Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-06
2003-08-12
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007230, C435S091100, C536S023100, C536S024300
Reexamination Certificate
active
06605436
ABSTRACT:
The present invention relates to a method for detecting an intrachromosome imbalance in interphase cell nuclei, to an appropriate kit which makes it possible to carry out this method as well as to the applications of this method and of this kit, in particular for the detection of cancer cells in a biological sample and the diagnosis of cancer pathologies.
The cytogenetic study of tumor cells has shown the existence, in these cells, of numerous chromosome rearrangements consisting in particular of losses and gains of genetic material which can lead to the disappearance of chromosome arms or, on the contrary, to the presence of supernumerary chromosome arms and which thus result in modifications of the normal ratio between the number and/or the length of the long and short arms of certain chromosomes which will be designated hereinafter by the term intrachromosome imbalances.
Thus, for example, DUTRILLAUX et al. (
Cancer Genet. Cytogenet.,
1990, 49, 203-217) have demonstrated, from 30 cases, the onset in breast cancers of multiple rearrangements affecting, at a particularly high frequently, chromosomes 1 and 16 and which manifest themselves mainly by a gain of the long arm of chromosome 1 and a loss of the long arm of chromosome 16, and, at a lower frequency, chromosomes 4, 6, 8, 9, 11, 13 and 17. Similarly, TESTA and SIEGFRIED (
Cancer Research,
1992, 52, 2702s-2706s) have also shown, from 30 cases, the presence, in non-small cell lung cancers, of numerous rearrangements preferentially affecting chromosome 7 (mainly in the form of a polysomy 7 or of a gain of all or part of the short arm of this chromosome), but also chromosomes 1, 3, 6, 7, 11, 13, 15, 17 and 19.
Indeed, it is now well established that the process of cancerogenesis is a multistage event requiring successive genetic modifications whose accumulation over time will lead to cells with transforming potential which will be increasingly aggressive, leading to a clinically detectable and possibly metastasizing tumor. Accordingly, a precise definition of the cytogenetic parameters characteristic of tumorigenesis appears to constitute an essential step both in the establishment of the positive diagnosis and in that of the evolutive diagnosis of tumors, thus allowing the use of the most appropriate therapies.
Currently, cytogeneticists have available mainly two approaches in the investigation of the genome and of its abnormalities:
on the one hand, conventional cytogenetics, which makes it possible to have access to the detailed karyotype of a cell and to apprehend the abnormalities in the structure and in the number of all the chromosomes which it contains and which consists, after a period of culture in vitro of cells whose lifespan varies with the type of cells studied, in blocking these cells in the metaphase and in treating the chromosomes according to various methods allowing observation under an optical microscope of bands extending over a few megabases, and it is thus generally designated by the Anglo-Saxon name banding. After photographing, the chromosomes are paired and ordered according to their length, the position of the centromer and the topography of the bands, and this classification results in the establishment of the karyotype.
This approach, of which the major interest is to give an overall “image” of the genome, has a number of limitations. The first relates to the fact that it requires prior culturing of the cells studied, so as to obtain a sufficient number of metaphase cells. However, it is now well established that, in the case of a polyclonal population, the carrying out of a cell culture is likely to introduce substantial bias into the results of a cytogenetic analysis, because of the fact that only the cells adapted to the culture conditions divide and give metaphases. Another limitation lies in the resolving power of conventional cytogenetics which makes it possible to apprehend only partially the often relatively complex chromosome rearrangements in solid tumors, which represent the majority of cancers. Finally, by virtue of its principle, this approach comes up against any possibility of an automated screening of complex and/or multiple chromosome abnormalities.
on the other hand, fluorescence in situ hybridization (FISH), which makes it possible to detect a chromosomal DNA sequence by means of a probe having a specific sequence which is homologous to that which is studied. Based on the complementarity existing between nucleotides (adenine-thymine, adenine-uridine, cytosine-guanine), it consists in hybridizing the target DNA with a probe labeled either directly or indirectly with a fluorochrome, and in using the fluorescence emitted by the latter as hybridization control.
Fluorescence in situ hybridization may be carried out on metaphase chromosomes, in which case it uses probes specific for a chromosome, for an arm, for a chromosomal region, or even for a given locus. The chromosomal abnormalities are revealed by visualization, under a microscope, of the fluorescent sites corresponding to the sites of the target DNA having hybridized with the probes. However, in routine pathological analysis, fluorescence in situ hybridization is carried out on interphase nuclei and it uses, in this case, probes specific for the centromer regions of the chromosomes. The chromosomal abnormalities are revealed by counting of the fluorescent spots either manually under a microscope or in an automated manner by means of an image cytometer, the number of spots being in all cases supposed to correspond to the number of copies of the chromosomes studied.
While the development of the FISH techniques has unquestionably improved the resolving power of cytogenetics (up to the order of a few kilobases), thus making it possible to better define the gains and losses of chromosomal material and the abnormalities in the structure of the chromosomes, these techniques as they currently exist, are not, however, completely satisfactory.
Indeed, as regards FISH on metaphase chromosomes a preliminary step of in vitro culture of the cells is essential, as in the case of conventional cytogenetics, in order to obtain a sufficient number of cells in the metaphase with the abovementioned disadvantages which that involves.
As regards FISH on interphase nuclei, while it has the advantage of avoiding the culturing step and, therefore, the potential selection of certain cells during this culture, it has the disadvantage that manual counting of these spots can only be carried out on a limited number of cells and is responsible for large errors when it is applied to a small population of abnormal cells (KIBBELAAR et al.,
Cytometry,
1993, 14, 716-724), while automated counting remains up until now highly imperfect, in spite of the efforts which have been made in order to improve the quality of the hybridization, the quality of the signals which it generates and the sensitivity of the cameras with which the image cytometers are equipped (TRUONG et al.,
Anal. Cell Path,
1997, 13, 137-146). What is more, it is the case that a number of chromosomal abnormalities do not affect the centromeric regions of the chromosomes, but their arms, and escape any possibility of detection by this technique.
The inventors therefore set themselves the objective of providing an appropriate method which allows the detection of an intrachromosome imbalance in the cells of a biological sample, and which is free, in general, from all the disadvantages of the methods of the prior state of the art.
More specifically, the inventors set themselves the objective of providing a method for detecting an intrachromosome imbalance which, while being sensitive, specific and reproducible so as to ensure the reliability of this detection, can:
on the one hand, be carried out on interphase cells so as to remove the need to subject beforehand the cells which have to be studied to an in vitro culture,
on the other hand, be applied not only to a monoclonal cell population, but also to a polyclonal cell population so as to allow the detection of the possible presence, in this p
Bourgeois Claire
Dutrillaux Bernard
Guilly Marie-Noëlle
Malfoy Bernard
Truong Khuong
Commissariat a l'Energie Atomique
Lu Frank
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Whisenant Ethan
LandOfFree
Method and kit for detecting intrachromosome imbalance in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method and kit for detecting intrachromosome imbalance in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and kit for detecting intrachromosome imbalance in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3117885