Novel assay for detecting integrated HPV. P. McGrath, P. Moen, D. Hochberg, E. O'Lear. One Cell Systems, Inc., Cambridge, MA.
   There is increasing consensus that integration of oncogenic HPV DNA into the host genome is the primary cause of cervical cancer and an assay that specifically detects viral integration would have high clinical utility. We are developing a method to detect integrated viral DNA after removing interfering episomal DNA. Using a model HPV cell line containing both episomal and integrated HPV-16 DNA, we developed experimental conditions for selectively removing episomal viral DNA without releasing integrated viral or cellular DNAs. Using in situ hybridization, the diffuse HPV DNA signal generated by episomal DNA present in control cells was absent in extracted cells. Conversely, hybridization signal from integrated viral DNA and an endogenous gene remained, indicating that the extraction conditions specifically removed protein-bound episomal DNA. Using a more sensitive PCR assay, we confirmed that, after extraction, the level of HPV DNA decreased in a cell line containing both episomal and integrated virus and remained essentially unchanged in two integrated cell lines. Successful application of this assay format to clinical samples will lead to precise detection of integrated HPV DNA and identification of patients who are likely to develop cervical cancer.