refractory and responsive cells.
  • Figure 32(b) contains scatter-plots which illustrate FLT3-induced and SCF- induced PI3K signaling response samples with high or low apoptosis responses to etoposide and staurosporine.
  • Figure 31(c) contains scatter-plots that compare a principle component representing PI3K pathway activity (derived from PCA of the nodes associated PI3K pathway).
  • Figure 32(d) tabulates the Pearson and Spearman correlations between staurosporine/etoposide response and individual nodes in the PI3K pathway.
  • Figure 33(a) and Figure 33(b) contain distribution plots that illustrate distinct subpopulations of AML samples and the differences in Etoposide, Staurosporine, FLT3L and G- CSF-induced signaling between the distinct subpopulations of AML.
  • Figure 34 depicts a model score vs. the predicted probability for the BBLRS model on the training data (unadjusted). Both the true outcome and the predicted probability (along with 95% confidence limits) of Complete Response (CR) are shown on the y-axis.
  • CR Complete Response
  • Figure 35 depicts FLT3 Ligand induced signaling of p-S6 at 5, 10, and 15 min time points in healthy bone marrow myeloblast (BM Mb, and leukemic blast from AML donors with or without FLT3-ITD mutation.
  • Figure 36 tabulates a list of stratifying nodes.
  • Figure 37 Characterization of biologic in vitro Ara-C/Dauno, GO, and SCF responses in primary AML samples.
  • Figure 37(a) indicates pPercent apoptosis induced by 24h Ara-C/Dauno or 48h GO treatment in: Adult (triangles) and Pediatric (crosses) peripheral blood (blue) and bone marrow (orange) AML samples. Arrows indicate pediatric samples resistant to Ara-C/Dauno and GO.
  • Figure 37(b) provides FACS plots of pediatric AML samples for SCF induced p-Akt vs. p-Erk or Ara-C/Dauno induced DNA damage (p-Chk2) vs. apoptosis (cleaved PARP). Quadrant frequencies are indicated.
  • Figure 38 Characterization of biologic in vitro Clofarabine (CLO), Decitabine (DEC), and Azacitidine (AZA) responses in primary AML samples.
  • Figure 38(a) shows results by drug (left panels) or by patient samples (right panels).
  • Metrics for DDR Log2Fold, upper plots
  • induced apoptosis lower plots
  • DDR data is gated on live cleaved PARP " blasts.
  • Figure 38(b) provides FACS plots showing DNA damage (pH2AX) and induced apoptosis (cleaved PARP+) of Ara- C/Dauno resistant pediatric AML samples incubated with CLO for 24 hours (left) or 48 hours (right). Quadrant frequencies are indicated.
  • Figure 38(c) provides FACS plots showing DNA damage (pH2AX) and induced apoptosis (cleaved PARP + ) of AML samples incubated with AZA or DEC for 48 hours. Quadrant frequencies are indicated.
  • FIG 39 provides a diagram of the work flow in Single Cell Network Profiling (SCNP) Technology: Bone marrow or blood cells (1) are modulated, fixed and permeabilized (2), then stained with an antibody cocktail containing antibodies directed against both cell surface markers as well as post-translational modifications of intra-cellular proteins(3). Cells are acquired using, e.g., cytometry such as multiparametric flow cytometry or mass cytometry (4) thus allowing quantification of intracellular pathway activity in cell subsets identified by gating on lineage surface markers (5). Various metrics to quantify basal and induced signaling and to assess association with biologic and clinical outcomes are applied.
  • SCNP Single Cell Network Profiling
  • Figure 40 shows a flow diagram for SWOG Patient Disposition, all patients enrolled onto the SWOG parent AML trials and rationale for exclusion of patients from the final analysis sets. Text boxes describe the characteristics of patients carried forward.
  • Figure 41 shows a flow diagram for ECOG Patient Disposition, all patients enrolled onto the parent ECOG AML trials and rationale for exclusion of patients from the final analysis sets. Text boxes describe the characteristics of patients carried forward.
  • Figure 42 shows a flowcart of the study design with descriptive schematics of the patient sets in the Training, Verification and Validation analysis sets.
  • SWOG samples were randomized into a Training and a Validation Analysis set and were sorted by tissue type (PB or BM).
  • An initial subset of classifiers was trained separately in PB and BM samples in the Training Analysis sets and then PB classifiers were applied to BM and BM classifiers were applied to PB. From this training process 5 candidate classifiers were selected and applied to the ECOG Verification Analysis set.
  • the final SCNP classifier was further refined and applied to 1) ECOG Verification Analysis set, 2) SWOG BM Validation Analysis set and 3) SWOG PB Validation Analysis set.
  • Figure 43 shows a schematic of the cell signaling pathways probed in the Training set.
  • An SCNP node consists of the combination of a modulator and the corresponding intracellular readout. Modulators are shown outside the cell initiating signaling pathways that produce an intracellular proteomic response (readouts shown below the curve indicating cell membrane).
  • Figure 44 is an illustration of gating to identify blast cell population and cPARP negative blast cells. Intact cells were identified using scatter. Amine Aqua was then used to identify viable cells. CD45 was then used to identify Blast Cells. In wells where short term signaling was assayed, the blast cells were gated using cPARP expression to identify healthy leukemic cells.
  • Figure 45 shows prediction accuracy of DX SCNP in various subgroups in the BM
  • patents and applications that are incorporated by reference include U.S. Patent Nos. 7,381,535, 7,393,656, 7,563,584, 7,695,924, 7,695,926, 7,939,278, 8,148,094, 8,187,885, 8,198,037, 8,206,939, 8,214,157, 8,227,202; U.S. Patent Applications Ser Nos. 11/338,957, 11/655,789, 12/061,565, 12/125,759, 12/125,763, 12/229,476, 12/432,239, 12/432,720,
  • 61/048,920 61/055,362, 61/079,537, 61/079,551, 61/079,579, 61/079,766, 61/085,789,
  • One embodiment of the present invention involves the classification, diagnosis, prognosis of disease or outcome after administering a therapeutic to treat the disease; exemplary diseases include AML, MDS and MPN. Another embodiment of the invention involves monitoring and predicting outcome of disease. Another embodiment is drug screening using some of the methods of the invention, to determine which drugs may be useful in particular diseases. In other embodiments, the invention involves the identification of new druggable targets, that can be used alone or in combination with other treatments. The invention allows the selection of patients for specific target therapies. The invention allows for delineation of subpopulations of cells associated with a disease that are differentially susceptible to drugs or drug combinations. In another embodiment, the invention allows to demarcate subpopulations of cells associated with a disease that have different genetic subclone origins.
  • the invention provides for the identification of a cell type, that in combination with other cell type(s), provide ratiometric or metrics that singly or coordinately allow for surrogate identification of subpopulations of cells associated with a disease, diagnosis, prognosis, disease stage of the individual from which the cells were derived, response to treatment, risk of relapse, monitoring and predicting outcome of disease.
  • Another embodiment involves the analysis of apoptosis, drug transport and/or drug metabolism. In performing these processes, one preferred analysis method involves looking at cell signals and/or expression markers.
  • One embodiment of cell signal analysis involves the analysis of phosphorylated proteins and the use of flow cytometers or mass spectrometers in that analysis.
  • a signal transduction- based classification of AML can be performed using clustering of phospho-protein patterns or biosignatures. See generally Figure 1.
  • the present invention provides methods for classification, diagnosis, prognosis of disease and outcome after administering a therapeutic to treat the disease by characterizing a plurality of pathways in a population of cells.
  • a treatment is chosen based on the characterization of plurality of pathways in single cells.
  • characterizing a plurality of pathways in single cells comprises determining whether apoptosis pathways, cell cycle pathways, signaling pathways, or DNA damage pathways are functional in an individual based on the activation levels of activatable elements within the pathways, where a pathway is functional if it is permissive for a response to a treatment.
  • the individual when the apoptosis, cell cycle, signaling, and DNA damage pathways are functional, the individual can respond to treatment, and when at least one of the pathways is not functional the individual can not respond to treatment. In some embodiments, if the apoptosis and DNA damage pathways are functional the individual can respond to treatment.
  • the characterization of pathways in conditions such as AML, MDS and MPN shows disruptions in cellular pathways that are reflective of increased proliferation, increased survival, evasion of apoptosis, insensitivity to anti-growth signals and other mechanisms.
  • the disruption in these pathways can be revealed by exposing a cell to one or more modulators that mimic one or more environmental cues.
  • Figure 1 shows an example of how biology determines response to therapy. For example, without intending to be limited to any theory, a responsive cell treated with Ara-C will undergo cell death through activation of DNA damage and apoptosis pathways. However, a non-responsive cell might escape apoptosis through disruption in one or more pathways that allows the cell to survive.
  • a non-responsive cell might have increased concentration of a drug transporter (e.g., MPR-1), which causes Ara-C to be removed from the cells.
  • a non-responsive cell might also have disruptions in one or more pathways involved in proliferation, cell cycle progression and cell survival that allows the cell to survive.
  • a non-responsive cell may have a DNA damage response pathway that fails to communicate with apoptosis pathways.
  • a non- responsive cell might also have disruptions in one or more pathways involve in proliferation, cell cycle progression and cell survival that allows the cell to survive. The disruptions in these pathways can be revealed, for example, by exposing the cell to a growth factor such as FLT3L or G-CSF.
  • the revealed disruptions in these pathways can allow for identification of target therapies that will be more effective in a particular patient and can allow the identification of new druggable targets, which therapies can be used alone or in combination with other treatments.
  • Expression levels of proteins, such as drug transporters and receptors may not be as informative by themselves for disease management as analysis of activatable elements, such as phosphorylated proteins. However, expression information may be useful in combination with the analysis of activatable elements, such as phosphorylated proteins.
  • SCNP Single cell network profiling
  • Single cell network profiling is a method that can be used to analyze activatable elements, such as phosphorylation sites of proteins, in signaling pathways in single cells in response to modulation by signaling agonists or inhibitors (e.g., kinase inhibitors).
  • activatable elements include an acetylation site, a ubiquitination site, a methylation site, a hydroxylation site, a SUMOylation site, or a cleavage site.
  • Activation of an activatable element can involve a change in cellular localization or conformation state of individual proteins, or change in ion levels, oxidation state, pH etc.
  • SCNP is one method that can be used in conjunction with an analysis of cell health, but there are other methods that may benefit from this analysis.
  • Embodiments of SCNP are shown in references cited herein. See for example, U.S. Patent No. 7,695,924.
  • SCNP can be used to generate a cell signaling profile.
  • SCNP can be used to measure apoptosis in cells stained with an antibody with specific affinity to cleaved PARP (cPARP or PARP+), for example, after the cells have been exposed to one or more modulators, such as chemotherapy drugs or other treatments.
  • cPARP or PARP+ cleaved PARP
  • the one or more cell health markers can be MCL-1 and/or cPARP.
  • the activation state or activation level of an activatable element in an untreated sample of cells may be attributable to cells undergoing apoptosis due to one or more reasons related to sample processing (e.g., shipment conditions, cryogenic storage, thawing of cryogenically stored cells, etc.).
  • apoptotic cells can negatively impact the measurement of treatment (e.g., with a modulator) induced activation of an activatable element, e.g., phosphorylation of a
  • embodiment of the present invention is a method to provide controls for a plurality of phases of the assay.
  • One or more control modules may be employed to monitor the process from start to finish. For example, one control module may span more than one step and others may span less steps.
  • One embodiment of the present invention uses the SCNP process in which samples are thawed, modulated, stained, and acquired. Some control processes described herein will be useful for all process steps and others will be more focused on one or two steps.
  • Hematopoietic cells are blood-forming cells in the body. Hematopoiesis (development of blood cells) begins in the bone marrow and depending on the cell type, further maturation occurs either in the periphery or in secondary lymphoid organs such as the spleen or lymph nodes. Hematopoietic disorders are recognized as clonal diseases, which are initiated by somatic and/or inherited mutations that cause dysregulated signaling in a progenitor cell. The wide range of possible mutations and accompanying signaling defects accounts for the diversity of disease phenotypes observed within this group of disorders.
  • Hematopoietic disorders fall into three major categories: Myelodysplasia syndromes (MDS), myeloproliferative disorders (MPN), and acute leukemias.
  • MDS Myelodysplasia syndromes
  • MPN myeloproliferative disorders
  • acute leukemias include non-B lineage derived, such as acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell acute lymphocytic leukemia (ALL), myelodysplastic disorders, myeloproliferative disorders, polycythemias, thrombocythemias, or non-B atypical immune lymphoproliferations.
  • AML acute myeloid leukemia
  • CML Chronic Myeloid Leukemia
  • ALL non-B cell acute lymphocytic leukemia
  • myelodysplastic disorders myeloproliferative disorders
  • polycythemias thrombocythemias
  • B-Cell or B cell lineage derived disorder examples include Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, Multiple Myeloma, acute lymphoblastic leukemia (ALL), B-cell pro-lymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • CLL Chronic Lymphocytic Leukemia
  • ALL acute lymphoblastic leukemia
  • B-cell pro-lymphocytic leukemia precursor B lymphoblastic leukemia
  • hairy cell leukemia or plasma cell disorders e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • AML will be further discussed below.
  • MDS and MPN are discussed in U.S. S.N. 12/910,769, 12/460,029 and 61/565,391 which are incorporated by reference
  • AML Acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • myeloproliferative neoplasms are examples of distinct myeloid hematopoietic disorders. However, it is recognized that these disorders share clinical overlap in that 30% of patients with MDS and 5-10% of patients with MPN will go on to develop AML. AML will be discussed as an example, but some of the advantages of the present methods, like the analysis methods, will be applicable to more than AML, MPN, MPD or hematopoetic diseases.
  • pediatric patients which are defined as less than 21 years old, typically measured at diagnosis. In another embodiment, pediatric is less than 18 years old.
  • the pathways depicted in Table 1 are characterized using the methods described herein by exposing cells to the modulators listed in the table and measuring the readout listed in the table, for each corresponding pathways. Disruption in one or more pathways can be revealed by exposing the cells to the modulators. This can then be used for classification, diagnosis, prognosis of AML, selection of treatment and/or predict outcome after administering a therapeutic.
  • hematopoiesis Receptors with intrinsic tyrosine kinase activity (RTKs) and those that do not contain their own enzymatic activity and often consist of heterodimers of a ligand-binding alpha subunit and a signal transducing beta subunit, which is frequently shared between a subset of cytokine receptors. Cytoplasmic tyrosine kinases phosphorylate cytokine receptors thereby creating docking sites for signaling molecules resulting in activation of a specific intracellular signaling pathway. Of the first class, Kit and FLt3 receptor have been shown to play an important role in the pathogenesis of AML.
  • the key signaling pathways such as the Ras/Raf/MAPK, PI3K/AKT, and JAK/STAT pathways.
  • the STATs have been shown to be critical for myeloid differentiation and survival, as well as for long-term maintenance of normal and leukemic stem cells.
  • Schoepers et al. STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells. Blood (2007) vol. 110 (8) pp. 2880-2888)
  • STAT signaling is activated by several cytokine receptors, which are differentially expressed depending on the cell type and the stage of differentiation.
  • Intrinsic or receptor-associated tyrosine kinases phosphorylate STAT proteins, causing them to form a homodimer.
  • the activated STAT dimer is able to enter the cell nucleus and activate the transcription of target genes, many of which are involved in the regulation of apoptosis and cell cycle progression.
  • target genes many of which are involved in the regulation of apoptosis and cell cycle progression.
  • growth factor receptors and signaling intermediates have been shown to play specific and important roles in myeloid differentiation.
  • G-CSF- or TPO-induced activation of the Ras-Raf-MAP Kinase pathway promotes myeloid or megakaryocyte differentiation in the respective progenitor cells by the activation of c/EBPa (frequently inactivated in myeloid leukemias) and GATA-1, respectively.
  • c/EBPa frequently inactivated in myeloid leukemias
  • GATA-1 GATA-1
  • Phosphatases One of the earliest events that occurs after engagement of myeloid receptors is the phosphorylation of cellular proteins on serine, threonine, and tyrosine residues 8, 9, 10. The overall level of phosphorylated tyrosine residues is regulated by the competing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs).
  • PTKs protein tyrosine kinases
  • PTPs protein tyrosine phosphatases
  • Decreases in the activity of tyrosine phosphatases may also contribute to an increase in cellular tyrosine phosphorylation following stimulation.
  • SHP-1 is a non-receptor protein tyrosine phosphatase that is expressed primarily in hematopoietic cells.
  • the enzyme is composed of two SH2 domains, a tyrosine phosphatase catalytic domain and a carboxy-terminal regulatory domain (Yi, T.L. et al. (1992) Mol Cell Biol 12, 836-46).
  • SHP-1 removes phosphates from target proteins to down regulate several tyrosine kinase regulated pathways.
  • the N-terminal SH2 domain of SHP-1 binds to tyrosine phosphorylated erythropoietin receptors (EpoR) to negatively regulate hematopoietic growth (Yi, T. et al. (1995) Blood 85, 87-95).
  • EpoR erythropoietin receptors
  • SHP-1 associates with IL-3R ⁇ chain and down regulates IL-3-induced tyrosine phosphorylation and cell proliferation (Yi, T. et al. (1993) Mol Cell Biol 13, 7577-86).
  • SHP-1 downregulates signaling pathways emanating from receptor tyrosine kinases, cytokine receptors, multi-chain recognition receptors and integrins, it is considered a potential tumor suppressor (Wu, C. et al. (2003) Gene 306, 1-12 , Bhattacharya, R. et al. (2008) J Mol Signal 3, 8).
  • SHP-2 (PTPN11) is a ubiquitously expressed, nonreceptor protein tyrosine phosphatase (PTP). It participates in signaling events downstream of receptors for growth factors, cytokines, hormones, antigens and extracellular matrices in the control of cell growth, differentiation, migration and death (Qu, C.K. (2000) Cell Res 10, 279-88). Activation of SHP-2 and its association with Gabl is critical for sustained Erk activation downstream of several growth factor receptors and cytokines (Maroun, C. R. et al. (2000) Mol Cell Biol 20, 8513-25.).
  • FLt3 ligand mutations During normal hematopoietic development, the FLT3 receptor functions in the differentiation and proliferation of multipotent stem cells and their progeny in the myeloid, B cell, and T cell lineages. (Gilliland, G.D., and Griffin, J.D. The roles of FLT3 in hematopoesis and leukemia. Blood (2002) 100: 1532-42).
  • FLT3 receptor expression is normally restricted to hematopoietic progenitors, and genetic ablation experiments have shown that FLT3 is required for the maturation of these early cells, but is not required in mature cells (Rosnet O., et al, Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia (1996) 10; 238-48; Mackarehtschian K., et al. Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity (1995) 3: 147-61).
  • the ITD-associated lengthening of the JMD appears to remove this hindrance, resulting in autodimerization and constitutive FLT3 kinase activity.
  • the second class of FLT3 mutation found in 5-10% of AML patients, comprises missense point mutations in exon 20— commonly in codons D835, 1836, N841, or Y842— which produce amino acid substitutions in the activation loop of the FLT3 tyrosine kinase domain (TKD) (Yamamoto Y., et al, Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood (2001) 97: 2434-39).
  • TKD tyrosine kinase domain
  • the AML-associated FLT3 mutations generally cause ligand-independent autophosphorylation of the FLT3 receptor and subsequent activation of downstream signaling pathways, such as PI3K, Ras, and JAK/STAT (Renneville, et al. (2008) 22: 915-31).
  • downstream signaling pathways such as PI3K, Ras, and JAK/STAT (Renneville, et al. (2008) 22: 915-31).
  • the FLT3-ITD and TKD mutations are associated with significant biological differences
  • FLT3-ITD mutations constitutively induce STAT5 phosphorylation, while FLT3-TKD mutations only weakly induce STAT5 phosphorylation (Choudry, C. et al. AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3-ITD mutations. Blood (2005) 106: 265-73). Furthermore, FLT3- ITD, but not TKD mutations suppress expression of the transcription factors, c/EBPa and Pu.l, which function in myeloid differentiation. Additionally, neither class of FLT3 mutation is sufficient to induce AML, suggesting that additional mechanisms may be involved (Renneville, et al.
  • FLT3-TKD mutations correlate with shorter clinical response duration and worse overall survival than for patients carrying the FLT3-TKD or wild-type alleles (Meshinchi, S and Applebaum, F Clin. Can. Res. (2009) 13: 4263- 4269; Frohling et al. Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood (2002) 100: 4372-80.).
  • the presence of the FLT3-ITD mutation and the ratio of the FLT3-ITD mutation to other FLT3 alleles are predictive of clinical response duration, cumulative incidence of relapse, and patient overall survival (Renneville, et al. (2008) 22: 915-31).
  • G-CSF promotes cell proliferation through activation of JAK/STAT signaling (Touw, LP., and Marijke, B., Granulocyte colony-stimulating factor: key factor or innocent bystander in the development of secondary myeloid malignancy? (2007). J. Natl. Cancer. Inst. 99: 183-186).
  • a class of AML-associated mutations produces truncated G- CSF receptor, and causes hyperreponsiveness to G-CSF stimulation (Gert-Jan, M. et al. G-CSF receptor truncations found in SCN/AML relieve SOCS3-controlled inhibition of STAT5 but leave suppression of STAT3 intact.
  • VEGF Vascular endothelial growth factor
  • VEGF Receptor R2 vascular endothelial growth factor (VEGF) and its cellular receptor KDR (VEGFR-2) in the bone marrow of patients with acute myeloid leukemia.
  • NPMl Mutations in the chaperone protein-encoding gene NPMl have been found in 30% of adults with de novo AML, but not in adults with secondary AML (Renneville, et al. (2008) 22: 915-31). Among patients with cytogenetically normal AML, NPMl mutations are predictive of higher rates of response to induction therapy and longer overall survival, but only in the absence of FLT3-ITD mutations. Mutations in the basic region leucine zipper-encoding gene CEBPA are found in 15-19% of AML patients, and are predictive of longer overall survival and longer complete response duration (Baldus, CD., et al. Clinical outcome of de novo acute myeloid leukemia patients with normal cytogenetics is affected by molecular genetic alterations: a concise review. British J. Haematology (2007) 137: 387-400).
  • Mutated genes that confer a non-favorable prognosis include ERG which encodes a transcription factor activated by signal transduction pathways that regulates cell differentiation, proliferation, and tissue invasion (Baldus, CD., et al. British J. Haematology (2007) 137: 387- 400.). Overexpression of ERG in AML patients is predictive of a higher rate of relapse and shorter overall survival (Marcucci et al, Overexpression of the ETS-related gene, ERG, predicts a worse outcome in acute myeloid leukemia with normal karyotype: a Cancer and Leukemia Group B study. J. Clinical Oncology (2005) 23: 9234-42).
  • Gain-of-function mutations in the receptor tyrosine kinase-encoding gene c-KIT are predictive of shorter overall complete response duration and overall survival in AML patients, and may also be predictive of response to treatment with tyrosine kinase inhibitors (Renneville, et al. (2008) 22: 915-31). Mutations in the Wlim 's Tumor 1 (WT1) gene are found in 10-15% of AML cases, and in cytogenetically normal AML patients, are predictive of failure to achieve complete response to chemotherapy (Renneville, et al. (2008) 22: 915-31). Point mutations in the RAS oncogenes are found in 10-20% of AML patients, but prognostic uses of these mutations have not yet been identified (Renneville, et al. (2008) 22: 915-31).
  • RAS mutations Ras proteins normally act as signaling switches, which alternate between the active (GTP-bound) and inactive (GDP-bound) states. Somatic point mutations in codons 12, 13 and 61 of the NRAS and KRAS genes occur in many myeloid malignancies, resulting in persistently active forms of the protein. Analyses of patients with MDS revealed a very high risk of transformation to AML in patients with N-RAS mutations, providing evidence that these mutations might represent an important progression factor in MDS. Under the two-hit model put forth by Gilliland et al, RAS mutations are likely to provide a growth advantage, which when combined with a secondary mutation that blocks differentiation, results in AML.
  • CBF-AML core binding factor AML
  • AML1-ETO AML1-ETO
  • CBF ⁇ -MYHl 1 gene fusions known to disrupt differentiation Boissel et al. Incidence and prognostic impact of c-Kit, FLT3 LIGAND, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML). Leukemia (2006) vol. 20 (6) pp. 965-970)
  • One embodiment of the invention will look at any of the cell signaling pathways described above in classifying diseases, such as AML. Modulators can be designed to investigate these pathways and any relevant parallel pathways. Other embodiments include diseases besides AML.
  • the invention provides a method for diagnosis, prognosis, determining progression, predicting response to treatment or choosing a treatment for AML, the method comprising the steps of (a) subjecting a cell population from the individual to a plurality of distinct modulators, (b) characterizing a plurality of pathways in one or more cells comprising determining an activation level of at least one activatable element in at least three pathways, where the pathways are selected from the group consisting of apoptosis, cell cycle, signaling, or DNA damage pathways, and (c) correlating the characterization with diagnosis, prognosis, determining progression, predicting response to treatment or choosing a treatment for AML, in an individual, where the pathways characterization is indicative of the diagnosis, prognosis, determining progression, response to treatment or the appropriate treatment for AML.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Tables 1, 1(a)- 1(e), 2, 3 or 5. In some embodiments, the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 12 and are used to predict response duration in an individual after treatment.
  • the modulator is selected from the group consisting of FLT3L, GM-CSF, SCF, G- CSF, SDFla, LPS, PMA, Thapsigargin, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, ZVAD, H 2 O 2 , Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • the individual has a predefined clinical parameter and the characterization of multiple pathways in combination with the clinical parameter is indicative of the diagnosis, prognosis, determining progression, predicting response to treatment or choosing a treatment for AML, in an individual.
  • predetermined clinical parameters include, but are not limited to, age, de novo acute myeloid leukemia patient, secondary acute myeloid leukemia patient, or a biochemical/molecular marker.
  • the individual is over 60 years old. In some embodiments, the individual is under 60 years old. In some embodiments, when the individual is under 60 years old the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 6. In some embodiments, where the individual is over 60 years the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 7. In some embodiments, where the individual is a secondary acute myeloid leukemia patient the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 8 and Table 9.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 10 and Table 11. In some embodiments, where the individual has a wild type FLT3 the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 13.
  • the activatable elements can demarcate AML cell
  • the activatable elements can demarcate AML subpopulations that, in combination with additional surface molecules, can allow for surrogate identification of AML cell subpopulations.
  • the activatable elements can demarcate AML subpopulations that can be used to determine other protein, epitope-based, RNA, mRNA, siRNA, or metabolic markers that singly or coordinately allow for surrogate identification of AML cell subpopulations, disease stage of the individual from which the cells were derived, diagnosis, prognosis, response to treatment, or new druggable targets.
  • the pathways characterization allows for the delineation of AML cell subpopulations that are differentially susceptible to drugs or drug combinations.
  • the cell types or activatable elements from a given cell type will, in combination with activatable elements in other cell types, provide ratiometric or metrics that singly or coordinately allow for surrogate identification of AML cell subpopulations, disease stage of the individual from which the cells were derived, diagnosis, prognosis, response to treatment, or new druggable targets.
  • Embodiments of the invention may be used to diagnose, predict or to provide therapeutic decisions for disease treatment, such as AML.
  • the invention may be used to identify new druggable targets and to design drug combinations. The following will discuss instruments, reagents, kits, and the biology involved with these and other diseases.
  • One aspect of the invention involves contacting a hematopoietic cell with a modulator; determining the activation states of a plurality of activatable elements in the cell; and classifying the cell based on said activation state.
  • this invention is directed to methods and compositions, and kits for analysis, drug screening, diagnosis, prognosis, for methods of disease treatment and prediction.
  • the present invention involves methods of analyzing experimental data.
  • the physiological status of cells present in a sample e.g. clinical sample
  • patient selection for therapy using some of the agents identified above, to monitor treatment, modify therapeutic regimens, and to further optimize the selection of therapeutic agents which may be administered as one or a combination of agents.
  • therapeutic regimens can be individualized and tailored according to the data obtained prior to, and at different times over the course of treatment, thereby providing a regimen that is individually appropriate.
  • a compound is contacted with cells to analyze the response to the compound.
  • the present invention is directed to methods for classifying a sample derived from an individual having or suspected of having a condition, e.g., a neoplastic or a hematopoietic condition.
  • a condition e.g., a neoplastic or a hematopoietic condition.
  • the invention allows for identification of prognostically and therapeutically relevant subgroups of conditions and prediction of the clinical course of an individual.
  • the methods of the invention provide tools useful in the treatment of an individual afflicted with a condition, including but not limited to methods for assigning a risk group, methods of predicting an increased risk of relapse, methods of predicting an increased risk of developing secondary complications, methods of choosing a therapy for an individual, methods of predicting duration of response, response to a therapy for an individual, methods of determining the efficacy of a therapy in an individual, and methods of determining the prognosis for an individual.
