Method and kit for detecting intrachromosome imbalance in interphase nuclei and their applications

Abstract

The invention concerns a method for detecting intrachromosome imbalance in interphase nuclei, characterised in that it consists in: (a) hybridising in situ the chromosome suspected to be affected by said imbalance using two probes marked with different fluorochromes and specific of said chromosome long arm and short arm respectively; (b) measuring the intensity of the fluorescence emitted in said nuclei with different emission wavelengths corresponding to each of the two fluorochromes; (c) calculating the ratio (Ra) between the two measured values of fluorescence intensity; and (d) comparing the resulting ratio value with at least one reference value. The invention also concerns a kit for implementing said method and uses of said kit and said method, in particular for detecting cancer cells in a biological sample and for diagnosing cancer pathologies.

1 . A method for detecting an intrachromosome imbalance in interphase cell nuclei, which is characterized in that it comprises: 
 (a) the in situ hybridization of the chromosome present in said nuclei and suspected of being affected by said imbalance using two probes labeled with two different fluorochromes, the first probe being specific for the long arm of this chromosome while the second probe is specific for the short arm of this same chromosome;    (b) measuring the intensity of the fluorescence emitted in said nuclei at the emission wavelength bands corresponding to each of the two fluorochromes;    (c) calculating the ratio (Ra) between the two fluorescence intensity values thus measured; and    (d) comparing the ratio value thus obtained with at least one reference value.    
     
     
         2 . The method as claimed in  claim 1 , characterized in that the probes used for the in situ hybridization of the chromosome consist of a set of nonrepeated probes covering all or part of the long arm or of the short arm of said chromosome.  
     
     
         3 . The method as claimed in  claim 1 , characterized in that the probes used for the in situ hybridization of the chromosome consist of a set of nonrepeated probes covering the entire long arm or short arm of the target chromosome.  
     
     
         4 . The method as claimed in any one of the preceding claims, characterized in that the labeling of the probes is carried out by coupling each of them to a hapten, and by an affinity reaction between this hapten and a ligand capable of binding specifically to said hapten and which is either conjugated with a fluorochrome, or which has been made fluorescent by reacting with a counterligand conjugated with a fluorochrome.  
     
     
         5 . The method as claimed in  claim 4 , characterized in that one of the probes is coupled to degoxigenin while the other is coupled to biotin.  
     
     
         6 . The method as claimed in any one of the preceding claims, characterized in that the two fluorochromes are chosen from fluorescein isothiocyanate (FITC), rhodamine derivatives such as rhodamine b isothiocyanate and sulforhodamine 101 sulfonate chloride, Cascade blue® or Bopidy® FL.  
     
     
         7 . The method as claimed in any one of the preceding claims, characterized in that one of the two fluorochromes is fluorescein isothiocyanate while the second fluorochrome is sulforhodamine 101 sulfonate chloride.  
     
     
         8 . The method as claimed in any one of the preceding claims, characterized in that it comprises, furthermore, counterstaining of the nuclei with a fluorochrome different from the fluorochromes used for labeling the probes.  
     
     
         9 . The method as claimed in any one of the preceding claims, characterized in that for the calculation of the ratio (R a ) between the two fluorescence intensity values measured in step b) , the mean of the intensity values of this fluorescence as measured for all the nuclei analyzed is determined, for each fluorescence, and the ratio between the two mean values thus obtained is calculated.  
     
     
         10 . The method as claimed in any one of  claims 1  to  8 , characterized in that for calculating the ratio (R a ) between the two fluorescence intensity values measured in step b), the ratio between the intensity values measured for each of the fluorescences is determined for each nucleus analyzed, and the mean of the values of this ratio as obtained for all the nuclei analyzed is calculated.  
     
     
         11 . The method as claimed in any one of the preceding claims, characterized in that the reference value corresponds to the ratio (R t ) between the two fluorescence intensity values measured for the nuclei of control cells treated exactly under the same conditions as the nuclei analyzed.  
     
