Methods and compositions for the detection of cervical disease

Abstract

Methods and compositions for identifying high-grade cervical disease in a patient sample are provided. The methods of the invention comprise detecting overexpression of at least one biomarker in a body sample, wherein the biomarker is selectively overexpressed in high-grade cervical disease. In particular claims, the body sample is a cervical smear or monolayer of cervical cells. The biomarkers of the invention include genes and proteins that are involved in cell cycle regulation, signal transduction, and DNA replication and transcription. In particular claims, the biomarker is an S-phase gene. In some aspects of the invention, overexpression of a biomarker of interest is detected at the protein level using biomarker-specific antibodies or at the nucleic acid level using nucleic acid hybridization techniques. Kits for practicing the methods of the invention are further provided.

1 . A kit comprising at least three antibodies, wherein each antibody specifically binds to a nuclear biomarker protein that is selectively overexpressed in high-grade cervical disease, and wherein a first and a second antibody in the kit specifically bind to the biomarker protein MCM2, and wherein a third antibody specifically binds to the biomarker protein MCM7. 
     
     
         2 . The kit of  claim 1 , wherein said kit further comprises a peroxidase blocking reagent, a protein blocking reagent, chemicals for the detection of antibody binding to said biomarker proteins, a counterstain, a bluing agent, and instructions for use. 
     
     
         3 . The kit of  claim 2 , wherein said chemicals for the detection of antibody binding comprise a chromogen and a secondary antibody conjugated to a labeled polymer, wherein the chromogen comprises 3′,3′-diaminobenzidine, and wherein the labeled polymer comprises horseradish peroxidase conjugated to a dextran polymer. 
     
     
         4 . The kit of  claim 2 , wherein said counterstain comprises hematoxylin. 
     
     
         5 . The kit of  claim 2 , wherein said bluing agent comprises a solution comprising Tris buffered saline, pH 7.4, Tween-20, and sodium azide. 
     
     
         6 . The kit of  claim 2  further comprising a positive control sample. 
     
     
         7 . The kit of  claim 6 , wherein said positive control sample comprises SiHa cells. 
     
     
         8 . The kit of  claim 1  further comprising reagents for Papanicolaou (Pap) staining. 
     
     
         9 . The kit of  claim 8 , wherein the reagents for Pap staining comprise EA50 and Orange G. 
     
     
         10 . The kit of  claim 1 , wherein the at least three antibodies are provided as separate reagents. 
     
     
         11 . The kit of  claim 1 , wherein the at least three antibodies are provided as a cocktail. 
     
     
         12 . A method for diagnosing high-grade cervical disease in a patient independent of the patient's HPV infection status, the method comprising:
 a) obtaining a body sample from the patient;   b) contacting the sample with at least three antibodies, wherein each of the antibodies specifically binds to a nuclear biomarker protein that is selectively overexpressed in high-grade cervical disease, wherein a first and a second antibody specifically bind to the biomarker protein MCM2, and wherein a third antibody specifically binds to the biomarker protein MCM7; and,   c) detecting binding of the antibodies to the biomarker proteins MCM2 and MCM7 to determine if the biomarker proteins are overexpressed in the sample, and thereby diagnosing high-grade cervical disease in the patient.   
     
     
         13 . The method of  claim 12 , wherein the method comprises performing immunocytochemistry. 
     
     
         14 . The method of  claim 12 , wherein the method is performed manually. 
     
     
         15 . The method of  claim 12 , wherein the method is performed in an automated manner. 
     
     
         16 . The method of  claim 12 , wherein the sample comprises cervical cells. 
     
     
         17 . The method of  claim 16 , wherein the sample comprises a monolayer of cervical cells. 
     
     
         18 . The method of  claim 12 , wherein the sensitivity of the method for diagnosing high-grade cervical disease is at least 90% and the specificity of the method for diagnosing high-grade cervical disease is at least 75%. 
     
     
         19 . The method of  claim 12 , wherein the method is performed in response to the patient having an abnormal Pap smear result. 
     
     
         20 . The method of  claim 12 , wherein the method is performed as a primary screen for high-grade cervical disease in a general patient population. 
     
     
         21 . The method of  claim 12  further comprising Papanicolaou (Pap) staining of the sample. 
     
     
         22 . The method of  claim 12 , wherein the antibodies are contacted with the sample sequentially as individual antibody reagents. 
     
     
         23 . The method of  claim 12 , wherein the antibodies are contacted with the sample simultaneously as an antibody cocktail.