Human papillomavirus (HPV) infection is associated with a variety of clinical conditions that range from innocuous lesions to cancer. Cervical cancer is the most common cancer caused by HPV. Other less common cancers affecting both men and women include anal, vulvar, vaginal, mouth/throat, and penile cancers. More than 200 types of HPV have been identified and classified as high-risk and low-risk. Among the high-risk HPV types, HPV 16 and HPV 18 are responsible for most HPV-related cancers.

We developed a multiplex method of nucleic acid and protein detection by combining in situ hybridization (ISH) application with AMPIVIEW® RNA probes and immunohistochemistry (IHC) applications. Powered by Enzo’s LoopRNA™ ISH technology, AMPIVIEW® RNA probes are uniquely designed with the precision of targeted, sequence-specific RNA, to deliver superior sensitivity for the detection and the expression analysis of key biomarkers in cells and tissue including formalin-fixed, paraffin-embedded (FFPE) tissue specimen.

In the progression of cervical lesions to cancer, expression of key markers of proliferation and checkpoint control such as Ki-67 and p16 can be dysregulated at the transcriptional and protein levels. In this study, we used IHC to detect the expression pattern of these key markers, as well as RNA ISH with AMPIVIEW® HPV RNA probes to spot integrated HPV DNA as well as HPV RNA.

Changes can be assessed between different infection levels and the severity and extent of marker gene dysregulation, thereby providing a proof of principle for AMPIVIEW®-based multiplexing as a tool to assist tracking the progression of cancer cells due to HPV infection.