Diagnostic and monitorings methods for cancer

Abstract

The invention is directed to diagnostic and monitoring methods (assays) for cancer and kits that may be used in such methods. More particularly, an aspect of the invention relates to the use of activated Stat5 for diagnosing and monitoring breast cancer and predicting the effectiveness of cancer treatment. The invention also relates to the use of screening assays for discovering compounds that effect levels of activated Stat5.

What is claimed is:  
     
         1 . A diagnostic or monitoring method comprising: 
 a) obtaining a sample of breast tissue from an individual in need of diagnosis or monitoring for breast cancer;    b) detecting levels of activated Stat5 antigen in said sample;    c) scoring said sample for activated Stat5 levels; and    d) comparing said scoring to that obtained from a control breast sample to determine the prognosis associated with said breast cancer.    
     
     
         2 . The diagnostic or monitoring method of  claim 1  wherein said breast cancer being diagnosed or monitored is node-negative breast cancer.  
     
     
         3 . A diagnostic or monitoring method comprising: 
 a) obtaining a sample from an individual in need of diagnosis or monitoring for breast cancer;    b) contacting said sample with an antibody or binding probe that detects activated Stat5,    c) detecting or measuring the level of activated Stat5, and    d) comparing the level of activated Stat5 to that obtained from a control breast sample.    
     
     
         4 . The method of  claim 1  or  3  wherein said detecting or measuring is selected from the group of methods consisting of immunoblotting, immunohistochemistry and immunocytochemistry.  
     
     
         5 . The method of  claim 1  or  3  wherein said detecting is done by Fluorescence-Activated Cell Sorting (FACS).  
     
     
         6 . The method of  claim 1  or  3 , wherein said sample is contacted or said activated Stat5 is detected by an antibody to tyrosine-phosphorylated StatS.  
     
     
         7 . A diagnostic or monitoring method comprising: 
 a) obtaining a sample of breast tissue from an individual in need of diagnosing or monitoring for breast cancer,    b) contacting said sample with an antibody that detects activated Stat    c) detecting or measuring the level of antibody binding to said sample; and    d) comparing the level of antibody binding in said sample to binding in a control breast sample.    
     
     
         8 . A methhod for screening compounds comprising: 
 a) obtaining compounds to be screened for use in breast cancer therapy;    b) contacting a cell or tissue sample of interest with said compound; and    c) determining the effect of said compound on the level of activated Stat5 in said cell or tissue sample relative to a control sample.    
     
     
         9 . The method of  claim 8 , wherein said sample is cells or tissue from a breast cancer.  
     
     
         10 . The method of  claim 7  wherein the effect of said compound is determined by the binding of an antibody to activated Stat5 to said sample relative to control cells or tissue.  
     
     
         11 . The method of  claim 10  wherein said activated Stat5 is tyrosine phosphorylated Stat5.  
     
     
         12 . A method for determining the effect of antiestrogen treatment comprising: 
 a) obtaining a cell or tissue sample from an individual in need of antiestrogen treatment;    b) measuring the levels of activated Stat5 in said cell or tissue sample; and    c) comparing said levels to that of a control breast cancer sample to predict the responsiveness of said breast cancer to antiestrogen treatment.    
     
     
         13 . A method for screening compounds comprising: 
 a) obtaining compounds to be screened for altering Stat5 activation levels;    b) contacting a cell or tissue sample of interest with said compound, and    c) determining the effect of said compound on the level of activated Stat5 in said cell or tissue sample relative to a control sample.    
     
     
         14 . A kit for determining the level of activated Stat5 in a mammalian biological sample wherein said activated Stat5 is an indicator of the prognosis of breast cancer, said kit comprising: 
 a) an antibody or binding probe to activated Stat5; and    b) a reagent useful for detecting the extent of interaction between said antibody or binding probe and activated Stat5;    c) a reagent for antigen retrieval; and    d) positive and negative control samples.    
     
     
         15 . A method for diagnosing a pathological condition or a susceptibility to a pathological condition comprising: 
 a) obtaining a sample from an individual in need of diagnosis for a pathological condition having altered levels of activated Stat5;    b) determining the amount or presence of activated Stat5 in said sample; and    c) diagnosing said pathological condition or a susceptibility to said pathological condition based on the presence or altered level of activated Stat5 relative to a control sample.    
     
     
         16 . The method of any one of claims  1 ,  3 ,  4  or  7  wherein the results are analyzed using univariate or multivariate analysis.  
     
     
         17 . The method of any one of claims  1 ,  3  or  7  wherein said sample is a tissue section sample.  
     
     
         18 . The method of any one of claims  1 ,  3 ,  7  or  12  further comprising analyzing the levels of activated Stat5 in conjunction with additional breast cancer markers.  
     
     
         19 . A diagnostic or monitoring method comprising: 
 a) obtaining a sample of breast tissue from an individual in need of diagnosis or monitoring for breast cancer;    b) treating said sample in a microwave oven or by other heat-based methods of antigen retrieval;    c) detecting levels of activated Stat5 antigen in said sample;    d) scoring said samples for activated Stat5 levels; and    e) comparing said scoring to that obtained from a control breast sample to determine the prognosis associated with said breast cancer.    
     
     
         20 . A diagnostic or monitoring method comprising: 
 a) obtaining a sample of breast tissue from an individual in need of diagnosis or monitoring for breast cancer;    b) treating said sample in a microwave oven or by other heat-based methods of antigen retrieval in an appropriate antigen retrieval solution;    c) detecting levels of activated Stat5 antigen in breast cells or breast tissue of said sample;    d) scoring said samples for activated Stat5 levels; and    e) comparing said scoring to that obtained from a control breast sample to determine the prognosis associated with said breast cancer.    
     
