In situ hybridization with biotinylated tyramide amplification: Detection of human papillomavirus DNA in cervical neoplastic lesions

A novel peroxidase-mediated amplification system, which is based on the deposition of biotinylated tyramide (BT) molecules, was recently described for immunohistochemistry and in situ hybridization (ISH). We report here a highly sensitive ISH system combined with a microwave prehybridization treatment and BT enhancement after hybridization. Our ISH with BT amplification can detect human papillomavirus (HPV) 16 DNA signals in the fixed SiHa cell, suggesting the ability to detect single or few copies of HPV DNA. To confirm its diagnostic usefulness, we used our ISH with BT amplification to detect HPV 16 DNA in 81 cases of cervical neoplastic lesions, which had been used for routine pathologic diagnosis, Of 81 tumors, 47 (58%) showed HPV 16 DNA, of which 29 and 18 were cervical intraepithelial neoplasia (CINs) and invasive cancers, respectively. In almost all of the invasive cancers positive for HPV 16 DNA, ISH revealed a basic dot signal pattern in the nuclei, suggesting that HPV DNA integrated into tumor cell DNA, On the other hand, low-grade CINs displayed a diffuse, mainly episomal, signal pattern, which decreased in frequency with increased grade of GIN. Our ISH with BT amplification is highly sensitive and can be used to detect various genes and their expressions.