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 comprising:
 reacting a formalin fixed paraffin embedded human specimen with a pair of primary antibodies consisting of an anti-human p16 INK4a  monoclonal antibody and an anti-human Ki-67 monoclonal antibody under conditions sufficient to specifically bind (i) the anti-human p16 INK4a  monoclonal antibody to human p16 INK4a  protein included in the formalin fixed paraffin embedded human specimen, and (ii) the anti-human Ki-67 monoclonal antibody to human Ki-67 protein included in the formalin fixed paraffin embedded human specimen;   reacting the anti-human p16 INK4a  monoclonal antibody bound to the formalin fixed paraffin embedded human specimen with a secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody, wherein the secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody is associated with a peroxidase enzyme;   reacting the formalin fixed paraffin embedded human specimen with a substrate for the peroxidase enzyme;   reacting the anti-human Ki-67 monoclonal antibody bound to the formalin fixed paraffin embedded human specimen with a secondary antibody specific to the anti-human Ki-67 monoclonal antibody, wherein the secondary antibody specific to the anti-human Ki-67 monoclonal antibody is associated with an alkaline phosphatase enzyme; and   reacting the formalin fixed paraffin embedded human specimen with a substrate for the alkaline phosphatase enzyme,   wherein a precipitate of the substrate of the peroxidase enzyme and a precipitate the substrate of the alkaline phosphatase enzyme comprise distinguishable chromogenic reporter signals.   
     
     
         2 . The method of  claim 1 , wherein the anti-human p16 INK4a  monoclonal antibody is a mouse monoclonal antibody, and wherein the anti-human Ki-67 monoclonal antibody is a rabbit monoclonal antibody. 
     
     
         3 . The method of  claim 1 , wherein the precipitate of the substrate of the peroxidase enzyme produces brown chromogenic reporter signals, and wherein the precipitate of the substrate of the alkaline phosphatase enzyme produces red chromogenic reporter signals. 
     
     
         4 . The method of  claim 3 , wherein the substrate for the peroxidase enzyme comprises 3,3′-diaminobenzidine, and wherein the substrate for the alkaline phosphatase enzyme comprises FastRed. 
     
     
         5 . The method of  claim 1 , wherein the formalin fixed paraffin embedded human specimen comprises cells of the cervix uteri. 
     
     
         6 . The method of  claim 1 , wherein the formalin fixed paraffin embedded human specimen comprises cells derived from an anogenital tract, a naso-pharyngeal space, or an oral cavity. 
     
     
         7 . The method of  claim 1 , wherein the secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody is conjugated to the peroxidase enzyme; and wherein the secondary antibody specific to the anti-human Ki-67 monoclonal antibody is conjugated to the alkaline phosphatase enzyme. 
     
     
         8 . A method of preparing a cytological specimen of the cervix uteri for diagnosis of cervical dysplasia, comprising:
 immunoenzymatically staining a cytological specimen of the cervix uteri for both human p16 INK4a  protein and human Ki-67 protein with agents that produce different chromogenic reporter signals such that individual cells of the cytological specimen of the cervix uteri that co-express both human p16 INK4a  protein and human Ki-67 protein appear as brown-stained cells having red-stained nuclei,   wherein the immunoenzymatic staining of the human b  16   INK4a  protein comprises reacting 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 the human p16 INK4a  protein included in the cytological specimen of the cervix uteri; and   wherein the immunoenzymatic staining of the human Ki-67 protein comprises reacting the cytological specimen of the cervix uteri with an anti-human Ki-67 monoclonal antibody under conditions sufficient to specifically bind the anti-human Ki-67 antibody to the human Ki-67 protein included with in the cytological specimen of the cervix uteri.   
     
     
         9 . The method of  claim 8 , wherein the immunoenzymatic staining of the human p16 INK4a  protein further comprises reacting the anti-human p16 INK4a  antibody bound to the human 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 reacting the peroxidase enzyme with a first set of agents comprising a 3,3′-diaminobenzidine (DAB) chromogenic dye under conditions sufficient to precipitate the DAB chromogenic dye on the cytological specimen of the cervix uteri. 
     
     
         10 . The method of  claim 9 , wherein the anti-human p16 INK4a  monoclonal antibody is a mouse monoclonal antibody. 
     
     
         11 . The method of  claim 8 , wherein the immunoenzymatic staining of the human Ki-67 protein further comprises reacting the anti-human Ki-67 antibody bound to the human Ki-67 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 Ki-67 antibody; and reacting the alkaline phosphatase enzyme bound to the anti-human Ki-67 antibody with a second set of agents comprising a FastRed chromogen under conditions sufficient to precipitate the FastRed chromogen on the cytological specimen of the cervix uteri. 
     
     
         12 . The method of  claim 11 , wherein the anti-human Ki-67 monoclonal antibody is a rabbit monoclonal antibody. 
     
     
         13 . The method of  claim 8 , wherein the cytological specimen of the cervix uteri is contacted with the anti-human p16 INK4a  monoclonal antibody and the anti-human Ki-67 monoclonal antibody simultaneously. 
     