  • the present invention provides methods that can serve as a prognostic indicator to predict the course of a condition, e.g.
  • the present invention provides information to a physician to aid in the clinical management of a patient so that the information may be translated into action, including treatment, prognosis or prediction.
  • the invention is directed to methods of characterizing a plurality of pathways in single cells.
  • Exemplary pathways include apoptosis, cell cycle, signaling, or DNA damage pathways.
  • the characterization of the pathways is correlated with diagnosing, prognosing or determining condition progression in an individual.
  • the characterization of the pathways is correlated with predicting response to treatment or choosing a treatment in an individual.
  • the characterization of the pathways is correlated with finding a new druggable target.
  • the pathways' characterization in combination with a predetermined clinical parameter is indicative of the diagnosis, prognosis or progression of the condition.
  • the pathways' characterization in combination with a predetermined clinical parameter is indicative of a response to treatment or of the appropriate treatment for an individual. In some embodiments, the characterization of the pathways in combination with a predetermined clinical parameter is indicative a new druggable target.
  • the invention is directed to methods for determining the activation level of one or more activatable elements in a cell upon treatment with one or more modulators.
  • the activation of an activatable element in the cell upon treatment with one or more modulators can reveal operative pathways in a condition that can then be used, e.g., as an indicator to predict course of the condition, to identify risk group, to predict an increased risk of developing secondary complications, to choose a therapy for an individual, to predict response to a therapy for an individual, to determine the efficacy of a therapy in an individual, and to determine the prognosis for an individual.
  • the operative pathways can reveal whether apoptosis, cell cycle, signaling, or DNA damage pathways are functional in an individual, where a pathway is functional if it is permissive for a response to a treatment. In some embodiments, when apoptosis, cell cycle, signaling, and DNA damage pathways are functional the individual can respond to treatment, and if at least one of the pathways is not functional the individual can not respond to treatment. In some embodiments, when the apoptosis and DNA damage pathways are functional the individual can respond to treatment. In some embodiments, the operative pathways can reveal new druggable targets.
  • the invention is directed to methods of determining a phenotypic profile of a population of cells by exposing the population of cells to a plurality of modulators in separate cultures, determining the presence or absence of an increase in activation level of an activatable element in the cell population from each of the separate culture and classifying the cell population based on the presence or absence of the increase in the activation of the activatable element from each of the separate culture.
  • at least one of the modulators is an inhibitor.
  • the presence or absence of an increase in activation level of a plurality of activatable elements is determined.
  • each of the activatable elements belongs to a particular pathway and the activation level of the activatable elements is used to characterize each of the particular pathways.
  • a plurality of pathways are characterized by exposing a population of cells to a plurality of modulators in separate cultures, determining the presence or absence of an increase in activation levels of a plurality of activatable elements in the cell population from each of the separate culture, wherein the activatable elements are within the pathways being characterized and classifying the cell population based on the characterizations of said multiple pathways.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Tables 1, 2, 3 or 5.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 12 and are used to predict response duration in an individual after treatment.
  • the invention is directed to methods for classifying a cell by determining the presence or absence of an increase in activation level of an activatable element in the, in combination with additional expression markers.
  • expression markers or drug transporters such as CD34, CD33, CD45, HLADR, CD1 IB, FLT3 Ligand, c- KIT, ABCG2, MDR1, BCRP, MRP1, LRP, and others noted below, can also be used for stratifying responders and non-responders.
  • the expression markers may be detected using many different techniques, for example using nodes from flow cytometry data (see the articles and patent applications referred to above).
  • the invention is directed to methods of classifying a cell population by contacting the cell population with at least one modulator that affects signaling mediated by receptors selected from the group comprising of growth factors, mitogens and cytokines. In some embodiments, the invention is directed to methods of classifying a cell population by contacting the cell population with at least one modulator that affects signaling mediated by receptors selected from the group comprising SDF-1 ⁇ , IFN- ⁇ , IFN- ⁇ , IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, and Thapsigargin; determining the activation states of a plurality of activatable elements in the cell comprising; and classifying the cell based on said activation states and expression levels.
  • the cell population is also exposed in a separate culture to at least one modulator that slows or stops the growth of cells and/or induces apoptosis of cells.
  • the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of, Etoposide, Mylotarg, AraC, daunorubicin, staurosporine, benzyloxycarbonyl-Val- Ala-Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, EPO, and azacitadine, decitabine.
  • the cell population is also exposed in a separate culture to at least one modulator that is an inhibitor.
  • the inhibitor is H 2 O 2 .
  • the expression of a growth factor receptor, cytokine receptor and/or a drug transporter is also measured.
  • the methods comprise determining the expression level at least one protein selected from the group comprising ABCG2, C-KIT receptor, and FLT3 LIGAND receptor.
  • Another embodiment of the invention further includes using the modulators IL-3, IL-4, GM-CSF, EPO, LPS, TNF- ⁇ , and CD40L.
  • the cell population in a hematopoietic cell population is directed to methods of correlating and/or classifying an activation state of an AML cell with a clinical outcome in an individual by subjecting the AML cell from the individual to a modulator, determining the activation levels of a plurality of activatable elements, and identifying a pattern of the activation levels of the plurality of activatable elements to determine the presence or absence of an alteration in signaling, where the presence of the alteration is indicative of a clinical outcome.
  • the activatable elements can demarcate AML cell subpopulations that have different genetic subclone origins.
  • the activatable elements can demarcate AML subpopulations that can be used to determine other protein, epitope-based, RNA, mRNA, siRNA, or metabolomic markers that singly or
  • the pathways characterization allows for the delineation of AML cell subpopulations that are differentially susceptible to drugs or drug combinations.
  • the cell types or activatable elements from a given cell type will, in combination with activatable elements in other cell types, provide ratiometric or metrics that singly or coordinately allow for surrogate identification of AML cell subpopulations, disease stage of the individual from which the cells were derived, diagnosis, prognosis, response to treatment, or new druggable targets.
  • kits for use in determining the physiological status of cells in a sample comprising one or more modulators, inhibitors, specific binding elements for signaling molecules, and may additionally comprise one or more therapeutic agents.
  • the above reagents for the kit are all recited and listed in the present application below.
  • the kit may further comprise a software package for data analysis of the cellular state and its physiological status, which may include reference profiles for comparison with the test profile and comparisons to other analyses as referred to above.
  • the kit may also include instructions for use for any of the above applications.
  • the invention provides methods, including methods to determine the physiological status of a cell, e.g., by determining the activation level of an activatable element upon contact with one or more modulators. In some embodiments, the invention provides methods, including methods to classify a cell according to the status of an activatable element in a cellular pathway. In some embodiments, the cells are classified by analyzing the response to particular modulators and by comparison of different cell states, with or without modulators. The information can be used in prognosis and diagnosis, including susceptibility to disease(s), status of a diseased state and response to changes, in the
  • the physiological status of the cells provided in a sample may be classified according to the activation of cellular pathways of interest.
  • the cells can also be classified as to their ability to respond to therapeutic agents and treatments.
  • the physiological status of the cells can provide new druggable targets for the development of treatments. These treatments can be used alone or in combination with other treatments.
  • the physiological status of the cells can be used to design combination treatments.
  • One or more cells or cell types, or samples containing one or more cells or cell types, can be isolated from body samples.
  • the cells can be separated from body samples by
  • centrifugation elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, solid supports (magnetic beads, beads in columns, or other surfaces) with attached antibodies, etc.
  • antibodies specific for markers identified with particular cell types a relatively homogeneous population of cells may be obtained.
  • a heterogeneous cell population can be used.
  • Cells can also be separated by using filters.
  • whole blood can also be applied to filters that are engineered to contain pore sizes that select for the desired cell type or class.
  • Rare pathogenic cells can be filtered out of diluted, whole blood following the lysis of red blood cells by using filters with pore sizes between 5 to 10 ⁇ , as disclosed in U.S. Patent Application No. 09/790,673.
  • Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short periods of time.
  • Methods to isolate one or more cells for use according to the methods of this invention are performed according to standard techniques and protocols well-established in the art. See also U.S.Serial. Nos. 12/432,720, 12/229,476, and 12/432,239. See also, the commercial products from companies such as BD and BCI as identified above.
  • the cells are cultured post collection in a media suitable for revealing the activation level of an activatable element (e.g. RPMI, DMEM) in the presence, or absence, of serum such as fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, or goat serum.
  • an activatable element e.g. RPMI, DMEM
  • serum such as fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, or goat serum.
  • serum is present in the media it could be present at a level ranging from 0.0001 % to 30%.
  • the cells are hematopoietic cells.
  • hematopoietic cells include but are not limited to pluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the term "patient” or “individual” as used herein includes humans as well as other mammals.
  • the methods generally involve determining the status of an activatable element.
  • the methods also involve determining the status of a plurality of activatable elements.
  • the invention provides a method of classifying a cell by determining the presence or absence of an increase in activation level of an activatable element in the cell upon treatment with one or more modulators, and classifying the cell based on the presence or absence of the increase in the activation of the activatable element.
  • the activation level of the activatable element is determined by contacting the cell with a binding element that is specific for an activation state of the activatable element.
  • a cell is classified according to the activation level of a plurality of activatable elements after the cell have been subjected to a modulator.
  • the activation levels of a plurality of activatable elements are determined by contacting a cell with a plurality of binding elements, where each binding element is specific for an activation state of an activatable element.
  • the classification of a cell according to the status of an activatable element can comprise classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome is the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition such as AML.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging examples include, but are not limited to, aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • the classification of a cell according to the status of an activatable element can comprise classifying a cell as a cell that is correlated to a patient response to a treatment.
  • the patient response is selected from the group consisting of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction.
  • the classification of a rare cell according to the status of an activatable element can comprise classifying the cell as a cell that can be correlated with minimal residual disease or emerging resistance. See U.S. Serial No. 12/432,720 which is incorporated by reference.
  • the classification of a cell according to the status of an activatable element can comprise selecting a method of treatment.
  • Method of treatments include, but are not limited to chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, and other therapy.
  • a modulator can be an activator, an inhibitor or a compound capable of impacting cellular signaling networks.
  • Modulators can take the form of a wide variety of environmental cues and inputs. Examples of modulators include but are not limited to growth factors, mitogens, cytokines, adhesion molecules, drugs, hormones, small molecules, polynucleotides, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulators, carbohydrates, proteases, ions, reactive oxygen species, radiation, physical parameters such as heat, cold, UV radiation, peptides, and protein fragments, either alone or in the context of cells, cells themselves, viruses, and biological and non-biological complexes (e.g.
  • modulators include but are not limited to SDF- ⁇ , IFN-a, IFN- ⁇ , IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, Thapsigargin, H 2 O 2 , Etoposide, Mylotarg, AraC, daunorubicin, staurosporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, EPO, azacitadine, decitabine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-a, and CD40L.
  • the modulator is an activator. In some embodiments the modulator is an inhibitor. In some embodiments, the invention provides methods for classifying a cell by contacting the cell with an inhibitor, determining the presence or absence of an increase in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the increase in the activation of the activatable element. In some embodiments, a cell is classified according to the activation level of a plurality of activatable elements after the cells have been subjected to an inhibitor. In some embodiments, the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell.
  • the inhibitor is a phosphatase inhibitor.
  • phosphatase inhibitors include, but are not limited to 3 ⁇ 4(3 ⁇ 4, siRNA, miRNA, Cantharidin, (-)-p- Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, ⁇ - Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP-1, N-(9
  • the methods of the invention provide methods for classifying a cell population or determining the presence or absence of a condition in an individual by subjecting a cell from the individual to a modulator and an inhibitor, determining the activation level of an activatable element in the cell, and determining the presence or absence of a condition based on the activation level.
  • the activation level of a plurality of activatable elements in the cell is determined.
  • the inhibitor can be an inhibitor as described herein.
  • the inhibitor is a phosphatase inhibitor.
  • the inhibitor is H2O2.
  • the modulator can be any modulator described herein.
  • the methods of the invention provides for methods for classifying a cell population by exposing the cell population to a plurality of modulators in separate cultures and determining the status of an activatable element in the cell population. In some embodiments, the status of a plurality of activatable elements in the cell population is determined. In some embodiments, at least one of the modulators of the plurality of modulators is an inhibitor. The modulator can be at least one of the modulators described herein.
  • At least one modulator is selected from the group consisting of SDF-1 ⁇ , IFN-a, IFN- ⁇ , IL-10, IL-6, IL- 27, G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, Thapsigargin, H 2 O 2 , Etoposide, Mylotarg, AraC, daunorubicin, staurosporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, EPO, azacitadine, decitabine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-a, and CD40L or a combination thereof.
  • the status of an activatable element is determined by contacting the cell population with a binding element that is specific for an activation state of the activatable element. In some embodiments, the status of a plurality of activatable elements is determined by contacting the cell population with a plurality of binding elements, where each binding element is specific for an activation state of an activatable element.
  • the methods of the invention provide methods for determining a phenotypic profile of a population of cells by exposing the population of cells to a plurality of modulators (recited herein) in separate cultures, determining the presence or absence of an increase in activation level of an activatable element in the cell population from each of the separate cultures and classifying the cell population based on the presence or absence of the increase in the activation of the activatable element from each of the separate culture.
  • the phenotypic profile is used to characterize multiple pathways in the population of cells.
  • Patterns and profiles of one or more activatable elements are detected using the methods known in the art including those described herein.
  • patterns and profiles of activatable elements that are cellular components of a cellular pathway or a signaling pathway are detected using the methods described herein.
  • patterns and profiles of one or more phosphorylated polypeptides are detected using methods known in art including those described herein.
  • cells e.g. normal cells
  • cells other than the cells associated with a condition e.g. cancer cells
  • a condition e.g. cancer cells
  • a combination of information from the cancer cell plus the immune cells in the blood that are responding to the disease, or reacting to the disease can be used for diagnosis or prognosis of the cancer. See U.S.S.N. 61/499,127 and PCT/US2011/01565 (incorporated by reference in its entirety) for a comparison to normal cells.
  • the invention provides methods to carry out multiparameter flow cytometry for monitoring phospho-protein responses to various factors in acute myeloid leukemia at the single cell level.
  • Phospho-protein members of signaling cascades and the kinases and phosphatases that interact with them are required to initiate and regulate proliferative signals in cells.
  • the effect of potential drug molecules on these network pathways was studied to discern unique cancer network profiles, which correlate with the genetics and disease outcome.
  • Single cell measurements of phospho- protein responses reveal shifts in the signaling potential of a phospho-protein network, enabling categorization of cell network phenotypes by multidimensional molecular profiles of signaling. See U.S. Patent No. 7,393,656. See also Irish et. al, Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell. 2004, vol. 118, p.1-20.
  • Cytokine response panels have been studied to survey altered signal transduction of cancer cells by using a multidimensional flow cytometry file which contained at least 30,000 cell events.
  • this panel is expanded and the effect of growth factors and cytokines on primary AML samples studied. See U.S. Patent Nos. 7,381,535 and 7,393,656. See also Irish et. al., CELL Jul 23; 118(2):217-28.
  • the analysis involves working at multiple characteristics of the cell in parallel after contact with the compound. For example, the analysis can examine drug transporter function; drug transporter expression; drug metabolism; drug activation; cellular redox potential; signaling pathways; DNA damage repair; and apoptosis.
  • the modulators include growth factors, cytokines,
  • the response panel is composed of at least one of: SDF- ⁇ , IFN-a, IFN- ⁇ , IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, Thapsigargin, H 2 O 2 , Etoposide, Mylotarg, AraC, daunorubicin,
  • lenalidomide EPO, azacitadine, decitabine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-a, and CD40L.
  • the response of each phospho-protein node is compared to the basal state and can be represented by calculating the log 2 fold difference in the Median Fluorescence Intensity (MFI) of the stimulated sample divided by the unstimulated sample.
  • MFI Median Fluorescence Intensity
  • the data can be analyzed using any of the metrics described herein including the metric described in Figure 2. However, other statistical methods may be used.
  • the growth factor and the cytokine response panel included detection of phosphorylated Statl, Stat3, Stat5, Stat6, PLC ⁇ 2, S6, Akt, Erkl/2, CREB, p38, and NF-KBp-65.
  • a diagnosis, prognosis, a prediction of outcome such as response to treatment or relapse is performed by analyzing the two or more phosphorylation levels of two or more proteins each in response to one or more modulators.
  • the phosphorylation levels of the independent proteins can be measured in response to the same or different modulators. Grouping of data points increases predictive value.
  • the AML panel of modulators is further expanded to examine the process of DNA damage, apoptosis, drug transport, drug metabolism, and the use of peroxide to evaluate phosphatase activity. Analysis can assess the ability of the cell to undergo the process of apoptosis after exposure to the experimental drug in an in vitro assay as well as how quickly the drug is exported out of the cell or metabolized.
  • the drug response panel can include but is not limited to detection of phosphorylated Chk2, Cleaved Caspase 3, Caspase 8, cleaved PARP and mitochondria-released Cytoplasmic Cytochrome C.
  • Modulators may include Stauro, Etoposide, Mylotarg, AraC, and daunorubicin. Analysis can assess phosphatase activity after exposure of cells to phosphatase inhibitors including but not limited to hydrogen peroxide ( ⁇ 2 0 2 ), H2O2 + SCF and H2O2 + IFNa.
  • the response panel to evaluate phosphatase activity can include but is not limited to the detection of phosphorylated Slp76, PLCg2, Lck, S6, Akt, Erk, Statl, Sta3, and Stat5. Later, the samples may be analyzed for the expression of drug
  • Another method of the present invention is a method for determining the prognosis and therapeutic selection for an individual with AML. Using the signaling nodes and
  • multiparametric flow could separate a patient into "cytarabine responsive", meaning that a cytarabine based induction regimen would yield a complete response or "cytarabine non-responsive", meaning that the patient is unlikely to yield a complete response to a cytarabine based induction regimen.
  • the individual's blood or marrow sample could reveal signaling biology that corresponds to either in-vivo or in-vitro sensitivity to a class of drugs including but not limited to direct drug resistance modulators, anti-Bcl-2 or pro-apoptotic drugs, proteosome inhibitors, DNA methyl transferase inhibitors, histone deacetylase inhibitors, anti-angiogenic drugs, farnesyl transferase inhibitors, FLt3 ligand inhibitors, or ribonucleotide reductase inhibitors.
  • An individual with AML with a complete response to induction therapy could further benefit from the present invention.
  • the individual's blood or marrow sample could reveal signaling biology that corresponds to likelihood of benefit from further cytarabine based chemotherapy versus myeloablative therapy followed by and stem cell transplant versus reduced intensity therapy followed by stem cell transplantation.
  • the invention provides a method for diagnosing, prognosing, determining progression, predicting response to treatment or choosing a treatment for AML in an individual where the individual has a predefined clinical parameter, the method comprising the steps of (a) subjecting a cell population from the individual to a plurality of distinct modulators in separate cultures, (b) characterizing a plurality of pathways in one or more cells from the separate cultures comprising determining an activation level of at least one activatable element in at least three pathways, where (i) the pathways are selected from the group consisting of apoptosis, cell cycle, signaling, or DNA damage pathways (ii) at least one of the pathways being characterized in at least one of the separate cultures is an apoptosis or DNA damage pathway, (iii) the distinct modulators independently activate or inhibit said one or more pathways being characterized, and (c) correlating the characterization with diagnosing, prognosing, determining progression, predicting response to treatment or choosing a treatment for AML in an individual,
  • predetermined clinical parameters include, but are not limited to, age, de novo acute myeloid leukemia patient, secondary acute myeloid leukemia patient, or a biochemical/molecular marker.
  • the individual is over 60 years old. In some embodiments, the individual is under 60 years old.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Tables 1, 1(a)- 1(e), 2, 3 or 5. In some embodiments, the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 12 and are used to predict response duration in an individual after treatment.
  • the modulator is selected from the group consisting of FLT3L, GM-CSF, SCF, G- CSF, SDFla, LPS, PMA, Thapsigargin, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, ZVAD, H 2 O 2 , Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 6.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 7.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 8 and Table 9. In some embodiments, where the individual is a de novo acute myeloid leukemia patient the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 10 and Table 11. In some embodiments, where the individual has a wild type FLT3 the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 13.
  • the invention provides a method for predicting a response to a treatment or choosing a treatment for AML in an individual, the method comprising the steps: (a) subjecting a cell population from the individual to at least two distinct modulators in separate cultures; (b) determining an activation level of at least one activatable element from each of at least three pathways selected from the group consisting of apoptosis, cell cycle, signaling, and DNA damage pathways in one or more cells from each said separate cultures, where at least one of the activatable elements is from an apoptosis or DNA damage pathway, and where the activatable elements measured in each separate culture are the same or the activatable elements measured in each separate culture are different; and (c) predicting a response to a treatment or choosing a therapeutic for AML in the individual based on the activation level of said activatable elements.
  • the method further comprises determining whether the apoptosis, cell cycle, signaling, or DNA damage pathways are functional in the individual based on the activation levels of the activatable elements, wherein a pathway is functional if it is permissive for a response to a treatment, where if the apoptosis, cell cycle, signaling, and DNA damage pathways are functional the individual can respond to treatment, and where if at least one of the pathways is not functional the individual can not respond to treatment.
  • the method further comprises determining whether the apoptosis, cell cycle, signaling, or DNA damage pathways are functional in the individual based on the activation levels of the activatable elements, wherein a pathway is functional if it is permissive for a response to a treatment, where if the apoptosis and DNA damage pathways are functional the individual can respond to treatment. In some embodiments, the method further comprises determining whether the apoptosis, cell cycle, signaling, or DNA damage pathways are functional in the individual based on the activation levels of the activatable elements, wherein a pathway is functional if it is permissive for a response to a treatment, where a therapeutic is chosen depending of the functional pathways in the individual.
  • the activatable elements and modulators are selected from the activatable elements and modulators listed in Tables 1, 2, 3 or 5. In some embodiments, the activatable elements and modulators are selected from the activatable elements and modulators listed in Table 12 and are used to predict response duration in an individual after treatment.
  • the modulator is selected from the group consisting of FLT3L, GM-CSF, SCF, G-CSF, SDFla, LPS, PMA, Thapsigargin, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, ZVAD, H 2 O 2 , Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • the invention provides a method of predicting a response to a treatment or choosing a treatment for AML in an individual, the method comprising the steps of: (a) subjecting a cell population from said individual to at least three distinct modulators in separate cultures, wherein: (i) a first modulator is a growth factor or mitogen, (ii) a second modulator is a cytokine, (iii) a third modulator is a modulator that slows or stops the growth of cells and/or induces apoptosis of cells or, the third modulator is an inhibitor; (b) determining the activation level of at least one activatable element in one or more cells from each of the separate cultures, where: (i) a first activatable element is an activatable element within the PI3K/AKT, or MAPK pathways and the activation level is measured in response to the growth factor or mitogen, (ii) a second activatable element is an activatable element within the STAT pathway and the activ
  • predefined clinical parameters include age, de novo acute myeloid leukemia patient, secondary acute myeloid leukemia patient, or a biochemical/molecular marker.
  • the cytokine is selected from the group consisting of G-CSF, IFNg, IFNa, IL-27, IL-3, IL-6, and IL-10.
  • the growth factor is selected from the group consisting of FLT3L, SCF, G-CSF, and SDFla.
  • the mitogen is selected from the group consisting of LPS, PMA, and Thapsigargin.
  • the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • activation levels of an activatable element within the STAT pathway higher than a threshold level in response to a cytokine are indicative that an individual can not respond to treatment.
  • a treatment is chosen based on the ability of the cells to respond to treatment.
  • the activatable element within the STAT pathway is selected from the group consisting of p-Stat3, p-Stat5, p-Statl, and p-Stat6 and the cytokine is selected from the group consisting of IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, and G-CSF.
  • the activatable element within the STAT pathway is Stat 1 and the cytokine is IL-27 or G-CSF.
  • PI3K/AKT, or MAPK pathway higher than a threshold level in response to a growth factor or mitogen is indicative that an individual can not respond to treatment.
  • a treatment is chosen based on the ability of the cells to respond to treatment with a modulator.
  • the activatable element within the PI3K/AKT, or MAPK pathway is selected from the group consisting of p-ERK, p38 and pS6 and the growth factor or mitogen is selected from the group consisting of FLT3L, SCF, G-CSF, SDFla, LPS, PMA, and Thapsigargin.
  • activation levels of an activatable element within the phospholipase C pathway higher than a threshold level in response to an inhibitor is indicative that an individual can respond to treatment.
  • a treatment is chosen based on the ability of the cells to respond to treatment.
  • the activatable element within the phospholipase C pathway is selected from the group consisting of p-Slp-76, and Plcg2 and the inhibitor is H2O2.
  • activation levels, of an activatable element within the apoptosis pathway, higher than a threshold in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells is indicative that an individual can respond to treatment.
  • a treatment is chosen based on the ability of the cells to respond to treatment.
  • the activatable element within the apoptosis pathway is selected from the group consisting of Parp+, Cleaved Caspase 8, and Cytoplasmic Cytochrome C
  • the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • activation levels of an activatable element within the apoptosis pathway higher than a threshold in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells and activation levels of an activatable element within the STAT pathway higher than a threshold level in response to a cytokine is indicative that an individual can not respond to treatment.
  • the activatable element within the apoptosis pathway is selected from the group consisting of Cleaved PARP, Cleaved Caspase 8, and Cytoplasmic Cytochrome C
  • the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • the activatable element within the STAT pathway is selected from the group consisting of p-Stat3, p-Stat5, p-Statl, and p-Stat6 and the cytokine is selected from the group consisting of IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, and G-CSF.
  • the activatable element within the STAT pathway is Stat 1 and the cytokine is IL-27 or G-CSF.
  • the methods of the invention further comprise determining an activation level of an activatable element within a DNA damage pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells.
  • the activatable element within a DNA damage pathway is selected from the group consisting of Chk2, ATM, ATR and 14-3-3 and the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • activation levels higher than a threshold of an activatable element within a DNA damage pathway and activation levels lower than a threshold of an activatable element within the apoptosis pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells are indicative of a communication breakdown between the DNA damage response pathway and the apoptotic machinery and that an individual can not respond to treatment.
  • a treatment is chosen based on the ability of the cells to respond to treatment.
  • the methods of the invention further comprise determining an activation level of an activatable element within a cell cycle pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells.
  • the activatable element within a DNA damage pathway is selected from the group consisting of Cdc25, p53, CyclinA-Cdk2, CyclinE-Cdk2, CyclinB-Cdkl, p21, and Gadd45and the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • the methods of the invention further comprise determining the levels of a drug transporter and/or a cytokine receptor.
  • the cytokine receptors or drug transporters are selected from the group consisting of MDR1, ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3, and c-kit.
  • levels higher than a threshold of the drug transporter and/or said cytokine receptor are indicative that an individual can not respond to treatment.
  • a treatment is chosen based on the ability of the cells to respond to treatment.
  • the methods of the invention further comprise determining the activation levels of an activatable element within the Akt pathway in response to an inhibitor, where activation levels higher that a threshold of the activatable element within the Akt pathway in response to the inhibitor are indicative that the individual can not respond to treatment.
  • a treatment is chosen based on the ability of the cells to respond to treatment.
  • activation levels higher than a threshold of an activatable element in the PI3K/AKT pathway in response to a growth factor is indicative that the individual can not respond to treatment.
  • the activatable element in the PI3K/Akt pathway is Akt and the growth factor is FLT3L.
  • activation levels higher than a threshold of an activatable element in the apoptosis pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells is indicative that the individual can respond to treatment.
  • the activatable element within the apoptosis pathway is Parp+ and the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • the invention provides a method of predicting a response to a treatment or choosing a treatment for AML in an individual where the individual is a secondary acute myeloid leukemia patient, the method comprising the steps of; (a) subjecting a cell population from the individual to IL-27 and G-CSF in separate cultures, (b) determining an activation level of pStatl in one or more cells from each separate culture, (c) predicting a response to a treatment or choosing a treatment for AML, in the individual, where if the activation levels of pStatl are higher than a threshold level in response to both IL-27 and G-CSF the individual can not respond to treatment and if the levels of pStatl are lower than a threshold in response to both IL-27 and G-CSF the individual can respond to treatment.
  • the treatment is a chemotherapy agent.
  • chemotherapy agents include, but are not limited to, cytarabine (ara-C), daunorubicin (Daunomycin), idarubicin (Idamycin), mitoxantrone and 6-thioguanine.
  • the treatment is allogeneic stem cell transplant or autologous stem cell transplant.
  • the invention provides a method of predicting a response to a treatment or choosing a treatment for AML in an individual, the method comprising the steps of: (a) subjecting a cell population from the individual to FLT3L, (b) determining an activation level of pAkt in one or more cells from the population (c) predicting a response to a treatment or choosing a treatment for AML in the individual, where if the activation levels of pAkt are higher than a predetermined threshold in response to FLT3L the individual can not respond to treatment.
