Method for the urinary detection of bladder cancer

Abstract

The invention relates to the diagnosis of bladder cancer and more specifically to the detection in urine samples of bladder carcinomas of the transitional type. The detection method according to the present invention enables, through the utilization of a DNA chip designed for this purpose, to determine the grade of the detected tumors.

1 . A method for in-vitro detection of bladder cancer in a patient, characterized in that it comprises the steps of:
 (i) extracting the DNA contained in a urine sample taken from said patient;   (ii) fragmenting the DNA extracted in step (i);   (iii) marking the obtained DNA fragments uniformly with a marking agent so as to form a pool of marked DNA;   (iv) forming at least one aliquot from the pool of marked DNA and bringing each aliquot into contact with a set of reference DNAs, said contact being carried out under conditions enabling specific hybridization of the marked DNA fragments with said reference DNAs; said reference DNAs comprising the DNA sequences included in each of the following loci present in the human chromosomes: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22;   (v) eliminating the marked DNA fragments which are not specifically hybridized to the reference DNAs;   (vi) determining the intensity of the signal produced by the marked fragments hybridized to each of the selected reference DNAs;   (vii) determining, for each reference DNA, the deviations between the signals obtained in comparison with those obtained with a control DNA from a healthy patient;   (viii) deriving the patient's cancer stage from said observed deviations for the loci 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22.   
     
     
         2 . A method according to  claim 1 , wherein the reference DNAs are DNA probes of 30 to 100 bases. 
     
     
         3 . A method according to  claim 2 , wherein said DNA probes are fixed on a microarray, on micro beads or on solid particles. 
     
     
         4 . A method according to  claim 1  wherein said reference DNAs comprise several sequences included in each of the following loci of the human chromosome: 1p, 3q, 8q22qter, 20, 5p12-p13, 9p, 9q, 18q12, 1q22-q24, 5p, 6q22, 7, 11q13, 12q15, 13q, 15, 16, 17q, 6q25-q27, 7q, 8p, 10q, 11p, 14q22-qter, 17p, 19 and 22. 
     
     
         5 . A method according to  claim 1 , wherein the reference DNAs are 1,000 to 200,000 bases in length. 
     
     
         6 . A method according to  claim 5 , wherein the reference DNAs consist of BAC clones of the human genome. 
     
     
         7 . A method according to  claim 1 , wherein a first and a second marker are used respectively for marking the urine DNA extracted fragments and the control DNA from healthy patient. 
     
     
         8 . A method according to  claim 7 , wherein the first and second markers are fluorescent markers emitting at different wavelengths. 
     
     
         9 . A method according to  claim 1 , wherein the grade of the tumor cells in step (viii) is determined as a function of the deviations observed with respect to the reference DNAs. 
     
     
         10 . A method according to  claim 1 , wherein the reference DNAs are deposited separately on a microarray, prior to hybridization with the marked DNA fragments. 
     
     
         11 . A method according to  claim 1 , wherein the DNA fragments taken from the patient in step (iii) are divided into two pools, one being marked with a first marker and the second with a second marker, the control DNA of step (vii) being also divided into two pools, one being marked with the first marker and the second with the second marker, a cross-determination of the signal deviation being carried out in step (vii) between each of the thus marked pools. 
     
     
         12 . A method according to  claim 2 , wherein a first and a second marker are used respectively for marking the urine DNA extracted fragments and the control DNA from healthy patient. 
     
     
         13 . A method according to  claim 12 , wherein the first and second markers are fluorescent markers emitting at different wavelengths. 
     
     
         14 . A method according to  claim 2 , wherein the grade of the tumor cells in step (viii) is determined as a function of the deviations observed with respect to the reference DNAs. 
     
     
         15 . A method according to  claim 2 , wherein the reference DNAs are deposited separately on a microarray, prior to hybridization with the marked DNA fragments. 
     
     
         16 . A method according to  claim 2 , wherein the DNA fragments taken from the patient in step (iii) are divided into two pools, one being marked with a first marker and the second with a second marker, the control DNA of step (vii) being also divided into two pools, one being marked with the first marker and the second with the second marker, a cross-determination of the signal deviation being carried out in step (vii) between each of the thus marked pools.