     
         12 . The method as claimed in  claim 11 , characterized in that the control cells are cells free of any chromosome abnormality.  
     
     
         13 . The method as claimed in any one of the preceding claims, characterized in that it comprises, in addition, a step consisting in determining, for the nuclei analyzed, an imbalance index (I).  
     
     
         14 . The method as claimed in  claim 13 , characterized in that the imbalance index (I) is determined by calculating the ratio between the ratio (R a ) and (R t ) values obtained for all the nuclei analyzed and all the nuclei of the control cells, respectively.  
     
     
         15 . The method as claimed in  claim 13 , characterized in that the imbalance index (I) is determined by calculating, for each nucleus analyzed, the ratio between the ratio (R a ) and (R t ) values, and retaining the value of this ratio corresponding to the largest number of nuclei analyzed.  
     
     
         16 . The method as claimed in any one of the preceding claims, characterized in that steps b), c), d) of this method as well as the step for determining the rearrangement index for the nuclei analyzed, are carried out using an apparatus which comprises: 
 a source of light capable of providing light in three different wavelength bands, it being possible for said source to be either a polychromatic source provided with filters, or a source consisting of a plurality of monochromatic sources,    means of measuring the intensity of the fluorescence emitted by fluorochromes in three different wavelength bands, and    means of treating and analyzing the fluorescence intensities thus measured.    
     
     
         17 . The method as claimed in any one of the preceding claims, characterized in that the intrachromosome imbalance which it is sought to detect results from a duplication of the long arm of human chromosome 1 and/or from a deletion of the short arm of this chromosome.  
     
     
         18 . A kit for implementing the method for detecting an intrachromosome imbalance in interphase intracellular nuclei as claimed in any one of  claims 1  to  17 , which is characterized in that it comprises: 
 an appropriate quantity of a first probe labeled with a first fluorochrome, said probe being specific for the long arm of the chromosome present in said nuclei and suspected of being affected by said imbalance,  
 an appropriate quantity of a second probe labeled with a second fluorochrome, said probe being specific for the short arm of this same chromosome,  
 one or more samples of a cell population free of any chromosome abnormality and, optionally,  
 one or more samples of a cell population consisting partly or completely of cells having the imbalance which it is desired to detect.  
 
     
     
         19 . The kit as claimed in  claim 18 , characterized in that the control cell samples are provided in the form of platings on slides ready to be subjected to an in situ hybridization.  
     
     
         20 . The kit as claimed in  claim 18  or  claim 19 , characterized in that the probes are coupled to a hapten, and the kit comprises, for each probe, an appropriate quantity of a ligand directed specifically against this hapten and which is conjugated with a fluorochrome or capable of being made fluorescent by a reaction with a counterligand conjugated with a fluorochrome, in which case the kit contains, furthermore, an appropriate quantity of said counterligand.  
     
     
         21 . The kit as claimed in any one of  claims 18  to  20 , characterized in that it comprises in addition: 
 an appropriate quantity of one or more reagents for fixing the cells; and/or  
 an appropriate quantity of a third fluorochrome for allowing counterstaining of the cell nuclei; and/or  
 an appropriate quantity of reagents (RNase, pepsine and the like) and buffer solutions (hybridization buffers, rinsing buffers and the like) for allowing in situ hybridization of the chromosome with the probes.  
 
     
     
         22 . The application of a method for detecting an intrachromosome imbalance in interphase nuclei as claimed in any one of  claims 1  to  17  and/or of a kit for implementing such a method as claimed in any one of  claims 18  to  21 , to the detection of cancer cells in a biological sample.  
     
     
         23 . The application of a method for detecting an intrachromosome imbalance in interphase nuclei as claimed in any one of  claims 1  to  17  and/or of a kit for implementing such a method as claimed in any one of  claims 18  to  21 , to the positive diagnosis and/or to the evolutive diagnosis of cancer pathologies in humans.