  • the method further comprises the steps of: (d) subjecting a cell population from said individual to IL-27 in a separate culture, (e) determining an activation level of Statl in one or more cells from the separate culture, (f) predicting a response to a treatment or choosing a treatment for AML in the individual, where if the activation levels of pStatl are higher than a predetermined threshold in response to IL-27 the individual can not respond to treatment.
  • the method further comprises the step of: (g) subjecting a cell population from the individual to H 2 O 2 in a separate culture, (h) determining an activation level of Plcg2 in one or more cells from the separate culture (i) predicting a response to a treatment or choosing a treatment for AML in the individual, wherein if the activation levels of Plcg2 are higher than a predetermined threshold in response to H 2 O 2 the individual can not respond to treatment.
  • the method further comprises the steps of (g) subjecting a cell population from said individual to Etoposide in a separate culture, (h) determining an activation level of Parp in one or more cells from the separate culture, and (i) predicting a response to a treatment for AML in said individual, where if the activation levels of Parp are higher than a predetermined threshold in response to Etoposide the individual can respond to treatment.
  • the treatment is a chemotherapy agent. Examples are shown above.
  • the invention provides methods of prediction response to a treatment and/or risk of relapse for AML in an individual, the method comprising the steps of: (a) subjecting a cell population from the individual to a modulator in the Tables 1(a) to 1(e) below, (b) determining an activation level of an activatable element in one or more cells from the population (c) predicting a response to a treatment, choosing a treatment or predicting risk of relapse for AML in the individual, where if the activation levels of the activatable elements are higher than a predetermined threshold in response to the modulator the individual can not respond to treatment or will have a higher probability of relapse.
  • a diagnosis, prognosis, a prediction of outcome such as response to treatment or relapse is performed by analyzing the two or more phosphorylation levels of two or more proteins each in response to one or more modulators.
  • the phosphorylation levels of the independent proteins can be measured in response to the same or different modulators. Grouping of data points increases predictive value.
  • the invention provides a method of diagnosing, prognosing or predicting a response to a treatment or choosing a treatment for AML in an individual, the method comprising the steps of: (a) subjecting a cell population from the individual in separate cultures to at least two modulators listed in la to le below; b) determining the activation level of at least three activatable elements listed in Tables 1(a) to 1(e) below; and (c) diagnosing, prognosing, or predicting a response to a treatment or choosing a treatment for AML based on the activation levels of the activatable elements.
  • the method further comprises determining the expression of a cytokine receptor or drug transporter selected from the group consisting of MDR1, ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3, and c-Kit.
  • the invention provides methods of diagnosing, prognosing, determining progression, predicting a response to a treatment or choosing a treatment for acute leukemia in an individual, the methods comprising the steps of: (1) classifying one or more hematopoietic cells associated with acute leukemia, in the individual by a method comprising: a) subjecting a cell population comprising the one or more hematopoietic cells from the individual to a modulator listed in Tables 1(a) to 1(e) below, b) determining an activation level of at least one activatable element selected from the group listed in Tables 1(a) to 1(e) below in one or more cells from the individual, and c) classifying the one or more hematopoietic cells based on the activation levels of the activatable element; and (2) making a decision regarding a diagnosis, prognosis, progression, response to a treatment or a selection of treatment for acute leukemia in the individual based on the classification of said one
  • the invention provides methods for predicting response to a treatment for AML, MDS or MPN, wherein the positive predictive value (PPV) is higher than 60, 70, 80, 90, 95, or 99.9 %. In some embodiments, the invention provides methods for predicting response to a treatment for AML, MDS or MPN, wherein the PPV is equal or higher than 95%. In some embodiments, the invention provides methods for predicting response to a treatment for AML, MDS or MPN, wherein the negative predictive value (NPV) is higher than 60, 70, 80, 90, 95, or 99.9 %. In some embodiments, the invention provides methods for predicting response to a treatment for AML, MDS or MPN, wherein the NPV is higher than 85 %.
  • the invention provides methods for predicting risk of relapse at 2 years, wherein the PPV is higher than 60, 70, 80, 90, 95, or 99.9 %. In some embodiments, the invention provides methods for predicting risk of relapse at 2 years, wherein the PPV is equal or higher than 95%. In some embodiments, the invention provides methods for predicting risk of relapse at 2 years, wherein the NPV is higher than 60, 70, 80, 90, 95, or 99.9 %. In some embodiments, the invention provides methods for predicting risk of relapse at 2 years, wherein the NPV is higher than 80 %.
  • the invention provides methods for predicting risk of relapse at 5 years, wherein the PPV is higher than 60, 70, 80, 90, 95, or 99.9 %. In some embodiments, the invention provides methods for predicting risk of relapse at 5 years, wherein the PPV is equal or higher than 95%. In some embodiments, the invention provides methods for predicting risk of relapse at 5 years, wherein the NPV is higher than 60, 70, 80, 90, 95, or 99.9 %. In some embodiments, the invention provides methods for predicting risk of relapse at 5 years, wherein the NPV is higher than 80 %.
  • the invention provides methods for predicting risk of relapse at 10 years, wherein the PPV is higher than 60, 70, 80, 90, 95, or 99.9 %. In some embodiments, the invention provides methods for predicting risk of relapse at 10 years, wherein the PPV is equal or higher than 95%. In some embodiments, the invention provides methods for predicting risk of relapse at 10 years, wherein the NPV is higher than 60, 70, 80, 90, 95, or 99.9 %. In some embodiments, the invention provides methods for predicting risk of relapse at 10 years, wherein the NPV is higher than 80 %.
  • the p value in the analysis of the methods described herein is below 0.05, 04, 0.03, 0.02, 0.01, 0.009, 0.005, or 0.001. In some embodiments, the p value is below 0.001.
  • the invention provides methods for diagnosing, prognosing, determining progression or predicting response for treatment of AML wherein the p value is below 0.05, 04, 0.03, 0.02, 0.01, 0.009, 0.005, or 0.001. In some embodiments, the p value is below 0.001.
  • the invention provides methods for diagnosing, prognosing, determining progression or predicting response for treatment of AML wherein the AUC value is higher than 0.5, 0.6, 07, 0.8 or 0.9. In some embodiments, the invention provides methods for diagnosing, prognosing, determining progression or predicting response for treatment of AML wherein the AUC value is higher than 0.7. In some embodiments, the invention provides methods for diagnosing, prognosing, determining progression or predicting response for treatment of AML wherein the AUC value is higher than 0.8. In some
  • the invention provides methods for diagnosing, prognosing, determining progression or predicting response for treatment of AML wherein the AUC value is higher than 0.9.
  • Another method of the present invention is a method for determining the prognosis and therapeutic selection for an individual with myelodysplasia or MDS.
  • multiparametric flow cytometry could separate a patient into one of five groups consisting of: "AML-like", where a patient displays signaling biology that is similar to that seen in acute myelogenous leukemia (AML) requiring intensive therapy, "Epo-Responsive", where a patient's bone marrow or potentially peripheral blood, shows signaling biology that corresponds to either in-vivo or in-vitro sensitivity to
  • erythropoietin "Lenalidomide responsive”, where a patient's bone marrow or potentially peripheral blood, shows signaling biology that corresponds to either in-vivo or in-vitro sensitivity to Lenalidomide, "Auto-immune”, where a patient's bone marrow or potentially peripheral blood, shows signaling biology that corresponds to sensitivity to cyclosporine A(CSA) and anti- thymocyte globulin(ATG).
  • the individual's blood or marrow sample could reveal signaling biology that corresponds to either in-vivo or in-vitro sensitivity to cytarabine or to a class of drugs including but not limited to direct drug resistance modulators, anti-Bcl-2 or pro-apoptotic drugs, proteosome inhibitors, DNA methyl transferase inhibitors, histone deacetylase inhibitors, anti-angiogenic drugs, farnesyl transferase inhibitors, FLt3 ligand inhibitors, or ribonucleotide reductase inhibitors.
  • direct drug resistance modulators anti-Bcl-2 or pro-apoptotic drugs
  • proteosome inhibitors include DNA methyl transferase inhibitors, histone deacetylase inhibitors, anti-angiogenic drugs, farnesyl transferase inhibitors, FLt3 ligand inhibitors, or ribonucleotide reductase inhibitors.
  • different gating strategies can be used in order to analyze only blasts in the sample of mixed population after treatment with the modulator. These gating strategies can be based on the presence of one or more specific surface marker expressed on each cell type.
  • the first gate eliminates cell doublets so that the user can focus on singlets. The following gate can differentiate between dead cells and live cells and subsequent gating of live cells classifies them into blasts, monocytes and lymphocytes.
  • a clear comparison can be carried out to study the effect of potential modulators, such as G-SCF on activatable elements in: ungated samples, blasts, monocytes, granulocytes and lymphocytes by using two-dimensional contour plot representations of Stat5 and Stat3 phosphorylation (x and Y axis) of patient samples.
  • the level of basal phosphorylation and the change in phosphorylation in both Stat3 and Stat5 phosphorylation in response to G-CSF can be compared.
  • G-CSF increases both STAT3 and STAT5 phosphorylation and this dual signaling can occur concurrently (subpopulations with increases in both pSTAT 3 and pSTAT5) or individually (subpopulations with either an increase in phospho pSTAT 3 or pSTAT5 alone).
  • the advantage of gating is to get a clearer picture and more precise results of the effect of various activatable elements on blasts.
  • a gate is established after learning from a responsive subpopulation. That is, a gate is developed from one data set. This gate can then be applied retrospectively or prospectively to other data sets (See Figures 5, 6, and 7).
  • the cells in this gate can be used for the diagnosis or prognosis of a condition.
  • the cells in this gate can also be used to predict response to a treatment or for treatment selection. The mere presence of cells in this gate may be indicative of a diagnosis, prognosis, or a response to treatment.
  • the presence of cells in this gate at a number higher than a threshold number may be indicative of a diagnosis, prognosis, or a response to treatment.
  • Some methods of analysis are: 1) measuring the difference in the log of the median fluorescence value between an unstimulated fluorochrome-antibody stained sample and a sample that has not been treated with a stimulant or stained (log (MFI Unstimulated Stained ) - log (MFI Gated Unstained )), 2) measuring the difference in the log of the median fluorescence value between a stimulated fluorochrome-antibody stained sample and a sample that has not been treated with a stimulant or stained (log (MFI Stimuiated stained ) - log(MFI Gated unstained )), 3) Measuring the change between the stimulated fluorochrome-antibody stained sample and the unstimulated fluorochrome-antibody stained sample log (MFI Stimuiated stained ) - log (MFI Unstimulated stained ), also called "fold change in median fluorescence intensity", 4) Measuring the percentage of cells in a Quadrant Gate of a contour plot which measures multiple populations in
  • % PARP+ cells positive for cleaved PARP
  • p-S6 and p-Akt See Figure 2B
  • measurements examining the changes in the frequencies of cells may be applied such as the Change in % PARP + which would measure the % PARP+ Stimulated stained - %
  • the AUC unstim metric also measures changes in population frequencies measuring the frequency of cells to become positive compared to an unstimulated condition (Figure 2B).
  • the metrics described in Figure 2B can be use to measure apoptosis. For example, these metrics can be applied to cleaved Caspase-3 and Caspase-8, e.g., Change in % Cleaved Caspase-3 or Cleaved Caspase-8.
  • third-color analysis (3D plots); percentage positive and relative expression of various markers; clinical analysis on an individual patient basis for various parameters, including, but not limited to age, race, cytogenetics, mutational status, blast percentage, CD34+ percentage, time of relapse, survival, etc. See Figure 2.
  • third color analysis 3D plots
  • Cytobank 2D plus third D in color.
  • the invention provides methods and compositions related to acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the invention provides a test to determine whether or not, or the likelihood that, an AML patient, e.g., an elderly AML patient, such as described elsewhere herein, will respond to induction therapy, e.g., therapy including administration of ara C, generally in conjunction with an anthracycline, such as daunorubicin.
  • induction therapy e.g., therapy including administration of ara C, generally in conjunction with an anthracycline, such as daunorubicin.
  • kits for use in such a test e.g., kits for use in such a test.
  • the invention relates to treating patients already diagnosed with AML, for example with standard induction therapy (i.e., a therapy that includes at least the administration of araC), on the basis of a test to determine likelihood of response to therapy, e.g., standard induction therapy.
  • standard induction therapy i.e., a therapy that includes at least the administration of araC
  • the invention relates to reviewing the results of the test and determining whether or not to treat a patient.
  • "likelihood of response to therapy" is likelihood of response immediately after therapy, e.g., likelihood of fewer than 5% blasts at the end of induction and no extramedullary blasts (EMB).
  • the test uses cells obtained from the patient, for example peripheral blood (PB) cells or bone marrow (BM) cells which are modulated, for example, with one or more apoptosis-inducing agents, and monitoring two or more, characteristics of the cells in response to the apoptosis-inducing agent or agents, where one of the characteristics is the level of a protein or other substance associated with apoptosis (a "marker"), such as an activatable element, in single cells.
  • the second characteristic may be, e.g., a level of expression of another marker, e.g., a surface marker related to maturity of the cells, for example maturity of blasts, in single cells.
  • the apoptosis-inducing agent is selected from the group consisting of etoposide, staurosporine, araC, daunorubicin, and combinations thereof. In certain embodiments, at least two apoptosis-inducing agents are used, for example, araC and
  • the apoptosis marker is pChk2, p-H2AX,Bcl-2, cytochrome c, c-caspase 3, c-caspase 8, or cPARP (c stands for "cleaved”).
  • cPARP is also designated “PARP .”
  • the marker of apoptosis is an activatable element selected from the group consisting of pChk2, p-H2AX, c-caspase 3, c-caspase 8, or cPARP.
  • the marker of apoptosis is cPARP.
  • the surface marker related to maturity is CD34.
  • the AML patient is an elderly patient, for example, a patient whose age is greater than (in some cases greater than or equal to) 55, 60, 65, or 69 at the time the test is performed.
  • the patient is a non-M3 AML patient.
  • the cells are prepared for the assay then modulated with the one or more apoptosis-inducing agents and allowed to incubate for a pre-determined time, then they are exposed to a cocktail containing one or more detectable binding elements that each binds to a characteristics to be detected, e.g., apoptosis marker, surface marker; generally, the cocktail to which any given cell sample is exposed includes detectable binding elements specific to all of the two or more characteristics, for example, a binding element to an apoptosis marker and a binding element to a surface marker.
  • the binding element may be an antibody, and may be detectable due to labeling, e.g., labeling with a fluorescent label or labeling with a mass tag (if detection is by mass cytometry).
  • the binding of the binding element(s) in each cell is detected by any suitable detection method, for example, flow cytometry or mass cytometry.
  • the signal detected will depend on the nature of the detector; for simplicity, it is described herein as fluorescence, as for a flow cytometer, but it will be appreciated that any detectable signal may be used, such as mass with a mass cytometer, and the analysis is essentially the same. Analysis of cells by flow cytometry or mass cytometry for characteristics such as surface markers and levels of activatable elements are known and described elsewhere herein.
  • samples are either cryopreserved or fresh. If a sample is cryopreserved, then it should be thawed and allowed to "rest" after thawing, for at least one hour and preferably for two hours. If a fresh sample is used, i.e., a sample that has not been frozen, the sample should be allowed to come to room temperature but the rest time may be shorter, e.g., less than two hours, or less than one hour, or less than 30 min, or no rest time may be required.
  • the time of incubation is important. If a marker of apoptosis that is relatively upstream is to be used, then shorter incubations may be desirable, e.g., 6-18, 6-20, 6-23 hours or even less than 6 hours. Such markers include pChk2, p-H2AX, Bcl-2, cytochrome c and the cleaved caspases, e.g., c-caspase 3 or c-caspase 8. If the marker of apoptosis is cPARP, which is relatively downstream, a longer incubation time may be desirable, e.g., 16-36 hours, such as 20- 30 hours, or in certain embodiments 22-28 hours, for example, 24 hours.
  • shorter time of incubation are acceptable even when using cPARP as a marker, such as 6-22 hours, or 6-20 hours, or 6-18 hours, or 6-16 hours, or 6-12 hours, or 6-10 hours, or 8-22 hours, or 8-20 hours, or 8-18 hours, or 8-16 hours, or 8-12 hours, or 8-10 hours, or 12-22 hours, or 12-20 hours, or 10- 18 hours, or 10-16 hours, or 10-12 hours, or 12-22 hours, or 12-20 hours, or 12-18 hours, or 12- 16 hours, or 14-22 hours, or 14-20 hours, or 14-18 hours, or 14-16 hours, or 16-22 hours, or 16- 20 hours, or 16-18 hours, or 18-22 hours, or 18-20 hours, or 20-22 hours or 20-23 hours.
  • the incubation time may be less than 6 hours when the marker is cPARP. At time periods longer than 24 hours, apoptosis may have progressed to the point where little or no meaningful data may be gathered, but in certain embodiments the incubation may be as long as 48 hour or even longer.
  • the incubation time may be 26-48 hours, or 26-42 hours, or 26-36 hours, or 26-30 hours, or 26-28 hours, or 28-48 hours, or 28-42 hours, or 28-36 hours, or 28-30 hours, or 30-48 hours, or 30-42 hours, or 30-36 hours, or 32-48 hours, or 32-42 hours, or 32-36 hours, or 34-48 hours, or 34-42 hours, or 34-36 hours, or 36-48 hours, or 36-42 hours, or 40-48 hours, or 42-48 hours. In certain embodiments, the incubation time may be longer than 48 hours.
  • the data for the cells from the patient is then gated to provide an estimate of viable cells in the original sample, then if the estimate is below a certain threshold value, for example, less than 15-50%, such as less than 20-40%), for example less than 20-30%), e.g., less than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30%>, the test results are not calculated further and no prediction is made as to response or non-response to therapy, e.g., induction therapy.
  • a certain threshold value for example, less than 15-50%, such as less than 20-40%), for example less than 20-30%), e.g., less than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30%>
  • the threshold is 25% viable cells.
  • cells deemed to be non-viable e.g., by some combination of scatter, Amine Aqua, and cPARP, may simply not be used in the analysis— however it will be appreciated that if the marker of apoptosis is cPARP, this approach cannot be taken.
  • One aspect of the invention is the manner in which viability is determined.
  • the manner and order in which the cells are gated can be important to the results of the test, e.g., results of viability analysis.
  • the gating is by side scatter and forward scatter (SSC and FSC) to eliminate cell debris, by Amine Aqua or other indicator of cell death and SSC to eliminate dead cells, by SSC and CD45 to select for blasts, and finally measures of the characteristics are taken (e.g., measure level of apoptosis and measure levels of immature blasts, CD34+.
  • Other cell markers useful in the invention include CD1 lb and CD117).
  • the measure of the apoptosis may be used in the preliminary viability gate; for example, cPARP levels as described in more detail herein.
  • the gating is done in the order above.
  • cells are gated for CD45+ before gating for Amine Aqua or other indicator of cell death.
  • cells are first gated by side scatter and forward scatter (SSC and FSC) to eliminate cell debris, then by Amine Aqua or other indicator of cell death and SSC to eliminate dead cells, then by SSC and CD45 to select for blasts, and finally measures of the characteristics are taken (e.g., measure level of apoptosis and measure levels of immature blasts, CD34+).
  • PH (number cells cPARP- in PI blasts (determined by CD45 gating)/total intact cells) X 100.
  • the number of intact cells is determined in the first gate, i.e., FSC vs. SSC, whereas the number of cPARP- cells is determined only in PI blasts, that is, after the third gate, i.e., CD45 vs. SSC.
  • cPARP- cells are cells whose cPARP reading, e.g., fluorescence of a fluorescently-labeled antibody in flow cytometry, is below a certain pre-determined threshold value.
  • viability is set too early, e.g., at FSC vs. SSC, or at Amine Aqua vs. SSC, cells will be included in the assay that are apoptotic or otherwise unhealthy or even dead, and if viability is set too late, e.g., after cPARP gating, then no meaningful data may be obtained.
  • the position in the gating at which viability is determined, and the order of the gating are thus important and not easily determined.
  • the viability gate is positive, the data is then analyzed to obtain a value that indicates the likelihood that the patient will respond to therapy, e.g., induction therapy.
  • the value is simply a threshold value, above which (or above or equal to) a patient is deemed to be responsive to therapy, e.g., induction therapy, and below which (or below or equal to) a patient is deemed to be non-repsonsive.
  • the value can be interpreted to give a probability of response to therapy, with no strict cutoff, so that the patient and/or the patient's healthcare provider(s) may make an informed decision as to whether or not to go ahead with therapy, e.g., induction therapy.
  • the invention provides for at least two characteristics to be used in the calculation of the test value, where one of the characteristics is the level of an marker of apoptosis, for example, level of a phosphorylated protein or level of a cleaved protein, such as a protein associated with apoptosis, in single cells, e.g., a level of cPARP, and the second characteristic is a level of expression of a marker, e.g., a surface marker related to maturity of the cells, for example maturity of blasts, in single cells, e.g., a level of CD34.
  • a marker of apoptosis for example, level of a phosphorylated protein or level of a cleaved protein, such as a protein associated with apoptosis
  • the second characteristic is a level of expression of a marker, e.g., a surface marker related to maturity of the cells, for example maturity of blasts, in single cells, e.g., a level of
  • the apoptosis marker cPARP is an indicator that the one or more apoptosis-inducing agents have caused the cell to enter apoptosis, and if enough cells, e.g., blasts, from the patient show this characteristic, then the patient is more likely to be responsive to induction therapy.
  • CD34 is another indication that the induction therapy will be successful that is relatively independent of apoptosis marker, that is, if after treatment with the one or more apoptosis-inducing agents the number of cells expressing CD34 (or expression above a certain threshold) decreases, this indicates a relative absence of immature blasts after the treatment (for example, immature blasts have been killed or rendered not viable by the treatment), and the patient will be more likely to respond to induction therapy.
  • Other useful cell surface markers in this regard include CD1 lb and CD 117. In both cases the characteristic is compared to cells that have not been treated with the apoptosis-inducing agent or agents.
  • the assay requires an early timepoint, e.g., 15 min, for cells that are untreated with apoptosis-inducing agents, then a later timepoint, e.g., 24 hours, or other suitable time as described herein, in which both untreated and apoptosis- inducing agent-treated cells are assayed. It is the change from the early timepoint to the late timepoint for each characteristic that is measured. Thus, there is an early timepoint and a later timepoint.
  • the early timepoint may be a time in a range from 0 min to 120 min, or 0 min to 90 min, or 0 min to 60 min, or 0 min to 45 min, or 0 min to 30 min, or 0 min to 25 min, or 0 min to 20 min, or 0 min to 15 min, or 0 min to 12 min, or 0 min to 10 min, or 0 min to 8 min, or 0 min to 5 min, or 2 min to 120 min, or 2 min to 90 min, or 2 min to 60 min, or 2 min to 45 min, or 2 min to 30 min, or 2 min to 25 min, or 2 min to 20 min, or 2 min to 15 min, or 2 min to 12 min, or 2 min to 10 min, or 2 min to 8 min, or 2 min to 5 min, or 5 min to 120 min, or 5 min to 90 min, or 5 min to 60 min, or 5 min to 45 min, or 5 min to 30 min, or 5 min to 25 min, or 5 min to 20 min, or 5 min to 90 min, or 5 min to 60
  • a first timepoint is taken, preferably in a sample that has not been treated with apoptosis-inducing agent or agents, and compared to a second timepoint for both untreated and treated cells, i.e., a total of three wells in, e.g., a 96-well plate (though other wells may be used as controls and for other reasons).
  • the early timepoint is used as the sample to determine cell viability.
  • any suitable metric may be used to express the change. Metrics are well-known and are described elsewhere herein.
  • the Uu (Mann- Whitney) metric is used. Uu has a range from 0.5 (no change between two samples) to 1.0 (maximum increase in characteristic measured) or to 0 (maximum decrease in characteristic measured). Because the change in cPARP or other apoptosis marker that is associated with probable response to treatment will be greater than 0.5, the apoptosis marker is evaluated as (Uu-0.5) (see Table 48, Example 22), and if Uu is less than or equal to 0.5 it is set at 0.
  • the coefficient for the apoptosis marker is at 1.25 to 5 times greater than the coefficient for the marker for cell maturity, e.g., CD34, or 1.5-5 times greater, or 1.75-5 times greater, or 2-5 times greater, or 2.25-5 times greater, or 2.5-5 times greater, or 2.75-5 times greater, or 3-5 times greater, or 3.5-5 times greater, or 4-5 times greater, or 1.25-4 times greater, or 1.5-4 times greater, or 1.75-4 times greater, or 2-4 times greater, or 2.25-4 times greater, or 2.5-4 times greater, or 2.75-4 times greater, or 3-4 times greater, or 3.5-4 times greater, or 4-5 times greater, , or 1.25-3.5 times greater, or 1.5-3.5 times greater, or 1.75-3.5 times greater, or 2-3.5 times greater, or 2.25-3.55 times greater, or 2.5-3.5 times greater, or 2.75-3.5 times greater, or 3-3.5 times greater, or , or 1.25-3 times greater, or 1.5-3.0 times
  • CD34 CD34 2 and ⁇ is the vector of the regression coefficients so that the overall continuous score is a value between 0 to 1. Note that in this example the values are squared before being multiplied by the appropriate coefficient and in certain embodiments one or more of the values is squared.
  • the decision to treat the patient is made by comparing this value to a threshold value, for example, 0.6 or similar value.
  • the value is related to a probability of response to treatment and the probability is used to determine whether or not to treat the patient.
  • a marker of apoptosis e.g., cPARP is used, after gating for viable cells; in certain embodiments, only a marker for cell maturity, e.g., CD34, may be used after gating for viable cells; however, the preferred embodiment is to use both markers, combined as above.
  • the test may also utilize one or more controls in order to ensure reliability.
  • one or more controls for example, in some embodiments, Rainbow Control Particles, as described in Example 22, are used in order to ensure cytometer reliability.
  • one or more control cell lines are used, also as described in Example 22, as a control for the end-to-end process.
  • lyophilized cells which have been treated with the appropriate modulator and known to contain certain levels of one or more elements, for example, activatable elements, may also be used to confirm that the test is measuring modulation properly.
  • the invention is directed to treating an elderly AML patient, such as a patient over 55 years old suffering from non-M3 AML by standard induction therapy, i.e., arac and an anthracycline such as daunorubicin.
  • the patient is a de novo or secondary AML patient and a sample is used which is a bone marrow sample.
  • the patient is a de novo AML patient and a sample is used which is a peripheral blood sample.
  • the decision to treat the patient is made by the patient and/or one or more healthcare provider is based at least in part on the results of a test as described above, giving the probability for complete remission immediately after induction therapy, in which cells from a sample from the patient, e.g., a bone marrow sample or a peripheral blood sample, are treated with a combination of araC and daunorubicin for a time period, e.g., 24 hours, or any other suitable time period, then fixed, permeabilized, and exposed to an antibody cocktail containing labeled antibody to cPARP and labeled antibody to CD34, as well as antibody to CD45, and also exposed to Amine Aqua, and an additional cell sample at an early timepoint, e.g., 15 minutes or any suitable timepoint as described herein, where the early timepoint sample is also fixed, permeabilized, and exposed to the cocktail and to Amine Aqua, and the levels of cPARP, CD34, CD45, and Amine Aqua or other cell death indicator are detected by a suitable
  • cPARP and CD34 are measured, and a decision to continue is based on a viability determination as described above. If the assay continues, the values for cPARP and CD34 at the later timepoint are compared to those at the earlier timepoint, using Uu metric.
  • the classifier is obtained by multiplying each parameter ((Uu-0.5) in the case of cPARP and ( 0.5-Uu) in the case of CD34, one or both of which may be squared) by a coefficient, where the coefficient for cPARP is at least 1.5 greater than the coefficient for CD34, in some embodiments at least 2.0 times greater and in some embodiments at least 2.5 times greater, and further modified as described in Table 48, Example 22, to obtain a value between 0 and 1 that correlates to the probability that the patient will respond to induction treatment, with a value of 0 being 0 probability of response and a value of 1 being 100% probability of response.
  • the patient is a de novo AML patient.
  • the patient is a secondary AML patient.
  • the decision to treat the patient may be made using other factors as well, such as patient age, gender, cytogenetics, health, especially cardiovascular health, and the like.
  • the invention also provides a system for treating an elderly patient (e.g., a patient greater than 55 years old, or as described elsewhere herein) suffering from AML with induction therapy, where the system includes the patient and one or more healthcare providers for the patient, a sample from the patient, such as a BM or PB sample, a transportation system for transporting the sample from the site of obtaining the sample to a test site, a test site for testing the sample to determine whether or not the patient will respond to induction therapy as described herein, a report-generating module for generating a report to communicate the results of the test to the patient and/or their healthcare provider(s), and a communication system for
  • the system may further include a site for administering induction therapy, for example, administering ara C to the patient and, generally, also administering an anthracycline such as daunorubicin to the patient.
