HPV E6, E7 mRNA assay and methods of use thereof

• US 7,524,631 B2
• Filed: 02/02/2005
• Issued: 04/28/2009
• Est. Priority Date: 02/02/2005
• Status: Active Grant

• Current Assignee: IncellDx, Inc.
• Original Assignee: Invirion, Inc.
• Inventors: Patterson, Bruce K.
• Primary Examiner(s): SWITZER, JULIET CAROLINE
• Application Number:
  US11/050,384
• Publication Number:
  US 20060172285A1
• Time in Patent Office: 1,546 Days
• Field of Search: None
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G16Z 99/00 Subject matter not provided for in other main groups of this subclass

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.

Claims

1. An assay for determining malignant transformation of cervical cells, the assay comprising the steps of:

(a) obtaining cervical cells from an individual;

(b) preparing the cervical cells as a liquid-based cytology (LBC) specimen;

(c) hybridizing the LBC cytology specimen using oligonucleotides specific for HPV E6, E7 mRNA;

(d) quantitating expression of HPV E6, E7 mRNA in the cervical cells; and

(e) identifying cervical cells that express 200 or more copies of HPV E6, E7 mRNA per cell,

wherein malignant transformation of the cervical cells is indicated by expression of 200 or more copies of HPV E6, E7 mRNA per cell and wherein the percentage of cells that express 200 or more copies of HPV E6, E7 mRNA per cell is quantitated.

2. The assay of claim 1, wherein the expression of HPV E6, E7 mRNA per cell is quantitated via flow cytometry.

3. 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).

4. A method of screening a patient for malignant transformation of cervical cells, the method comprising:

(a) obtaining cervical cells from the patient;

(b) quantitating expression of HPV E6, E7 mRNA in the cervical cells; and

(c) identifying cervical cells that express 200 or more copies of HPV E6, E7 mRNA per cell,

wherein malignant transformation of cervical cells in the patient is indicated by expression of 200 or more copies of HPV E6, E7 mRNA per cell and wherein the percentage of cells that express 200 or more copies of HPV E6, E7 mRNA per cell is quantitated.

5. The method of claim 4, 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).

6. The method of claim 4, wherein the HPV E6, E7 mRNA is identified using in situ hybridization with oligonucleotides specific for HPV E6, E7 mRNA.

7. The method of claim 4, wherein the HPV E6, E7 mRNA is quantitated using flow cytometry.

8. An assay for identifying one or more 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;

(d) hybridizing the LBC specimen using oligonucleotides specific for HPV E6, E7 mRNA,

(e) quantitating expression of HPV E6, E7 mRNA in the cervical cells;

(f) identifying cells that express 200 or more copies of HPV E6, E7 mRNA per cell,

wherein neutrophils are identified by CD16.sup.+ staining, columnar endocervical cells are identified by CD16.sup.−

, CAM 5.2.sup.+ staining, and squamous ectocervical cells are identified by CD16.sup.−

, CAM 5.2.sup.−

staining, and malignant transformation of one or more of the cervical cell types is indicated by expression of 200 or more copies of HPV E6, E7 mRNA per cell and wherein the percentage of cells that express 200 or more copies of HPV E6, E7 mRNA per cell is quantitated.

9. The assay of claim 8, wherein expression of HVP E6, E7 mRNA is quantitated via flow cytometry.