Method of determining a chemotherapeutic regimen based on
The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing TS and/or ERCC1 expression levels in fixed or fixed and paraffin embedded tissues and prognosticate the probable resistance of a patient's tumor to treatment with 5-FU and oxaliplatin-based therapies by examination of the amount of TS and/or ERCC1 mRNA in a patient's tumor cells and comparing it to a predetermined threshold expression level for those genes. More specifically, the invention provides to oligonucleotide primer pairs ERCC1 and TS and methods comprising their use for detecting levels of ERCC1 and TS mRNA, respectively.
1. A method for determining a chemotherapeutic regimen comprising 5-fluorouracil, oxaliplatin, or combination thereof for treating a tumor in a patient comprising:
(a) isolating mRNA from a fixed tumor tissue sample;
(b) subjecting the mRNA to reverse transcriptase-polymerase chain reaction (RT-PCR) amplification using a pair of oligonucleotide primers capable of amplifying a region of the Excision Repair Cross-Complementing (ERCC1) gene, and a pair of oligonucleotide primers capable of amplifying a region of the Thymidylate Synthase (TS) gene, to obtain an ERCC1 and a TS amplified sample;
(c) determining the amount of TS and ERCC1 mRNA in the amplified sample;
(d) comparing the amount of TS and ERCC1 mRNA from step (c) to an amount of mRNA of an internal control gene; and
(e) determining a chemotherapeutic regime comprising 5-flurouracil, oxaliplatin, or combination thereof based on the amount of TS and/or ERCC1 mRNA in the amplified sample and the threshold level for TS and/or ERCC1 gene expression.
2. The method of claim 1 , wherein mRNA isolation is carried out by:
(a) heating the tumor tissue sample in a solution comprising an effective concentration of a chaotropic compound to a temperature in the range of about 50 to about 100° C. for a time period of about 5 to about 120 minutes; and
(b) recovering the mRNA from the chaotropic solution.
3. The method of claim 2 , wherein the temperature range is about 75 to about 100° C.
4. The method claim 1 , wherein:
(a) for amplifying the region of the ERCC1 gene the oligonucleotide primer pair consists of an oligonucleotide having the sequence of SEQ ID NO:1 or a sequence at least 90% identical thereto and an oligonucleotide having the sequence of SEQ ID NO:2 or a sequence at least 90% identical thereto; and
(b) for amplifying the region of the TS gene the oligonucleotide primer pair consists of an oligonucleotide having the sequence of SEQ ID NO:3 or a sequence at least 90% identical thereto and an oligonucleotide having the sequence of SEQ ID NO:4 or a sequence at least 90% identical thereto.
5. The method of claim 4 , wherein for amplifying the region of the TS gene the oligonucleotide primer pair consists of oligonucleotides having the sequences of SEQ ID NO:3 and SEQ ID NO:4, and for amplifying the region of the ERCC1 gene the oligonucleotide primer pair consists of oligonucleotides having the sequences of SEQ ID NO:1 and SEQ ID NO:2.
6. The method of claim 1 , wherein the tumor is a colorectal adenocarcinoma tumor.
7. The method of claim 1 , wherein the internal control gene is the β-actin gene.
8. The method of claim 7 , wherein the threshold level of ERCC1 gene expression is about 4.9 times the expression level of the β-actin gene.
9. The method of claim 7 , wherein the threshold level of TS gene expression is about 7.5 times the expression level of the β-actin gene.