  • a site for administering induction therapy for example, administering ara C to the patient and, generally, also administering an anthracycline such as daunorubicin to the patient.
  • kits are provided by the invention.
  • the kits can provide at least two agents for inducing apoptosis in test cells, such as two agents selected from etoposide, ara C, daunorubicin, and staurosporine, for example, ara C and daunorubicin; a detectable binding element for detecting a marker of apoptosis where the marker is selected from pChk2, p- H2AX,Bcl-2, cytochrome c, c-caspase 3, c-caspase 8, and cPARP, for example, cPARP; at least two detectable binding elements for cell surface markers, such as at least two of CD34, CD1 lb, CD45, and CD117, for example, CD45 and CD34; and instructions for use, where the instructions for use may be physically included with the other elements of the kit or may be supplied separately for use with the kit by electronic or physical delivery to an end user of the kit.
  • the detectable binding element can be an antibody or antibody fragment, as described elsewhere herein, for example, a labled antibody such as a fluorescently labeled antibody or an antibody labeled with a mass tag.
  • the kit can further include one or more reagents for detecting dead cells, such as Amine Aqua.
  • the kit can further include at least one, or at least two control cell lines for maintaining consistency of the assay, such as one or more of the control cell lines described in Example 22.
  • the kit can further include Rainbow Control Particles, such as those described in Example 22.
  • the kit can further include cells that have been modulated then lyophilized, as controls to ensure that the assay is working for a particular modulator and activatable element.
  • the kit can further include software, either in physical form, e.g., as tangible electronically readable medium, or delivered to the end user electronically, e.g., cloud- based.
  • the kit may include buffers, equipment, apparatus, and the like as necessary or desirable to run the test in an optimal fashion.
  • the methods of the invention are applicable to any condition in an individual involving, indicated by, and/or arising from, in whole or in part, altered physiological status in a cell.
  • physiological status includes mechanical, physical, and biochemical functions in a cell.
  • the physiological status of a cell is determined by measuring characteristics of cellular components of a cellular pathway.
  • Cellular pathways are well known in the art.
  • the cellular pathway is a signaling pathway.
  • Signaling pathways are also well known in the art (see, e.g., Hunter T., Cell 100(1): 113-27 (2000); Cell Signaling Technology, Inc., 2002 Catalogue, Pathway Diagrams pgs. 232-253).
  • a condition involving or characterized by altered physiological status may be readily identified, for example, by determining the state in a cell of one or more activatable elements, as taught herein.
  • the present invention is directed to methods for classifying one or more cells in a sample derived from an individual having or suspected of having a condition.
  • Example conditions include AML.
  • the invention allows for identification of prognostically and therapeutically relevant subgroups of the conditions and prediction of the clinical course of an individual.
  • the invention provides methods of classifying a cell according to the activation levels of one or more activatable elements in a cell from an individual having or suspected of having a condition.
  • the classification includes classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome can be the prognosis and/or diagnosis of a condition, and/or staging or grading of a condition.
  • the classifying of the cell includes classifying the cell as a cell that is correlated with a patient response to a treatment. In some embodiments, the classifying of the cell includes classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • the methods and compositions of the invention may be employed to examine and profile the status of any activatable element in a cellular pathway, or collections of such activatable elements.
  • Single or multiple distinct pathways may be profiled (e.g. sequentially or simultaneously), or subsets of activatable elements within a single pathway or across multiple pathways can be examined (e.g. sequentially or simultaneously).
  • apoptosis, signaling, cell cycle and/or DNA damage pathways are characterized in order to classify one or more cells in an individual.
  • the characterization of multiple pathways can reveal operative pathways in a condition that can then be used to classify one or more cells in an individual.
  • the classification includes classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome can be the prognosis and/or diagnosis of a condition, and/or staging or grading of a condition.
  • the classifying of the cell includes classifying the cell as a cell that is correlated with a patient response to a treatment. In some embodiments, the classifying of the cell includes classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance. In some embodiments, the cell classification includes correlating a response to a potential drug treatment. In another embodiment, the present invention includes a method for drug screening. See also U.S. Ser. Nos. 12/432,720 and 61/048,886 for activatable elements.
  • activation can result in a change in the activatable protein that is detectable by some indication (termed an "activation state indicator"), e.g. by altered binding of a labeled binding element or by changes in detectable biological activities (e.g., the activated state has an enzymatic activity which can be measured and compared to a lack of activity in the non-activated state).
  • an activation state indicator e.g. by altered binding of a labeled binding element or by changes in detectable biological activities (e.g., the activated state has an enzymatic activity which can be measured and compared to a lack of activity in the non-activated state.
  • two or more activation states e.g. "off and "on”
  • the activation state of an individual activatable element can be in the on or off state.
  • an individual phosphorylatable site on a protein can activate or deactivate the protein.
  • Phosphorylation of an adapter protein can promote its interaction with other components/proteins of distinct cellular signaling pathways.
  • the difference in enzymatic activity in a protein can reflect a different activation state.
  • the terms "on” and "off,” when applied to an activatable element that is a part of a cellular constituent, are used here to describe the state of the activatable element, and not the overall state of the cellular constituent of which it is a part.
  • the activation state of an individual activatable element can be represented as continuous numeric values representing a quantity of the activatable element or can be discretized into categorical variables. For instance, the activation state may be discretized into a binary value indicating that the activatable element is either in the on or off state.
  • an individual activatable element can be represented as continuous numeric values representing a quantity of the activatable element or can be discretized into categorical variables. For instance, the activation state may be discretized into a binary value indicating that the activatable element is either in the on or off state.
  • phosphorylatable site on a protein will either be phosphorylated and then be in the "on” state or it will not be phosphorylated and hence, it will be in the "off state. See Blume- Jensen and Hunter, Nature, vol 411, 17 May 2001, p 355-365. [00182] Typically, a cell possesses a plurality of a particular protein or other constituent with a particular activatable element and this plurality of proteins or constituents usually has some proteins or constituents whose individual activatable element is in the on state and other proteins or constituents whose individual activatable element is in the off state.
  • each activatable element can be measured through the use of a binding element that recognizes a specific activation state, only those activatable elements in the specific activation state recognized by the binding element, representing some fraction of the total number of activatable elements, will be bound by the binding element to generate a measurable signal.
  • the measurable signal corresponding to the summation of individual activatable elements of a particular type that are activated in a single cell can be the "activation level" for that activatable element in that cell.
  • Activation levels for a particular activatable element may vary among individual cells so that when a plurality of cells is analyzed, the activation levels follow a distribution.
  • the distribution may be a normal distribution, also known as a Gaussian distribution, or it may be of another type. Different populations of cells may have different distributions of activation levels that can then serve to distinguish between the populations.
  • specific activatable elements, signaling pathways, and drug transporters see U.S. Ser. No. 61/350,864 or U.S. Pub. No. 2009/0269773, which are hereby incorporated by reference in their entireties.
  • the basis for classifying cells is that the distribution of activation levels for one or more specific activatable elements will differ among different phenotypes.
  • a certain activation level, or more typically a range of activation levels for one or more activatable elements seen in a cell or a population of cells, is indicative that that cell or population of cells belongs to a distinctive phenotype.
  • the activation level or levels of one or more activatable elements optionally in conjunction with levels of one or more levels of biomolecules that may or may not contain activatable elements, of cell or a population of cells may be used to classify a cell or a population of cells into a class.
  • the activation level of intracellular activatable elements of individual single cells is known they can be placed into one or more classes, e.g., a class that corresponds to a phenotype.
  • a class encompasses a class of cells wherein every cell has the same or substantially the same known activation level, or range of activation levels, of one or more intracellular activatable elements. For example, if the activation levels of five intracellular activatable elements are analyzed, predefined classes of cells that encompass one or more of the intracellular activatable elements can be constructed based on the activation level, or ranges of the activation levels, of each of these five elements. It is understood that activation levels can exist as a distribution and that an activation level of a particular element used to classify a cell may be a particular point on the distribution but more typically may be a portion of the distribution.
  • the basis for classifying cells may use the position of a cell in a contour or density plot.
  • the contour or density plot represents the number of cells that share a characteristic such as the activation level of activatable proteins in response to a modulator.
  • a characteristic such as the activation level of activatable proteins in response to a modulator.
  • the number of cells that have a specific activation level e.g. specific amount of an activatable element
  • a cell can be classified according to its location within a given region in the contour or density plot.
  • the basis for classifying cells may use a series of population clusters whose centers, centroids, boundaries, relative positions describe the state of a cell, the diagnosis or prognosis of a patient, selection of treatment, or predicting response to treatment or to a combination of treatments, or long term outcome.
  • the basis for classifying cells may use an N-dimensional Eigen map that describe the state of a cell, the diagnosis or prognosis of a patient, selection of treatment, or predicting response to treatment or to a combination of treatments, or long term outcome.
  • the basis for classifying cells may use a Bayesian inference network of activatable elements interaction capabilities that together, or in part, describe the state of a cell, the diagnosis or prognosis of a patient, selection of treatment, or predicting response to treatment or to a combination of treatments, or long term outcome. See U.S. publication no. 2007/0009923 entitled Use of Bayesian Networks for Modeling Signaling Systems, incorporated herein by reference on its entirety.
  • cellular redox signaling nodes are analyzed for a change in activation level.
  • Reactive oxygen species ROS
  • ROS can modify many intracellular signaling pathways including protein phosphatases, protein kinases, and transcription factors. This activity may indicate that the majority of the effects of ROS are through their actions on signaling pathways rather than via non-specific damage of
  • Reactive oxygen species can be measured.
  • One example technique is by flow cytometry. See Chang et al, Lymphocyte proliferation modulated by glutamine: involved in the endogenous redox reaction; Clin Exp Immunol. 1999 September; 117(3): 482-488.
  • Redox potential can be evaluated by means of an ROS indicator, one example being 2',7'- dichlorofluorescein-diacetate (DCFH-DA) which is added to the cells at an exemplary time and temperature, such as 37°C for 15 minutes.
  • DCFH-DA 2',7'- dichlorofluorescein-diacetate
  • DCF peroxidation can be measured using flow cytometry. See Yang KD, Shaio MF. Hydroxyl radicals as an early signal involved in phorbol ester-induced monocyte differentiation of HL60 cells. Biochem Biophys Res Commun.
  • other characteristics that affect the status of a cellular constituent may also be used to classify a cell. Examples include the translocation of biomolecules or changes in their turnover rates and the formation and disassociation of complexes of biomolecule.
  • Such complexes can include multi-protein complexes, multi-lipid complexes, homo- or hetero-dimers or oligomers, and combinations thereof.
  • proteolytic cleavage e.g. from exposure of a cell to an extracellular protease or from the intracellular proteolytic cleavage of a biomolecule.
  • cellular pH is analyzed. See June, CH and Moore, and JS, Curr Protoc Immulon, 2004 Dec;Chapter 5:Unit 5.5; Leyval, D et al., Flow cytometry for the intracellular pH measurement of glutamate producing Corynebacterium glutamicum, Journal of Microbiological Methods, Volume 29, Issue 2, 1 May 1997, Pages 121-127; Weider, ED, et al, Measurement of intracellular pH using flow cytometry with carboxy-SNARF-1. Cytometry, 1993 Nov;14(8):916-21; and Valli, M, et al., Intracellular pH Distribution in Saccharomyces cerevisiae Cell Populations, Analyzed by Flow Cytometry, Applied and Environmental
  • the activatable element is the phosphorylation of
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • An immunoreceptor tyrosine-based inhibition motif (ITIM) is a conserved sequence of amino acids (S/I/V/LxYxxI/V/L) that is found in the cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM- possessing inhibitory receptors interact with their ligand, their ITIM motif becomes
  • these phospho-tyrosine residues provide docking sites for the Shps which may result in transmission of inhibitory signals and effect the signaling of neighboring membrane receptor complexes (Paul et al., Blood (2000 96:483). ITIMs can be analyzed by flow cytometry.
  • Additional elements may also be used to classify a cell, such as the expression level of extracellular or intracellular markers, nuclear antigens, enzymatic activity, protein expression and localization, cell cycle analysis, chromosomal analysis, cell volume, and morphological characteristics like granularity and size of nucleus or other distinguishing characteristics.
  • B cells can be further subdivided based on the expression of cell surface markers such as CD 19, CD20, CD22 or CD23.
  • predefined classes of cells can be aggregated or grouped based upon shared characteristics that may include inclusion in one or more additional predefined class or the presence of extracellular or intracellular markers, similar gene expression profile, nuclear antigens, enzymatic activity, protein expression and localization, cell cycle analysis,
  • chromosomal analysis cell volume, and morphological characteristics like granularity and size of nucleus or other distinguishing cellular characteristics.
  • the physiological status of one or more cells is determined by examining and profiling the activation level of one or more activatable elements in a cellular pathway.
  • a cell is classified according to the activation level of a plurality of activatable elements.
  • a hematopoietic cell is classified according to the activation levels of a plurality of activatable elements.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more activatable elements may be analyzed in a cell signaling pathway.
  • hematopoietic cell are correlated with a condition.
  • the activation levels of one or more activatable elements of a hematopoietic cell are correlated with a neoplastic or hematopoietic condition as described herein.
  • Examples of hematopoietic cells include, but are not limited to, AML cells.
  • the activation level of one or more activatable elements in single cells in the sample is determined.
  • Cellular constituents that may include activatable elements include without limitation proteins, carbohydrates, lipids, nucleic acids and
  • the activatable element may be a portion of the cellular constituent, for example, an amino acid residue in a protein that may undergo phosphorylation, or it may be the cellular constituent itself, for example, a protein that is activated by translocation, change in
  • a change occurs to the activatable element, such as covalent modification of the activatable element (e.g., binding of a molecule or group to the activatable element, such as phosphorylation) or a conformational change.
  • covalent modification of the activatable element e.g., binding of a molecule or group to the activatable element, such as phosphorylation
  • a conformational change e.g., binding of a molecule or group to the activatable element, such as phosphorylation
  • Such changes generally contribute to changes in particular biological, biochemical, or physical properties of the cellular constituent that contains the activatable element.
  • the state of the cellular constituent that contains the activatable element is determined to some degree, though not necessarily completely, by the state of a particular activatable element of the cellular constituent.
  • a protein may have multiple activatable elements, and the particular activation states of these elements may overall determine the activation state of the protein; the state of a single activatable element is not necessarily determinative. Additional factors, such as the binding of other proteins, pH, ion concentration, interaction with other cellular constituents, and the like, can also affect the state of the cellular constituent.
  • the activation levels of a plurality of intracellular activatable elements in single cells are determined. In some embodiments, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 intracellular activatable elements are determined.
  • Activation states of activatable elements may result from chemical additions or modifications of biomolecules and include biochemical processes such as glycosylation, phosphorylation, acetylation, methylation, biotinylation, glutamylation, glycylation,
  • biomolecules include the formation of protein carbonyls, direct modifications of protein side chains, such as o-tyrosine, chloro-, nitrotyrosine, and dityrosine, and protein adducts derived from reactions with carbohydrate and lipid derivatives. Other modifications may be non-covalent, such as binding of a ligand or binding of an allosteric modulator.
  • a covalent modification is the substitution of a phosphate group for a hydroxyl group in the side chain of an amino acid (phosphorylation).
  • phosphorylation A wide variety of proteins are known that recognize specific protein substrates and catalyze the phosphorylation of serine, threonine, or tyrosine residues on their protein substrates. Such proteins are generally termed "kinases.”
  • kinases Substrate proteins that are capable of being phosphorylated are often referred to as phosphoproteins (after phosphorylation). Once phosphorylated, a substrate phosphoprotein may have its phosphorylated residue converted back to a hydroxyl one by the action of a protein phosphatase that specifically recognizes the substrate protein.
  • Protein phosphatases catalyze the replacement of phosphate groups by hydroxyl groups on serine, threonine, or tyrosine residues. Through the action of kinases and phosphatases a protein may be reversibly phosphorylated on a multiplicity of residues and its activity may be regulated thereby. Thus, the presence or absence of one or more phosphate groups in an activatable protein is a preferred readout in the present invention.
  • Another example of a covalent modification of an activatable protein is the acetylation of histones.
  • histone acetylation and histone deactelyation have been linked with malignant progression. See Nature, 2004 May 27; 429(6990): 457-63.
  • Another form of activation involves cleavage of the activatable element.
  • one form of protein regulation involves proteolytic cleavage of a peptide bond. While random or misdirected proteolytic cleavage may be detrimental to the activity of a protein, many proteins are activated by the action of proteases that recognize and cleave specific peptide bonds. Many proteins derive from precursor proteins, or pro-proteins, which give rise to a mature isoform of the protein following proteolytic cleavage of specific peptide bonds. Many growth factors are synthesized and processed in this manner, with a mature isoform of the protein typically possessing a biological activity not exhibited by the precursor form.
  • proteolytically activated proteins are relatively short-lived proteins, and their turnover effectively results in deactivation of the signal. Inhibitors may also be used.
  • enzymes that are proteolytically activated are serine and cysteine proteases, including cathepsins and caspases respectively.
  • the activatable enzyme is a caspase.
  • the caspases are an important class of proteases that mediate programmed cell death (referred to in the art as "apoptosis").
  • Caspases are constitutively present in most cells, residing in the cytosol as a single chain proenzyme. These are activated to fully functional proteases by a first proteolytic cleavage to divide the chain into large and small caspase subunits and a second cleavage to remove the N- terminal domain. The subunits assemble into a tetramer with two active sites (Green, Cell 94:695-698, 1998).
  • Many other proteolytically activated enzymes known in the art as
  • zymogens also find use in the instant invention as activatable elements.
  • the activation of the activatable element involves prenylation of the element.
  • prenylation and grammatical equivalents used herein, is meant the addition of any lipid group to the element.
  • prenylation include the addition of farnesyl groups, geranylgeranyl groups, myristoylation and palmitoylation. In general these groups are attached via thioether linkages to the activatable element, although other attachments may be used.
  • activation of the activatable element is detected as intermolecular clustering of the activatable element.
  • clustering or “multimerization”, and grammatical equivalents used herein, is meant any reversible or irreversible association of one or more signal transduction elements.
  • Clusters can be made up of 2, 3, 4, etc., elements.
  • Clusters of two elements are termed dimers.
  • Clusters of 3 or more elements are generally termed oligomers, with individual numbers of clusters having their own designation; for example, a cluster of 3 elements is a trimer, a cluster of 4 elements is a tetramer, etc.
  • Clusters can be made up of identical elements or different elements. Clusters of identical elements are termed “homo” dimers, while clusters of different elements are termed “hetero” clusters. Accordingly, a cluster can be a homodimer, as is the case for the p 2 -adrenergic receptor.
  • a cluster can be a heterodimer, as is the case for GABA B R.
  • the cluster is a homotrimer, as in the case of TNFa, or a heterotrimer such the one formed by membrane-bound and soluble CD95 to modulate apoptosis.
  • the cluster is a homo-oligomer, as in the case of Thyrotropin releasing hormone receptor, or a hetero-oligomer, as in the case of TGFpi .
  • the activation or signaling potential of elements is mediated by clustering, irrespective of the actual mechanism by which the element's clustering is induced.
  • elements can be activated to cluster a) as membrane bound receptors by binding to ligands (ligands including both naturally occurring and synthetic ligands), b) as membrane bound receptors by binding to other surface molecules, or c) as intracellular (non- membrane bound) receptors binding to ligands.
  • the activatable element is a protein.
  • proteins that may include activatable elements include, but are not limited to kinases, phosphatases, lipid signaling molecules, adaptor/scaffold proteins, cytokines, cytokine regulators, ubiquitination enzymes, adhesion molecules, cytoskeletal/contractile proteins, heterotrimeric G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activating proteins, caspases, proteins involved in apoptosis, cell cycle regulators, molecular chaperones, metabolic enzymes, vesicular transport proteins, hydroxylases, isomerases, deacetylases, methylases, demethylases, tumor suppressor genes, proteases, ion channels, molecular transporters, transcription factors/DNA binding factors, regulators of transcription, and regulators of translation.
  • the protein that may be activated is selected from the group consisting of HER receptors, PDGF receptors, FLT3 receptor, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, erythropoetin receptor, thromobopoetin receptor, CD114, CD116, TIE1, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot
  • MKPs Dual Specificity phosphatases
  • DUSPs Dual Specificity phosphatases
  • CDC25 phosphatases Low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PP1, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins, phosphoinositide kinases, phopsholipases, prostaglandin synthases, 5- lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated
  • the activatable element is a nucleic acid.
  • Activation and deactivation of nucleic acids can occur in numerous ways including, but not limited to, cleavage of an inactivating leader sequence as well as covalent or non-covalent modifications that induce structural or functional changes.
  • many catalytic RNAs e.g. hammerhead ribozymes, can be designed to have an inactivating leader sequence that deactivates the catalytic activity of the ribozyme until cleavage occurs.
  • An example of a covalent modification is methylation of DNA. Deactivation by methylation has been shown to be a factor in the silencing of certain genes, e.g. STAT regulating SOCS genes in lymphomas.
  • the activatable element is a small molecule, carbohydrate, lipid or other naturally occurring or synthetic compound capable of having an activated isoform.
  • activation of these elements need not include switching from one form to another, but can be detected as the presence or absence of the compound.
  • activation of cAMP cyclic adenosine mono-phosphate
  • cAMP cyclic adenosine mono-phosphate
  • the methods described herein are employed to determine the activation level of an activatable element, e.g., in a cellular pathway.
  • Methods and compositions are provided for the classification of a cell according to the activation level of an activatable element in a cellular pathway.
  • the cell can be a hematopoietic cell.
  • hematopoietic cells include but are not limited to pluripotent hematopoietic stem cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, macrophage lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the cell is classified according to the activation level of an activatable element, e.g., in a cellular pathway comprises classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome is the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging examples include, but are not limited to, aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70 and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • methods and compositions are provided for the classification of a cell according to the activation level of an activatable element, e.g., in a cellular pathway wherein the classification comprises classifying a cell as a cell that is correlated to a patient response to a treatment.
  • the patient response is selected from the group consisting of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction.
  • methods and compositions are provided for the classification of a cell according to the activation level of an activatable element, e.g., in a cellular pathway wherein the classification comprises classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • methods and compositions are provided for the classification of a cell according to the activation level of an activatable element, e.g., in a cellular pathway wherein the classification comprises selecting a method of treatment.
  • Method of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, and watchful waiting.
  • the methods of the invention involve determining the activation levels of an activatable element in a plurality of single cells in a sample.
  • the methods of the invention are employed to determine the status of an activatable element in a signaling pathway.
  • a cell is classified, as described herein, according to the activation level of one or more activatable elements in one or more signaling pathways. Signaling pathways and their members have been described. See (Hunter T. Cell Jan. 7, 2000;100(1): 13-27; Weinberg, 2007; and Blume- Jensen and Hunter, Nature, vol 411, 17 May 2001, p 355-365 cited above).
  • Exemplary signaling pathways include the following pathways and their members: the JAK-STAT pathway including JAKs, STATs 2,3 4 and 5, the FLT3L signaling pathway, the The MAP kinase pathway including Ras, Raf, MEK, ERK and Elk; the PI3K/Akt pathway including PI-3-kinase, PDK1, Akt and Bad; the NF- ⁇ pathway including IKKs, IkB and NF- ⁇ , and the Wnt pathway including frizzled receptors, beta-catenin, APC and other co-factors and TCF (see Cell Signaling Technology, Inc. 2002 Catalog pages 231-279 and Hunter T., supra.).
  • the correlated activatable elements being assayed are members of the MAP kinase, Akt, NFkB, WNT, STAT and/or PKC signaling pathways. See the description of signaling pathways in U.S. S.N. 12/910,769 which is incorporated by reference in its entirety.
  • the status of an activatable element within the PI3K/AKT, or MAPK pathways in response to a growth factor or mitogen is determined.
  • the activatable element within the PI3K/AKT or MAPK pathway is selected from the group consisting of Akt, p-Erk, p38 and pS6 and the growth factor or mitogen is selected from the group consisting of FLT3L, SCF, G-CSF, SCF, G-CSF, SDFla, LPS, PMA and Thapsigargin.
  • the status of an activatable element within JAK/STAT pathways in response to a cytokine is determined.
  • the activatable element within the JAK/STAT pathway is selected from the group consisting of p-Stat3, p-Stat5, p-Statl, and p-Stat6 and the cytokine is selected from the group consisting of IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, and G-CSF.
  • the activatable element within the STAT pathway is Stat 1 and the cytokine is IL-27 or G-CSF.
  • the status of an activatable element within the phospholipase C pathway in response to an inhibitor is determined.
  • the activatable element within the phospholipase C pathway is selected from the group consisting of p-Slp-76, and Plcg2 and the inhibitor is H 2 O 2 .
  • the status of a phosphatase in response to an inhibitor is determined.
  • the inhibitor is H 2 O 2 .
  • the methods of the invention are employed to determine the status of a signaling protein in a signaling pathway known in the art including those described herein.
  • Exemplary types of signaling proteins within the scope of the present invention include, but are not limited to kinases, kinase substrates (i.e. phosphorylated substrates), phosphatases, phosphatase substrates, binding proteins (such as 14-3-3), receptor ligands and receptors (cell surface receptor tyrosine kinases and nuclear receptors)).
  • kinases and protein binding domains for example, have been well described (see, e.g., Cell Signaling Technology, Inc., 2002
  • Nuclear Factor-kappaB (NF- ⁇ ) Pathway Nuclear factor-kappaB (NF-kappaB) transcription factors and the signaling pathways that activate them are central coordinators of innate and adaptive immune responses. More recently, it has become clear that NF-kappaB signaling also has a critical role in cancer development and progression. NF-kappaB provides a mechanistic link between inflammation and cancer, and is a major factor controlling the ability of both pre-neoplastic and malignant cells to resist apoptosis-based tumor-surveillance mechanisms.
  • NF- ⁇ complexes In mammalian cells, there are five NF- ⁇ family members, RelA (p65), RelB, c- Rel, p50/p105 (NF-KB1) and p52/pl00 (NF-KB2) and different NF- ⁇ complexes are formed from their homo and heterodimers. In most cell types, NF- ⁇ complexes are retained in the cytoplasm by a family of inhibitory proteins known as inhibitors of NF- ⁇ (IKBS). Activation of NF-KB typically involves the phosphorylation of ⁇ by the ⁇ kinase (IKK) complex, which results in ⁇ ubiquitination with subsequent degradation. This releases NF- ⁇ and allows it to translocate freely to the nucleus.
  • IKBS inhibitory proteins
  • NF- ⁇ The genes regulated by NF- ⁇ include those controlling programmed cell death, cell adhesion, proliferation, the innate- and adaptive-immune responses, inflammation, the cellular-stress response and tissue remodeling.
  • the expression of these genes is tightly coordinated with the activity of many other signaling and transcription- factor pathways. Therefore, the outcome of NF- ⁇ activation depends on the nature and the cellular context of its induction. For example, it has become apparent that NF- ⁇ activity can be regulated by both oncogenes and tumor suppressors, resulting in either stimulation or inhibition of apoptosis and proliferation. See Perkins, N. Integrating cell-signaling pathways with NF-KB and IKK function. Reviews: Molecular Cell Biology.
  • receptor tyrosine kinases include but are not limited to FLT3 LIGAND, EGFR, IGF-1R, HER2/neu, VEGFR, and PDGFR.
  • the lipid second messengers generated by PBKs regulate a diverse array of cellular functions.
  • Akt a serine/threonine kinase, which is activated when its PH domain interacts with PI3, 4P 2 and PI3,4,5P 3 resulting in recruitment of Akt to the plasma membrane.
  • Akt is phosphorylated at threonine 308 by 3-phosphoinositide-dependent protein kinase- 1 (PDK-1) and at serine 473 by several PDK2 kinases.
  • Akt then acts downstream of PI3K to regulate the phosphorylation of a number of substrates, including but not limited to forkhead box O transcription factors, Bad, GSK-3P, ⁇ - ⁇ , mTOR, MDM-2, and S6 ribosomal subunit. These phosphorylation events in turn mediate cell survival, cell proliferation, membrane trafficking, glucose homeostasis, metabolism and cell motility.
  • Deregulation of the PI3K pathway occurs by activating mutations in growth factor receptors, activating mutations in a PI3-K gene (e.g. PIK3CA), loss of function mutations in a lipid phosphatase (e.g.
