HPV E6, E7 MRNA Assay and Methods of Use Thereof

Abstract

Provided is an HPV E6, E7 mRNA assay, referenced herein as the “In Cell HPV Assay,” that is capable of sensitive and specific detection of normal cervical cells undergoing malignant transformation as well as abnormal cervical cells with pre-malignant or malignant lesions. The In Cell HPV Assay identifies HPV E6, E7 mRNA via in situ hybridization with oligonucleotides specific for HPV E6, E7 mRNA and quantitates the HPV E6, E7 mRNA via flow cytometry. The In Cell HPV Assay can be carried out in less than three hours directly from liquid-based cervical (“LBC”) cytology specimens. The In Cell HPV Assay provides an efficient and highly sensitive alternative to the Pap smear for determining abnormal cervical cytology.

1 .- 28 . (canceled) 
     
     
         29 . A method of detecting a precancerous squamous cell in a cellular sample obtained from a subject, the method comprising:
 fixing and permeabilizing the cells of the cellular sample to produce a prepared cellular sample;   staining the cells of the prepared cellular sample with an oligonucleotide probe for a nucleic acid analyte expressed by the precancerous squamous cell;   
       imaging the stained cells; and
 quantifying the nucleic acid analyte present in the imaged cells on a per cell basis to detect the presence or absence of the precancerous squamous cell in the cellular sample. 
 
     
     
         30 . The method according to  claim 29 , wherein the nucleic acid analyte is an expression product of a viral oncogene. 
     
     
         31 . The method according to  claim 29 , wherein the staining further comprises contacting the cells of the prepared cellular sample with an antibody that specifically binds a marker expressed by the precancerous squamous cell. 
     
     
         32 . The method according to  claim 29 , wherein the staining further comprises contacting the cells of the prepared cellular sample with a fluorescent nuclear stain. 
     
     
         33 . The method according to  claim 29 , wherein the oligonucleotide probe comprises a detectable label. 
     
     
         34 . The method according to  claim 33 , wherein the detectable label comprises a fluorophore. 
     
     
         35 . The method according to  claim 29 , further comprising per cell signal amplification using an amplifier to detect the oligonucleotide probe. 
     
     
         36 . The method according to  claim 29 , wherein the method further comprises obtaining the cellular sample from the subject. 
     
     
         37 . The method according to  claim 29 , wherein the cellular sample is a liquid cellular sample. 
     
     
         38 . The method according to  claim 29 , wherein the fixing and permeabilizing comprises contacting the cells of the cellular sample with a composition comprising a fixation reagent and a permeabilization reagent. 
     
     
         39 . The method according to  claim 29 , wherein the precancerous squamous cell is detected when the quantified per cell amount of nucleic acid analyte is above a predetermined cut-off point. 
     
     
         40 . The method according to  claim 29 , wherein the cells are stained using a cocktail of oligonucleotide probes for at least two nucleic acid analytes expressed by the precancerous squamous cell. 
     
     
         41 . The method according to  claim 29 , further comprising determining the percentage of cells of the cellular sample expressing the nucleic acid analyte. 
     
     
         42 . The method according to  claim 29 , wherein the imaging comprises microscopic imaging or confocal imaging. 
     
     
         43 . A method comprising:
 contacting a cellular sample with fixing and permeabilizing reagents to produce a fixed and permeabilized cellular sample;   combining the fixed and permeabilized cellular sample with:
 a nucleic acid probe to a RNA biomarker; and 
 an antibody to a protein biomarker; 
   to produce a labeled sample; and   flow cytometrically assaying the labeled sample to detect hybridized nucleic acid probe and bound antibody.   
     
     
         44 . The method according to  claim 43 , wherein the protein biomarker is an intracellular protein biomarker. 
     
     
         45 . The method according to  claim 44 , wherein the protein biomarker is a cell surface protein biomarker. 
     
     
         46 . The method according to  claim 43 , wherein the method further comprises combining the fixed and permeabilized cellular sample with a second nucleic acid probe to a second RNA biomarker. 
     
     
         47 . The method according to  claim 46 , wherein the combining comprises contacting the fixed and permeabilized cellular sample with a hybridization cocktail comprising the first and second nucleic acid probes. 
     
     
         48 . The method according to  claim 43 , wherein the method comprises combining the fixed and permeabilized cellular sample with a second antibody to a second protein biomarker.