High-Throughput Cervical Cancer Screening Using Intracellular Human Papillomavirus E6 and E7 mRNA Quantification by Flow Cytometry

Abstract

The Papanicolaou (Pap) test, based solely on the morphologic examination of exfoliated cells from the cervix, has reduced deaths due to cervical cancer by 74% in the United States during the past 40 years. During that time, the molecular mechanisms of cervical cancer have largely been elucidated. Taken together, these observations have identified a need for a high-throughput cervical cancer screening assay. We report the development of a high-throughput assay consisting of simultaneous immunophenotyping and ultrasensitive in situ hybridization for human papillomavirus (HPV) E6 and E7 messenger RNA (mRNA). This assay can be performed in less than 3 hours directly from liquid-based cervical cytology specimens. Overall, HPV fluorescence in situ hybridization (FISH) for E6 and E7 mRNA demonstrated 83.3% sensitivity and 91.3% specificity for high-grade squamous intraepithelial lesions compared with the Pap test in 231 liquid-based cytology samples from 2 cohorts. In a subset of these samples, HPV FISH demonstrated higher sensitivity and specificity than Hybrid Capture (Digene, Gaithersburg, MD) for high-risk genotypes.

Intracellular Human Papillomavirus E6, E7 mRNA Quantification Predicts CIN 2+ in Cervical Biopsies Better Than Papanicolaou Screening for Women Regardless of Age

“…In cell E6, E7 mRNA quantification was performed by resuspending the cells in hybridization mixture and a cocktail of oligonucleotide probes (HPV OncoTect; IncellDx). 6,7,16,17 Hybridization was performed at 43uC for 30 minutes and was followed by a 5-minute high stringency wash and a 15-minute high stringency wash. The cells were resuspended in PBS, pH 7.4, with 2% fetal calf serum for flow cytometric analysis.…”

“…5 To improve the performance of cervical cancer screening especially in women younger than 30 years, we investigated the use of a quantitative E6, E7 mRNA assay (HPV OncoTect, Incell DX, Menlo Park, California) that determines oncogene overexpression on a cell by cell basis using high throughput flow cytometry. 6,7 The E6 and E7 oncogenes drive cervical cell transformation leading to cancer. The expression of the HPV E6 gene leads to degradation of the tumor suppressor gene p53, and similarly, the HPV E7 gene associates with the tumor suppressor gene pRB.…”

Monitoring viral RNA in infected cells with LNA flow-FISH

“…The flow-FISH method described here utilizes one LNA probe and one fluorophore in contrast to many other flow-FISH methods, which need two or more oligonucleotide probes for detection (Borzì et al 1996;Crouch et al 1997;Just et al 1998;Stowe et al 1998;Narimatsu and Patterson 2005). With small changes such as the addition of a second biotin at the 39 end of the probe or the addition of a second or third LNA probe complementary to the same RNA, the fluorescence signal and sensitivity could be increased dramatically.…”

Human Papillomavirus: Flow Cytometry Detection and PCR Analysis, A Comparison Study

“…progress to high-grade lesions, detects the HPV oncogenic activity analyzing cervical cells that over express oncoproteins E6 and E7, and can be used at any time of woman life because there is no low age limit, and also exclude the false negative or unsatisfactory results [11,12]. The specificity and positive predictive value are also higher than other HPV detection techniques such as PCR analysis.…”