  • Wnt Pathway The Wnt signaling pathway describes a complex network of proteins well known for their roles in embryogenesis, normal physiological processes in adult animals, such as tissue homeostasis, and cancer. Further, a role for the Wnt pathway has been shown in self-renewal of hematopoietic stem cells (Reya T et al., Nature. 2003 May 22;423(6938):409- 14). Cytoplasmic levels of ⁇ -catenin are normally kept low through the continuous proteosomal degradation of ⁇ -catenin controlled by a complex of glycogen synthase kinase 3 ⁇ (GSK-3 ⁇ ), axin, and adenomatous polyposis coli (APC).
  • GSK-3 ⁇ glycogen synthase kinase 3 ⁇
  • APC adenomatous polyposis coli
  • PKC Protein Kinase C
  • the mammalian PKC superfamily consists of 13 different isoforms that are divided into four subgroups on the basis of their structural differences and related cofactor requirements cPKC (classical PKC) isoforms (a, ⁇ , ⁇ and ⁇ ), which respond both to Ca2+ and DAG (diacylglycerol), nPKC (novel PKC) isoforms ( ⁇ , ⁇ , ⁇ and ⁇ ), which are insensitive to Ca2+, but dependent on DAG, atypical PKCs (aPKCs, ⁇ / ⁇ , ⁇ ), which are responsive to neither co-factor, but may be activated by other lipids and through protein-protein interactions, and the related PKN (protein kinase N) family (e.g.
  • PKC phosphoinositide-dependent kinase 1
  • the phospholipid DAG has a central role in the activation of PKC by causing an increase in the affinity of classical PKCs for cell membranes accompanied by PKC activation and the release of an inhibitory substrate (a pseudo-substrate) to which the inactive enzyme binds.
  • PKC Activated PKC then phosphorylates and activates a range of kinases.
  • the downstream events following PKC activation are poorly understood, although the MEK-ERK (mitogen activated protein kinase kinase-extracellular signal-regulated kinase) pathway is thought to have an important role.
  • MEK-ERK mitogen activated protein kinase kinase-extracellular signal-regulated kinase
  • PKC isoforms probably form part of the multi-protein complexes that facilitate cellular signal transduction.
  • MAPK Mitogen Activated Protein
  • MAPKs are activated by protein kinase cascades consisting of three or more protein kinases in series: MAPK kinase kinases (MAP3Ks) activate MAPK kinases (MAP2Ks) by dual phosphorylation on S/T residues; MAP2Ks then activate MAPKs by dual phosphorylation on Y and T residues MAPKs then phosphorylate target substrates on select S/T residues typically followed by a proline residue. In the ERK1/2 cascade the MAP3K is usually a member of the Raf family.
  • MAP3Ks reside upstream of the p38 and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/ SAPK) MAPK groups, which have generally been associated with responses to cellular stress. Downstream of the activating stimuli, the kinase cascades may themselves be stimulated by combinations of small G proteins,
  • MAP4Ks MAP4Ks, scaffolds, or oligomerization of the MAP3K in a pathway.
  • Ras family members usually bind to Raf proteins leading to their activation as well as to the subsequent activation of other downstream members of the pathway.
  • the MAPK pathway has been a focus of intense investigation for therapeutic targeting.
  • Many receptor tyrosine kinases are capable of initiating MAPK signaling. They do so after activating phosphorylation events within their cytoplasmic domains provide docking sites for src-homology 2 (SH2) domain-containing signaling molecules.
  • SH2 src-homology 2
  • adaptor proteins such as Grb2 recruit guanine nucleotide exchange factors such as SOS- 1 or CDC25 to the cell membrane.
  • the guanine nucleotide exchange factor is now capable of interacting with Ras proteins at the cell membrane to promote a conformational change and the exchange of GDP for GTP bound to Ras.
  • Ras isoforms have been described, including K-Ras, N-Ras, and H-Ras. Termination of Ras activation occurs upon hydrolysis of Ras GTP to Ras GDP. Ras proteins have intrinsically low GTPase activity. Thus, the GTPase activity is stimulated by GTPase-activating proteins such as NF-1 GTPase-activating protein/neurofibromin and pi 20 GTPase activating protein thereby preventing prolonged Ras stimulated signaling. Ras activation is the first step in activation of the MAPK cascade.
  • Raf (A- Raf, B-Raf, or Raf-1) is recruited to the cell membrane through binding to Ras and activated in a complex process involving phosphorylation and multiple cofactors that is not completely understood.
  • Raf proteins directly activate MEKl and MEK2 via phosphorylation of multiple serine residues.
  • MEKl and MEK2 are themselves tyrosine and threonine/ serine dual-specificity kinases that subsequently phosphorylate threonine and tyrosine residues in Erkl and Erk2 resulting in activation.
  • Erk has no known targets besides Erk proteins, Erk has multiple targets including Elk-1, c-Etsl, c-Ets2, p90RSKl, MNK1, MNK2, and TOB .
  • the cellular functions of Erk are diverse and include regulation of cell proliferation, survival, mitosis, and migration. McCubrey, J. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochimica et Biophysica Acta. 2007; 1773: 1263-1284, hereby fully incorporated by reference in its entirety for all purposes, Friday and Adjei, Clinical Cancer Research (2008) 14, p342-346.
  • JNK c-Jun N-terminal kinase
  • SAPK stress-activated protein kinase
  • JNKs The c-Jun N-terminal kinases (JNKs) were initially described as a family of serine/threonine protein kinases, activated by a range of stress stimuli and able to phosphorylate the N-terminal transactivation domain of the c- Jun transcription factor. This phosphorylation enhances c-Jun dependent transcriptional events in mammalian cells. Further research has revealed three JNK genes (JNKl, JNK2 and JNK3) and their splice-forms as well as the range of external stimuli that lead to JNK activation. JNKl and JNK2 are ubiquitous, whereas JNK3 is relatively restricted to brain.
  • MAP3Ks upstream of JNK are MEK4 (MKK4) and MEK7 (MKK7).
  • MAP3Ks with the capacity to activate JNK/SAPKs include MEKKs (MEKK1, -2, -3 and -4), mixed lineage kinases (MLKs, including MLK1-3 and DLK), Tpl2, ASKs, TAOs and TAKl .
  • MLKs mixed lineage kinases
  • Tpl2ASKs TAOs
  • TAKl TAKl
  • Knockout studies in several organisms indicate that different MAP3Ks predominate in JNK/SAPK activation in response to different upstream stimuli.
  • the wiring may be comparable to, but perhaps even more complex than, MAP3K selection and control of the ERK1/2 pathway.
  • JNK/SAPKs are activated in response to inflammatory cytokines;
  • JNKs phosphorylate transcription factors c-Jun, ATF-2, p53, Elk-1, and nuclear factor of activated T cells (NFAT), which in turn regulate the expression of specific sets of genes to mediate cell proliferation, differentiation or apoptosis. JNK proteins are involved in cytokine production, the inflammatory response, stress- induced and developmentally programmed apoptosis, actin reorganization, cell transformation and metabolism. Raman, M. Differential regulation and properties ofMAPKs. Oncogene. 2007; 26: 3100-3112, hereby fully incorporated by reference in its entirety for all purposes.
  • p38 Map kinases Several independent groups identified the p38 Map kinases, and four p38 family members have been described ( ⁇ , ⁇ , ⁇ , ⁇ ). Although the p38 iso forms share about 40% sequence identity with other MAPKs, they share only about 60%> identity among themselves, suggesting highly diverse functions. p38 MAPKs respond to a wide range of extracellular cues particularly cellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines and less often growth factors. Responding to osmotic shock might be viewed as one of the oldest functions of this pathway, because yeast p38 activates both short and long-term homeostatic mechanisms to osmotic stress.
  • cellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines and less often growth factors.
  • osmotic shock might be viewed as one of the oldest functions of this pathway, because yeast p38 activates both short and long-term homeostatic mechanisms to
  • p38 is activated via dual phosphorylation on the TGY motif within its activation loop by its upstream protein kinases MEK3 and MEK6.
  • MEK3/6 are activated by numerous MAP3Ks including MEKK1-4, TAOs, TAK and ASK.
  • p38 MAPK is generally considered to be the most promising MAPK therapeutic target for rheumatoid arthritis as p38 MAPK iso forms have been implicated in the regulation of many of the processes, such as migration and accumulation of leucocytes, production of cytokines and pro-inflammatory mediators and angiogenesis, that promote disease pathogenesis. Further, the p38 MAPK pathway plays a role in cancer, heart and neurodegenerative diseases and may serve as promising therapeutic target. Cuenda, A.
  • Src Family Kinases Src is the most widely studied member of the largest family of nonreceptor protein tyrosine kinases, known as the Src family kinases (SFKs). Other SFK members include Lyn, Fyn, Lck, Hck, Fgr, Blk, Yrk, and Yes.
  • the Src kinases can be grouped into two sub-categories, those that are ubiquitously expressed (Src, Fyn, and Yes), and those which are found primarily in hematopoietic cells (Lyn, Lck, Hck, Blk, Fgr). (Benati, D. Src Family Kinases as Potential Therapeutic Targets for Malignancies and Immunological
  • SFKs are key messengers in many cellular pathways, including those involved in regulating proliferation, differentiation, survival, motility, and angiogenesis.
  • the activity of SFKs is highly regulated intramolecularly by interactions between the SH2 and SH3 domains and intermolecularly by association with cytoplasmic molecules. This latter activation may be mediated by focal adhesion kinase (FAK) or its molecular partner Crk-associated substrate (CAS), which plays a prominent role in integrin signaling, and by ligand activation of cell surface receptors, e.g. epidermal growth factor receptor (EGFR).
  • FAK focal adhesion kinase
  • CAS molecular partner Crk-associated substrate
  • Src can also be activated by dephosphorylation of tyrosine residue Y530. Maximal Src activation requires the autophosphorylation of tyrosine residue Y419 (in the human protein) present within the catalytic domain. Elevated Src activity may be caused by increased transcription or by deregulation due to overexpression of upstream growth factor receptors such as EGFR, HER2, platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor, ephrins, integrin, or FAK.
  • upstream growth factor receptors such as EGFR, HER2, platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor, ephrins, integrin, or FAK.
  • Janus kinase (JAK)/ Signal transducers and activators of transcription (STAT) pathway The JAK/STAT pathway plays a crucial role in mediating the signals from a diverse spectrum of cytokine receptors, growth factor receptors, and G-protein-coupled receptors.
  • Signal transducers and activators of transcription (STAT) proteins play a crucial role in mediating the signals from a diverse spectrum of cytokine receptors growth factor receptors, and G-protein- coupled receptors.
  • STAT directly links cytokine receptor stimulation to gene transcription by acting as both a cytosolic messenger and nuclear transcription factor.
  • JAK Janus Kinase
  • STAT Janus Kinase
  • JFK JAK family kinase
  • Tyrosine phosphorylated STAT forms a dimer, translocates to the nucleus, and binds to specific DNA elements to activate target gene transcription, which leads to the regulation of cellular proliferation, differentiation, and apoptosis.
  • the entire process is tightly regulated at multiple levels by protein tyrosine phosphatases, suppressors of cytokine signaling and protein inhibitors of activated STAT.
  • JAKs contain two symmetrical kinase-like domains; the C-terminal JAK homology 1 (JH1) domain possesses tyrosine kinase function while the immediately adjacent JH2 domain is enzymatically inert but is believed to regulate the activity of JH1.
  • JAK family members JAK1, JAK2, JAK3 and tyrosine kinase 2 (Tyk2). Expression is ubiquitous for JAK1, JAK2 and TYK2 but restricted to hematopoietic cells for JAK3.
  • JAK3 e.g. JAK3A572V, JAK3V722I, JAK3P132T
  • fusion JAK2 e.g. ETV6-JAK2, PCM1- JAK2, BCR-JAK2 mutations
  • JAK2 V617F, JAK2 exon 12 mutations
  • MPL MPL W515 L/K/S MPLS505N
  • JAK2 mutations primarily JAK2V617F, are invariably associated with polycythemia vera (PV). This mutation also occurs in the majority of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) (Tefferi n., Leukemia & Lymphoma, March 2008; 49(3): 388 - 397). STATs can be activated in a JAK-independent manner by src family kinase members and by oncogenic FLt3 ligand-ITD (Hayakawa and Naoe, Ann N Y Acad Sci.
  • STAT5 Activation mechanisms of STAT5 by oncogenic FLt3 ligand-ITD. Blood (2007) vol. 110 (1) pp. 370-4).
  • mutations of STATs have not been described in human tumors, the activity of several members of the family, such as STAT1, STAT3 and STAT5, is dysregulated in a variety of human tumors and leukemias.
  • STAT3 and STAT5 acquire oncogenic potential through constitutive phosphorylation on tyrosine, and their activity has been shown to be required to sustain a transformed phenotype.
  • STAT5 phosphorylation was also shown to be required for the long-term maintenance of leukemic stem cells.
  • STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells. Blood (2007) vol. 110 (8) pp. 2880-2888
  • STAT1 negatively regulates cell proliferation and angiogenesis and thereby inhibits tumor formation.
  • STAT1 and its downstream targets have been shown to be reduced in a variety of human tumors (Rawlings, J. The JAK/STAT signaling pathway. J of Cell Science. 2004; 117 (8):1281-1283, hereby fully incorporated by reference in its entirety for all purposes).
  • ATP -binding cassette (ABC) superfamily of proteins consists of 49 membrane proteins that transport a diverse array of substrates, including sugars, amino acids, bile salts lipids, sterols, nucleotides, endogenous metabolites, ions, antibiotics drugs and toxins out of cells using the energy of hydrolysis of ATP.
  • ATP -binding-cassette (ABC) transporters are evolutionary extremely well-conserved transmembrane proteins that are highly expressed in hematopoietic stem cells (HSCs).
  • ABC proteins are multidrug efflux pumps that not only protect the body from exogenous toxins, but also play a role in uptake and distribution of therapeutic drugs. Expression of these proteins in target tissues causes resistance to treatment with multiple drugs. (Gillet et al, Biochimica et Biophysica Acta (2007) 1775, p237, Sharom (2008) Pharmacogenomics 9 pl05). A more detailed discussion of the ABC family members with critical roles in resistance and poor outcome to treatment is discussed below
  • the second class of plasma membrane transporter proteins that play a role in the uptake of nucleoside-derived drugs are the Concentrative and Equilibrative Nucleoside
  • Transporters (CNT and ENT, respectively), encoded by gene families SLC28 and SLC29 (Pastor-Anglada (2007) J. Physiol. Biochem 63, p97). They mediate the uptake of natural nucleosides and a variety of nucleoside-derived drugs, mostly used in anti-cancer therapy. In vitro studies, have shown that one mechanism of nucleoside resistance can be mediated through mutations in the gene for ENT1/SLC29A1 resulting in lack of detectable protein (Cai et al., Cancer Research (2008) 68, p2349).
  • ABC transporter family three family members account for most of the multiple drug resistance (MDR) in humans; P-gycoprotein (Pgp/MDR1/ABCB1), MDR - associated protein (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2 or MXR).
  • Pgp/MDRl and ABCG2 can export both unmodified drugs and drug conjugates, whereasMRP1 exports glutathione and other drug conjugates as well as unconjugated drugs together with free glutathione. All three ABC transporters demonstrate export activity for a broad range of structurally unrelated drugs and display both distinct and overlapping specificities.
  • MRP1 promotes efflux of drug-glutathione conjugates, vinca alkaloids, camptothecin, but not taxol.
  • drugs exported by ABCG2 include mitoxantrone, etoposide, daunorubicin as well as the tyrosine kinase inhibitors Gleevec and Iressa.
  • ABCG2 examples of drugs exported by ABCG2 include mitoxantrone, etoposide, daunorubicin as well as the tyrosine kinase inhibitors Gleevec and Iressa.
  • Expression levels of drug transporters and receptors may not be as informative by themselves for disease management as analysis of activatable elements, such as phosphorylated proteins. However, expression information may be useful in combination with the analysis of activatable elements, such as phosphorylated proteins. In some embodiments, the methods described herein analyze the expression of drug transporters and receptors in combination with the analysis of one or more activatable elements for the diagnosis, prognosis, selection of treatment, or predicting response to treatment for a condition.
  • the response to DNA damage is a protective measure taken by cells to prevent or delay genetic instability and tumorigenesis. It allows cells to undergo cell cycle arrest and gives them an opportunity to either: repair the broken DNA and resume passage through the cell cycle or, if the breakage is irreparable, trigger senescence or an apoptotic program leading to cell death (Wade Harper et al, Molecular Cell , (2007) 28 p739 - 745, Bartek J et al, Oncogene (2007)26 p7773-9). See also U.S. S.N. 61/436,534 and PCT/US2011/48332 which are both incorporated by reference in their entireties for all purposes.
  • DNA damage sensor protein complexes are positioned at strategic points within the DNA damage response pathway and act as sensors, transducers or effectors of DNA damage.
  • checkpoint kinases phosphorylating a multitude of substrates. Both checkpoint kinases have overlapping and distinct roles in orchestrating the cell's response to DNA damage.
  • Chk2 Maximal kinase activation of Chk2 involves phosphorylation and homo-dimerization with ATM-mediated phosphorylation of T68 on Chk2 as a preliminary event. This in turn activates the DNA repair. As mentioned above, in order for DNA repair to proceed, there must be a delay in the cell cycle. Chk2 seems to have a role at the G1/S and G2/M junctures and may have overlapping functions with Chkl . There are multiple ways in which Chkl and Chk2 mediate cell cycle suspension.
  • Chk2 phosphorylates the CDC25A and CDC25C phosphatases resulting in their removal from the nucleus either by proteosomal degradation or by sequestration in the cytoplasm by 14-3-3. These phosphatases are no longer able to act on their nuclear CDK substrates. If DNA repair is successful cell cycle progression is resumed (Antoni et al, Nature reviews cancer (2007) 7, p925-936).
  • Chk2 substrates that operate in a p53 -independent manner include the E2F1 transcription factor, the tumor suppressor promyelocytic leukemia (PML) and the polo-like kinases 1 and 3 (PLK1 and PLK3).
  • E2F1 drives the expression of a number of apoptotic genes including caspases 3, 7, 8 and 9 as well as the pro-apoptotic Bcl-2 related proteins (Bim, Noxa, PUMA).
  • the p53 In its response to DNA damage, the p53 activates the transcription of a program of genes that regulate DNA repair, cell cycle arrest, senescence and apoptosis.
  • the overall functions of p53 are to preserve fidelity in DNA replication such that when cell division occurs tumorigenic potential can be avoided. In such a role, p53 is described as "The Guardian of the Genome (Riley et al., Nature Reviews Molecular Cell Biology (2008) 9 p402-412).
  • the diverse alarm signals that impinge on p53 result in a rapid increase in its levels through a variety of post translational modifications. Worthy of mention is the phosphorylation of amino acid residues within the amino terminal portion of p53 such that p53 is no longer under the regulation of Mdm2.
  • the responsible kinases are ATM, Chkl and Chk2.
  • the subsequent stabilization of p53 permits it to transcriptionally regulate multiple pro-apoptotic members of the Bcl-2 family, including Bax, Bid, Puma, and Noxa (discussion below).
  • Bcl-2 family Key regulators of apoptosis are proteins of the Bcl-2 family.
  • the founding member, the Bcl-2 proto-oncogene was first identified at the chromosomal breakpoint of t(14:18) bearing human follicular B cell lymphoma. Unexpectedly, expression of Bcl-2 was proved to block rather than promote cell death following multiple pathological and physiological stimuli (Danial and Korsemeyer, Cell (2204) 116, p205-219).
  • the Bcl-2 family has at least 20 members which are key regulators of apoptosis, functioning to control mitochondrial permeability as well as the release of proteins important in the apoptotic program.
  • the ratio of anti- to pro-apoptotic molecules constitutes a rheostat that sets the threshold of susceptibility to apoptosis for the intrinsic pathway, which utilizes organelles such as the mitochondrion to amplify death signals.
  • the family can be divided into 3 subclasses based on structure and impact on apoptosis.
  • Family members of subclass 1 including Bcl-2, BC1-XL and Mcl-1 are
  • BH3-only proteins include Noxa, Puma, Bid, Bad and Bim. They function to promote apoptosis either by activating the pro-apoptotic members of group 2 or by inhibiting the anti-apoptotic members of subclass 1 (Er et al., Biochimica et Biophysica Act (2006) 1757, pl301-1311, Fernandez-Luna Cellular Signaling (2008) Advance Publication Online).
  • Caspase 9 is one member of a family of cysteine aspartyl-specific proteases; genes encoding 11 of these proteases have been mapped in the human genome.
  • PARP poly(ADP-ribose) polymerase 1
  • IAPs inhibitors of apoptosis
  • caspase proteases with aspartate specificity play significant roles in both inflammation and apoptosis.
  • Caspases exhibit catalytic and substrate recognition motifs that have been highly conserved. These characteristic amino acid sequences allow caspases to interact with both positive and negative regulators of their activity. The substrate preferences or specificities of individual caspases have been exploited for the development of peptides that successfully compete for caspase binding.
  • the catalytic domains of the caspases require at least four amino acids to the left of the cleavage site with P4 as the prominent specificity-determining residue.
  • WEHD, VDVAD, and DEVD are examples of peptides that preferentially bind caspase- 1, caspase-2 and caspase-3, respectively. It is possible to generate reversible or irreversible inhibitors of caspase activation by coupling caspase-specific peptides to certain aldehyde, nitrile or ketone compounds. These caspase inhibitors can successfully inhibit the induction of apoptosis in various tumor cell lines as well as normal cells. Fluoromethyl ketone (FMK)-derivatized peptides act as effective irreversible inhibitors with no added cytotoxic effects.
  • FMK Fluoromethyl ketone
  • Inhibitors synthesized with a benzyloxycarbonyl group also known as BOC or Z
  • BOC or Z benzyloxycarbonyl group
  • Benzyloxycarbonyl- Val-Ala-Asp (OMe) fluoromethylketone (ZVAD) is a caspase inhibitor. See Misaghi, et al., z-VAD-fmk inhibits peptide :N-glycanase and may result in ER stress Cell Death and Differentiation (2006) 13, 163-165.
  • Figure 3 shows the role of apoptosis in AML.
  • the status of an activatable element within an apoptosis pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells is determined.
  • the activatable element within the apoptosis pathway is selected from the group consisting of Cleaved PARP (PARP+), Cleaved Caspase 8, and Cytoplasmic Cytochrome C
  • the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • the status of an activatable element within a DNA damage pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells is determined.
  • the activatable element within a DNA damage pathway is selected from the group consisting of Chkl, Chk2, ATM, and ATR and the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • interrogation of the apoptotic machinery will also be performed by etoposide with or without ZVAD, an inhibitor of caspases, or a combination of Cytarabine and Daunorubicin at clinically relevant concentrations based on peak plasma drug levels.
  • Daunorubicin at 25 mg/m2 yields a peak plasma concentration of 50 ng/ml and at 50 mg /m2 yields a peak plasma concentration of 200 ng/ml.
  • Our in vitro apoptosis assay will use concentrations of Cytarabine up to 2 uM, and concentrations of Daunorubicin up to 200 ng/ml.
  • the cell cycle is the series of events that take place in a cell leading to its division and duplication (replication).
  • the cell cycle consists of five distinct phases: G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis).
  • M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's chromosomes are divided between the two daughter cells, and cytokinesis, in which the cell's cytoplasm divides forming distinct cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called GO phase.
  • Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division.
  • the molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.
  • cyclins and cyclin-dependent kinases determine a cell's progress through the cell cycle.
  • Many of the genes encoding cyclins and CDKs are conserved among all eukaryotes, but in general more complex organisms have more elaborate cell cycle control systems that incorporate more individual components.
  • Many of the relevant genes were first identified by studying yeast, especially Saccharomyces cerevisiae genetic nomenclature in yeast dubs many these genes cdc (for "cell division cycle") followed by an identifying number, e.g., cdc25.
  • Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin.
  • CDKs When activated by a bound cyclin, CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle.
  • Different cyclin-CDK combinations determine the downstream proteins targeted.
  • CDKs are constitutively expressed in cells whereas cyclins are synthesised at specific stages of the cell cycle, in response to various molecular signals.
  • G1 cyclin-CDK complexes Upon receiving a pro-mitotic extracellular signal, G1 cyclin-CDK complexes become active to prepare the cell for S phase, promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication.
  • the G1 cyclin-CDK complexes also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination. Once a protein has been ubiquitinated, it is targeted for proteolytic degradation by the proteasome. Active S cyclin-CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during G1 phase on DNA replication origins.
  • the phosphorylation serves two purposes: to activate each already- assembled pre-replication complex, and to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once. The reason for prevention of gaps in replication is fairly clear, because daughter cells that are missing all or part of crucial genes will die. However, for reasons related to gene copy number effects, possession of extra copies of certain genes would also prove deleterious to the daughter cells. [00262] Mitotic cyclin-CDK complexes, which are synthesized but inactivated during S and G2 phases, promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly.
  • a critical complex activated during this process is an ubiquitin ligase known as the anaphase-promoting complex (APC), which promotes degradation of structural proteins associated with the chromosomal kinetochore.
  • APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed.
  • Interphase generally lasts at least 12 to 24 hours in mammalian tissue. During this period, the cell is constantly synthesizing RNA, producing protein and growing in size. By studying molecular events in cells, scientists have determined that interphase can be divided into 4 steps: Gap 0 (G0), Gap 1 (G1), S (synthesis) phase, Gap 2 (G2).
  • Cyclin D is the first cyclin produced in the cell cycle, in response to extracellular signals (e.g. growth factors). Cyclin D binds to existing CDK4, forming the active cyclin D- CDK4 complex. Cyclin D-CDK4 complex in turn phosphorylates the retinoblastoma
  • Rb susceptibility protein
  • the hyperphosphorylated Rb dissociates from the E2F/DPl/Rb complex (which was bound to the E2F responsive genes, effectively "blocking” them from transcription), activating E2F.
  • Activation of E2F results in transcription of various genes like cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc. Cyclin E thus produced binds to CDK2, forming the cyclin E-CDK2 complex, which pushes the cell from G1 to S phase ( G1/S transition).
  • Cyclin B along with cdc2 (cdc2 - fission yeasts (CDK1 - mammalia)) forms the cyclin B-cdc2 complex, which initiates the G2/M transition. Cyclin B-cdc2 complex activation causes breakdown of nuclear envelope and initiation of prophase, and subsequently, its deactivation causes the cell to exit mitosis.
  • the Cip/Kip family includes the genes p21, p27 and p57. They halt cell cycle in G1 phase, by binding to, and inactivating, cyclin-CDK complexes.
  • p21 is a p53 response gene (which, in turn, is triggered by DNA damage eg. due to radiation).
  • p27 is activated by
  • TGF ⁇ Transforming Growth Factor ⁇ (TGF ⁇ ), a growth inhibitor.
  • the INK4a/ARF family includes pl6INK4a, which binds to CDK4 and arrests the cell cycle in G1 phase, and pl4arf which prevents p53 degradation.
  • Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle. Checkpoints prevent cell cycle progression at specific points, allowing
  • G1/S checkpoint is a rate-limiting step in the cell cycle and is also known as restriction point.
  • An alternative model of the cell cycle response to DNA damage has also been proposed, known as the postreplication checkpoint.
  • p53 plays an important role in triggering the control mechanisms at both G1/S and G2/M checkpoints.
  • a disregulation of the cell cycle components may lead to tumor formation.
  • some genes like the cell cycle inhibitors, RB, p53 etc. when they mutate, may cause the cell to multiply uncontrollably, forming a tumor.
  • the duration of cell cycle in tumor cells is equal to or longer than that of normal cell cycle, the proportion of cells that are in active cell division (versus quiescent cells in GO phase) in tumors is much higher than that in normal tissue.
  • the number of cells that die by apoptosis or senescence remains the same.
  • the status of an activatable element within a cell cycle pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells is determined.
  • the activatable element within a DNA damage pathway is selected from the group consisting of, Cdc25, p53, CyclinA-Cdk2, CyclinE- Cdk2, CyclinB-Cdkl, p21, and Gadd45.
  • the modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
  • the methods and composition utilize a modulator.
  • a modulator can be an activator, a therapeutic compound, an inhibitor or a compound capable of impacting a cellular pathway. Modulators can also take the form of environmental cues and inputs.
  • Modulation can be performed in a variety of environments.
  • cells are exposed to a modulator immediately after collection.
  • purification of cells is performed after modulation.
  • whole blood is collected to which a modulator is added.
  • cells are modulated after processing for single cells or purified fractions of single cells.
  • whole blood can be collected and processed for an enriched fraction of lymphocytes that is then exposed to a modulator.
  • Modulation can include exposing cells to more than one modulator. For instance, in some embodiments, a sample of cells is exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more modulators. See U.S. Patent Applications 12/432,239 and 12/910,769 which are incorporated by reference in their entireties. See also U.S. Patent Nos. 7,695,926 and 7,381,535 and U.S. Pub. No. 2009/0269773.
  • cells are cultured post collection in a suitable media before exposure to a modulator.
  • the media is a growth media.
