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 determining the activation state of a signal transduction pathway signaling protein in a leukocyte-containing sample comprising:
 a) activating the activatable proteins of at least one signal transduction pathway in the leukocytes of the sample by exposing the leukocyte-containing sample to a pan-kinase activator;   b) preserving the activated sample with a preservative;   c) unmasking intracellular epitopes of the preserved leukocytes in the sample;   d) contacting the unmasked intracellular epitopes of the preserved leukocytes with a plurality of fluorescently labeled capture molecules, said plurality of capture molecules comprising at least two different capture molecules capable of binding to the activated state of at least two different unmasked intracellular epitopes of preserved, activated leukocytes in the sample and at least one control capture molecule, wherein the control capture molecule binds to an epitope on the preserved leukocytes that is unactivated by the pan-kinase activator;   e) detecting fluorescence of the preserved, activated leukocytes captured by the binding of the capture molecules to the activated state of the unmasked intracellular epitopes;   f) detecting fluorescence of the preserved leukocytes captured by the binding of the control capture molecule; and   g) comparing the fluorescence of the detected preserved, activated leukocytes captured by the capture molecules to the fluorescence of the detected preserved leukocytes captured by the control capture molecule.   
     
     
         15 . The method of  claim 14 , further comprising: h) evaluating the compared fluorescence measured in step g) against compared fluorescence measured in an unactivated reference sample. 
     
     
         16 . The method of  claim 15 , wherein the unactivated reference sample is a second aliquot of the sample. 
     
     
         17 . The method of  claim 15 , wherein the unactivated reference sample is a standardized reference sample. 
     
     
         18 . The method of  claim 14 , wherein the activation is performed for about 1 minute to about 10 minutes. 
     
     
         19 . The method of  claim 14 , wherein the activation is performed for at least about 30 minutes. 
     
     
         20 . The method of  claim 15 , wherein the sample is from a patient and the evaluation of the fluorescence indicates that the patient has a signal transduction associated disease or condition when the fluorescence of the activated and unactivated samples are approximately comparable. 
     
     
         21 . The method of  claim 20 , wherein the signal transduction associated disease or condition is inflammation, autoimmune, allergic, fever, sepsis, cancer, diabetes, or heart failure. 
     
     
         22 . The method of  claim 21 , wherein the signal transduction associated disease or condition is sepsis. 
     
     
         23 . The method of  claim 21 , further comprising repeating steps a) to g) with a sample from the patient after the patient has received a therapeutic agent to treat the inflammation, fever, sepsis, cancer, diabetes, or heart failure and monitoring the effectiveness of that therapeutic agent by monitoring for a change in the detected fluorescence between the activated and unactivated samples. 
     
     
         24 . The method of  claim 21 , wherein the sample is from a patient receiving a kinase inhibitor. 
     
     
         25 . The method of  claim 24 , wherein the evaluating of the compared fluorescence indicates that the kinase inhibitor is effective in treating the signal transduction associated disease or condition patient when a change is determined in the detected fluorescence between the activated and unactivated samples. 
     
     
         26 . The method of  claim 15 , wherein the sample has been exposed to a putative kinase inhibitor and the method further comprises ascertaining the effectiveness of the kinase inhibitor when the activated sample does not demonstrate a change in fluorescence of the activatable proteins of the at least one signal transduction pathway. 
     
     
         27 . The method of  claim 26 , wherein the putative kinase inhibitor is a putative inhibitor of ERK or PI3K, further comprising monitoring for the inhibition of ribosomal S6, wherein inhibition of both ERK and PI3K are required for ribosomal S6 inhibition by: i) exposing the sample to a known ERK inhibitor and a putative PI3K inhibitor and monitoring for ribosomal S6 inhibition; or ii) exposing the sample to a known PI3K inhibitor and a putative ERK inhibitor and monitoring for ribosomal S6 inhibition. 
     
     
         28 . The method of  claim 14 , comprising measuring the activity of at least a second signal transduction pathway. 
     
     
         29 . The method of  claim 14 , wherein said intracellular epitopes comprise phosphorylated epitopes. 
     
     
         30 . The method of  claim 14 , wherein said unmasking comprises contacting the fixed cells with an alcohol and a detergent. 
     
     
         31 . The method of  claim 30 , wherein said alcohol is added at a concentration between approximately 25 percent and approximately 90 percent. 
     
     
         32 . The method of  claim 31 , wherein said alcohol is selected from the group consisting of ethanol and methanol. 
     
     
         33 . The method of  claim 14 , wherein said preservative is aldehyde, paraformaldehyde, or formaldehyde. 
     
     
         34 . The method of  claim 19 , wherein said detergent is at a concentration between approximately 0.1 percent and approximately 10 percent. 
     
     
         35 . The method of  claim 34 , wherein said detergent is selected from the group consisting of Triton X-100, Nonidet P-40 (NP-40), and Brij-58. 
     
     
         36 . The method of  claim 14 , wherein said detection is accomplished by cytometry. 
     
     
         37 . The method of  claim 14 , wherein said signal transduction pathway protein is selected from the group consisting of P 13K, ribosomal S6 protein, p44/42 MAP kinase, TYK2, p38 MAP kinase, PKC, PKA, SAPK, ELK, JNK, cJun, RAS, Raf, MEK 1/2, MEK 3/6, MEK 4/7, ZAP-70, LAT, SRC, LCK, ERK 1/2, Rsk 1, PYK2, SYK, PDK1, GSK3, FKHR, AFX, PLCg, PLCy, FAK, CREB, aIII.beta.3, FcsRI, BAD, p70S6K, STAT1, STAT2, STAT3, STATS, STATE, and combinations thereof. 
     