     
         21 . The method of  claim 20 , wherein said appropriate antigen retrieval solution is an aqueous buffer with a pH of about 7-10, e.g. phosphate buffered saline, pH 7.4.  
     
     
         22 . The method of  claim 12  further comprising analysis of the activated Stat5 levels between said sample and control by univariate statistical analysis.  
     
     
         23 . A method for predicting disease-free survival and overall survival in patients with node-negative breast cancer comprising: 
 a) obtaining a sample of breast cancer tissue from an individual with node-negative breast cancer,    b) detecting levels of activated Stat5 antigen in breast cancer cells or breast cancer tissue of said sample;    c) scoring said samples for activated Stat5 levels; and    d) comparing said scoring to that obtained from a control breast sample to determine likelihood of disease-free survival and overall survival associated with said breast cancer.    
     
     
         24 . The method of  claim 23 , wherein said scoring comprises using a scale of 0 to 4, where 0 is negative (no detectable activated Stat5 in cell nuclei), and 4 is high intensity staining in the majority of cell nuclei and wherein a score of 1 to 4 (i.e. a positive score) indicates a better prognosis for disease free and overall survival in patients with node-negative breast cancer.  
     
     
         25 . A method for predicting disease-free survival and overall survival in patients who have not received adjuvant hormone or chemotherapy comprising: 
 a) obtaining a sample of breast tissue from an individual with breast cancer who has not received adjuvant hormone or chemotherapy;    b) detecting levels of activated Stat5 antigen in breast cells or breast tissue of said sample;    c) scoring said sample for activated Stat5 levels; and    d) comparing said scoring to that obtained from a control breast sample to determine the likelihood of disease-free survival and overall survival associated with said breast cancer.    
     
     
         26 . The method of  claim 25 , wherein said scoring comprises using a scale of 0 to 4, where 0 is negative (no detectable activated Stat5 in cell nuclei), and 4 is high intensity staining in the majority of cell nuclei and wherein a score of 1 to 4 (i.e. a positive score) indicates a better prognosis for disease free and overall survival in patients with breast cancer.  
     
     
         27 . A method for treating breast cancer comprising: 
 a) obtaining a sample of breast tissue from a patient in need of treatment of breast cancer;    b) determining the level of activated Stat5 in said breast tissue sample;    c) treating said patient with a therapeutic regime known to improve the prognosis for breast cancer;    d) repeating steps “′a” and “b”   e) adjusting the therapeutic regime known to improve the prognosis for breast cancer;    f) repeating steps a-e as frequently as deemed appropriate.    
     
     
         28 . A method for screening for metastatic potential of breast tumors comprising: 
 a) obtaining a sample of breast tissue from an individual in need of screening for metastatic potential of a breast tumor;    b) reacting an antibody to activated Stat5 with tumor tissue from said patient;    c) detecting the extent of binding of said antibody to said tissue; and    d) correlating the extent of binding of said antibody with metastatic potential.    
     
     
         29 . The method of  claim 28  wherein said tumor is a node-negative breast cancer.  
     
     
         30 . The kit of  claim 14  wherein said antibody is a monoclonal antibody.  
     
     
         31 . The kit of  claim 14  wherein said antibody is a polyclonal antibody.  
     
     
         32 . The kit of  claim 14  further comprising a second indicator antibody linked to an indicator reagent.  
     
     
         33 . The kit of  claim 32  wherein said indicator reagent is selected from the group consisting of fluorescent, calorimetric, immunoperoxidase and isotopic reagents.  
     
     
         34 . A diagnostic or monitoring method comprising: 
 a) obtaining a sample of tissue from an individual in need of diagnosis or monitoring for cancer;    b) detecting levels of activated Stat5 antigen in said sample,    c) scoring said sample for activated Stat5 levels; and    d) comparing said scoring to that obtained from a control tissue sample to determine the prognosis associated with said cancer.    
     
     
         35 . The diagnostic or monitoring method of  claim 34  wherein said cancer is selected from the group consisting of cancer of the ovary, uterus, thyroid, prostate, skin and large bowel.  
     
     
         36 . A kit for determining the level of activated Stat5 in a mammalian biological sample wherein said activated Stat5 is an indicator of the prognosis of cancer, said kit comprising: 
 a) an antibody or binding probe to activated Stat5; and    b) a reagent useful for detecting the extent of interaction between said antibody or binding probe and activated Stat5;    c) a reagent for antigen retrieval; and    d) positive and negative control samples.    
     
     
         37 . The kit of  claim 36 , wherein said cancer is a cancer selected from the group consisting of ovarian cancer, colorectal cancer, bowel cancer, uterine cancer, endometrial cancer, thyroid gland cancer, prostayic cancer and skin cancer.  
     
     
         38 . The method of  claim 21  wherein said antigen-retrieval buffer is 1 mM Tris at pH 10.  
     
     
         39 . A monoclonal antibody, wherein said antibody: 
 a) is generated against the phosphopeptide KAVDG(phospho Y)VPQIK);    b) specifically recognizes tyrosine phosphorylated isoforms of of Stat5, but not unphosphorylated isoforms;    c) does not recognize Stat5 mutants in which the Tyr694 residue has been substituted with phenylalanine; and    d) recognizes phosphorylated Stat5 following an antigen retrieval treatment that does not use a protease.    
     
     
         40 . The kit of  claim 14 , wherein said antibody is a monoclonal antibody, wherein said antibody: 
 e) is generated against the phosphopeptide KAVDG(phospho Y)VPQIK);    f) specifically recognizes tyrosine phosphorylated isoforms of of Stat5, but not unphosphorylated isoforms;    g) does not recognize Stat5 mutants in which the Tyr694 residue has been substituted with phenylalanine; and    h) recognizes phosphorylated Stat5 following an antigen retrieval treatment that does not use a protease.