  • the growth media is a complex media that may include serum.
  • the growth media comprises serum.
  • the serum is selected from the group consisting of fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, and goat serum.
  • the serum level ranges from 0.0001% to 30%, about 0.001% to 30%, about 0.01% to 30%, about 0.1% to 30% or 1% to 30%.
  • the growth media is a chemically defined minimal media and is without serum.
  • cells are cultured in a differentiating media.
  • Modulators include chemical and biological entities, and physical or environmental stimuli. Modulators can act extracellularly or intracellularly. Chemical and biological modulators include growth factors, mitogens, cytokines, drugs, immune modulators, ions, neurotransmitters, adhesion molecules, hormones, small molecules, inorganic compounds, polynucleotides, antibodies, natural compounds, lectins, lactones, chemotherapeutic agents, biological response modifiers, carbohydrate, proteases and free radicals. Modulators include complex and undefined biologic compositions that may comprise cellular or botanical extracts, cellular or glandular secretions, physiologic fluids such as serum, amniotic fluid, or venom.
  • Physical and environmental stimuli include electromagnetic, ultraviolet, infrared or particulate radiation, redox potential and pH, the presence or absences of nutrients, changes in temperature, changes in oxygen partial pressure, changes in ion concentrations and the application of oxidative stress.
  • Modulators can be endogenous or exogenous and may produce different effects depending on the concentration and duration of exposure to the single cells or whether they are used in combination or sequentially with other modulators. Modulators can act directly on the activatable elements or indirectly through the interaction with one or more intermediary biomolecule. Indirect modulation includes alterations of gene expression wherein the expressed gene product is the activatable element or is a modulator of the activatable element.
  • the modulator is selected from the group consisting of growth factors, mitogens, cytokines, adhesion molecules, drugs, hormones, small molecules, polynucleotides, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulators, carbohydrates, proteases, ions, reactive oxygen species, peptides, and protein fragments, either alone or in the context of cells, cells themselves, viruses, and biological and non-biological complexes (e.g. beads, plates, viral envelopes, antigen presentation molecules such as major histocompatibility complex).
  • the modulator is a physical stimuli such as heat, cold, UV radiation, and radiation. Examples of modulators, include but are not limited to Growth factors, such as Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs),Brain-derived neurotrophic factor
  • BDNF Epidermal growth factor
  • EPO Erythropoietin
  • FGF Fibroblast growth factor
  • Glial cell line-derived neurotrophic factor GDNF
  • G- CSF Granulocyte colony-stimulating factor
  • G-CSF Granulocyte macrophage colony-stimulating factor
  • GDF9 Growth differentiation factor-9
  • HGF Hepatocyte growth factor
  • HDGF Hepatoma-derived growth factor
  • IGF Insulin-like growth factor
  • GDF-8 Nerve growth factor
  • NGF Nerve growth factor
  • PDGF Platelet-derived growth factor
  • SDGF Stromal Derived Growth Factor
  • TPO Transforming growth factor alpha
  • TGF- ⁇ Transforming growth factor beta
  • TGF- ⁇ Tumour necrosis factor-alpha
  • TNF- a Tumour necrosis factor-alpha
  • Vascular endothelial growth factor Keratin Derived
  • T cells Activates T cells, IL-2- T-cell growth factor. Stimulates IL-1 synthesis. Activates B-cells and NK cells, IL-3- Stimulates production of all non-lymphoid cells, IL-4- Growth factor for activated B cells, resting T cells, and mast cells,IL-5- Induces differentiation of activated B cells and eosinophils, IL-6- Stimulates Ig synthesis. Growth factor for plasma cells, and IL-7- Growth factor for pre-B cells.
  • Cell motility factors such as peptide growth factors, (e.g., EGF, PDGF, TGF-beta), substrate-adhesion molecules (e.g., fibronectin, laminin), cell adhesion molecules (CAMs), and metalloproteinases, hepatocyte growth factor (HGF) or scatter factor (SF), autocrine motility factor (AMF), and migration-stimulating factor (MSF).
  • peptide growth factors e.g., EGF, PDGF, TGF-beta
  • substrate-adhesion molecules e.g., fibronectin, laminin
  • CAMs cell adhesion molecules
  • metalloproteinases e.g., hepatocyte growth factor (HGF) or scatter factor (SF), autocrine motility factor (AMF), and migration-stimulating factor (MSF).
  • HGF hepatocyte growth factor
  • SF scatter factor
  • AMF autocrine motility factor
  • MSF migration-stimulating factor
  • modulators include SDF- ⁇ , IFN-a, IFN- ⁇ , IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, Thapsigargin, H 2 O 2 , Etoposide, Mylotarg, AraC, daunorubicin, staurosporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, EPO, azacitadine, decitabine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-a, and CD40L.
  • the modulator is an activator. In some embodiments the modulator is an inhibitor. In some embodiments, cells are exposed to one or more modulators. In some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators. In some embodiments, cells are exposed to at least two modulators, wherein one modulator is an activator and one modulator is an inhibitor. In some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators, where at least one of the modulators is an inhibitor.
  • the cross-linker is a molecular binding entity.
  • the molecular binding entity is a monovalent, bivalent, or multivalent is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates in a cellular pathway (e.g. signaling cascade) in the cell.
  • the inhibitor is a phosphataseor a tyrosine kinase inhibitor.
  • phosphatase inhibitors include, but are not limited to H2O2, siRNA, miRNA, Cantharidin, (-)-p- Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, ⁇ - Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP-1, N-(9,10- Dioxo-9, 10-dihydro-phenanthren-2-
  • the activation level of an activatable element in a cell is determined by contacting the cell with an inhibitor and a modulator, where the modulator can be an inhibitor or an activator. In some embodiments, the activation level of an activatable element in a cell is determined by contacting the cell with an inhibitor and an activator. In some embodiments, the activation level of an activatable element in a cell is determined by contacting the cell with two or more modulators.
  • a phenotypic profile of a population of cells is determined by measuring the activation level of an activatable element when the population of cells is exposed to a plurality of modulators in separate cultures.
  • the modulators include H 2 O 2 , PMA, SDF1 a, CD40L, IGF-1, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and/or a combination thereof.
  • a population of cells can be exposed to one or more, all or a combination of the following combination of modulators: H 2 O 2; PMA; SDF1 ⁇ ; CD40L; IGF-1; IL-7; IL-6; IL-10; IL-27; IL-4; IL-2; IL-3; thapsigargin;.
  • the phenotypic profile of the population of cells is used to classify the population as described herein.
  • the modulator is etoposide phosphate.
  • Etoposide phosphate brand names: Eposin, Etopophos, Vepesid, VP- 16
  • Eposin brand names: Eposin, Etopophos, Vepesid, VP- 16
  • enzyme topoisomerase II can inhibit enzyme topoisomerase II.
  • the modulator is Mylotarg.
  • Mylotarg® (gemtuzumab ozogamicin for Injection) is a chemotherapy agent composed of a recombinant humanized IgG4, kappa antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin, isolated from fermentation of a bacterium, Micromonospora echinospora subsp. calichensis.
  • the antibody portion of Mylotarg can bind specifically to the CD33 antigen, a sialic acid-dependent adhesion protein found on the surface of leukemic blasts and immature normal cells of myelomonocytic lineage, but not on normal hematopoietic stem cells. See U.S. Patent Nos. 7,727,968, 5,773,001, and 5,714,586.
  • the modulator is staurosporine.
  • Staurosporine antioxidant AM- 2282 or STS
  • Staurosporine can have biological activities ranging from anti-fungal to antihypertensive. See e.g.,Ruegg UT, Burgess GM. (1989) Staurosporine, K-252 and UCN-01 : potent but nonspecific inhibitors of protein kinases. Trends in Pharmacological Science 10 (6): 218-220.
  • Staruosporine can be an anticancer treatment. Staurosporine can inhibit protein kinases through the prevention of ATP binding to the kinase.
  • Staurosporine is a prototypical ATP-competitive kinase inhibitor in that it can bind to many kinases with high affinity, though with little selectivity. Staurosporine can be used to induce apoptosis.
  • One way in which staurosporine can induce apoptosis is by activating caspase-3.
  • the modulator is AraC.
  • Ara-C cytosine arabinoside or cytarabine
  • cytosine arabinoside triphosphate which damages DNA when the cell cycle holds in the S phase (synthesis of DNA). Rapidly dividing cells, which require DNA replication for mitosis, are therefore affected by treatment with cytosine arabinoside.
  • Cytosine arabinoside can also inhibit both DNA and RNA polymerases and nucleotide reductase enzymes needed for DNA synthesis.
  • Cytarabine can be used in the treatment of acute myeloid leukaemia, acute lymphocytic leukaemia (ALL) and in lymphomas where it is the backbone of induction chemotherapy.
  • the modulator is daunorubicin. Daunorubicin or
  • daunomycin (daunomycin cerubidine) is a chemotherapeutic of the anthracycline family that can be given as a treatment for some types of cancer. It can be used to treat specific types of leukaemia (acute myeloid leukemia and acute lymphocytic leukemia). It was initially isolated from Streptomyces peucetius. Daunorubicin can also used to treat neuroblastoma. Daunorubicin has been used with other chemotherapy agents to treat the blastic phase of chronic myelogenous leukemia. On binding to DNA, daunomycin can intercalate, with its daunosamine residue directed toward the minor groove.
  • a user may analyze the signaling in subpopulations based on surface markers. For example, the user could look at: "stem cell populations" by CD34+ CD38- or CD34+ CD33- expressing cells; drug transporter positive cells; i.e. FLT3 LIGAND+ cells; or multiple leukemic subclones based on CD33, CD45, HLA-DR, CD1 lb and analyzing signaling in each subpopulation.
  • a user may analyze the data based on intracellular markers, such as transcription factors or other intracellular proteins; based on a functional assay (i.e. dye negative "side population” aka drug transporter + cells, or fluorescent glucose uptake, or based on other fluorescent markers.
  • a gate is established after learning from a responsive subpopulation. That is, a gate is developed from one data set after finding a population that correlates with a clinical outcome. This gate can then be applied retrospectively or prospectively to other data sets.
  • the populations of interest and the method for characterizing these populations are determined prior to analyzing of data. For instance, there are at least two general ways of identifying populations for data analysis: (i) "Outside -in" comparison of Parameter sets for individual samples or subset (e.g., patients in a trial). In this more common case, cell populations are homogenous or lineage gated in such a way as to create distinct sets considered to be homogenous for targets of interest.
  • An example of sample-level comparison would be the identification of signaling profiles in tumor cells of a patient and correlation of these profiles with non-random distribution of clinical responses.
  • Each of these techniques capitalizes on the ability of flow cytometry to deliver large amounts of multiparameter data at the single cell level.
  • a condition e.g. neoplastic or hematopoietic condition
  • a third "meta-level" of data exists because cells associated with a condition (e.g. cancer cells) are generally treated as a single entity and classified according to historical techniques.
  • These techniques have included organ or tissue of origin, degree of differentiation, proliferation index, metastatic spread, and genetic or metabolic data regarding the patient.
  • the present invention uses variance mapping techniques for mapping condition signaling space. These methods represent a significant advance in the study of condition biology because it enables comparison of conditions independent of a putative normal control.
  • Traditional differential state analysis methods e.g., DNA microarrays, subtractive Northern blotting
  • They rely on multiple clusterings and reclusterings to group and then further stratify patient samples according to phenotype.
  • variance mapping of condition states compares condition samples first with themselves and then against the parent condition population.
  • activation states with the most diversity among conditions provide the core parameters in the differential state analysis. Given a pool of diverse conditions, this technique allows a researcher to identify the molecular events that underlie differential condition pathology (e.g., cancer responses to chemotherapy), as opposed to differences between conditions and a proposed normal control.
  • the activation level of an activatable element is determined.
  • One embodiment makes this determination by contacting a cell from a cell population with a binding element that is specific for an activation state of the activatable element.
  • binding element includes any molecule, e.g., peptide, nucleic acid, small organic molecule which is capable of detecting an activation state of an activatable element over another activation state of the activatable element. Binding elements and labels for binding elements are shown in U.S.S.N. 12/432,720, 12/229,476, 12/460,029, 12/730,120, 12/617,438 and 12/910,769.
  • the binding element is a peptide, polypeptide, oligopeptide or a protein.
  • the peptide, polypeptide, oligopeptide or protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein include both naturally occurring and synthetic amino acids.
  • homo-phenylalanine, citrulline and noreleucine are considered amino acids.
  • the side chains may be in either the (R) or the (S) configuration.
  • the amino acids are in the (S) or L-configuration.
  • non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
  • Proteins including non-naturally occurring amino acids may be synthesized or in some cases, made recombinantly; see van Hest et al., FEBS Lett 428:(l-2) 68-70 May 22, 1998 and Tang et al, Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which are expressly incorporated by reference herein.
  • Methods described herein may be used to detect any particular activatable element in a sample that is antigenically detectable and antigenically distinguishable from other activatable element which is present in the sample.
  • activation state-specific antibodies can be used in the present methods to identify distinct signaling cascades of a subset or subpopulation of complex cell populations and the ordering of protein activation (e.g., kinase activation) in potential signaling hierarchies.
  • protein activation e.g., kinase activation
  • activation state-specific antibody or “activation state antibody” or grammatical equivalents thereof, can refer to an antibody that specifically binds to a corresponding and specific antigen.
  • the corresponding and specific antigen can be a specific form of an activatable element.
  • the binding of the activation state- specific antibody can be indicative of a specific activation state of a specific activatable element.
  • the binding element is an antibody. In some embodiment, the binding element is an activation state-specific antibody.
  • antibody includes full length antibodies and antibody fragments, and can refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below.
  • antibody fragments as are known in the art, such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
  • Fab fragment antigen-binding subsequences of antibodies
  • antibody comprises monoclonal and polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing, inhibitory, or stimulatory. They can be humanized, glycosylated, bound to solid supports, and posses other variations. See U.S.S. Nos U.S. S.N. 12/432,720, 12/229,476, 12/460,029, and 12/910,769 for more information about antibodies as binding elements.
  • Activation state specific antibodies can be used to detect kinase activity; however additional means for determining kinase activation are provided herein.
  • substrates that are specifically recognized by protein kinases and phosphorylated thereby are known.
  • Antibodies that specifically bind to such phosphorylated substrates but do not bind to such non- phosphorylated substrates can be used to determine the presence of activated kinase in a sample.
  • the antigenicity of an activated isoform of an activatable element can be any antigenicity.
  • an activated isoform of an element possesses an epitope that is absent in a non-activated isoform of an element, or vice versa.
  • this difference is due to covalent addition of a moiety to an element, such as a phosphate moiety, or due to a structural change in an element, as through protein cleavage, or due to an otherwise induced conformational change in an element which causes the element to present the same sequence in an antigenically distinguishable way.
  • such a conformational change causes an activated isoform of an element to present at least one epitope that is not present in a non-activated isoform, or to not present at least one epitope that is presented by a non-activated isoform of the element.
  • the epitopes for the distinguishing antibodies are centered around the active site of the element, although as is known in the art, conformational changes in one area of an element may cause alterations in different areas of the element as well.
  • proteins that can be analyzed with the methods described herein include, but are not limited to, kinases, HER receptors, PDGF receptors, FLT3 receptor, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, erythropoetin receptor, thromobopoetin receptor, CD114, CD116, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGFP receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Me
  • phosphatases Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PP1, PPS, inositol phosphatases, PTEN, SHIPs,
  • myotubularins myotubularins, lipid signaling, phosphoinositide kinases, phopsholipases, prostaglandin synthases, 5 -lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated binder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, cytokines, IL-2, IL-4, IL-8, IL-6, interferon f, interferon a, cytokine regulators, suppressors of cytokine signaling (SOCs), ubiquitination enzymes, Cbl, SCF ubiquitination ligas
  • Rho-GAPs caspases, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, proteins involved in apoptosis, Be 1-2, Mcl-1, Bel -XL, Bcl-w, Bcl-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPs, XIAP, Smac, cell cycle regulators, Cdk4, Cdk 6, Cdk 2, Cdkl, Cdk 7, Cyclin D, Cyclin E, Cyclin A, Cyclin B, Rb, pl6, pl4Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP citrate lyase, nitric oxide synthase, ve
  • an epitope-recognizing fragment of an activation state antibody rather than the whole antibody is used.
  • the epitope-recognizing fragment is immobilized.
  • the antibody light chain that recognizes an epitope is used.
  • a recombinant nucleic acid encoding a light chain gene product that recognizes an epitope can be used to produce such an antibody fragment by recombinant means well known in the art.
  • aromatic amino acids of protein binding elements can be replaced with other molecules. See U.S. S. Nos. U.S. S.N. 12/432,720, 12/229,476, 12/460,029, 12/730,120, 12/617,438 and 12/910,769.
  • the activation state-specific binding element is a peptide comprising a recognition structure that binds to a target structure on an activatable protein.
  • recognition structures are well known in the art and can be made using methods known in the art, including by phage display libraries (see e.g., Gururaja et al. Chem. Biol.
  • fluorophores can be attached to such antibodies for use in the methods described herein.
  • the activation state-specific antibody is a protein that only binds to an isoform of a specific activatable protein that is phosphorylated and does not bind to the isoform of this activatable protein when it is not phosphorylated or nonphosphorylated.
  • the activation state-specific antibody is a protein that only binds to an isoform of an activatable protein that is intracellular and not extracellular, or vice versa.
  • the recognition structure is an anti-laminin single-chain antibody fragment (scFv) (see e.g., Sanz et al, Gene Therapy (2002) 9: 1049-53; Tse et al, J. Mol. Biol. (2002) 317:85-94, each expressly incorporated herein by reference).
  • scFv anti-laminin single-chain antibody fragment
  • the binding element is a nucleic acid.
  • nucleic acid include nucleic acid analogs, for example, phosphoramide (Beaucage et al, Tetrahedron
  • the detection of the status of the one or more activatable elements can be carried out by a person, such as a technician in the laboratory. Alternatively, the detection of the status of the one or more activatable elements can be carried out using automated systems. In either case, the detection of the status of the one or more activatable elements for use according to the methods described herein can be performed according to standard techniques and protocols well-established in the art.
  • One or more activatable elements can be detected and/or quantified by any method that detects and/or quantitates the presence of the activatable element of interest. Such methods may include flow cytometry, mass cytometry, radioimmunoassay (RIA) or enzyme linked immunoabsorbance assay (ELISA), immunohistochemistry, immunofluorescent histochemistry with or without confocal microscopy, reversed phase assays, homogeneous enzyme
  • immunoassays and related non-enzymatic techniques, Western, Northern, and Southern blots, PCR, nucleic acid sequencing, whole cell staining , immunoelectronmicroscopy, nucleic acid amplification, gene array, protein array, mass spectrometry, patch clamp, 2-dimensional gel electrophoresis, differential display gel electrophoresis, microsphere-based multiplex protein assays, label-free cellular assays and flow cytometry, etc.
  • U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for modified protein parameters. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules.
  • the present invention provides methods for determining an activatable element's activation profile for a single cell.
  • the methods may comprise analyzing cells by flow cytometry on the basis of the activation level of at least two activatable elements.
  • Binding elements e.g. activation state-specific antibodies
  • binding elements are used to analyze cells on the basis of activatable element activation level, and can be detected as described below.
  • non- binding elements systems as described above can be used in any system described herein.
  • Detection of cell signaling states may be accomplished using binding elements and labels.
  • Cell signaling states may be detected by a variety of methods known in the art. They generally involve a binding element, such as an antibody, and a label, such as a fluorochrome to form a detection element. Detection elements do not need to have both of the above agents, but can be one unit that possesses both qualities. These and other methods, instruments and devices are well described in U.S. Patent Nos. 7,381535 and 7,393,656 and U.S.S.Nos.
  • fluorescent monitoring systems e.g., cytometric measurement device systems
  • flow cytometric systems are used or systems dedicated to high throughput screening, e.g. 96 well or greater microtiter plates.
  • Methods of performing assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in:
  • Fluorescence in a sample can be measured using a fluorimeter.
  • excitation radiation from an excitation source having a first wavelength, passes through excitation optics.
  • the excitation optics deliver the excitation radiation to excite the sample.
  • fluorescent proteins in the sample emit radiation that has a wavelength that is different from the excitation wavelength.
  • Collection optics then collect the emission from the sample.
  • the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned.
  • a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed.
  • the multi- axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer.
  • the computer also can transform the data collected during the assay into another format for presentation.
  • known robotic systems and components can be used.
  • the activation level of an activatable element is measured using Inductively Coupled Plasma Mass Spectrometer (ICP-MS).
  • ICP-MS Inductively Coupled Plasma Mass Spectrometer
  • a binding element that has been labeled with a specific element binds to the activatable.
  • the elemental composition of the cell, including the labeled binding element that is bound to the activatable element is measured.
  • the presence and intensity of the signals corresponding to the labels on the binding element indicates the level of the activatable element on that cell (Tanner et al. Spectrochimica Acta Part B: Atomic Spectroscopy, 2007 Mar;62(3):188-195.).
  • the detecting, sorting, or isolating step of the methods of the present invention can entail fluorescence-activated cell sorting (FACS) techniques, where FACS is used to select cells from the population containing a particular surface marker, or the selection step can entail the use of magnetically responsive particles as retrievable supports for target cell capture and/or background removal.
  • FACS fluorescence-activated cell sorting
  • a variety of FACS systems are known in the art and can be used in the methods described herein (see e.g., W099/54494, filed Apr. 16, 1999; U.S. Ser. No.
  • a FACS cell sorter e.g. a FACSVantageTM Cell Sorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.
  • FACSVantageTM Cell Sorter Becton Dickinson Immunocytometry Systems, San Jose, Calif.
  • Other flow cytometers that are commercially available include the LSR II and the Canto II both available from Becton Dickinson others are available from Attune Acoustic Cytometer (Life Technologies, Carlsbad, CA) and the CyTOF (DVS Sciences, Sunnyvale, CA). See Shapiro, Howard M., Practical Flow Cytometry, 4th Ed., John Wiley & Sons, Inc., 2003 for additional information on flow cytometers.
  • the cells are first contacted with fluorescent-labeled activation state-specific binding elements (e.g. antibodies) directed against specific activation state of specific activatable elements.
  • the amount of bound binding element on each cell can be measured by passing droplets containing the cells through the cell sorter. By imparting an electromagnetic charge to droplets containing the positive cells, the cells can be separated from other cells. The positively selected cells can then be harvested in sterile collection vessels.
  • Fluorescent compounds such as Daunorubicin and Enzastaurin are problematic for flow cytometry based biological assays due to their broad fluorescence emission spectra. These compounds get trapped inside cells after fixation with agents like paraformaldehyde, and are excited by one or more of the lasers found on flow cytometers. The fluorescence emission of these compounds is often detected in multiple PMT detectors which complicates their use in multiparametric flow cytometry. A way to get around this problem is to compensate out the fluorescence emission of the compound from the PMT detectors used to measure the relevant biological markers.
  • positive cells can be sorted using magnetic separation of cells based on the presence of an isoform of an activatable element.
  • cells to be positively selected are first contacted with specific binding element (e.g., an antibody or reagent that binds an isoform of an activatable element).
  • the cells are then contacted with retrievable particles (e.g., magnetically responsive particles) that are coupled with a reagent that binds the specific element.
  • the cell-binding element-particle complex can then be physically separated from non-positive or non-labeled cells, for example, using a magnetic field.
  • the positive or labeled cells can be retained in a container using a magnetic field while the negative cells are removed.
  • methods for the determination of a receptor element activation state profile for a single cell comprise providing a population of cells and analyze the population of cells by flow cytometry. Preferably, cells are analyzed on the basis of the activation level of at least two activatable elements. In some embodiments, a multiplicity of activatable element activation-state antibodies is used to simultaneously determine the activation level of a multiplicity of elements.
  • cell analysis by flow cytometry on the basis of the activation level of at least two elements is combined with a determination of other flow cytometry readable outputs, such as the presence of surface markers, granularity and cell size to provide a correlation between the activation level of a multiplicity of elements and other cell qualities measurable by flow cytometry for single cells.
  • methods described herein also provide for the ordering of element clustering events in signal transduction.
  • the methods described herein allow the artisan to construct an element clustering and activation hierarchy based on the correlation of levels of clustering and activation of a multiplicity of elements within single cells. Ordering can be accomplished by comparing the activation level of a cell or cell population with a control at a single time point, or by comparing cells at multiple time points to observe subpopulations arising out of the others.
  • the methods described herein provide a valuable method of determining the presence of cellular subsets within cellular populations. Ideally, signal transduction pathways are evaluated in homogeneous cell populations to ensure that variances in signaling between cells do not qualitatively nor quantitatively mask signal transduction events and alterations therein. As the ultimate homogeneous system is the single cell, the present invention allows the individual evaluation of cells to allow true differences to be identified in a significant way.
  • the invention provides methods of distinguishing cellular subsets within a larger cellular population.
  • these cellular subsets often exhibit altered biological characteristics (e.g. activation levels, altered response to modulators) as compared to other subsets within the population.
  • the methods described herein allow the identification of subsets of cells from a population such as primary cell populations, e.g. peripheral blood mononuclear cells that exhibit altered responses (e.g. response associated with presence of a condition) as compared to other subsets.
  • this type of evaluation distinguishes between different activation states, altered responses to modulators, cell lineages, cell differentiation states, etc.
  • these methods provide for the identification of distinct signaling cascades for both artificial and stimulatory conditions in complex cell populations, such a peripheral blood mononuclear cells, or naive and memory lymphocytes.
  • a suitable protease e.g. collagenase, dispase, etc; and the like.
  • An appropriate solution is used for dispersion or suspension.
  • Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hanks balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES1 phosphate buffers, lactate buffers, etc.
  • the cells may be fixed, e.g.
  • a methanol dispensing instrument is used to permeabilize the cells. It is important to ensure that the correct volume of methanol is being dispensed into the wells, otherwise the labeling reagents will not have access to their targets. To ensure that the appropriate amount of methanol is dispensed, the dispenser is charged beforehand with methanol or is charged with methanol either manually or automatically.
  • the methanol dispensing heads in the instrument can be stored with methanol or air in the dispensing channels. Air can be drawn through the dispensing heads, then an alcohol solution and then stored air dried or with methanol. Upon reuse of the instrument or any restart of the process, the dispensing heads are recharged with methanol. A bleeder valve can be used to fill up the head with the correct amount of methanol. In one embodiment, the instrument dispenser is charged by flushing several methanol washes through the dispenser head. In one embodiment, 2, 3, 4, 5, 6, washes are used to fill and clean the head.
  • the present invention uses platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • One embodiment uses microtiter plates and reference will be made to this embodiment as a representative of those articles that can contain samples to be analyzed.
  • one or more cells are contained in a well of a 96 well plate or other commercially available multiwell plate.
  • the reaction mixture or cells are in a cytometric measurement device.
  • Other multiwell plates useful in the present invention include, but are not limited to 384 well plates and 1536 well plates. Still other vessels for containing the reaction mixture or cells and useful for the present invention will be apparent to the skilled artisan. Methods to automate the analysis are shown in U.S. Ser. No. 12/606,869 which is hereby incorporated by reference in its entirety.
  • the activation level of an activatable element is measured using Inductively Coupled Plasma Mass Spectrometer (ICP-MS).
  • ICP-MS Inductively Coupled Plasma Mass Spectrometer
  • a binding element that has been labeled with a specific element binds to the activatable.
  • the elemental composition of the cell, including the labeled binding element that is bound to the activatable element is measured.
  • the presence and intensity of the signals corresponding to the labels on the binding element indicates the level of the activatable element on that cell (Tanner et al. Spectrochimica Acta Part B: Atomic Spectroscopy, 2007 Mar;62(3):188-195.).
  • compositions find use in a variety of other assay formats in addition to flow cytometry analysis.
  • DNA microarrays are commercially available through a variety of sources
  • Such methods and materials may be adapted for the purpose of affixing activation state binding elements to a chip in a prefigured array.
  • a chip comprises a multiplicity of element activation state binding elements, and is used to determine an element activation state profile for elements present on the surface of a cell.
  • a chip comprises a multiplicity of the "second set binding elements," in this case generally unlabeled.
  • sample preferably cell extract
  • a second multiplicity of binding elements comprising element activation state specific binding elements is used in the sandwich assay to simultaneously determine the presence of a multiplicity of activated elements in sample.
  • each of the multiplicity of activation state-specific binding elements is uniquely labeled to facilitate detection.
  • confocal microscopy can be used to detect activation profiles for individual cells.
  • Confocal microscopy relies on the serial collection of light from spatially filtered individual specimen points, which is then electronically processed to render a magnified image of the specimen.
  • the signal processing involved confocal microscopy has the additional capability of detecting labeled binding elements within single cells, accordingly in this embodiment the cells can be labeled with one or more binding elements.
