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