Discovery and validation of cancer biomarkers using a protein analysis methodology to analyze specimens

Abstract

Methods are provided for the analysis, including the serial analysis, of very small samples of tissue. The methods utilize a nanofluidic proteomic immunoassay (NIA) to quantify total and low-abundance protein isoforms in a small amount of lysate. NIA detection accurately measure oncoprotein expression and activation in limited clinical specimens, including isoforms that differ in post-translational modifications, such as phosphorylation, and the like. The NIA detection method combines isoelectric protein focusing and antibody detection in a nanofluidic system.

1 .- 14 . (canceled) 
     
     
         15 . A method for serial monitoring of specific changes in protein isoforms in clinical tumor microbiopsy specimens, the method comprising;
 performing nanofluidic proteomic immunoassay (NIA) on a tumor microbiopsy cellular sample from an individual for simultaneous quantification of multiple isoforms of a single protein;   determining the ratio of isoforms of the protein in said cellular sample;   comparing the ratio of isoforms in said cellular sample with a paired sample from said individual, wherein the paired sample from said individual comprises two samples taken from the same tumor at different time points; and   identifying from the comparison of isoform ratios in said paired samples a determination of specific changes in protein isoforms, as measured by isoelectric focusing.   
     
     
         16 . The method of  claim 15 , wherein the microbiopsy cellular sample is less than 100,000 cells. 
     
     
         17 . The method of  claim 15 , wherein the microbiopsy cellular sample is less than 100 cells. 
     
     
         18 . The method of  claim 15 , wherein the protein is an oncoprotein. 
     
     
         19 . The method of  claim 18 , wherein the isoform differs from alternative isoforms of the same oncoprotein by total level of phosphorylation or percent phosphorylation relative to the paired sample. 
     
     
         20 . The method of  claim 19 , wherein the oncoprotein is ERK2. 
     
     
         21 . The method of  claim 15 , wherein multiple time points from a single tumor in response to treatment are compared. 
     
     
         22 . The method of  claim 15 , wherein the cellular sample was previously frozen. 
     
     
         23 . The method of  claim 15 , wherein said cellular sample comprises blood cells. 
     
     
         24 . The method of  claim 15 , wherein said microbiopsy cellular sample comprises kidney cancer cells. 
     
     
         25 . The method of  claim 15 , wherein said microbiopsy cellular sample comprises head and neck cancer cells. 
     
     
         26 . The method of  claim 15 , wherein the microbiopsy cellular sample is less than 1000 cells. 
     
     
         27 . The method of  claim 15 , wherein two or more oncoproteins are analyzed, and the two or more oncoproteins are selected from pSTAT3, pSTAT5, myc, bcl2, MEK1, MEK2, pJNK, akt isoforms, total ERK1/2, phospho-ERK1/2, unphosphorylated ERK1/2, total ERK1, pERK1, ppERK1, unphosphorylated ERK1, total ERK2, pERK2, ppERK2, unphosphorylated ERK2; and ERK relative ratios for: % phospho-ERK1/2, % unphosphorylated ERK1/2, % phos-pho-ERK1, % unphosphorylated ERK1, % phospho-ERK2, % unphosphorylated ERK2, % pERK1, % ppERK1, % pERK2, % ppERK2, % phospho-MEK1 and % phospho-MEK2. 
     
     
         28 . The method of  claim 15 , wherein said cellular sample is obtained by fine needle aspiration (FNA). 
     
     
         29 . A method for serial monitoring of specific changes in ERK2 protein isoforms in clinical microbiopsy specimens or blood samples to determine response to a tyrosine kinase inhibitor, the method comprising;
 performing nanofluidic proteomic immunoassay (NIA) on a clinical microbiopsy specimen or blood sample from an individual for simultaneous quantification of multiple isoforms of a single protein;   determining the ratio of isoforms of the protein in said clinical microbiopsy specimen or blood sample; and   comparing the ratio of isoforms in said clinical microbiopsy specimen or blood sample with a paired sample from said individual, wherein the paired sample from said individual comprises two samples taken at different time; and   identifying from the comparison of isoform ratios in said paired samples a determination of specific changes in protein isoforms, as measured by isoelectric focusing, wherein the percentage of the single phosphorylated form is down regulated in a clinical microbiopsy specimen or blood sample from a patient that is responsive to the tyrosine kinase inhibitor.   
     
     
         30 . The method of  claim 29 , wherein the tyrosine kinase inhibitor is imatinib. 
     
     
         31 . The method of  claim 29 , wherein the microbiopsy specimens or blood samples were previously frozen. 
     
     
         32 . The method of  claim 29 , wherein the microbiopsy specimens or blood samples comprise less than 100,000 cells. 
     
     
         33 . The method of  claim 29 , wherein the microbiopsy specimens or blood samples comprise less than 100 cells. 
     
     
         34 . The method of  claim 29 , wherein said clinical microbiopsy specimen or blood sample is a clinical CML microbiopsy specimen or blood sample.