Abstract 3786: A study of HPV induced anal cancer progression in a raft tissue culture system

Abstract

Abstract Introduction: Anal cancer is one of few malignancies for which the incidence continues to rise in the US. It is highly associated with high risk (HR) HPV infection with HPV 16 and 18 accounting for the vast majority of cases. Understanding the molecular mechanisms underlying anal cancer progression would be valuable for the purposes of developing novel clinical management strategies. However, there remains a paucity of experimental models allowing for the study of HPV-associated anal cancer development. Herein, we describe an in vitro raft tissue culture system model to study anal squamous neoplastic progression via anal intraepithelial neoplasa (AIN) induced by HPV infection. Methods: Primary human anal keratinocyte (HAK) cells were transfected with either HPV16 or 18 DNA and grown in a raft tissue culture system. The progression of dysplastic changes was sequentially examined at ~5 passage intervals by histologic review including hematoxylin-eosin (H&E) staining, immunohistochemistry (p16, Ki67, cytokeratins CK5/6, CK8/18 and CK10) and in situ hybridization (ISH) with an HPV16/18 RNA probe cocktail. The degree of dysplasia was determined by the degree of disorganization of the epithelial architecture, presence of mitotic/dysplastic cells, p16 positivity, diffuse Ki67 staining, increased number of basal-parabasal type cells (CK5/6 and CK10 markers) and lack of epithelial differentiation. Results: Advanced dysplastic changes (corresponding to AIN2-3 lesions) were already apparent at the earliest of initial passages of transfected HAK cells (passage 8 for HPV16 and 7 for HPV 18). Later passages suggested development of AIN3/squamous cell carcinoma in situ in both lines with HPV16 HAK cells showing progressively increased labeling of CK8/18 and HPV18 HAK cells demonstrating increasing cell keratinization and epithelial disintegration. In both cell lines, HPV was present in both episomal and integrated forms throughout all stages of progression as suggested by ISH signal patterns. Conclusions: A raft tissue culture model system has been developed to study HPV-induced dysplastic progression in human anal keratinocytes. Importantly, this system is of great interest as an in vitro tool to characterize the sequential molecular alterations associated with the development of anal cancer which in turn, may have implications for novel screening, prevention and treatment strategies. Citation Format: Irina V. Getun, Abidemi Ajidahun, Janice Milici, Sreejata Chatterjee, Jorge Solares, Abul Elahi, Leah E. Hendrick, Evan S. Glazer, Louisa Balazs, Craig Meyers, David Shibata. A study of HPV induced anal cancer progression in a raft tissue culture system [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 3786.