Methods for diagnosis, prognosis and methods of treatment

Abstract

The present invention provides an approach for the determination of the activation states of a plurality of proteins in single cells. This approach permits the rapid detection of heterogeneity in a complex cell population based on activation states, expression markers and other criteria, and the identification of cellular subsets that exhibit correlated changes in activation within the cell population. Moreover, this approach allows the correlation of cellular activities or properties. In addition, the use of modulators of cellular activation allows for characterization of pathways and cell populations. Several exemplary diseases that can be analyzed using the invention include AML, MDS, and MPN.

1 .- 38 . (canceled) 
     
     
         39 . A method of treating a patient suffering from AML comprising
 (i) determining that the patient will likely respond to treatment, based at least in part on the results of an assay comprising
 (a) contacting a population of cells from a sample from the patient to modulator selected from the group consisting of staurosporine, etoposide, mylotarg, daunorubicin and araC, and combinations thereof; 
 (b) determining, on a single cell basis, the level of an activated form of an activatable element in the apoptosis pathway in the population of cells, wherein the activatable element is selected from the group consisting of Bcl-2, Mcl-1, PARP, Caspse 3, Caspase 7 and Caspase 8, and combinations thereof and 
   (ii) treating the patient.   
     
     
         40 . The method of  claim 39  further comprising comparing the level of the activated form of the activatable element with a threshold level. 
     
     
         41 . The method of  claim 39  wherein the patient is 60 years old or older. 
     
     
         42 . The method of  claim 39  wherein the treatment comprises a treatment selected from the group consisting of 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, and combinations thereof. 
     
     
         43 . The method of  claim 42  wherein the treatment comprises induction therapy. 
     
     
         44 . The method of  claim 39  wherein the sample comprise a peripheral blood mononuclear cell (PBMC) sample or a bone marrow mononuclear cell (BMMC) sample. 
     
     
         45 . The method of  claim 39  wherein the determining of the level of the activated form of the activatable element comprises permeabilizing the cell, contacting the cell with a detectable binding element specific to the activated form of the activatable element, and detecting the binding element by flow cytometry or mass cytometry. 
     
     
         46 . The method of  claim 39  wherein the population of cells is a leukemic blast cell population. 
     
     
         47 . The method of  claim 46  wherein the leukemic blast population is determined by gating using CD34, CD11b, or a combination thereof. 
     
     
         48 . The method of  claim 39  wherein the activatable element in the apoptosis pathway comprises PARP. 
     
     
         49 . A method of treating a patient suffering from AML with a cytarabine-based induction therapy comprising
 (i) determining that the patient will likely respond to the cytarabine-based induction therapy, based at least in part on the results of an assay comprising
 (a) contacting a population of cells from a sample from the patient to modulator or combination of modulators that slows or stops the growth of cells and/or induces apoptosis in cells; 
 (b) determining, on a single cell basis, the level of an activated form of an activatable element in the apoptosis pathway in the population of cells and 
   (ii) treating the patient, wherein the treatment comprises a cytarabine-based induction therapy.   
     
     
         50 . The method of  claim 49  further comprising comparing the level of the activated form of the activatable element with a threshold level. 
     
     
         51 . The method of  claim 49  wherein the patient is 60 years old or older. 
     
     
         52 . The method of  claim 49  wherein the sample comprise a peripheral blood mononuclear cell (PBMC) sample or a bone marrow mononuclear cell (BMMC) sample. 
     
     
         53 . The method of  claim 49  wherein the determining of the level of the activated form of the activatable element comprises permeabilizing the cell, contacting the cell with a detectable binding element specific to the activated form of the activatable element, and detecting the binding element by flow cytometry or mass cytometry. 
     
     
         54 . The method of  claim 49  wherein the population of cells is a leukemic blast cell population. 
     
     
         55 . The method of  claim 54  wherein the leukemic blast population is determined by gating using CD34, CD11b, or a combination thereof. 
     
     
         56 . The method of  claim 49  wherein the activatable element in the apoptosis pathway comprises PARP. 
     
     
         57 . A method of diagnosing, prognosing, determining progression, predicting a response to a treatment or choosing a treatment in an individual suffering from AML, said method comprising:
 (i) classifying one or more hematopoietic cells in said individual by a method comprising:
 a) subjecting a cell population comprising said one or more hematopoietic cells from said individual to: no modulator, Daunorubicin, AraC, Etoposide Staurosporine, ZVAD or a combination thereof; 
 b) determining an activation level of a marker of apoptosis in one or more cells from said individual, wherein said determining is performed on single cells by a method comprising
 (1) permeabilizing the cell; 
 (2) contacting cell with activation state-specific binding agent; 
 (3) determining the level of the marker of apoptosis by flow cytometry or mass spectroscopy; and 
 
 c) classifying said one or more hematopoietic cells based on said activation levels of the marker of apoptosis; and 
   (ii) making a decision regarding a diagnosis, prognosis, progression, response to a treatment or a selection of treatment in said individual based on said classification of said one or more hematopoietic cells.   
     
     
         58 . A kit for diagnosing, prognosing, determining progression, predicting a response to a treatment or choosing a treatment in an individual suffering from AML, wherein the kit comprises
 (i) at least two modulators selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, AraC, G-CSF, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, FLT3L, SCF, G-CSF, SCF, G-CSF, SDF1a, LPS, PMA, Thapsigargin and H2O2;   (ii) at least one binding element specific to a particular activation state of an activatable element selected from the group consisting of p-Slp-76, p-Plcg2, p-Stat3, p-Stat5, p-Stat1, p-Stat6, P-Creb, Parp+, Chk2, Rel-A (p65-NFKB), p-AKT, p-S6, p-ERK, Cleaved Caspase 8, Cytoplasmic Cytochrome C, and p38; and   (iii) instructions for diagnosis of AML, prognosis of AML, determining AML progression and/or predicting response to a treatment for AML in an individual.   
     
     
         59 . The kit of  claim 58  further comprising
 (iv) a binding element for a cell surface marker selected from the group consisting of CD11b, CD33, CD34, CD45, and combinations thereof. 
 
     
     
         60 . The kit of  claim 59  wherein the cell surface marker comprises CD34.