     
         14 . A method of staining a formalin fixed paraffin embedded (FFPE) specimen comprising:
 immunoenzymatically staining human p16 INK4a  protein in the FFPE specimen, wherein the immunoenzymatic staining of the human p16 INK4a  protein comprises reacting the FFPE specimen with first agents that produce brown chromogenic reporter signals; and   immunoenzymatically staining human Ki-67 protein in the FFPE specimen, wherein the immunoenzymatic staining of the human Ki-67 protein comprises reacting the FFPE specimen with second agents that produce red chromogenic reporter signals,   wherein the brown chromogenic reporter signals and the red chromogenic reporter signals are distinguishable.   
     
     
         15 . The method of  claim 14 , wherein the first agents comprise 3,3′-diaminobenzidine, and wherein the second agents comprise FastRed. 
     
     
         16 . The method of  claim 14 , wherein the immunoenzymatic staining of the human p16 INK4a  protein comprises reacting the FFPE specimen with an anti-human p16 INK4a  monoclonal antibody under conditions sufficient to specifically bind the anti-human p16 INK4a  antibody to the human p16 INK4a  protein included in the FFPE specimen. 
     
     
         17 . The method of  claim 16 , wherein the immunoenzymatic staining of the human p16 INK4a  protein further comprises: (i) reacting the anti-human p16 INK4a  antibody bound to the p16 INK4a  protein in the specimen with at least a peroxidase enzyme such that the peroxidase enzyme binds to the anti-human p16 INK4a  antibody; and (ii) reacting the peroxidase enzyme with at least 3,3′-diaminobenzidine (DAB) under conditions sufficient to precipitate the DAB. 
     
     
         18 . The method of  claim 14 , wherein the immunoenzymatic staining of the human Ki-67 protein comprises reacting the FFPE specimen with an anti-human Ki-67 monoclonal antibody under conditions sufficient to specifically bind the anti-human Ki-67 antibody to the human Ki-67 protein included in the FFPE specimen. 
     
     
         19 . The method of  claim 18 , wherein the immunoenzymatic staining of the human Ki-67 protein further comprises: (i) reacting the anti-human Ki-67 antibody bound to the Ki-67 protein in the specimen with at least an alkaline phosphatase enzyme such that the alkaline phosphatase enzyme binds to the anti-human Ki-67 antibody; and (ii) reacting the alkaline phosphatase enzyme bound to the anti-human Ki-67 antibody with at least FastRed under conditions sufficient to precipitate the FastRed on the FFPE specimen. 
     
     
         20 . The method of  claim 14 , wherein the formalin fixed paraffin embedded specimen comprises human cells of the cervix uteri. 
     
     
         21 . The method of  claim 14 , wherein the formalin fixed paraffin embedded specimen comprises human cells derived from an anogenital tract, a naso-pharyngeal space, or an oral cavity. 
     
     
         22 . The method of  claim 14 , wherein the formalin fixed paraffin embedded specimen comprises a histopathology specimen. 
     
     
         23 . A method comprising:
 reacting a 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  monoclonal antibody to human p16 INK4a  protein in the cytological specimen of cervix uteri;   reacting the cytological specimen of the cervix uteri with a peroxidase enzyme under conditions sufficient to specifically bind the peroxidase enzyme to the anti-human p16 INK4a  monoclonal antibody bound to the cytological specimen of cervix uteri;   reacting the peroxidase enzyme bound to the anti-human p16 INK4a  monoclonal antibody with a first set of agents comprising 3,3′-diaminobenzidine (DAB) under conditions sufficient to precipitate the DAB onto cells of the cytological specimen of cervix uteri that express the human p16 INK4a  protein;   reacting the cytological specimen of the cervix uteri with an anti-human Ki-67 monoclonal antibody under conditions sufficient to specifically bind the anti-human Ki-67 monoclonal antibody to human Ki-67 protein in the cytological specimen of cervix uteri;   reacting the cytological specimen of the cervix uteri with an alkaline phosphatase enzyme under conditions sufficient to specifically bind the alkaline phosphatase enzyme to the anti-human Ki-67 monoclonal antibody bound to the cytological specimen of cervix uteri; and   reacting the alkaline phosphatase enzyme bound to the anti-human Ki-67 monoclonal antibody with a second set of agents comprising a FastRed chromogen under conditions sufficient to precipitate the FastRed chromogen onto cells of the cytological specimen of cervix uteri that express the human Ki-67 protein.   
     
     
         24 . The method of  claim 23 , wherein the anti-human p16 INK4a  monoclonal antibody is a mouse monoclonal antibody, and wherein the anti-human Ki-67 monoclonal antibody is a rabbit monoclonal antibody. 
     
     
         25 . The method of  claim 23 , wherein the biological specimen is contacted with the anti-human p16 INK4a  monoclonal antibody and the anti-human Ki-67 monoclonal antibody simultaneously. 
     
     
         26 . The method of  claim 23 , wherein the presence of at least one individual brown stained cell having a red stained nucleus n the cytological specimen of the cervix uteri is indicative of a dysplastic state of the cell.