Method of determining a chemotherapeutic regimen based on ERCCI expression

A gene expression and protocol technology, applied in the medical field, can solve problems such as failure to obtain

Inactive Publication Date: 2005-07-06
RESPONSE GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, methods for quantifying ERCC1 expression in fixed and paraffin-embedded (FPE) tissues have not been successfully obtained in the art

Method used

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  • Method of determining a chemotherapeutic regimen based on ERCCI expression
  • Method of determining a chemotherapeutic regimen based on ERCCI expression
  • Method of determining a chemotherapeutic regimen based on ERCCI expression

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Experimental program
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Effect test

Embodiment 4

[0034] The "predetermined threshold level" is further defined as the corrected relative ERCC1 expression level of the tumor. Above this level, patients receiving platinum-based chemotherapy may have a low survival rate. When the corrected relative ERCC1 expression level of the tumor in patients receiving platinum-based chemotherapy regimens is lower than this domain level, it is associated with high patient survival rates. The domain correction expressed as the ratio of ERCC1:β-actin relative to ERCC1 expression is about 6.7×10 -3 . Figure 1, see Example 4. However, the present invention is not limited to the use of β-actin as an internal control.

[0035] When performing the method of this embodiment of the present invention, it is preferable to isolate the patient's tumor cells. Solid or lymphoma or part of it is removed from the patient by surgery or obtained by routine biopsy. RNA isolated from frozen or fresh tumor samples is extracted from cells by any typical method in the ...

Embodiment 1

[0076] Isolate RNA from FPE tissue

[0077] RNA is extracted from paraffin-embedded tissue through the following general process.

[0078] A. Deparaffinization and hydration of sections

[0079] (1) Put a part of the about 10 μM slice in a 1.5 mL plastic centrifuge tube.

[0080] (2) Add 600 μL of xylene, and shake the mixture vigorously at room temperature (about 20-25° C.) for about 10 minutes.

[0081] (3) At room temperature, centrifuge the sample for about 7 minutes at the maximum speed of the bench top centrifuge (about 10-20,000xg).

[0082] (4) Repeat steps 2 and 3 until most of the paraffin is dissolved. Depending on the amount of paraffin contained in the original sample part, it usually needs to be repeated 2 or more times.

[0083] (5) Use lower alcohol, preferably 100% ethanol (about 600 μL) to vigorously shake for about 3 minutes to remove the xylene solution.

[0084] (6) Centrifuge the test tube for about 7 minutes according to step (3). Pour out and discard the su...

Embodiment 2

[0097] mRNA reverse transcription and PCR

[0098]Reverse transcription: as exemplified in Example 1 and described in US Patent Application US09 / 469,338 filed on December 20, 1999 (incorporated here as a reference in its entirety), RNA is from microdissection or non-microdissection Isolated from formalin-fixed paraffin-embedded (FPE) tissue. After ethanol precipitation and centrifugation, the RNA pellets were dissolved in 50 μL of 5 mM Tris / Cl pH 8.0. M-MLV reverse transcriptase can extend an oligonucleotide primer that hybridizes to a single-stranded RNA or DNA template in the presence of deoxynucleotides to produce a complementary strand. The resulting RNA was reverse transcribed using M-MLV reverse transcriptase from Life Technologies and random hexamers. Reverse transcription is performed by mixing 25 μL of RNA solution with 25.5 μL of "Reverse Transcription Mix" (see below). Place the reactants in a thermal cycler, at 26°C for 8 minutes (for binding random hexamers to RNA), a...

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Abstract

The present invention relates to predictive methods for use in medicine, in particular cancer chemotherapy. The purpose of the present invention is to provide a method for evaluating the expression level of ERCC1 in fixed or fixed and paraffin-embedded tissues by detecting the amount of ERCC1 mRNA in tumor cells of a patient and comparing it with a predetermined threshold expression level, and determining the platinum-based Methods of chemotherapy. More particularly, the present invention provides oligonucleotide primer pairs for ERCC1, and methods for detecting ERCC1 mRNA levels using them.

Description

Invention field [0001] The present invention relates to a predictive method that is effectively used in medicine, especially cancer chemotherapy. More particularly, the present invention relates to assessing the gene expression of tumor cells in a patient. By detecting mRNA expressed by genes involved in DNA repair in humans, the survival of patients treated with chemotherapeutic agents that target DNA, especially agents that destroy DNA in the form of platinum preparations, is determined. Background of the invention [0002] Cancer occurs when normal cells undergo oncogenic transformation and become malignant cells. Transformed (malignant) cells escape the normal physiological controls that can dictate cell phenotype and inhibit cell proliferation. The transformed cells in the individual's body thus proliferate and form tumors. When a tumor is found, the clinical goal is to selectively destroy malignant cells while reducing any damage to normal cells during the treatment of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7068G01N33/50A61K33/24A61P35/00C12N15/09C12QC12Q1/68G01N33/574
CPCC12Q1/6886C12Q2600/158A61P35/00C12Q1/68
Inventor K·D·达南伯格
Owner RESPONSE GENETICS
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