Signal-Amplified Colorimetric In Situ Hybridization for Assessment of Human Papillomavirus Infection in Cervical Lesions

Detection and typing of human papillomavirus (HPV) infection may have a major impact in cervical-screening and follow-up. In this study various commercially available techniques for the detection of HPV were evaluated. HPV-status was determined in 86 samples of cervical cancer by PCR and direct sequencing, catalyzed signal amplified colorimetric DNA in situ hybridization (CSAC- ISH) (GenPoint system, DAKO), immunohistochemistry (IHC) and in 12 selected cases also by conventional, non-amplified ISH. Twenty-one samples of cervical intraepithelial neoplasias grade III (CIN III) were investigated by CSAC-ISH, conventional ISH and by IHC, in corresponding PAP smears HPV-detection and typing was performed by CSAC-ISH and Hybrid Capture test II (HC). In additional 20 PAP smears HPV typing was performed using HC and a novel immunocytochemical system for HPV detection and-typing. CSAC-ISH showed good correlation with PCR analysis in cervical cancers: In 87% of PCR positive cases, HPV infection was also detected by CSAC- ISH (66/76). HPV 16 was detected in 75% of PCR-positive cases (44/59), HPV 18 in 71% of PCR positive cases (5/7). CSAC-ISH detected HPV 31 in only 29% of PCR positive cases (2/7), and HPV 33 in 64% of PCR-positive cases (23/36). Nevertheless, CSAC-ISH- false negative cases for HPV 31 or 33 were nearly always combined infections with other HPV types, which were detectable by CSAC-ISH in most cases. CSAC-ISH revealed HPV infection in 20 of 21 HC-positive cervical smears, while in corresponding biopsies (CIN III) CSAC-ISH detected 100% of HPV infections. Conventional, non-amplified ISH showed significantly lower sensitivity compared with CSAC-ISH, and immunocyto- and -histochemistry were of very low sensitivity for detection of HPV. CSAC-ISH is an easy-to-handle method for detection and typing of cervical HPV infection, and shows sufficient sensitivity for clinical practice.