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 . An assay for determining malignant transformation of cervical cells comprising the steps of:
 (a) obtaining cervical cells from an individual;   (b) preparing the cervical cells as a liquid-based cytology (LBC) specimen; and   (c) hybridizing the LBC specimen using oligonucleotides specific for HPV E6, E7 mRNA, wherein expression of HPV E6, E7 mRNA indicates malignant transformation of the cervical cells.   
     
     
         2 . The assay of  claim 1 , further comprising:
 (d) quantitating the expression of HPV E6, E7 mRNA per cell via flow cytometry.   
     
     
         3 - 6 . (canceled) 
     
     
         7 . The assay of  claim 1 , wherein the cervical cells are selected from the group consisting of normal cells, abnormal squamous cells of undetermined significance (ASCUS), cells with low grade squamous intraepithelial lesions (LGSILs), and cells with high grade intraepithelial lesions (HGSILs). 
     
     
         8 . A method of screening a patient for malignant transformation of cervical cells comprising identifying expression of HPV E6, E7 mRNA in the cervical cells. 
     
     
         9 . The method of  claim 8 , wherein the cervical cells are selected from the group consisting of normal cells, abnormal squamous cells of undetermined significance (ASCUS), cells with low grade squamous intraepithelial lesions (LGSILs), and cells with high grade intraepithelial lesions (HGSILs). 
     
     
         10 . The method of  claim 8 , wherein the HPV E6, E7 mRNA is identified using in situ hybridization with oligonucleotides specific for HPV E6, E7 mRNA. 
     
     
         11 . The method of  claim 10 , wherein the HPV E6, E7 mRNA is quantitated using flow cytometry. 
     
     
         12 - 15 . (canceled) 
     
     
         16 . The method of  claim 8 , wherein the HPV E6, E7 mRNA is identified using in situ hybridization with oligonucleotides specific for HPV E6, E7 mRNA. 
     
     
         17 - 20 . (canceled) 
     
     
         21 . An assay for identifying malignantly transformed cervical cell types comprising the steps of:
 (a) obtaining cervical cells from an individual;   (b) preparing the cervical cells as a liquid-based cytology (LBC) specimen;   (c) staining the cervical cells with a combination of CD16 and CAM 5.2; and   (d) hybridizing the LBC specimen using oligonucleotides specific for HPV E6, E7 mRNA, wherein neutrophils are identified by CD16 +  staining, columnar endocervical cells are identified by CD16 − , CAM 5.2 +  staining, and squamous ectocervical cells are identified by CD16 − , CAM 5.2 +  staining, and expression of HPV E6, E7 mRNA indicates malignant transformation of the cervical cells.   
     
     
         22 . The assay of  claim 21 , further comprising:
 (e) quantitating the expression of HPV E6, E7 mRNA per cell via flow cytometry.   
     
     
         23 - 26 . (canceled) 
     
     
         27 . The assay of  claim 21 , wherein malignantly transformed columnar endocervical cells indicate a pre-adenocarcinoma or adenocarcinoma cervical condition. 
     
     
         28 . The assay of  claim 27 , wherein malignantly transformed squamous ectocervical cells indicate pre-squamous cell carcinoma or squamous cell carcinoma cervical condition.