Methods for improved diagnosis of dysplasias
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. Particularly 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 characterizing the proliferation properties of the respective cell. The characterization 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 for a specific diagnosis of dysplasias in histological and cytological specimens.
1. A method of discriminating cervical pre-neoplastic, neoplastic, and/or dysplastic lesions from metaplastic lesions associated with infection by papilloma virus in a human subject, comprising:
(a) obtaining a cervical tissue sample from a subject;
(b) measuring an expression level of HPV16 in the sample;
(c) measuring expression levels of a p16 INK4a protein marker and a human Ki67 protein marker within single cells in the sample;
(d) determining that the tissue has pre-neoplastic, neoplastic, and/or dysplastic lesions associated with infection by papilloma virus if there is a detectable level of HPV16 in the sample and there is at least one cell in the sample simultaneously expressing the p16 INK4a protein marker and the Ki67 protein marker; and
(e) determining that the tissue has metaplastic lesions associated with infection by papilloma virus if there is a detectable level of HPV16 in the sample and if no cell in the sample co-expresses the p16 INK4a protein marker and the Ki67 protein marker; thereby discriminating between pre-neoplastic, neoplastic, and/or dysplastic lesions from metaplastic lesions.
2. The method of claim 1 , wherein the method is performed in a course of a cytological testing procedure.
3. The method of claim 1 , wherein measuring the expression levels in step (c) comprises using a probe specifically recognizing the p16 INK4a protein marker and a probe specifically recognizing the Ki67 protein marker.
4. The method of claim 3 , wherein each probe is an antibody probe.
5. The method of claim 4 , wherein each antibody probe is recognized by a detectably labeled agent.
6. The method of claim 5 , wherein the detectably labeled agent is labeled with a label selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.
7. The method of claim 3 , wherein measuring the expression levels in step (c) comprises a cytochemical staining procedure rendering either a chromogenic or a fluorescent signal.