Determination of responders to chemotherapy

Abstract

The present invention relates to a method of determining whether a biological sample comprising human lung cancer cells is sensitive to a combination of an epidermal growth factor receptor inhibitor and a chemotherapeutic agent by determining the overexpression of a phosphorylated AKT protein and/or a phosphorylated MAPK protein in the biological sample. The invention is also related to methods for deriving a candidate agent or for selecting a composition for inhibiting the progression of lung cancer in a patient wherein a phosphorylated AKT protein and/or a phosphorylated MAPK protein is used.

1 . A method of determining whether a sample comprising human lung cancer is sensitive to a combination of an epidermal growth factor receptor (EGFR) inhibitor and a chemotherapeutic agent, the method comprising determining the overexpression of a phosphorylated AKT protein and/or a phosphorylated MAPK protein in the sample, wherein the overexpression of phosphorylated AKT protein and/or phosphorylated MAPK protein in the sample is determined by
 a) determining the level of expression of phosphorylated AKT protein and/or phosphorylated MAPK protein in the sample:   b) determining the level of expression of phosphorylated AKT protein and/or phosphorylated MAPK protein in a sample comprising human lung cancer that are not sensitive to a combination of a epidermal growth factor inhibitor and a chemotherapeutic agent; and   c) determining the difference of the level of expression of phosphorylated AKT protein and/or phosphorylated MAPK protein determined in step a) and b) thereby determining the overexpression of phosphorylated AKT protein and/or phosphorylated MAPK protein.   whereby the overexpression of the phosphorylated AKT protein and/or the phosphorylated MAPK protein is an indication that the sample comprising human lung cancer is sensitive to a combination of a epidermal growth factor receptor inhibitor and a chemotherapeutic agent.   
     
     
         2 . The method of  claim 1  whereby the difference of the level of expression of phosphorylated AKT protein and/or phosphorylated MAPK protein determined in step a) and b) is at least 10%. 
     
     
         3 . The method of  claim 2  whereby the difference of the level of expression of phosphorylated AKT protein and/or phosphorylated MAPK protein determined in step a) and b) is at least 25%. 
     
     
         4 . The method of  claim 1 , wherein the sample is a primary lung tumor or a metastasis. 
     
     
         5 . The method of  claim 1  wherein the EGFR inhibitor is erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine). 
     
     
         6 . The method according to any of the  claims 1  to  6  wherein the chemotherapeutic agent is selected from the group consisting of gemcitabine or cis-platin. 
     
     
         7 . The method of  claim 1 , wherein the overexpression of the phosphorylated AKT protein or of the phosphorylated MAPK protein is determined using a reagent which specifically binds the phosphorylated protein. 
     
     
         8 . The method of  claim 7 , wherein the reagent which specifically binds the phosphorylated protein is an antibody, antibody derivative, or an antibody fragment. 
     
     
         9 . The method of  claim 1 , wherein the phosphorylated AKT protein is phosphorylated at an amino acid position corresponding to amino acid position 473 of the AKT1 protein or the MAPK protein is phosphorylated at amino acid positions corresponding to amino acid positions 202 and 204 of MAPK1. 
     
     
         10 . The method of  claim 1  wherein the amino acid sequence of the MAPK protein is selected from the group consisting of the amino acid sequence SEQ ID NO: 1 or 2 and the amino acid sequence of the AKT protein is selected from the group consisting of amino acid sequence SEQ ID NO: 6, 7 or 8. 
     
     
         11 . The method of  claim 1  wherein the overexpression of phosphorylated AKT protein and/or phosphorylated MAPK protein is determined by
 a) immunohistochemically staining the sample,   b) assigning a grade selected from the numbers 1, 2, 3 and 4 for the level of expression of the phosphorylated AKT protein and/or the phosphorylated MAPK protein upon visual inspection of the staining of the cells in the sample whereby the highest detectable grade for the level of expression is assigned,   c) determining the percentage of cells with the highest detectable grade in the immunohistochemically stained biological sample,   d) multiplying the assigned grade with the percentage of cells with the highest detectable grade in the immunohistochemically stained biological sample and with the number 100, and   e) determining overexpression of phosphorylated AKT protein and/or phosphorylated MAPK protein in the biological sample when the result of the multiplication in step d) is above 100.   
     
     
         12 . A method of selecting a composition for inhibiting the progression of lung cancer in a patient, the method comprising:
 a) separately exposing aliquots of a sample comprising lung cancer that are sensitive to a combination of an EGFR inhibitor and a chemotherapeutic agent from the patient in the presence of a plurality of test compositions;   b) comparing the level of expression of a phosphorylated AKT protein and/or a phosphorylated MAPK protein in the aliquots of the sample contacted with the test compositions and the level of expression of the phosphorylated AKT protein and/or the phosphorylated MAPK protein in an aliquot of the sample not contacted with the test compositions,   c) selecting one of the test compositions which alters the level of expression of the phosphorylated AKT protein and/or a phosphorylated MAPK protein in the aliquot containing that test composition, relative to the aliquot not contacted with the test composition wherein an at least 10% difference between the level of expression of the phosphorylated AKT protein and/or the phosphorylated MAPK protein in the aliquot of the sample contacted with the test composition and the level of expression of the phosphorylated AKT protein and/or the phosphorylated MAPK protein in the aliquot of the sample not contacted with the test composition is an indication for the selection of the test composition.   
     
     
         13 . A kit comprising an antibody against phosphorylated MAPK and/or phosphorylated AKT protein.