Methods and compositions for risk stratification

Abstract

The present invention provides an approach for the simultaneous 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, 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 potentiators of cellular activation allows for characterization of such pathways and cell populations.

1 - 13 . (canceled) 
     
     
         14 . A method for establishing a composite marker profile for a sample derived from an individual suspected having a neoplastic condition, the method comprising the steps of: (a) reacting the biological sample with a collection of binding molecules, wherein the collection of binding molecules contains two or more groups of binding molecules specific for distinct cell population associated markers, wherein marker levels and marker combinations identify normal and neoplastic cell populations internal to the sample, (b) identifying the presence of normal and neoplastic cell populations internal to the sample by detecting the marker levels and marker combinations that identify the normal and neoplastic cell populations internal to the sample, and (c) correlating the marker levels across each of the normal and neoplastic cell populations with the presence of a target protein to establish a composite marker profile for a sample derived from an individual suspected having a neoplastic condition. 
     
     
         15 . The method of  claim 14 , wherein said target protein is an activation-phosphorylated signal transduction protein. 
     
     
         16 . The method of  claim 14 , further comprising a determination of target protein modification. 
     
     
         17 . The method of  claim 14 , wherein the cancer is selected from the group consisting of Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), and Acute Lymphocytic Leukemia (ALL). 
     
     
         18 . The method of  claim 14 , wherein the cell population associated markers comprise cell-surface markers selected from the group consisting of CD3, CD5, CD10, CD11b, CD13, CD15, CD14, CD15, CD16, CD19, CD22, CD23, CD56, CD45, CD33, CD34, CD15, CD16, MPL (myeloperoxidase), CD64, CD79a, CD79b, and CD117 (c-kit receptor). 
     
     
         19 . The method of  claim 18 , wherein the markers further include kappa and lambda immunoglobulin light chains to determine clonality. 
     
     
         20 . The method of  claim 17 , wherein said cancer is B-Cell Chronic Lymphocytic Leukemia (B-CLL). 
     
     
         21 . The method of  claim 20 , wherein said target protein is selected from the group consisting of ZAP-70, Activation Induced C-type Lectin (AICL), Lipoprotein Lipase and IM68532. 
     
     
         22 . The method of  claim 21 , wherein the B-Cell Chronic Lymphocytic Leukemia (B-CLL) is Ig-mutated B-CLL. 
     
     
         23 . The method of  claim 20 , wherein the cell population associated markers are selected from the group consisting of CD3, CD5, CD1 g, CD 23, CD38, CD56, CD 79b, and fmc7. 
     
     
         24 . The method of  claim 23 , wherein the cell population associated markers identify a cell population comprising leukemic B cells. 
     
     
         25 . The method of  claim 24 , wherein the leukemic B cells are ZAP-70 positive. 
     
     
         26 . The method of  claim 24 , wherein one or more of the cell populations comprise ZAP-70 negative cell populations. 
     
     
         27 . The method of  claim 24 , wherein one or more of the cell populations comprise normal B cells. 
     
     
         28 . The method of  claim 26 , wherein the ZAP-70 negative cell population comprises granulocytes. 
     
     
         29 . A method for predicting the clinical course of Ig-unmutated B-cell Chronic Lymphocytic Leukemia (CLL) in an individual, the method comprising the steps of: (a) providing a biological sample derived from the individual; (b) reacting the biological sample with a collection of binding molecules, wherein the collection of binding molecules contains two or more groups of binding molecules specific for distinct cell population associated markers, wherein marker levels and marker combinations identify normal and neoplastic cell populations internal to the sample, (c) identifying the presence of normal and neoplastic cell populations internal to the sample by detecting the marker levels and marker combinations that identify the normal and neoplastic cell populations internal to the sample, (d) correlating the marker levels across each of the normal and neoplastic cell populations with the presence of at least one signal transduction protein to establish a composite marker profile for a sample derived from an individual suspected having B-Cell Chronic Lymphocytic Leukemia (B-CLL), wherein the composite profile represents a relative quantification of levels of said signal transduction protein; and (e) comparing the composite profile to one or more reference composite profiles, wherein the comparison allows for predicting the clinical course of Ig-unmutated B-cell Chronic Lymphocytic Leukemia (CLL). 
     
     
         30 . A method for establishing a composite marker profile for a sample derived from an individual suspected having Chronic Myelogenous Leukemia (CML), the method comprising the steps of: (a) reacting the biological sample with a collection of binding molecules, wherein the collection of binding molecules contains two or more groups of binding molecules specific for distinct cell population associated markers, wherein marker levels and marker combinations identify normal and neoplastic cell populations internal to the sample, (b) identifying the presence of normal and neoplastic cell populations internal to the sample by detecting the marker levels and marker combinations that identify the normal and neoplastic cell populations internal to the sample, and (c) correlating the marker levels across each of the normal and neoplastic cell populations with the presence of at least one target protein selected from the group consisting of activation-phosphorylated signal transduction proteins, proliferation markers, differentiation markers and apoptosis markers to establish a composite marker profile for a sample derived from an individual suspected having Chronic Myelogenous Leukemia (CML). 
     
     
         31 . The method of  claim 30 , wherein the cell population associated markers are selected from the group consisting of CD45, CD34, CD11b, CD13, CD15, CD14, CD33, CD79a, CD79b, CD22, CD10, CD16, Bcr/Abl and TdT. 
     
     
         32 . The method of  claim 30 , the target protein is an activation-phosphorylated signal transduction protein. 
     
     
         33 . The method of  claim 32 , the activation-phosphorylated signal transduction protein is selected from the group consisting of Abl, CRKL, Hck, STAT1, STAT3, STAT5, Akt/PKB, and S6. 
     