  • the binding elements used in connection with confocal microscopy are antibodies conjugated to fluorescent labels, however other binding elements, such as other proteins or nucleic acids are also possible.
  • the methods and compositions of the instant invention can be used in conjunction with an "In-Cell Western Assay.”
  • an assay cells are initially grown in standard tissue culture flasks using standard tissue culture techniques. Once grown to optimum confluency, the growth media is removed and cells are washed and trypsinized. The cells can then be counted and volumes sufficient to transfer the appropriate number of cells are aliquoted into microwell plates (e.g., NuncTM 96 MicrowellTM plates). The individual wells are then grown to optimum confluency in complete media whereupon the media is replaced with serum- free media. At this point controls are untouched, but experimental wells are incubated with a modulator, e.g. EGF.
  • a modulator e.g. EGF.
  • the plates can be scanned using an imager such as the Odyssey Imager (LiCor, Lincoln Nebr.) using techniques described in the Odyssey Operator's Manual vl .2., which is hereby incorporated in its entirety. Data obtained by scanning of the multiwell plate can be analyzed and activation profiles determined as described below.
  • an imager such as the Odyssey Imager (LiCor, Lincoln Nebr.) using techniques described in the Odyssey Operator's Manual vl .2., which is hereby incorporated in its entirety. Data obtained by scanning of the multiwell plate can be analyzed and activation profiles determined as described below.
  • the detecting is by high pressure liquid chromatography (HPLC), for example, reverse phase HPLC, and in a further aspect, the detecting is by mass spectrometry.
  • HPLC high pressure liquid chromatography
  • Flow cytometry or capillary electrophoresis formats can be used for individual capture of magnetic and other beads, particles, cells, and organisms.
  • the software program modules allow creation, modification, and running of methods.
  • the system diagnostic modules allow instrument alignment, correct connections, and motor operations.
  • Customized tools, labware, and liquid, particle, cell and organism transfer patterns allow different applications to be performed.
  • Databases allow method and parameter storage. Robotic and computer interfaces allow communication between instruments.
  • the methods described herein include the use of liquid handling components.
  • the liquid handling systems can include robotic systems comprising any number of components.
  • any or all of the steps outlined herein may be automated; thus, for example, the systems may be completely or partially automated. See U.S. Ser. Nos. 12/606,869 and 12/432,239.
  • Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications.
  • This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration.
  • These manipulations are cross-contamination-free liquid, particle, cell, and organism transfers.
  • This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.
  • chemically derivatized particles, plates, cartridges, tubes, magnetic particles, or other solid phase matrix with specificity to the assay components are used.
  • the binding surfaces of microplates, tubes or any solid phase matrices include non-polar surfaces, highly polar surfaces, modified dextran coating to promote covalent binding, antibody coating, affinity media to bind fusion proteins or peptides, surface-fixed proteins such as recombinant protein A or G, nucleotide resins or coatings, and other affinity matrix are useful in this invention.
  • platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • the methods described herein include the use of a plate reader.
  • thermocycler and thermoregulating systems are used for stabilizing the temperature of heat exchangers such as controlled blocks or platforms to provide accurate temperature control of incubating samples from 0° C to 100° C.
  • interchangeable pipet heads with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms.
  • Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.
  • the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay.
  • useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation.
  • the robotic apparatus includes a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, as outlined below, this may be in addition to or in place of the CPU for the multiplexing devices described herein.
  • a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • input/output devices e.g., keyboard, mouse, monitor, printer, etc.
  • this may be in addition to or in place of the CPU for the multiplexing devices described herein.
  • the general interaction between a central processing unit, a memory, input/output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.
  • the computer program can execute some or all of the following functions: (i) exposing reference population of cells to one or more modulators, (ii) exposing reference population of cells to one or more binding elements, (iii) detecting the activation levels of one or more activatable elements, (iv) characterizing one or more cellular pathways and/or , (v) classifying one or more cells into one or more classes based on the activation level (vi) determining cell health status of a cell, (vii) determining the percentage of viable cells in a sample; (viii) determining the percentage of healthy cells in a sample; (ix) determining a cell signaling profile; (x) adjusting a cell signaling profile based on the percentage of healthy cells in a sample; (xi) adjusting a cell signaling profile for an individual cell based on the health of the cell; (xii) excluding or including a cell or population of cells in a cell signaling analysis based on the health of the cell or population of cells; (xiii) as
  • the computer executable logic can work in any computer that may be any of a variety of types of general-purpose computers such as a personal computer, network server, workstation, or other computer platform now or later developed.
  • a computer program product is described comprising a computer usable medium having the computer executable logic (computer software program, including program code) stored therein.
  • the computer executable logic can be executed by a processor, causing the processor to perform functions described herein.
  • some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts.
  • the program can provide a method of determining the status of an individual by accessing data that reflects the activation level of one or more activatable elements in the reference population of cells.
  • the fluorescent intensity raw data from the detector is subject to processing using metrics outlined below.
  • the data is fed to a model, such as machine learning, data mining, classification, or regression to provide a model for an outcome.
  • a model such as machine learning, data mining, classification, or regression to provide a model for an outcome.
  • models There is also a selection of models to produce an outcome, which can be a prediction or a prognosis.
  • the data can also be processed by using characteristics of cell health and cell maturity. Information on how to use cell health to analyze cells is shown in U.S.S.No.
  • a method is provided to analyze cells comprising obtaining cells, determining if the cell is undergoing apoptosis and then excluding cells from a final analysis that are undergoing apoptosis.
  • One way to determine if a cell is undergoing apoptosis is by measuring the intracellular level of one or more activatable elements related to cell health such as cleaved PARP, MCL-1, or other compounds whose activation state or activation level correlate to a level of apoptosis within single cells.
  • Indicators for cell health can include molecules and activatable elements within molecules associated with apoptosis, necrosis, and/or autophagy, including but not limited to caspases, caspase cleavage products such as dye substrates, cleaved PARP, cleaved cytokeratin 18, cleaved caspase, cleaved caspase 3, cytochrome C, apoptosis inducing factor (AIF), Inhibitor of Apoptosis (IAP) family members, as well as other molecules such as Bcl-2 family members including anti-apoptotic proteins (MCL-1, BCL-2, BCL-XL), BH3-only apoptotic sensitizers (PUMA, NOXA, Bim, Bad), and pro-apoptotic proteins (Bad, Bax) (see below), p53, c-myc proto-oncogene, APO-l/Fas/CD95, growth stimulating genes, or
  • Another general method for analyzing cells takes into account the maturity level of the cells.
  • cells that are immature are included in the analysis and mature cells are not included.
  • the analysis can include all the patient's cells if they go above a certain threshold for the entire sample, for example, a call will be made on the basis of the entire sample.
  • samples having greater than 50, 60, 65, 70, 75, 80, 85, 90, or 95% immature cells can be classified as immature as a whole.
  • only those specific cells which are classified as immature are included in the analysis, irrespective of the total number of immature cells, for example, only those cells that are classified as immature will be part of the analysis for each sample.
  • a combination of the two methods could be employed, such as the counting of individual immature cells for samples that exceed a threshold related to cell maturity.
  • Cells may be classified as mature or immature manually or automatically. Methods for determining maturity are shown in Stelzer and Goodpasture, Immunophenotyping, 2000 Wiley-Liss Inc. which is incorporated by reference in its entirety. See also JOHN M.
  • maturity may be determined by surface marker expression which can be applied to individual cells or at the sample level.
  • the FAB system may also be used and applied to samples as a whole. For example, in one embodiment, samples as a whole are classified in the FAB system as M4, M5, or M7 are mature.
  • the cells may be analyzed by a variety of methods and markers, such as side scatter (SSC), CD1 lb, CD 117, CD45 and CD34. Generally, higher side scatter, and populations of CD45 or CD 1 lb cells will indicate mature cells and generally lower populations of CD34 and CD117 will indicate mature cells. Immature populations are classified in the FAB system as M0, Ml, M2 and M6.
  • PB peripheral blood
  • BM bone marrow
  • the use of the cell maturity analysis is combined with the analysis of cell health, in which immature blasts that are not apoptosing, are used in the analysis.
  • this method is used to model relapse, one endpoint is a complete continuous response with duration of > lyear (CCR1) another is survival.
  • cells are classified as mature or immature and then the immature cells are analyzed using a classifier.
  • the sample is classified as mature or immature and then the immature samples are analyzed using a classifier. See example 19.
  • the metrics that are employed can relate to absolute cell counts, fluorescent intensity, frequencies of cellular populations (univariate and bivariate), relative fluorescence readouts (such as signal above background, etc.), and measurements describing relative shifts in cellular populations.
  • raw intensity data is corrected for variances in the instrument. Then the biological effect can be measured, such as measuring how much signaling is going on using the basal, fold, total and delta metrics. Also, a user can look at the number of cells that show signaling using the Mann Whitney model below.
  • flow cytometry experiments are performed and the results are expressed as fold changes using graphical tools and analyses, including, but not limited to a heat map or a histogram to facilitate evaluation.
  • graphical tools and analyses including, but not limited to a heat map or a histogram to facilitate evaluation.
  • One common way of comparing changes in a set of flow cytometry samples is to overlay histograms of one parameter on the same plot.
  • Flow cytometry experiments ideally include a reference sample against which experimental samples are compared. Reference samples can include normal and/or cells associated with a condition (e.g. tumor cells). See also U.S.Ser. No. 12/501,295 for visualization tools.
  • the patients are stratified based on nodes that inform the clinical question using a variety of metrics.
  • a prioritization of the nodes can be made according to statistical significance (such as p-value from a t-test or Wilcoxon test or area under the receiver operator characteristic (ROC) curve) or their biological relevance.
  • Figure 2 shows four methods to analyze data, such as from AML patients. Other characteristics such as expression markers may also be used.
  • the fold over isotype can be used (e.g., log2(MFIstain)-Log2(MFIisotype)) or % positive above Isotype.
  • Figure 2 shows the use of four metrics used to analyze data from cells that may be subject to a disease, such as AML.
  • the "basal” metric is calculated by measuring the autofluorescence of a cell that has not been stimulated with a modulator or stained with a labeled antibody.
  • the “total phospho” metric is calculated by measuring the autofluorescence of a cell that has been stimulated with a modulator and stained with a labeled antibody.
  • the "fold change” metric is the measurement of the total phospho metric divided by the basal metric.
  • the quadrant frequency metric is the frequency of cells in each quadrant of the contour plot
  • a user may also analyze multimodal distributions to separate cell populations.
  • metrics can be used for analyzing bimodal and spread distribution.
  • a Mann- Whitney U Metric is used.
  • metrics that calculate the percent of positive above unstained and metrics that calculate MFI of positive over untreated stained can be used.
  • a user can create other metrics for measuring the negative signal. For example, a user may analyze a "gated unstained” or ungated unstained autofluorescence population as the negative signal for calculations such as "basal” and “total". This is a population that has been stained with surface markers such as CD33 and CD45 to gate the desired population, but is unstained for the fluorescent parameters to be quantitatively evaluated for node determination. However, every antibody has some degree of nonspecific association or "stickyness" which is not taken into account by just comparing fluorescent antibody binding to the autofluorescence. To obtain a more accurate "negative signal", the user may stain cells with isotype-matched control antibodies.
  • a user may block with unlabeled antibodies. This method uses the same antibody clones of interest, but uses a version that lacks the conjugated fluorophore. The goal is to use an excess of unlabeled antibody with the labeled version.
  • a user may block other high protein concentration solutions including, but not limited to fetal bovine serum, and normal serum of the species in which the antibodies were made, i.e. using normal mouse serum in a stain with mouse antibodies. (It is preferred to work with primary conjugated antibodies and not with stains requiring secondary antibodies because the secondary antibody will recognize the blocking serum).
  • a user may treat fixed cells with phosphatases to enzymatically remove phosphates, then stain.
  • One embodiment of the present invention is software to examine the correlations among phosphorylation or expression levels of pairs of proteins in response to stimulus or modulation.
  • the software examines all pairs of proteins for which phosphorylation and/or expression was measured in an experiment.
  • the Total phosho metric (sometimes called
  • “FoldAF”) is used to represent the phosphorylation or expression data for each protein; this data is used either on linear scale or log2 scale. See Figure 2, metric 3 for Total Phospho.
  • Delta CRNR unstim the difference between Pearson correlation coefficients for each protein pair for the responding patients and for the non-responding patients in the basal or unstimulated state.
  • Delta CRNR stim the difference between Pearson correlation coefficients for each protein pair for the responding patients and for the non-responding patients in the stimulated or treated state.
  • DeltaDelta CRNR the difference between Delta CRNRstim and Delta CRNRunstim.
  • All pair data is plotted as a scatter plot with axes representing phosphorylation or expression level of a protein.
  • Data for each sample (or patient) is plotted with color indicating whether the sample represents a responder (generally blue) or non-responder (generally red).
  • Each graph is annotated with the Pearson correlation coefficient and linear regression parameters for the individual classes and for the data as a whole.
  • the resulting plots are saved in PNG format to a single directory for browsing using Picassa. Other visualization software can also be used.
  • a Mann Whitney statistical model is used for describing relative shifts in cellular populations.
  • a Mann Whitney U test or Mann Whitney Wilcoxon (MWW) test is a non parametric statistical hypothesis test for assessing whether two independent samples of observations have equally large values. See Wikipedia at http(colon)(slashslash)en.wikipedia.org(slash)wiki/Mann%E2%80%93Whitney_U .
  • the U metric may be more reproducible in some situations than Fold Change in some applications.
  • U u is a measure of the proportion of cells that have an increase (or decrease) in protein levels upon modulation from the basal state. It is computed by dividing the scaled Mann- Whitney U statistic
  • Modulated (m) and modulated (u) populations are being compared
  • n m number of cells in the modulated population
  • n u number of cells in the unmodulated population
  • U i is another value that is the same as U u except that the isotype control is used as the reference instead of the unmodulated well.
  • Each protein pair can be further annotated by whether the proteins comprising the pair are connected in a "canonical" pathway.
  • canonical pathways are defined as the pathways curated by the NCI and Nature Publishing Group. This distinction is important; however, it is likely not an exclusive way to delineate which protein pairs to examine.
  • High correlation among proteins in a canonical pathway in a sample may indicate the pathway in that sample is "intact" or consistent with the known literature.
  • One embodiment of the present invention identifies protein pairs that are not part of a canonical pathway with high correlation in a sample as these may indicate the non-normal or pathological signaling. This method will be used to identify stimulator/modulator-stain-stain combinations that distinguish classes of patients.
  • nodes and/or nodes/metric combinations can be analyzed and compared across sample for their ability to distinguish among different groups (e.g., CR vs. NR patients) using classification algorithms.
  • Any suitable classification algorithm known in the art can be used. Examples of classification algorithms that can be used include, but are not limited to, multivariate classification algorithms such as decision tree techniques: bagging, boosting, random forest, additive techniques: regression, lasso, bblrs, stepwise regression, nearest neighbors or other methods such as support vector machines.
  • nodes and/or nodes/metric combinations can be analyzed and compared across sample for their ability to distinguish among different groups (e.g., CR vs. NR patients) using random forest algorithm.
  • Random forest or random forests is an ensemble classifier that consists of many decision trees and outputs the class that is the mode of the class's output by individual trees.
  • the algorithm for inducing a random forest was developed by Leo Breiman (Breiman, Leo (2001). "Random Forests”. Machine Learning 45 (1): 5-32.
  • nodes and/or nodes/metric combinations can be analyzed and compared across sample for their ability to distinguish among different groups (e.g., CR vs. NR patients) using lasso algorithm.
  • the method of least squares is a standard approach to the approximate solution of overdetermined systems, i.e. sets of equations in which there are more equations than unknowns. "Least squares" means that the overall solution minimizes the sum of the squares of the errors made in solving every single equation. The best fit in the least-squares sense minimizes the sum of squared residuals, a residual being the difference between an observed value and the fitted value provided by a model.
  • nodes and/or nodes/metric combinations can be analyzed and compared across sample for their ability to distinguish among different groups (e.g., CR vs. NR patients) using BBLRS model building methodology.
  • Best subsets selection of main effects is used to identify the combination of predictors that yields the largest score statistic among models of a given size in each bootstrap sample. Models having from 1 to 2xN/10 are typically entertained at this stage, where N is the number of observations. This is much larger than the number of predictors generally recommended when building a generalized linear prediction model (Harrell, 2001) but subsequent model building rules are applied to reduce the likelihood of over-fitting. At the conclusion of this step, there will be a "best" main effects model of each size for each bootstrap sample, though the number of unique models of each size may be considerably fewer.
  • each of the unique "best" models of each size, identified in the previous step are fit to each of a subset of the bootstrap samples, where the number of bootstrap samples in the subset is under the control of the user (i.e. a tuning parameter) so that the processing time required at this step can be controlled.
  • the median SBC of the "best" models of the same size is calculated and the model size yielding the lowest median SBC in that bootstrap sample is identified.
  • the optimal model size is then determined as the size for which the median SBC is smallest most often over the subset of bootstrap samples.
  • the procedure described here results in the selection of the effects (main effects and possibly two-way interactions) to be included in the final model, but not specification of the model itself.
  • the latter includes the effects and the specific regression coefficients associated with the intercept and each of the model effects.
  • Another method of the present invention relates to display of information using scatter plots.
  • Scatter plots are known in the art and are used to visually convey data for visual analysis of correlations. See U. S. Patent No. 6,520,108.
  • the scatter plots illustrating protein pair correlations can be annotated to convey additional information, such as one, two, or more additional parameters of data visually on a scatter plot.
  • scatter plots used equal size plots to denote all events.
  • two additional parameters can be visualized as follows. First, the diameter of the circles representing the phosphorylation or expression levels of the pair of proteins may be scaled according to another parameter. For example they may be scaled according to expression level of one or more other proteins such as transporters (if more than one protein, scaling is additive, concentric rings may be used to show individual contributions to diameter).
  • additional shapes may be used to indicate subclasses of patients. For example they could be used to denote patients who responded to a second drug regimen or where CRp status. Another example is to show how samples or patients are stratified by another parameter (such as a different stim-stain-stain combination). Many other shapes, sizes, colors, outlines, or other distinguishing glyphs may be used to convey visual information in the scatter plot.
  • the size of the dots is relative to the measured expression and the box around a dot indicates a NRCR patient that is a patient that became CR (Responsive) after more aggressive treatment but was initially NR (Non-Responsive). Patients without the box indicate a NR patient that stayed NR.
  • the Total Phospho metric for p-Akt and p-Statl are correlated in response to peroxide ("H 2 O 2 ") treatment.
  • Total phoshpho is calculated as shown in Fig. 2, metric #3).
  • the Pearson correlation coefficient for p-Akt and p-Statl in response to HOOH for samples from patients who responded to first treatment is 0.89 and the p-value for linear regression line fit is 0.0075.
  • Table 3(a) below shows nodes identified by a fold change metric.
  • Table 3(b) below shows node identified by a variety of methods. In some embodiments, the nodes depicted in Tables 3(a) and 3(b) are used according to the methods described herein for classification, diagnosis, prognosis of AML or for the selection of treatment and/or predict outcome after administering a therapeutic.
  • analyses are performed on healthy cells.
  • the health of the cells is determined by using cell markers that indicate cell health.
  • cells that are dead or undergoing apoptosis will be removed from the analysis.
  • cells are stained with apoptosis and/or cell death markers such as PARP or Aqua dyes.
  • Cells undergoing apoptosis and/or cells that are dead can be gated out of the analysis.
  • apoptosis is monitored over time before and after treatment.
  • the percentage of healthy cells can be measured at time zero and then at later time points and conditions such as: 24h with no modulator, and 24h with Ara- C/Daunorubicin.
  • the measurements of activatable elements are adjusted by measurements of sample quality for the individual sample, such as the percent of healthy cells present.
  • a regression equation will be used to adjust raw node readout scores for the percentage of healthy cells at 24 hours post-thaw.
  • means and standard deviations will be used to standardize the adjusted node readout scores.
  • x is the raw node-metric signal readout
  • b 0 and b x are the coefficients from the regression equation used to adjust for the percentage of healthy cells ⁇ pcthealthy )
  • residual _ mean and residual _ sd are the mean and standard deviation, respectively, for the adjusted signal readouts in the training set data.
  • b 0 , b x , residual _ mean, and residual _ sd for each node-metric are included in the embedded object below, with values of the latter two parameters stored in variables by the same name.
  • the values of the b 0 and b x parameters are contained on separate records in the variable named
  • the value for b 0 is contained on the record where the variable "parameter” is equal to "Intercept” and the value for b x is contained on the record where the variable "parameter” is equal to "percenthealthy24Hrs”.
  • the value of pcthealthy ' will be obtained for each sample as part of the standard assay output.
  • the SCNP classifier will be applied to the z values for the node- metrics to calculate the continuous SCNP classifier score and the binary induction response assignment (pNR or pCR) for each sample.
  • the measurements of activatable elements are adjusted by measurements of sample quality for the individual cell populations or individual cells, based on markers of cell health in the cell populations or individual cells. Examples of analysis of healthy cells can be found in U.S. application serial number 61/374,613 filed August 18, 2010, the content of which is incorporated herein by reference in its entirety for all purposes.
  • the invention provides methods of diagnosing, prognosing, determining progression, predicting a response to a treatment or choosing a treatment for AML in an individual, the method comprising: (1) classifying one or more hematopoietic cells associated with AML in said individual by a method comprising: a) subjecting a cell population comprising said one or more hematopoietic cells from said individual to modulator conditions , b) determining an activation level of activatable elements in one or more cells from said individual, and c) classifying said one or more hematopoietic cells based on said activation levels in response to modulator conditions using multivariate classification algorithms such as decision tree techniques: bagging, boosting, random forest, additive techniques: regression, lasso, bblrs, stepwise regression, nearest neighbors or other methods such as support vector machines (2) making a decision regarding a diagnosis, prognosis, progression, response to a treatment or a selection of treatment for AML in said individual based on said multivariate classification algorithms such as decision
  • classifying further comprises identifying a difference in kinetics of said activation level.
  • the measurements of activatable elements are made only in healthy cells as determined using markers of cell health.
  • the measurements of activatable elements are adjusted by measurements of sample quality for the individual sample, such as the percent of healthy cells present.
  • Another embodiment of the present invention is a method for screening drugs that are in development and indicated for patients that have been diagnosed with acute myelogenous leukemia (AML), myelodysplasia (MDS) or myelodyspastic syndrome (MPN).
  • AML acute myelogenous leukemia
  • MDS myelodysplasia
  • MPN myelodyspastic syndrome
  • multiparametric flow cytometry could be used in-vitro to predict both on and off-target cell signaling effects.
  • the bone marrow or peripheral blood obtained from a patient diagnosed with AML could be divided and part of the sample subjected to a therapeutic.
  • Modulators e.g. GM-CSF or PMA
  • Activatable elements e.g. JAKs/STATs/AKT
  • JAKs/STATs/AKT including the proposed target of the therapeutic, or those that may be affected by the therapeutic (off-target) can then be assessed for an activation state. This activation state can be used to predict the therapeutics' potential for on and off target effects prior to first in human studies.
  • one embodiment of the present invention could be used after in-vivo exposure to a therapeutic in development for patients that have been diagnosed with AML to determine both on and off-target effects.
  • the bone marrow or peripheral blood fresh, frozen, ficoll purified, etc.
  • Activatable elements e.g. JAKs/STATs/AKT
  • This activation state can then be used to determine the on and off target signaling effects on the bone marrow or blast cells.
  • the apoptosis and peroxide panel study may reveal new biological classes of stratifying nodes for drug screening.
  • Some of the important nodes could include changes on levels of p-Lck, pSlp-76, p PLC ⁇ 2, in response to peroxide alone or in combination with growth factors or cytokines.
  • These important nodes are induced Cleaved Caspase 3 and Cleaved Caspase 8, and etoposide induced p-Chk2, peroxide (H 2 O 2 ) induced p-SLP-76, peroxide (H 2 O 2 ) induced p-PLC ⁇ 2 and peroxide (H 2 O 2 ) induced P-Lck.
  • the apoptosis panel may include but is not limited to, detection of changes in phosphorylation of Chk2, changes in amounts of cleaved caspase 3, cleaved caspase 8, cleaved poly (ACP ribose) polymerase PARP, cytochrome C released from the mitochondria these apoptotic nodes are measured in response to agents that included but are not limited to DNA damaging agents such as Etoposide, Mylotarg, AraC and daunorubicin either alone or in combination as well as to the global kinase inhibitor
  • multiparametric flow cytometry could be used to find new target for treatment (e.g. new druggable targets).
  • the bone marrow or peripheral blood obtained from a patient diagnosed with AML could be divided and part of the sample subjected to one or more modulators (e.g. GM-CSF or PMA).
  • Activatable elements e.g. JAKs/STATs/AKT
  • This activation state can be used to predict find new target molecule for new existing therapeutics.
  • These therapeutics can be used alone or in combination with other treatments for the treatment of AML, MDS or MPN.
  • kits provided by the invention may comprise one or more of the state-specific binding elements described herein, such as phospho-specific antibodies.
  • a kit may also include other reagents that are useful in the invention, such as modulators, fixatives, containers, plates, buffers, therapeutic agents, instructions, and the like.
  • a kit can be used to assay for one or more cell health markers.
  • a kit can be used to assay for one or more markers of apoptosis and/or necrosis. See U.S. Patent No. 8,242,248.
  • the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of PI3-Kinase (p85, pi 10a, pi 10b, pi lOd), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nek, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP, PTEN, She, Grb2, PDK1 , SGK, Aktl, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7
  • the kit comprises one or more of the phospho- specific antibodies specific for the proteins selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, Cbl, PLC ⁇ 2, Akt, RelA, p38, S6.
  • the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of Akt 1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erkl/2, Syk, ZAP70, Btk, BLNK, Lck, PLC ⁇ , PLC ⁇ 2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, NF- ⁇ , GSK3 ⁇ , CARMA/Bc110 and Tcl-1.
  • the proteins selected from the group consisting of Akt 1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erkl/2, Syk, ZAP70, Btk, BLNK, Lck, PLC ⁇ , PLC ⁇ 2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, NF- ⁇ , GSK3 ⁇ , CARMA/Bc110 and Tcl-1.
  • kits having the following reagents: Phenotyping, DNA content, and signaling reagents.
  • Phenotyping including
  • CytoKeratin FITC EpCAM PerCP-Cy5.5, CD45 PE-Cy7; DNA Content, including DAPI;
  • Apoptosis including cPARP AF700; and Intracellular Signaling including, pERK PE, pAKT AF647.
  • the state-specific binding element described herein can be conjugated to a solid support and to detectable groups directly or indirectly.
  • the reagents can also include ancillary agents such as buffering agents and stabilizing agents, e.g., polysaccharides and the like.
  • the kit can further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
  • the kit can be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.
  • kits enable the detection of activatable elements by sensitive cellular assay methods, such as IHC and flow cytometry, which are suitable for the clinical detection, prognosis, and screening of cells and tissue from patients, such as leukemia patients, having a disease involving altered pathway signaling.
  • sensitive cellular assay methods such as IHC and flow cytometry
  • kits can additionally comprise one or more therapeutic agents.
  • the kit can further comprise a software package for data analysis of the physiological status, which can include reference profiles for comparison with the test profile.
  • kits can also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information can be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits can also, in some embodiments, be marketed directly to the consumer.
  • the invention provides a kit comprising: (a) at least two modulators selected from the group consisting of Staurosporine, Etoposide, Mylotarg,
  • the kit further comprises a binding element specific for a cytokine receptor or drug transporter are selected from the group consisting of MDR1, ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3, and c-kit.
  • the binding element is an antibody.
  • the present illustrative example represents how to analyze cells in one embodiment of the present invention.
  • There are several steps in the process such as the stimulation step, the staining step and the flow cytometry step.
  • the stimulation step of the phospho-flow procedure can start with vials of frozen cells and end with cells fixed and permeabilized in methanol. Then the cells can be stained with an antibody directed to a particular protein of interest and then analyzed using a flow cytometer.
  • the materials used in this invention include thawing medium which comprises PBS- CMF + 10% FBS + 2mM EDTA; 70 urn Cell Strainer (BD); anti-CD45 antibody conjugated to Alexa 700 (Invitrogen) used at 1 ul per sample; propidium iodide (PI) solution (Sigma 10ml, 1 mg/ml) used at 1 ug/ml; RPMI + 1% FBS medium; media A comprising RPMI + 1% FBS + IX Penn/Strep; Live/Dead Reagent, Amine Aqua (Invitrogen); 2 ml, 96-Deep Well, U-bottom polypropylene plates (Nunc); 300ul 96-Channel Extended-Length D.A.R.T.
  • thawing medium which comprises PBS- CMF + 10% FBS + 2mM EDTA; 70 urn Cell Strainer (BD); anti-CD45 antibody conjugated to Alexa 700 (Invitrogen) used at 1
  • Frozen cells are thawed in a 37°C water bath and gently resuspended in the vial and transferred to the 15 mL conical tube.
