Abstract 1401: Sensitive detection of integrated HPV in carcinoma cells
Authors: Mauro Maurizio , Bovi Richard Delle , Coleman Jack 
Published: August 2019

Abstract
It is estimated that 79 million people in the US are currently infected with human papillomavirus (HPV). There are more than 200 different HPV types that are common worldwide. HPV types are categorized as either high risk (HR) or low risk (LR). LR types, such as HPV 6 and HPV 11, can cause genital warts or squamous papillomas of the oral cavity, while persistent infection with HR HPV types can induce cervical, anal, or head and neck cancers. Episomal HPV DNA can be detected in advanced stages of cancer. HPV integration in the host genome drives oncogenesis by the expression of the two major viral oncogenes, E6 and E7, which abrogate cell cycle checkpoints, promote cellular proliferation, and cause progressive genetic instability. Hence, screening for the presence of E6/E7 RNA transcripts expression is considered the gold standard for confirming active HPV infection. Early detection of active HPV is critical for better understanding of prognosis and therapeutics. So far, detection of integrated HPV DNA in tissue specimens has been challenging. A novel, cost-effective, in situ hybridization (ISH) detection assay has been developed using two sets of HPV probes with enhanced signaling, termed hyper probes. The Hyper HPV D probe detects HR HPV DNA and the Hyper HPV DR probe can simultaneously detect DNA and E6/E7 RNA of HR HPV subtypes in cancer cell lines and formalin fixed paraffin embedded (FFPE) biopsy specimens. The probes were tested on cervical carcinoma SiHa cells containing 1-3 copies of integrated DNA of HPV 16 and HPV negative cervical cancer C33A cell line. The Hyper HPV DR probe resulted in 1-3 puncta in more than 90% of SiHa cell nuclei, indicating integrated copies of HPV DNA into the host genome. There was also the additional presence of a diffuse signal which was abolished by pretreating SiHa cells with RNase. These data suggest the diffuse signal corresponds to E6/E7 mRNA expression. No signals were detected in HPV negative C33A cells. When cells were stained with Hyper HPV D probes, only punctate signals in nuclei of SiHa cells was present and no signal was evident in C33A control cells. Additionally, human cervical and anal FFPE biopsies were analyzed. Simultaneous presence of both punctate signals and diffuse signals were detected when using Hyper HPV DR probes in HR HPV infected tissues, while only punctate signal was detected when using HPV D hyper probes. The signal from the hyper probes was significantly stronger when compared to other commercially available HPV detection kits. Moreover, other commercially available kits failed to detect low copy of integrated HPV. In conclusion, these data demonstrate the Hyper HPV probes can reliably detect low copies of HPV DNA integrated in the host genome and the presence of HPV mRNA using ISH in HPV infected cells.
Citation Format: Maurizio Mauro, Richard Delle Bovi, Jack Coleman. Sensitive detection of integrated HPV in carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1401.