Persistent infection with high-risk human papillomavirus (hrHPV) types is a prerequisite for development of cervical dysplasia and cancer. During progression, deregulation and overexpression of viral proteins E6 and E7 occur, leading to loss of cell cycle control and neoplastic transformation. Current cervical cancer screening methods rely on cytological analyses compromised by frequent false-negative results and thus low sensitivity. HPV DNA-based tests pick up frequently infections without underlying disease leading to a low specificity. A more effective and reliable screening approach may involve exploitation of the oncoproteins E6 and E7 for specific detection of cervical dysplasia.
RabMabs with high specificity and sensitivity against the E7 protein of different hrHPV types were generated and a hrHPV E7 sandwich ELISA – recomWell HPV 16/18/45 - was developed for detection of the three hrHPV types with highest carcinogenicity (HPV 16, 18, and 45).
Cervical samples were collected to test the feasibility of recomWell HPV 16/18/45 in normal patients as well as in patients showing various stages of cervical intraepithelial neoplasia (CIN). Suitable for measurement of E7 protein are liquid-based cytological samples in PreserveCyte (ThinPrep).
Validation of recomWell HPV 16/18/45 with recombinant proteins of 12 hrHPV types showed specific detection with minimal cross-reaction. Validation with cell lysates of cervical cancer cell lines CaSki (HPV16+), HeLa (HPV18+), and MS751 (HPV45+) detected the E7 protein in the background of other cellular proteins. As part of the technical validation, the detection limit for HPV positive cell lines was determined with at least 1250 cells for CaSki and MS751 and 500 cells for HeLa and the inter- and intra-assay variance was calculated.
The proof of concept was shown by measurement of well characterized clinical samples collected in PreserveCyte. Furthermore, sensitivity, specificity, and positive and negative predictive value (PPV/NPV) were calculated with the clinical samples.
E7 detection of hrHPV by ELISA is a feasible method to detect hrHPV infection and dysplasia. Sensitivity of E7 detection seems high enough as shown by testing of cell lines and clinical samples. Clinical sensitivity and specificity is under investigation.
As there is evidence to suggest that E7 expression is up-regulated in high-grade dysplasia, the recomWell HPV 16/18/45 may have a high potential for detection of dysplasia with a higher specificity for disease than HPV DNA or RNA-based tests and could be a means of molecular triage in reflex testing of hrHPV positive screening results with HPV 16/18 genotyping.