Method for improved diagnosis of dysplasias

Abstract

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 preparing a cytological specimen of the cervix uteri for cytological analysis, the method comprising immunoenzymatically staining human p16 INK4a  protein with a first chromogenic dye and immunoenzymatically staining human Ki67 protein with a second chromogenic dye, wherein the first chromogenic dye and the second chromogenic dye are selected such that an individual cell of the stained cytological specimen of the cervix uteri that co-expresses both human p16 INK4a  protein and human Ki67 protein appears as a brown-stained cell with a red-stained nucleus. 
     
     
         2 . The method of  claim 1 ,
 (a) wherein the first chromogenic dye comprises 3,3′-diaminobenzidine (DAB) and immunoenzymatically staining human p16 INK4a  protein comprises:
 (a1) contacting the cytological specimen of the cervix uteri with an anti-human p16 INK4a  monoclonal antibody under conditions sufficient to specifically bind the anti-human p16 INK4a  antibody to human p16 INK4a  protein contained in the cytological specimen of the cervix uteri; 
 (a2) reacting the anti-human p16 INK4a  antibody bound to p16 INK4a  protein in the cytological specimen of the cervix uteri with a first set of reagents sufficient to bind a peroxidase enzyme to the anti-human p16 INK4a  antibody; and 
 (a3) reacting the peroxidase enzyme with a set of agents comprising DAB under conditions sufficient to precipitate the DAB on the cytological specimen of the cervix uteri; and 
   (b) wherein the second chromogenic dye comprises a FastRed chromogen and immunoenzymatically staining human Ki67 protein comprises:
 (b1) contacting the cytological specimen of the cervix uteri with an anti-human Ki67 monoclonal antibody under conditions sufficient to specifically bind the anti-human Ki67 antibody to human Ki67 protein contained in the cytological specimen of the cervix uteri; 
 (b2) reacting the anti-human Ki67 antibody bound to Ki67 protein in the cytological specimen of the cervix uteri with a second set of reagents sufficient to bind an alkaline phosphatase enzyme to the anti-human Ki67 antibody; and 
 (b3) reacting the alkaline phosphatase enzyme bound to the anti-human Ki67 antibody with a set of agents comprising a FastRed substrate chromogen under conditions sufficient to precipitate the FastRed chromogen on the cytological specimen of the cervix uteri. 
   
     
     
         3 . The method of  claim 2 , wherein
 (a1) the anti-human p16 INK4a  antibody is a mouse anti-human p16 INK4a  monoclonal antibody, and   (b1) the anti-human Ki67 antibody is a rabbit anti-human Ki67 monoclonal antibody.   
     
     
         4 . The method of  claim 3 , wherein
 (a1) the first set of reagents further comprises an anti-mouse secondary antibody; and   (b1) the second set of reagents further comprises an anti-rabbit secondary antibody.   
     
     
         5 . The method of  claim 4 , wherein the anti-mouse secondary antibody is linked to the peroxidase enzyme and the anti-rabbit secondary antibody is linked to the alkaline phosphatase enzyme. 
     
     
         6 . The method of  claim 1 , wherein the cytological specimen of the cervix uteri is positive for an infection with a high-risk human papillomavirus (HR-HPV) selected from the group consisting of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV 58, HPV 59, HPV 66, and HPV68. 
     
     
         7 . A method comprising of staining a cytological specimen of the cervix uteri, the method comprising:
 (a) obtaining a microscope slide having deposited thereon the cytological specimen of the cervix uteri;   (b) immunoenzymatically staining the cytological specimen of the cervix uteri deposited on the microscope slide for human p16 INK4a  protein with an anti-human p16 INK4a  antibody and a set of agents that produce a first chromogenic reporter signal; and   (c) immunoenzymatically staining the cytological specimen of the cervix uteri deposited on the microscope slide for human Ki67 protein with an anti-human   Ki67 antibody and a set of agents that produce a second chromogenic reporter signal, wherein the first chromogenic reporter signal and the second chromogenic reporter signal are distinguishable.   
     
     
         8 . The method of  claim 7 , wherein the first chromogenic reporter signal is generated by a brown chromogenic dye and the second chromogenic reporter signal is generated by a red chromogenic dye. 
     
     
         9 . The method of  claim 7 , wherein the set of agents that produce the first chromogenic reporter signal comprise 3,3′-diaminobenzidine (DAB), and wherein the set of agents that produce the second chromogenic reporter signal comprise a FastRed chromogen substrate. 
     
     
         10 . The method of  claim 7 , wherein:
 (b) immunoenzymatically staining the cytological specimen of the cervix uteri deposited on the microscope slide for human p16 INK4a  comprises:
 (b1) contacting the cytological specimen of the cervix uteri with the anti-human p16 INK4a  monoclonal antibody under conditions sufficient to enable specific binding between the anti-human p16 INK4a  antibody and human p16 INK4a  protein in the cytological specimen of the cervix uteri; 
 (b2) reacting the anti-human p16 INK4a  antibody bound to the cytological specimen of the cervix uteri with the set of agents that produce the first chromogenic reporter signal, wherein the set agents that produce the first chromogenic reporter signal comprises a peroxidase enzyme and 3,3′-diaminobenzidine (DAB), wherein:
 (b2a) the anti-human p16 INK4a  antibody is reacted with the peroxidase enzyme under conditions sufficient to bind the peroxidase enzyme to the anti-human p16 INK4a  antibody; and 
 (b2b) the peroxidase enzyme bound to the anti-human p16 INK4a  antibody is reacted with DAB under conditions sufficient to cause DAB to precipitate onto the cytological specimen of the cervix uteri; and 
 