     
         34 . The method of  claim 30 , the target protein is a proliferation marker or an apoptosis marker 
     
     
         35 . The method of  claim 34 , wherein the target protein is a proliferation marker is selected from the group consisting of Cyclin D1 and Cyclin A2. 
     
     
         36 . The method of  claim 34 , wherein the target protein is an apoptosis marker selected from the group consisting of Caspase-3 and Bcl-XI. 
     
     
         37 . A method for predicting the clinical course of Chronic Myelogenous Leukemia (CML) in an individual, the method comprising the steps of: (a) providing a biological sample derived from the individual; (b) reacting the biological sample with a collection of binding molecules, wherein the collection of binding molecules contains two or more groups of binding molecules specific for distinct cell population associated markers, wherein marker levels and marker combinations identify normal and neoplastic cell populations internal to the sample, (c) identifying the presence of normal and neoplastic cell populations internal to the sample by detecting the marker levels and marker combinations that identify the normal and neoplastic cell populations internal to the sample, (d) correlating the marker levels across each of the normal and neoplastic cell populations with the presence of at least one target protein selected from the group consisting of activation-phosphorylated signal transduction proteins, proliferation markers and apoptosis markers to establish a composite marker profile for a sample derived from an individual suspected having Chronic Myelogenous Leukemia (CML), and (e) comparing the composite profile to one or more reference composite profiles, wherein the comparison allows for predicting the clinical course of Chronic Myelogenous Leukemia (CML). 
     
     
         38 . A method for establishing a composite marker profile for a sample derived from an individual suspected having Acute Myelogenous Leukemia (AML), the method comprising the steps of: (a) reacting the biological sample with a collection of binding molecules, wherein the collection of binding molecules contains two or more groups of binding molecules specific for distinct cell population associated markers, wherein marker levels and marker combinations identify normal and neoplastic cell populations internal to the sample, (b) identifying the presence of normal and neoplastic cell populations internal to the sample by detecting the marker levels and marker combinations that identify the normal and neoplastic cell populations internal to the sample, and (c) correlating the marker levels across each of the normal and neoplastic cell populations with the presence of at least one target protein selected from the group consisting of activation-phosphorylated signal transduction proteins, proliferation markers and apoptosis markers to establish a composite marker profile for a sample derived from an individual suspected having Acute Myelogenous Leukemia (AML). 
     
     
         39 . The method of  claim 38 , wherein the cell population associated markers are selected from the group consisting of CD45, CD33, CD34, CD11b, CD13, CD14, CD15, CD16, MPL (myeloperoxidase), CD64 and CD117 (c-kit receptor). 
     
     
         40 . The method of  claim 39 , wherein the cell population associated markers identify a cell population comprising leukemic granulocytes or leukemic monocytes. 
     
     
         41 . The method of  claim 38 , wherein the target protein is an activation-phosphorylated signal transduction protein. 
     
     
         42 . The method of  claim 38 , wherein the activation-phosphorylated signal transduction protein is selected from the group consisting of Abl, CRKL, Hck, STAT1, STAT3, STAT5, Akt/PKB, and S6. 
     
     
         43 . The method of  claim 38 , wherein the target protein is a proliferation marker or an apoptosis marker. 
     
     
         44 . The method of  claim 43 , wherein the target protein is a proliferation marker selected from the group consisting of Cyclin D1 and Cyclin A2. 
     
     
         45 . The method of  claim 43 , wherein the target protein is an apoptosis marker selected from the group consisting of Caspase-3 and Bcl-XI. 
     
     
         46 . A method for predicting the clinical course of Acute Myelogenous Leukemia (AML) in an individual, the method comprising the steps of: (b) reacting the biological sample obtained from the individual with a collection of binding molecules, wherein the collection of binding molecules contains two or more groups of binding molecules specific for distinct cell population associated markers, wherein marker levels and marker combinations identify normal and neoplastic cell populations internal to the sample, (c) identifying the presence of normal and neoplastic cell populations internal to the sample by detecting the marker levels and marker combinations that identify the normal and neoplastic cell populations internal to the sample, (d) correlating the marker levels across each of the normal and neoplastic cell populations with the presence of at least one target protein selected from the group consisting of activation-phosphorylated signal transduction proteins, proliferation markers and apoptosis markers to establish a composite marker profile for a sample derived from an individual suspected having Acute Myelogenous Leukemia (AML); and (e) comparing the composite profile to one or more reference composite profiles, wherein the comparison allows for predicting the clinical course of Acute Myelogenous Leukemia (AML). 
     
     
         47 . A kit for establishing a composite marker profile according to  claim 14 , the kit comprising: (a) a collection of binding molecules, wherein the collection of binding molecules contains two or more groups of binding molecules specific for distinct cell population associated markers, wherein marker levels and marker combinations identify normal and neoplastic cell populations internal to the sample, and (b) at least one binding molecule specific for a target protein to establish a composite marker profile for a sample derived from an individual suspected having a neoplastic condition. 
     
     
         48 . The kit of  claim 47 , wherein the neoplastic condition is selected from the group consisting of leukemia, lymphoma and multiple myeloma. 
     
     
         49 . The kit of  claim 48 , wherein the leukemia is selected from the group consisting of Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), and Acute Lymphocytic Leukemia (ALL). 
     
     
         50 . The kit of  claim 47 , wherein the cell population associated markers comprise cell-surface markers. 
     
     
         51 . The method of  claim 50 , wherein the cell-surface markers are selected from the group consisting of CD3, CD5, CD10, CD11b, CD13, CD15, CD14, CD15, CD16, CD19, CD22, CD56, CD45, CD33, CD34, CD15, CD16, MPL (myeloperoxidase), CD64, CD79a, CD79b, and CD117 (c-kit receptor).