     
         38 . The method of  claim 37 , wherein said signal transduction pathway proteins are P38 and ERK and PI3K or ribosomal S6. 
     
     
         39 . The method of  claim 37 , wherein said signal transduction pathway proteins are P38, ERK, and ribosomal S6. 
     
     
         40 . The method of  claim 37 , wherein the first protein is JNK and the second protein is ribosomal S6. 
     
     
         41 . The method of  claim 14 , wherein said pan-kinase activator is a toll-like receptor 4 (TLR4) activator or lipopolysaccharide (LPS). 
     
     
         42 . The method of  claim 14 , wherein the capture molecule is an antibody or antigen binding fragment thereof. 
     
     
         43 . The method of  claim 42 , wherein said antibody is specific for a phosphorylation state of said signal transduction pathway protein. 
     
     
         44 . The method of  claim 43 , wherein said phosphorylation-state-specific antibody is selected from the group consisting of anti-phospho-p44/42 MAP kinase (Thr202/Tyr204), anti-phospho-TYK2 (Tyr1054/1055), anti-phospho-p38 MAP kinase (Thr180/Tyr182), phospho-PKC-PAN substrate antibody, phospho-PKA-substrate antibody, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-phospho-tyrosine (P-tyr-100), anti-p44/42 MAPK, anti-phospho-MEK1/2 (Ser217/221), anti-phospho-p90RSK (Ser381), anti-p38 MAPK, anti-JNK/SAPK, anti-phospho-Raf1 (Ser259), anti-phosphoElk-1 (Ser383), anti-phospho-CREB (Ser133), anti-phosphoSEK1/MKK4 (Thr261), anti-phospho-Jun (Ser 63), anti-phosphoMKK3/MKK6 (Ser189/207), anti-AKT, anti-phospho FKHR, anti-FKHR, anti-phospho-Gsk3 alp21, anti-pAFX, anti-PARP, anti-BAD, anti-BADser 112, anti-BADser 136, anti-phospho-BADser 155, anti-p27, anti-p21, anti-cFLIP, antiMYC, anti-p53, anti-NFKB, anti-Ikk.alpha., anti-Ikk.beta., anti-phospho-tyrosine, and anti-phospho-threonine. 
     
     
         45 . The method of  claim 14 , wherein said fluorescent label is selected from the group consisting of a chemiluminescent label and FRET label. 
     
     
         46 . The method of  claim 14 , wherein the sample is whole blood. 
     
     
         47 . The method of  claim 14 , wherein the sample comprises the leukocytes isolated from a whole blood sample. 
     
     
         48 . A kit for monitoring the activation state of a signal transduction pathway comprising: a) a pan-kinase activator; and b) at least two different capture molecules that bind at least one signal transduction pathway protein selected from the group consisting of P38, ERK, PI3K, JNK, and ribosomal S6, wherein at least one of the capture molecules binds to either PI3K, JNK, or ribosomal S6. 
     
     
         49 . The kit of  claim 48 , wherein said pan kinase activator is a toll-like receptor 4 activator or LPS. 
     
     
         50 . The kit of  claim 48  wherein at least one of the capture molecules binds to either PI3K, JNK, or P38 and another of the capture molecules binds to ribosomal S6. 
     
     
         51 . A method of detecting the activation state of at least a first and a second activatable protein in single cells, said method comprising the steps of:
 a) providing a population of cells comprising said first and said second activatable proteins, wherein said first and second activatable proteins are distinct proteins that each have at least an activated isoform, and a non-activated isoform;   b) permeabilizing said population of cells;   c) contacting said permeabilized population of cells with at least two distinguishably labeled activation state-specific antibodies,   wherein a first of said at least two distinguishably labeled activation state-specific antibodies is specific for said activated isoform of said first activatable protein; and a second of said at least two distinguishably labeled activation state-specific antibodies is specific for said activated isoform of said second activatable protein; and   d) using flow cytometry to detect binding of said first and said second distinguishably labeled activation state-specific antibodies to their corresponding activated isoform of said first and second activatable proteins in single cells of said population of cells, wherein said binding of said first distinguishably labeled activation state-specific antibody is indicative of the activation state of said first activatable protein, and said binding of said second distinguishably labeled activation state-specific antibody is indicative of the activation state of said second activatable protein.   
     
     
         52 . The method according to any one of  claim 51 , wherein step a) further comprises contacting said population of cells with an agent that induces the activation of at least said first activatable protein. 
     
     
         53 . A method of determining a signaling phenotype of a cell population, said method comprising:
 contacting said cell population with at least two distinct potentiators in separate cultures;   determining signaling node states of a plurality of signaling nodes in one or more cells from each of said separate cultures, wherein at least two of said plurality of signaling nodes belong to the same signaling pathway comprising:
 permeabilizing said one or more cells from each of said separate cultures; 
 contacting said permeabilized cells from each of said separate cultures with at least one detectible state-specific binding element for each of said plurality of signaling nodes, wherein said detectible state-specific binding elements are distinguishably labeled; and 
 detecting the presence or absence of binding of each of said distinguishably labeled state-specific binding elements to said plurality of signaling nodes in each of said permeabilized cells from each of said separate cultures; 
   creating a response panel for said cell population comprising said determined signaling node states; and   determining a signaling phenotype of said cell population based on said response panel.   
     
     
         54 . The method according to  claim 53 , wherein said determining step further comprises contacting said cell population with a detectible cell specific binding element that binds to a subset of cells of interest in said cell population. 
     
     
         55 . The method according to  claim 54 , wherein a signaling phenotype of said subset of cells of interest is determined.