| Method for improved diagnosis of dysplasias -> Monitor Keywords |
|
|
 
Method for improved diagnosis of dysplasiasRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidMethod for improved diagnosis of dysplasias description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128599, Method for improved diagnosis of dysplasias. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a National Stage of International Application PCT/EP03/050738, filed Oct. 21, 2003, published May 6, 2004, under PCT Article 21(2) in English; which claims the priority of EP 02024030.5, filed Oct. 28, 2002; and EP 03100584.6, filed Mar. 7, 2003. FIELD OF THE INVENTION [0002] The present invention relates to a method for improved diagnosis of dysplasias based on simultaneous detection of INK4a gene products and at least one marker for cell proliferation. In particular, the present invention provides a method for discriminating dysplastic cells over-expressing INK4a gene products from cells over-expressing INK4a gene products without being dysplastic by detection of a marker suitable for characterising the proliferation properties of the respective cell. The characterisation of the proliferation properties may comprise the detection of a marker or a set of markers characteristic for active cell proliferation and/or a marker or a set of markers characteristic for retarded or ceased cell proliferation. The method presented herein thus enables a specific diagnosis of dysplasias in histological and cytological specimens. [0003] The detection of the over-expression of p16.sup.INK4a in biological samples has proven as a useful marker in the detection of anogenital lesions, such as carcinoma of the uterine cervix (see WO00/01845; Klaes et al., Int. J. Cancer: 92, 276-284 (2001)). The method based on p16.sup.INK4a-specific immuno-chemical staining allows for a sensitive and specific identification of dysplastic cells in tissue sections and in cytological samples. [0004] In immuno-histochemical examinations of tissues, dysplastic and neoplastic cells can be stained using a p16.sup.INK4a specific antibody mediated staining procedure. The histological diagnosis of neoplastic lesions can thus be supported by a staining procedure based on a molecular marker characteristic for transformation of cells in anogenital lesions. The diagnosis, whether or not cells are neoplastic, in these procedures is not solely based on the p16.sup.INK4a specific staining, but does also rely on the histological information. [0005] This is due to the fact that, in about 20-30% of samples, metaplastic cells show some immuno-reactivity with p16.sup.INK4a specific antibodies, and thus are stained in the course of the procedures. Yet the staining pattern of these metaplastic cells differs from the pattern of neoplastic lesions. [0006] Metaplastic cells give rise to a patchy or focal staining pattern, whereas neoplastic lesions give rise to diffuse staining pattern. Moreover, the staining intensities of metaplastic cells are predominantly less than that of neoplastic cells. [0007] The common methods used in screening tests for the early detection of dysplasias and/or neoplasias do not employ histology based tests, but rather rely on cytological testing procedures. Yet especially in cases when there is no histological information available concerning the architecture of tissues, such as, for example, in cytological examinations, testing for p16.sup.INK4a over-expression alone may lead to false positive results. This is due to the fact that those fractions of metaplastic cells expressing p16.sup.INK4a at detectably elevated levels may not be differentiated by means of a histologic criteria. [0008] The percentage of cells showing over-expression of p16.sup.INK4a increases in the course of emergence of dysplasias. So, in neoplastic or pre-neoplastic stages, when only a restricted population of neoplastic or pre-neoplastic cells is present in samples, the immunoreactivity of p16.sup.INK4a may be weak. This weak immuno-reactivity may be of about the level as the level caused by metaplastic cells. In later stages of dysplasias, the overall immunoreactivity of p16.sup.INK4a is stronger, so neoplastic lesions are easily discernible from metaplasias even in a cytological testing format. This might lead to cases where the presence of metaplastic cells expressing p16.sup.INK4a might be confused with the presence of neoplastic cells, and thus produce a false positive result. [0009] Especially in screening tests, where the detection of early stages of neoplasias is desirable, this condition is quite unpleasant. This is especially true, as the p16.sup.INK4a based diagnosis has proven to be a valuable tool in histological examinations and the application in cytological based screening procedures would be able to enhance these established procedures. [0010] To reduce false positive results in cytological testing formats and thereby further enhance the fidelity of the p16.sup.INK4a mediated diagnosis of anogenital lesions, a method for discriminating the metaplasias from neoplastic and dysplastic lesions would be desirable. A method for the discrimination of metaplasias from neoplastic and pre-neoplastic lesions is provided within the embodiments claimed according to the present invention. [0011] For supporting the discrimination of metaplasias from neoplastic lesions in testing procedures based on the over-expression of p16.sup.INK4a, a marker molecule would be desirable that is expressed in neoplastic and/or pre-neoplastic cells and tissues and which is not expressed simultaneously with INK4a gene products in one single metaplastic cell. [0012] A solution to the problem present in the art is provided by the methods claimed according to this invention. In the course of the experiments leading to the present invention, the inventors have found that a combination of detection of the presence or absence and/or the level of p16.sup.INK4a, in combination