Method of Determining Chemotherapy of Metastatic Tumor by Assaying DPD Gene Expression in Primary Tumors

Abstract

The invention relates to a method for determining a chemotherapeutic regimen for an individual, comprising obtaining a mRNA sample from a primary tumor specimen; determining a gene expression level for a tumor gene determinant in the specimen; comparing the gene expression level for the tumor gene determinant with a predetermined threshold value for that gene; and providing a chemotherapeutic regimen comprising a chemotherapeutic agent appropriate for the tumor gene determinant to treat the tumor metastases.

1 - 16 . (canceled) 
     
     
         17 . A method for determining a chemotherapeutic regimen for treating a metastatic tumor in an individual having a primary and metastatic tumor comprising:
 (a) obtaining a primary tumor specimen;   (b) determining a level of gene expression of Dihydropyrimidine Dehydrogenase (DPD) in a primary tumor comprising
 (1) determining the level of DPD mRNA in the specimen; 
 (2) comparing said level of DPD mRNA to a level of mRNA of an internal control gene; and 
   (c) determining a chemotherapeutic regimen for treating the metastatic tumor in the individual based on the level of DPD mRNA in the primary tumor specimen and a predetermined threshold level for the DPD mRNA.   
     
     
         18 . The method of  claim 17 , wherein the primary tumor specimen is fixed; and
 wherein the level of DPD mRNA in the specimen in step (b)(1) is determined by a process comprising:   (1) isolating DPD mRNA from the fixed tumor specimen by heating in the presence of a solution comprising an effective concentration of a chaotropic agent at a temperature in the range of about 50° C. to about 100° C. and recovering the mRNA from the chaotropic solution;   (2) subjecting the mRNA to amplification using a pair of oligonucleotide primers capable of amplifying a region of DPD to obtain an amplified sample; and   (3) determining the quantity of DPD mRNA in the amplified sample.   
     
     
         19 . The method of  claim 18 , wherein the fixed primary tumor specimen is a fixed, paraffin-embedded (FPE) primary tumor specimen, and said method further comprises deparaffinizing the FPE primary tumor specimen to obtain a deparaffinized sample before step (b), and wherein step (b) is performed by determining a level of gene expression for DPD in the deparaffinized sample comprising
 (1) isolating DPD mRNA from the deparaffinized sample by heating in the presence of a solution comprising an effective concentration of a chaotropic agent at a temperature in the range of about 50° C. to about 100° C. and recovering the mRNA from the chaotropic solution;   (2) subjecting the mRNA to amplification using a pair of oligonucleotide primers capable of amplifying a region of DPD to obtain an amplified sample; and   (3) determining the quantity of DPD mRNA in the amplified sample.   
     
     
         20 . The method of  claim 18 , wherein the primer pair consists of a first oligonucleotide having the sequence set forth in SEQ ID: 4 or a sequence at least 90% identical thereto and a second oligonucleotide having the sequence set forth in SEQ ID NO: 5 or a sequence at least 90% identical thereto. 
     
     
         21 . The method of  claim 19 , wherein the primer pair consists of a first oligonucleotide having the sequence set forth in SEQ ID: 4 or a sequence at least 90% identical thereto and a second oligonucleotide having the sequence set forth in SEQ ID NO: 5 or a sequence at least 90% identical thereto. 
     
     
         22 . The method of  claim 18 , wherein the heating occurs at a temperature in the range of about 75° C. to about 100° C. for a period of about 5 to about 120 minutes. 
     
     
         23 . The method of  claim 19 , wherein the heating occurs at a temperature in the range of about 75° C. to about 100° C. for a period of about 5 to about 120 minutes. 
     
     
         24 . The method of  claim 17 , wherein determining the expression level of DPD comprises a fluorescence based real-time detection method. 
     
     
         25 . The method of  claim 18 , wherein determining the expression level of DPD comprises a fluorescence based real-time detection method. 
     
     
         26 . The method of  claim 19 , wherein determining the expression level of DPD comprises a fluorescence based real-time detection method.