   (c) immunoenzymatically staining the cytological specimen of the cervix uteri deposited on the microscope slide for human Ki67 protein with the FastRed chromogen comprises:
 (c1) contacting the cytological specimen of the cervix uteri with the anti-human Ki67 monoclonal antibody under conditions sufficient to enable specific binding between the anti-human Ki67 antibody and human Ki67 protein in the cytological specimen of the cervix uteri; 
 (c2) reacting the anti-human Ki67 antibody bound to the cytological specimen of the cervix uteri with the set of agents that produce the second chromogenic reporter signal, wherein the set agents that produce the second chromogenic reporter signal comprises a peroxidase enzyme and a FastRed chromogen substrate, wherein:
 (c2a) the anti-human Ki67 antibody is reacted with the peroxidase enzyme under conditions sufficient to bind the peroxidase enzyme to the anti-human Ki67 antibody; and 
 (c2b) the peroxidase enzyme bound to the anti-human Ki67 antibody is reacted with the FastRed chromogen substrate under conditions sufficient to cause a FastRed chromogen to precipitate onto the cytological specimen of the cervix uteri. 
 
   
     
     
         11 . The method of  claim 10 , wherein
 (b1) the anti-human p16 INK4a  antibody is a mouse anti-human p16 INK4a  monoclonal antibody, and   (c1) the anti-human Ki67 antibody is a rabbit anti-human Ki67 monoclonal antibody.   
     
     
         12 . The method of  claim 11 ,
 (b1) wherein the set of agents that produce the first chromogenic reporter signal further comprises an anti-mouse secondary antibody conjugated to the peroxidase enzyme; and   (c1) wherein the set of agents that produce the second chromogenic reporter signal further comprises an anti-rabbit secondary antibody.   
     
     
         13 . The method of  claim 7 , wherein the cytological specimen of the cervix uteri is positive for an infection with a high-risk human papillomavirus (HR-HPV). 
     
     
         14 . The method of  claim 13 , wherein the HR-HPV is selected from the group consisting of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV 58, HPV 59, HPV 66, and HPV68. 
     
     
         15 . The method of  claim 13 , wherein the cytological specimen of the cervix uteri is determined to be positive for an infection with the HR-HPV by a method employing a nucleic acid amplification reaction. 
     
     
         16 . A method of evaluating a cytological specimen of the cervix uteri infected with a high-risk human papillomavirus (HR-HPV) for the presence of dysplastic cells, the method comprising:
 (a) immunoenzymatically staining human p16 INK4a  protein with a first chromogenic dye and immunoenzymatically staining human Ki67 protein with a second chromogenic dye, wherein the first chromogenic dye and the second chromogenic dye are selected such that an individual cell of the stained cytological specimen of the cervix uteri that co-expresses both human p16 INK4a  protein and human Ki67 protein appears as a brown-stained cell with a red-stained nucleus, and   (b) detecting individual brown-stained cells with a red stained nucleus in the immunoenzymatically-stained cytological specimen of the cervix uteri, wherein the presence of at least one individual brown stained cell with a red stained nucleus is indicative of a dysplastic cell.   
     
     
         17 . The method of  claim 16 , wherein
 (a1) the first chromogenic dye comprises 3,3′-diaminobenzidine (DAB) and immunoenzymatically staining human p16 INK4a  protein comprises:
 (a1a) contacting the cytological specimen of the cervix uteri with an anti-human p16 INK4a  monoclonal antibody under conditions sufficient to specifically bind the anti-human p16 INK4a  antibody to human p16 INK4a  protein contained in the cytological specimen of the cervix uteri; 
 (a1b) reacting the anti-human p16 INK4a  antibody bound to p16 INK4a  protein in the cytological specimen of the cervix uteri with a first set of reagents sufficient to bind a peroxidase enzyme to the anti-human p16 INK4a  antibody; and 
 (a1c) reacting the peroxidase enzyme with a set of agents comprising DAB under conditions sufficient to precipitate the DAB on the cytological specimen of the cervix uteri; and 
   (a2) the second chromogenic dye comprises a FastRed chromogen and immunoenzymatically staining human Ki67 protein comprises:
 (a2a) contacting the cytological specimen of the cervix uteri with an anti-human Ki67 monoclonal antibody under conditions sufficient to specifically bind the anti-human Ki67 antibody to human Ki67 protein contained in the cytological specimen of the cervix uteri; 
 (a2b) reacting the anti-human Ki67 antibody bound to Ki67 protein in the cytological specimen of the cervix uteri with a second set of reagents sufficient to bind an alkaline phosphatase enzyme to the anti-human Ki67 antibody; and 
 (a2c) reacting the alkaline phosphatase enzyme bound to the anti-human Ki67 antibody with a set of agents comprising a FastRed substrate chromogen under conditions sufficient to precipitate the FastRed chromogen on the cytological specimen of the cervix uteri. 
   
     
     
         18 . The method of  claim 17 , wherein
 (a1a) the anti-human p16 INK4a  antibody is a mouse anti-human p16 INK4a  monoclonal antibody;   (a1b) the first set of reagents further comprises an anti-mouse secondary antibody;   (a2a) the anti-human Ki67 antibody is a rabbit anti-human Ki67 monoclonal antibody; and   (a2b) the second set of reagents further comprises an anti-rabbit secondary antibody.   
     
     
         19 . The method of  claim 18 , wherein the anti-mouse secondary antibody is linked to the peroxidase enzyme and the anti-rabbit secondary antibody is linked to the alkaline phosphatase enzyme. 
     
     
         20 . The method of  claim 16 , wherein the cytological specimen of the cervix uteri is positive for an infection with a high-risk human papillomavirus (HR-HPV) selected from the group consisting of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV 58, HPV 59, HPV 66, and HPV68.