with at least one marker for cell proliferation, such as e.g. Ki67, Ki-S2, mcm5 or mcm2, may solve the problem present in the art. [0013] In the art, a couple of documents pertaining to the use of a combination of molecular markers for improved diagnosis of dysplasias is presented. In WO0208764, a method for improved diagnosis of cervical malignancies is disclosed using a combination of an HPV marker and a marker for cell proliferation or viral activity. p16.sup.INK4a is mentioned in the context of this invention as a viral activity marker to be combined with HPV markers. [0014] In EP1217377, a method for automated detection of cervical malignancies is disclosed that is mediated by detection of more than one marker molecules. Some defined marker combinations are named within the document. There is no disclosure in this document relating to the choice of suitable markers for a combination. The purpose of the combination in this application is improved fidelity of the automated analysis of staining patterns in biological cytological specimens. This document mentions combination of p16.sup.INK4a with other tumor markers. [0015] WO02059616 discloses a method for detection of cell-cycle disturbances for improved diagnosis of cervical malignancies. The document discloses that dysplastic cells exhibit disturbances in cell cycle control, and may thus be identified by means of detection of cyclin E type proteins together with post G1 substances in cells. [0016] Jeffrey Keating in "Ki67, Cyclin E, and p16.sup.INK4a Are Complimentary Surrogate Biomarkers for human papilloma Virus-Related Cervical Neoplasia" (American Journal of Surgical Pathology 25(7): 884-891, (2001)) discloses the complementarity of the use of p16.sup.INK4a, Ki67, and Cyclin E in the course of diagnosis of cervical dysplasias. The document refers to the problems of each single marker in the detection of dysplasias, and states that p16.sup.INK4a in combination with Cyclin E may be suitable to overcome the drawbacks of the single marker molecules, especially when cytological specimens are under examination. No disclosure teaching use of p16.sup.INK4a in concert with a marker characteristic of proliferating cells such as Ki67 is given. The document does not teach the combination of p16.sup.INK4a for use in diagnostic methods; moreover, the disclosure pertains to a restricted use of Ki67 in cervical differential diagnosis, and thus, teaches away from the use of this marker in diagnosis of cervical malignancies. [0017] In the art, there is no disclosure teaching the use of a combination of p16.sup.INK4a with a marker characteristic of cell proliferation for use in a diagnostic method for improved discrimination of p16.sup.INK4a positive non-dysplastic cells from p16.sup.INK4a positive dysplasias. WO02059616 does not give a hint as to the use of p16.sup.INK4a in the detection of dysplastic cell proliferation. EP1217377 does not teach a purpose for the combination of tumor markers in the course of the detection other than the automation of the analysis process. There is no disclosure pertaining to the advantage of combinations for specific discrimination purposes such as discrimination of dysplastic cells from e.g. metaplastic cells in cervical specimens. SUMMARY OF THE INVENTION [0018] The inventors of the present invention sought to overcome the drawback present in the art that p16.sup.INK4a, over-expressed in various dysplasias, may also be detected in some other non-dysplastic cells. The discrimination between non-dysplastic p16.sup.INK4a positive cells and dysplastic cells over-expressing p16.sup.INK4a may be based on the proliferation characteristics of the respective cells. In normal control cells, p16.sup.INK4a inhibits cdk4, and thus inhibits proliferation. In contrast, in dysplastic cells, this regulation is impaired. Thus, p16.sup.INK4a does not lead--despite its uncommonly high expression level--to an inhibition of the cell proliferation. [0019] The inventors of the present invention found that dysplastic cells may be discriminated from cells exhibiting control cell proliferation by the simultaneous detection of p16.sup.INK4a with a marker characteristic for cell proliferation. Due to the fact that, in normal cells, elevated levels of p16.sup.INK4a inhibit cell proliferation, cells over-expressing p16.sup.INK4a may be classified as being dysplastic provided they are exhibiting the characteristics of active cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS [0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with drawing(s) will be provided by the Office upon request and payment of the necessary fee. [0021] FIG. 1 is an example of fluorescent staining of a histological specimen of the cervix uteri. FIG. 1 shows staining of a severe dysplasia using antibodies directed against p16.sup.INK4a. For experimental details, see Example 1. Immunoreactivity for p16.sup.INK4a renders green fluorescence. Almost all cells of the lesion are diffusely positive stained in the cytoplasm and in the nuclei. Continue reading about Method for improved diagnosis of dysplasias... Full patent description for Method for improved diagnosis of dysplasias Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for improved diagnosis of dysplasias patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Method for improved diagnosis of dysplasias or other areas of interest. ### Previous Patent Application: Method for distinguishing aml subtypes with different gene dosages Next Patent Application: Method of isolation and self-assembly of small protein particles from blood and other biological materials Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Method for improved diagnosis of dysplasias patent info. IP-related news and info Results in 0.16719 seconds Other interesting Feshpatents.com categories: Software: Finance , AI , Databases , Development , Document , Navigation , Error 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
