Oral Presentation Australasian Cytometry Society 41st Annual Conference

Application and validation of flow cytometry-based E6/E7 mRNA quantification  for the detection of high-risk HPV infection in cervical cancer screening (24175)

PRANAY TANWAR 1 , Neerja Bhatla 1 , Shachi Vashist 1 , Sandeep Mathur 1 , Imran Haider 1
  1. ALL INDIA INSTITUTE OF MEDICAL SCIENCES, NEW DELHI, DELHI, India

Background: Persistent infection with high-risk human papillomavirus (HR-HPV) is the necessary cause of cervical carcinoma. HPV DNA testing is used for primary screening and co-testing. The role of E6/E7 mRNA needs to be defined.

Materials & Methods: Women presenting with intermenstrual bleeding, postcoital bleeding, prolonged vaginal discharge or an unhealthy cervix underwent VIA and collection of cervical samples in ThinPrep® PreservCyt® Solution (Hologic MA, USA) for HR-HPV DNA testing (HC2, Qiagen), cytology HPV mRNA (HPV OncoTect 3Dx (IncellDx, CA, USA), colposcopy and biopsy.

Result: 302 women underwent screening. Lesions detected were: CIN1 (n=39), CIN2 (n=3), CIN3 (n=3) and invasive cancer (n=3). VIA and HC2 showed the best sensitivity. HPV OncoTect 3Dx test standardization was done. Samples were hybridized with oligonucleotide probes for E6/E7 mRNA and counterstained with a nuclear dye for cell cycle analysis. Cells were analyzed on a CytoFLEX (Beckman Coulter, Inc. CA). Ectocervical cells were differentiated from endocervical cells, inflammatory cells and debris using forward and side light scatter properties, depending on the size of cell and cytoplasmic complexity. It quantitatively detected both E6/E7 mRNA overexpression and cell proliferation in intact cervical cells.

Conclusion: E6/E7 mRNA detection allows identification of a transcriptionally active virus genome. A larger study is needed for standardisation of the morphological cut-off to differentiate between transient and persistent HPV infections, recognize high-grade CIN and establish its role in the screening strategy