  • the 15 mL tube is centrifuged at 930 RPM (200xg) for 8 minutes at room temperature. The supernatant is aspirated and the pellet is gently resuspended in 1 mL media A.
  • the cell suspension is filtered through a 70 um cell strainer into a new 15 mL tube.
  • the cell strainer is rinsed with 1 mL media A and another 12ml of media A into the 15 mL tube.
  • the cells are mixed into an even suspension.
  • a 20 aliquot is immediately removed into a 96-well plate containing 180 PBS + 4% FBS + CD45 Alexa 700 + PI to determine cell count and viability post spin.
  • the 15 mL tubes are centrifuged at 930 RPM (200xg) for 8 minutes at room temperature. The supernatant is aspirated and the cell pellet is gently resuspended in 4 mL PBS + 4 Amine Aqua and incubated for 15 min in a 37°C incubator. 10 mL RPMI + 1% FBS is added to the cell suspension and the tube is inverted to mix the cells.
  • the 15 mL tubes are centrifuged at 930 RPM (200xg) for 8 minutes at room
  • the cells are resuspended in Media A at the desired cell concentration (1.25x 10 6 /mL).
  • the cells are resuspended in up to 15 mL media.
  • the volume is raised to 10 mL with media A and the desired volume is transferred to a new 15 mL tube, and the cell concentration is adjusted to 1.25 x 10 6 cells/ml. 1.6mL of the above cell suspension
  • a concentration for each stimulant that is five folds more (5X) than the final concentration is prepared using Media A as diluent.
  • 5X stimuli are arrayed into wells of a standard 96 well v-bottom plate that correspond to the wells on the plate with cells to be stimulated.
  • Preparation of fixative Stock vial contains 16% paraformaldehyde which is diluted with PBS to a concentration that is 1.5X. The stock vial is placed in a 37°C water bath. [00424] Adding the stimulant: The cell plate(s) are taken out of the incubator and placed in a 37°C water bath next to the pipette apparatus. The cell plate is taken from the water bath and gently swirled to resuspend any settled cells. With pipettor, the stimulant is dispensed into the cell plate and vortexed at "7" for 5 seconds. The deep well plate is put back into the water bath.
  • Adding Fixative 200 ⁇ 1 of the fixative solution (final concentration at 1.6%) is dispensed into wells and then mixed on the titer plate shaker on high for 5 seconds.
  • the plate is covered with foil sealer and incubated in a 37°C water bath for 10 minutes.
  • the plate is spun for 6 minutes at 2000 rpm at room temperature.
  • the cells are aspirated using a 96 well plate aspirator (VP Scientific).
  • the plate is vortexed to resuspend cell pellets in the residual volume. The pellet is ensured to be dispersed before the Methanol step (see cell permeabilization) or clumping will occur.
  • Cell Permeabilization Permeability agent, for example methanol, is added slowly and while the plate is vortexing. To do this, the cell plate is placed on titer plate shaker and made sure it is secure. The plate is set to shake using the highest setting. A pipetter is used to add 0.6 mis of 100% methanol to the plate wells. The plate(s) are put on ice until this step has been completed for all plates. Plates are covered with a foil seal using the plate roller to achieve a tight fit. At this stage the plates can be stored at -80°C. c. Staining Protocol
  • Reagents for staining include FACS/Stain Buffer-PBS + 0.1% Bovine serum albumen (BSA) + 0.05% Sodium Azide; Diluted Bead Mix-lmL FACS buffer + 1 drop anti- mouse Ig Beads + 1 drop negative control beads.
  • BSA Bovine serum albumen
  • the general protocol for staining cells is as follows, although numerous variations on the protocol may be used for staining cells:
  • FACS/stain buffer is added to the plate and the plate is covered and incubated on a plate shaker for 5 minutes at room temperature. Repeat the above two steps and resuspend the cells in 75 ⁇ FACS/stain buffer.
  • the cells are analyzed using a flow cytometer, such as a LSRII (Becton Disckinson). All wells are selected and Loader Settings are described below: Flow Rate: 2 uL/sec; Sample Volume: 40 uL; Mix volume: 40 uL; Mixing Speed: 250uL/sec; # Mixes: 5; Wash Volume: 800uL; STANDARD MODE. When a plate has completed, a Batch analysis is performed to ensure no clogging. d. Gating protocol
  • Flow cytometry data can be analyzed using several commercially available software programs including FACSDivaTM, Flow Jo, and WinlistTM.
  • the initial gate is set on a two- parameter plot of forward light scatter (FSC) versus side light scatter (SSC) to gate on "all cells” and eliminate debris and some dead cells from the analysis.
  • a second gate is set on the "live cells” using a two-parameter plot of Amine Aqua (a dye that brightly stains dead cells, commercially available from Invitrogen) versus SSC to exclude dead cells from the analysis.
  • Subsequent gates are be set using antibodies that recognize cell surface markers and in so doing define cell sub-sets within the entire population.
  • a third gate is set to separate lymphocytes from all myeloid cells (acute myeloid leukemia cells reside in the myeloid gate). This is done using a two-parameter plot of CD45 (a cell surface antigen found on all white blood cells) versus SSC. The lymphocytes are identified by their characteristic high CD45 expression and low SSC. The myeloid population typically has lower CD45 expression and a higher SSC signal allowing these different populations to be discriminated.
  • the gated region containing the entire myeloid population is also referred to as the PI gate.
  • the antibodies used to identify subpopulations of AML blast cells are CD34, CD33, and CD11b.
  • the CD34 + CD11b " blast population represents the most immature phenotype of AML blast cells. This population is gated on CD34 high and CD11b negative cells using a two- parameter plot of CD34 versus CD11b.
  • the CD33 and CD11b antigens are used to identify AML blast cells at different stages of monocytic differentiation. All cells that fall outside of the CD34 + CD11b " gate described above (called “Not CD34+”) are used to generate a two-parameter plot of CD33 versus CD11b.
  • the CD33 + CD11b hl myeloid population represents the most differentiated monocytic phenotype.
  • the CD33 + CD11b intermediate and CD33 + CD11b 10 populations represent less differentiated monocytic phenotypes.
  • the data can then be analyzed using various metrics, such as basal level of a protein or the basal level of phosphorylation in the absence of a stimulant, total phosphorylated protein, or fold change (by comparing the change in phosphorylation in the absence of a stimulant to the level of phosphorylation seen after treatment with a stimulant), on each of the cell populations that are defined by the gates in one or more dimensions.
  • These metrics are then organized in a database tagged by: the Donor ID, plate identification (ID), well ID, gated population, stain, and modulator.
  • These metrics tabulated from the database are then combined with the clinical data to identify nodes that are correlated with a pre-specified clinical variable (for example; response or non response to therapy) of interest.
  • Multi-parameter flow cytometric analysis was performed on peripheral blasts taken at diagnosis from 9 AML patients who achieved a complete response (CR) and 24 patients who were non-responders (NR) to one cycle of standard 7 + 3 induction therapy (100-200mg/m2 cytarabine and 60mg/m2 daunorubicin).
  • the signaling nodes were organized into 4 biological categories: 1) Protein expression of receptors and drug transporters 2) Response to cytokines and growth factors, 3) Phosphatase activity, and 4) Apoptotic signaling pathways.
  • peroxide H 2 O 2
  • H2O2 peroxide, (H 2 O 2 ) a physiologic phosphatase inhibitor revealed increased phosphatase activity in CRs versus NRs for some signaling molecules and increased phosphatase activity in NRs versus CRs for others.
  • CRs had lower levels of phosphorylated PLC ⁇ 2 and SLP-76 versus NRs, and attained higher levels of phosphorylated PLC ⁇ 2 and SLP-76 upon H2O2 treatment.
  • H2O2 revealed higher levels of p-Akt in NR patients versus CR patients.
  • NR patient blasts failed to undergo cell death, as determined by cleaved PARP and cleaved Caspase 8.
  • these agents did promote an increase in phosphorylated Chk2 suggesting a communication breakdown between the DNA damage response pathway and the apoptotic machinery.
  • blasts from CR patients showed significant populations of cells with cleaved PARP and caspase 8 consistent with their clinical outcomes.
  • Figure 4 shows different activation profiles for NR patients. The operative pathways in these patients can be used to predict response to a treatment or to choose a specific treatment for the patients.
  • Figure 4 shows that NR patients in subset 1 have high levels of p-Stat3 and p-Stat5 in response to G-CSF. This suggests that JAK, Src and other new therapeutics could be good candidates for the treatment of these patients.
  • Figure 4 shows that NR patients in subset 2 have high levels of p-Akt and p-S6 in response to FLT3L.
  • Figure 4 also shows that NR patients in subset 2 have high levels of p-Stat3 and p-Stat5 in response to G-CSF, high levels of p-Akt and p-S6 in response to FLT3L, and high levels of p-Akt and p-S6 in response to SCF. This suggests that inhibitors to JAK, Src, FLT3R, PI-3K/mTor, RKT inhibitors and other new therapeutics could be good candidates for the treatment of these patients.
  • Phospho-Flow technology allows detection of multiple signaling subpopulations within the AML blast population which could be instrumental in disease monitoring and following rare populations after therapy. See Figure 4 and not shown.
  • phospho-flow identifies patient subgroups of AML with different clinical outcomes to induction therapy, reveals mechanisms of potential pathophysiology, and provides a tool for personalized treatment options based on unique patient-specific signaling networks and for disease monitoring under therapeutic pressure.
  • An analysis of a heterogeneous population of AML patients may be conducted as outlined above.
  • the results may show the following.
  • univariate analysis is performed on relatively homogeneous clinical groups, such as patents over 60 years old, patients under 60 years old, de novo AML patients, and secondary AML patients.
  • the groups may be molecularly homogeneous groups, such as Flt-3-ITD WT.
  • NRs may have a higher H 2 O 2 response than CRs and/or a higher FLT3L response than CRs.
  • NRs may have a higher IL-27 response than CRs and/or CRs may induce apoptosis to Etoposide or Ara-C/Daunorubicin more than NRs.
  • CRs may induce apoptosis (cleaved PARP) in response to Etoposide or Ara- C/Daunorubicin, they may have higher total p-S6 levels than NRs, or NRs may have higher H 2 O 2 response than CRs.
  • NRs may have higher H 2 O 2 responses than CRs, NRs may have higher FLT3L, SCF response than CRs, NRs may have higher G-CSF, IL-27 response than CRs, and there may be overlapping nodes with the over 60 patient set.
  • the following tables may illustrate the above.
  • the tables show the node, metric, and patient subpopulations.
  • the node can be shown as the node (readout) followed by the stimulant/modulator, and in some instances the receptor through which they act (Table 11 also lists some labels that can be employed in the readout).
  • the metric is the way the result may be calculated (see definitions above and in the figures; ppos is percent positive).
  • Multi-parameter flow cytometric analysis was performed on BMMC samples taken at diagnosis from 61 AML patients. The samples were balanced for complete response (CR) and non-responders (NR) after 1 to 3 cycles of induction therapy and de novo versus secondary AML. Nodes in Tables 2 to 10 were examined. [00443] 10 nodes are common in stratifying NR and CR between the studies in Example 2 and these studies. Table 14 shows the common stratifying nodes.
  • NRs have higher H2O2 and FLT3L responses than CRs.
  • NRs have higher IL-27 response than CRs.
  • CRs induce apoptosis to Etoposide or Ara-C/Daunorubicin more than NRs.
  • Individual nodes can be combined for analysis. Several methods can be used for the analysis.
  • the nodes can be analyzed using additive linear models to discover combinations that provide better accuracy of prediction for response to induction therapy than the individual nodes.
  • These models can also include clinical covariates like age, gender, secondary AML that may already be predictive of the outcome. Only nodes that add to the accuracy of the model after accounting for these clinical covariates are considered to be useful.
  • the formula below is an example of how additive linear models can be used
  • CI and C2 are the clinical covariates that are considered to be predictive of response
  • Nodel and Node2 are the two nodes from the biological data.
  • Several such models can be explored to find combinations of nodes that are complimentary. Examples of methods for exploring multiple such models include bootstrapping, and stepwise regression.
  • Analysis methods can include additive lineal models, such as the model represented in the following equations
  • the nodes can be analyzed using independent combinations of nodes. This method seeks threshold along different node axes independently. This model among clinical sub-groups improves predictive value (not shown).
  • the nodes can be analyzed using decision trees model. This model involves the hierarchical splitting of data. This model might mimic a more natural decision process. Each node is evaluated on sub-set of data at each level of the tree.
  • Results from the BMMC samples were compared with PBMC samples from the same patients in 10 of the patients. The samples were compared for sub-populations and signaling. The same phenotypic sub-populations are present in PBMC and BMMC, but in different percentage. It was observed that 2/3 of nodes correlate (i.e. Pearson > 0.8 or
  • SCNP single cell network profiling
  • SCF:pAkt/p-S6 subpopulation was independent from c-Kit (SCF receptor) expression levels on the AML blasts and from patient age, cytogenetics and FLT-3 mutational status.
  • This example shows that longitudinal SCNP analysis can provide unique insights into the nature of AML chemoresistance allowing for the identification of subpopulations of cells present at diagnosis with unique signaling characteristics predictive of higher rates of relapse.
  • BMMC bone marrow mononuclear cells
  • BMMC samples collected at diagnosis from AML patients with a disease response of CR after high dose cytarabine based chemotherapy was used to assess the ability of the identified signaling profiles to predict disease relapse.
  • SCNP assay measured response to growth factors and cytokines involved in hematopoietic progenitor or myeloid biology (SCF, FLT3L, G-CSF, IL-27), drug transporter (ABCG2, MRP-1) and chemokine receptors (CXCR4) associated with adverse disease prognosis in AML, and the c-Kit growth factor receptor for SCF.
  • the SCF and FLT3L- mediated PI3K/Akt and MAPK pathway is important for maintaining the hematopoietic stem cell pool;
  • G- CSF- mediated Jak/STAT pathway activation is important for neutrophilic differentiation of hematopoietic progenitor cells;
  • interleukin (IL)- 27 mediated Jak/STAT pathway activation is important in regulating proliferation and differentiation of hematopoietic stem cells;
  • CXCR4 expression is associated with disease relapse and decreased survival; and drug transporter expression levels (i.e. ABCG2 and MRP-1) are known to be associated with adverse prognosis in AML. All together, approximately 20 signaling nodes were evaluated in each sample.
  • SCNP assays were performed as described previously. Cryopreserved samples were thawed at 37°C and washed once in warm PBS containing 10% FBS (HyClone, Waltham, MA, USA) and 2 mM EDTA. The cells were re-suspended, filtered to remove debris and washed in RPMI 1640 (MediaTech, Manassas, VA, USA) cell culture media containing 1% FBS before staining with Aqua LIVE/DEAD viability dye (Invitrogen, Carlsbad, CA, USA) to distinguish non-viable cells. The cells were re-suspended in RPMI containing 1% FBS, aliquoted to
  • FLT3L fms-like tyrosine kinase receptor-3 ligand
  • G-CSF granulocyte colony-stimulating factor
  • IL-27 interleukin-27
  • SCF stem cell factor
  • FACS buffer containing phosphate buffered saline (PBS, Fisher Scientific, Waltham, MA, USA), 0.5%> bovine serum albumin (BSA, Ankeny, IA, USA), 0.05% NaN3 (Mallinckrodt, Hazelwood, MO, USA), pelleted and stained with cocktails of fluorochrome-conjugated antibodies.
  • BSA bovine serum albumin
  • NaN3 NaN3
  • Antibodies were available from commercial vendors such as BD, Bechman Coulter, Invitrogen and R&D Systems.
  • Flow cytometry data was acquired on an LSR II and/or CANTO II flow cytometer using the FACS DIVA software (BD Biosciences, San Jose, CA). All flow cytometry data were gated using either Flow Jo (TreeStar Software, Ashland, OR), or WinList (Verity House
  • Gating applied to the second data set to assay SCF, FLT3L, G-CSF, and IL-27 responsiveness was defined by the basal state (unstimulated) fluorescence of downstream readouts (e.g. p-Akt, p-S6, STAT3) .
  • This gating was performed on healthy BM samples which were run in each study as controls since absolute values were not comparable between the studies due to differences in experimental configurations (e.g. reagent and cytometer
  • Modulated SCNP was evaluated on longitudinally paired diagnosis and relapse AML samples from two patients with AML. Clinical characteristics of the patients are shown in Table 15. Both patients received high dose cytarabine based induction chemotherapy with disease response of CR followed by relapse within one year. Cytogenetic analysis revealed
  • the data also supports the concept that the cells that give rise to resistance are selected from amongst the diversity of leukemic blasts present at diagnosis, as opposed to induction of cells with new characteristics. This implies that recognition of resistance prone characteristics at diagnosis could be used to select and apply therapies that target these cells mechanistically on an individualized basis at the time of diagnosis. Thus, the results described herein coul be used to prevent chemoresistance from emerging and improve clinical outcome.
  • PI3K/Akt signaling is known to play a fundamental role in opposing apoptosis and has been shown to be associated with resistance to a variety of chemotherapeutic agents, including those used to induce remission in AML and with inferior survival in AML.
  • the prognostic value of the presence of the SCF:p-Akt/p-S6 profile was independent from other known prognostic factors for relapse in AML including age and the presence of FLT3 ITD mutation.
  • the combination of the SCF:p-Akt/p-S6 phenotype with FLT-3 ITD mutational status resulted in higher predictive value for disease relapse than that either marker alone. Further studies are warranted to determine whether these findings, including the significance of this phenotype occurring predominantly in early myeloid (FAB M1-M2) leukemia, hold true in larger independent sample sets.
  • Kit The receptor tyrosine kinase Kit and its ligand SCF are expressed on early hematopoietic cells and are essential for the proliferation and survival of these cells.
  • Kit is expressed on over 70% of pediatric and adult AML and activating mutations of c-Kit are associated with poor outcome in the core binding factor subset of adult AML. While this study did not examine molecular aberrations aside from FLT-3 mutational status, we show that c-Kit expression could not substitute for the poor prognostic SCF:p-Akt/p-S6 phenotype.
  • Example 6 a Exposure of AML blasts in vitro to staurosporine and etoposide reveals three distinct apoptosis profiles
  • Jak/Stat and PI3 kinase pathways are tied to cancer cell survival. For this reason, apoptotic proficiency in AML samples was determined in response to etoposide and
  • the data showed three distinct apoptosis responses of AML blasts after in vitro exposure to staurosporine and etoposide (not shown).
  • the metric used to analyze this data was "Apoptosis" and is a measure of apoptosis and cell death induced by a drug.
  • a viable cell will be Aqua negative and PARP negative and a measure of cell death is PARP and/or Aqua positivity.
  • Apoptosis % of PARP- Aqua- unstim - % of PARP- Aqua- Drug .
  • staurosporine a multi-kinase inhibitor and inducer of apoptosis
  • Staurosporine Resistant profile Samples responsive to staurosporine were then classified by their responses to Etoposide, a topoisomerase 11 inhibitor which identified a second signature in which AML blasts were competent to undergo an apoptotic response to staurosporine but not to etoposide (Etoposide Resistant Profile).
  • the third profile described AML blasts that were competent to undergo apoptosis in response to both agents (Apoptosis Competent Profile).
  • JAK/STAT and PI3K/S6 pathway activity in leukemic samples was analyzed in the context of the apoptotic profiles described above. While some differences in the basal unstimulated levels of phosphorylated STAT proteins were observed between apoptotic signature groups, stimulation with cytokines revealed variable JAK/STAT activity among the apoptosis categories described above.
  • cytokines and chemokines known to stimulate the PI3K/S6 and pathway are G-CSF, GM-CSF, and SDF- ⁇ .
  • G-CSF myeloid cytokines and chemokines known to stimulate the PI3K/S6 and pathway
  • GM-CSF myeloid cytokines and chemokines known to stimulate the PI3K/S6 and pathway
  • SDF- ⁇ myeloid cytokines and chemokines known to stimulate the PI3K/S6 and pathway.
  • G-CSF GM-CSF
  • SDF- ⁇ SDF- ⁇
  • Pathway characterization of AML blasts highlights the different signaling mechanisms utilized to evade apoptosis (for example: sample 8093, NR, "Etoposide resistant”, induced Jak/Stat signaling elevated, sample 0521, NR, “Etoposide Resistant”, induced PI3K/S6 signaling elevated, sample 4353, NR, "Staurosporine Resistant”, induced Jak/Stat and PI3K/S6 pathways elevated c.
  • FLT3-ITD patients had higher basal p-Stat5 and cytokine induced p-Stat5 levels than FLT3-WT patients although a large spread of responses was seen in either FLT3-ITD or FLT3-WT patients. Also, FLT3-ITD patients had lower basal and FLT3L induced p-S6 than FLT3-WT patients. Again a spread of responses was seen within FLT3 WT or FLT3-ITD subgroups demonstrating how single cell network profiling can further characterize samples within a molecularly-defined patient subgroup
  • a 49 year-old individual presents to their primary medical doctor with the chief complaint of fatigue and bruising.
  • a complete blood count reveals increased white blood cells, decreased hemoglobin and hematocrit, low platelets and circulating blasts.
  • a bone marrow aspirate is obtained and flow cytometry reveals an immature myeloid blast population.
  • the patient is diagnosed with acute myeloid leukemia and the physician and patient must determine the best course of therapy.
  • the bone marrow or peripheral blood of the patient might be removed and modulators such as GMCSF or PMA added.
  • Activatable elements such asp- Stat3, p-Stat5 and p-Akt might classify this patient as one of the 25% of patients diagnosed with AML less than 60 years old who will not benefit from cytarabine based induction therapy.
  • This invention may also reveal signaling biology within this patient's blasts population that suggests to the physician that the patient should be treated with a DNA methyl transferase inhibitor. With this invention, the patient would then be spared the toxicities associated with cytarabine therapy and could be placed on a clinical trial where he would receive a therapy from which he would likely benefit.
  • a 52 year-old female presents to her primary medical doctor with the chief complaint of fatigue and bruising.
  • a complete blood count reveals normal numbers of white blood cells, decreased hemoglobin and hematocrit, and low platelets.
  • a bone marrow aspirate and biopsy is obtained and flow cytometry and histology reveals tri-lineage myelodysplasia.
  • the patient is diagnosed with MDS.
  • the bone marrow or peripheral blood of the patient might be removed and modulators such as GMCSF or PMA added.
  • Activatable elements such as STAT3, STAT5 and AKT might reveal that the biology associated with this patient's MDS is likely of auto-immune origin.
  • the physician promptly places this patient on CSA and ATG. Within 6 weeks she shows complete normalization of her complete blood count.
  • This example relates to the publication "Dynamic Single-Cell Network Profiles in Acute Myelogenous Leukemia Are Associated with Patient Response to Standard Induction Therapy". Kornblau SM, Minden MD, Rosen DB, Putta S, Cohen A, Covey T, Spellmeyer DC, Fantl WJ, Gayko U, Cesano A. Clinical Cancer Research. 2010 Jul 15; 16(14): 3721-33 Jan 31. This publication is incorporate herein by reference in its entirety for all purposes.
  • AML acute myeloid leukemia
  • SCNP single cell network profiling
  • Intracellular signaling profiles from two sequential training cohorts of diagnostic non-M3 AML patient samples showed high reproducibility (Pearson correlation coefficients > 0.8).
  • univariate analysis identified multiple "nodes” (modulated readouts of proteins in signaling pathways) relevant to myeloid biology and correlated with disease response to conventional induction therapy (i.e. AUC of ROC >0.66; p ⁇ 0.05).
  • AML Acute Myeloid Leukemia
  • cytogenetic and molecular aberrations resulting in downstream effects on gene expression, protein function and cell signal transduction pathways, ultimately affecting proliferation and cellular differentiation.
  • morphology and cytochemical stains historically have formed the basis for AML classification, and emerging technologies such as gene expression profiling, microRNA profiling, epigenetic profiling and more recently proteomic profiling have been used to elucidate the biologic heterogeneity of AML, and have provided useful insights into the disease biology and its correlation with clinical outcomes.
  • SCNP Single cell network profiling
  • This approach interrogates the physiology of signaling pathways by measuring network properties beyond those detected in resting cells (e.g. failure of a pathway to become activated, hyper/hyposensitivity of the pathway to physiologic stimulators, altered response kinetics and rewiring of canonical pathways), thus revealing otherwise unseen functional heterogeneity in apparently
  • mapping signaling networks When applied to pathways shown to be important in disease pathology, this method of mapping signaling networks has potential applications in the development of predictive/diagnostic tests for therapeutic response and for improved efficiency of drug development. (Irish et al. Cell. 2004; 118:217-228; Irish et al. Nat Rev Cancer. 2006;6: 146-155; Krutzik et al. Nat Methods. 2006;3:361-368; and Sachs et al. Science. 2005;308:523-529.)
  • PBMC peripheral blood mononuclear cell
  • BMMC bone marrow mononuclear cell
  • induction chemotherapy consisted of at least one cycle of standard cytarabine-based induction therapy (i.e. daunorubicin 60 mg/m 2 x 3 days, cytarabine 100-200 mg/m 2 continuous infusion x 7 days); responses were measured after one cycle of induction therapy.
  • cytarabine 200mg/m 2 to 3g/m 2
  • anthracycline daunorubicin or idarubicin
  • an additional anti-metabolite e.g. fludarabine or troxacitabine
  • ⁇ Poor prognosis is defined as having one or more of the following high risk features: age ⁇ 60 years, unfavorable cytogenetics, FLT3 ITD positive or secondary AML [00521] SCNP Assays
  • phosphorylated intracellular signaling molecules phosphorylated intracellular signaling molecules, cell lineage markers, and drug transporters in AML cells. Measurements were taken at basal state and after extracellular modulation with growth factors or cytokines.
  • a pathway "node” (Figure 1) was defined as a combination of specific proteomic readout in the presence or absence of a specific modulator. Up to 147 nodes (including eight surface receptors and transporters) using 27 modulators were assessed in the two studies (Table 17).
  • Each modulator and read-out combination is a node. Unmodulated, basal levels were also measured. In #1, there were 18 basal, 121 modulated, and 8 surface markers for a total node count of 147. In #2, there were 16 basal, 69 modulated, and 5 surface markers for a total node count of 90.
  • Akt indicates protein kinase B; APC, allophyco-cyanin; Ara-C, cytarabine; ATP-binding cassette, subfamily G, member 2; BCL, CD, cluster of differentiation; c-, cleaved-; CCG, cytokine, chemokine, growth factor; C-kit, CD117; CREB, cAMP response element binding; CXCR, CXC chemokine receptor; EPO, erythropoietin; Erk, Extracellular signal-regulated kinase; FITC, fluorescein isothiocyanate; FLT3, fms-like tyrosine kinase; G-CSF, granulocyte colony stimulating factor; GM-CSF, granulocyte macrophage stimulating factor; H 2 O 2 , hydrogen peroxide; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; M-CSF, macrophage colony stimulating factor
  • MFI median fluorescence intensity
  • Basal MFI log 2 (MFI Unmodulated stained ) - log2(MFI Gated unstained (Autofluoresence )) , designed to measure the basal levels of a certain protein under unmodulated conditions.
  • Fold Change MFI log 2 (MFI Modulated stained ) - log2(MFI Unmodulated stained ), a measure of the change in the activation state of a protein under modulated conditions.
  • TotalPhospho MFI log 2 (MFI Modulated stained ) - log 2 (MFI Gated Unstained (Autofluorescence )), & measure of the total levels of ⁇ protein under modulated conditions.
  • Relative Protein Expression log2(MFIstain) - log 2 (MFI control ), a measure of the levels of surface marker staining relative to control antibody staining.
  • Percent Cell Positivity a measure of the frequency of cells that have surface markers staining at an intensity level greater than the 95 th percentile for isotype control antibody staining.
  • Quadrant a measure of the percentage of cells in a flow cytometry quadrant region defined by p-Chk2 and c- PARP i.e. the % of cells that are both p-Chk2- and C-PARP+.

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Abstract

La présente invention concerne une approche pour la détermination des états d'activation d'une pluralité de protéines dans des cellules individuelles. Cette approche permet la détection rapide d'hétérogénéité dans une population cellulaire complexe sur la base d'états d'activation, de marqueurs d'expression et d'autres critères, et l'identification de sous-ensembles cellulaires qui présentent des modifications corrélées d'activation au sein de la population cellulaire. De plus, cette approche permet la corrélation d'activités ou de propriétés cellulaires. En outre, l'utilisation de modulateurs d'activation cellulaire permet une caractérisation de voies et de populations cellulaires.
EP15838003.0A 2014-09-02 2015-09-02 Procédés de diagnostic, de pronostic et procédés de traitement Withdrawn EP3189161A4 (fr)

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