The Value of a Novel Panel of Cervical Cancer Biomarkers for Triage of HPV Positive Patients and for Detecting Disease Progression.

In the era of primary vaccination against HPV and at the beginning of the low prevalence of cervical lesions, introduction of screening methods that can distinguish between low- and high-grade lesions is necessary in order to maintain the positive predictive value of screening. This case-control study included 562 women who attended cervical screening or were referred for colposcopy and 140 disease free controls, confirmed by histology and/or cytology. The cases were stratified by age. Using routine exfoliated liquid based cytological samples RT-PCR measurements of biomarker genes, high-risk HPV testing and liquid based cytology were performed and used to evaluate different testing protocols including sets of genes/tests with different test cut-offs for the diagnostic panels. Three new panels of cellular biomarkers for improved triage of hrHPV positive women (diagnostic panel) and for prognostic assessment of CIN lesions were proposed. The diagnostic panel (PIK3AP1, TP63 and DSG3) has the potential to distinguish cytologically normal hrHPV+ women from hrHPV+ women with CIN2+. The prognostic gene panels (KRT78, MUC5AC, BPIFB1 and CXCL13, TP63, DSG3) have the ability to differentiate hrHPV+ CIN1 and carcinoma cases. The diagnostic triage panel showed good likelihood ratios for all age groups. The panel showed age-unrelated performance and even better diagnostic value under age 30, a unique feature among the established cervical triage tests. The prognostic gene-panels demonstrated good discriminatory power and oncogenic, anti-oncogenic grouping of genes. The study highlights the potential for the gene expression panels to be used for diagnostic triage and lesion prognostics in cervical cancer screening.

Human papillomavirus detection and genotyping, by HC2, full-spectrum HPV and molecular beacon real-time HPV assay in an Irish colposcopy clinic.

Cervical screening programmes are moving towards HPV testing as part of the screening process and as a triage for colposcopy. Three HPV detection methods were evaluated using cervical cytology specimens from colposcopy patients. PreservCyt™ liquid based cytology specimens from 241 women attending colposcopy clinics with greater than 2 persistently abnormal smears were recruited through the Coombe Women and Infants University Hospital, Dublin. HPV DNA was detected by Hybrid Capture (HC2) for 13 high-risk HPV types, Full-Spectrum HPV (FS-HPV) for 49 high and low-risk types and Molecular Beacon Real-Time HPV assay (MBRT-HPV) for 16 high and low-risk types. HPV genotyping was performed using Linear Array HPV Assay (LA-HPV). HPV was detected in 83.3% (195/234), 91.9% (217/236) and 80.1% (169/211) of cytology specimens by HC2, FS-HPV and MBRT-HPV, HPV DNA detection assays. The sensitivity of the assays for the detection of high-risk HPV in cytology specimens that had a Cervical Intraepithelial Neoplasia Grade 2+ result by histology were, 98%, 97% and 94% for HC2, FS-HPV and MBRT-HPV assays with positive predictive values of 94.1%, 94.1% and 97.3%. The most common HPV genotypes were HPV 16, 31, 33, 58, 42, 61 and 53, and the most common high-risk HPV genotypes were HPV 16, 31, 33, 58, 18, 45, 59, 51, 56 and 39, with detection of multiple infections in 57.7% of all cases. FS-HPV and MBRT-HPV are highly sensitive and have a similarly high PPV as the HC2 assay for detection of HPV in patients with Cervical Intraepithelial Neoplasia Grade 2+ disease. HPV genotyping of women with persistent abnormalities is warranted prior to the introduction of HPV DNA testing in a colposcopy setting.

Molecular beacon-based real-time PCR method for detection of 15 high-risk and 5 low-risk HPV types.

Detection of HPV infections requires a robust time-effective single-step method for efficient screening. A molecular beacon-based one-step multiplex real-time PCR system was developed to detect 15 high-risk (HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and 5 low-risk HPV types (HPV types 6, 11, 42, 43, 44). Molecular beacons detecting high-risk types are 5'-FAM-3'-DABCYL-labelled, molecular beacons for low-risk detection are 5'-TET-3'-DABCYL-labelled, while the internal control added before sample DNA extraction is detected by a 5'-FAM-TexasRed-3'-DABCYL wavelength-shifting molecular beacon. Accordingly, fluorescent data for HPV detection are collected at 530 nm for high-risk types, 560 nm in case of low-risk types and the reaction internal control is detected at 610 nm on a Roche LightCycler 2.0 instrument. The sensitivity for detected types varies between 22 and 700 copies/reaction. The clinical performance was tested on 161 clinical sample DNAs. The MB-RT PCR results were compared to the typing results obtained by the L1F/L1R PCR and hybridization-based system described previously, and the concordance rate between the two systems was 89.44%. The favorable characteristics shown by this multiplex single-step real-time HPV detection system make this promising approach worthy for further development and application for clinical screening.