Abstract 4567: Demonstration of pharmacodynamic effects of Notch and PI3Kinase inhibitors using robust immunohistochemical assays on human skin explant models

Abstract

Assessment of pharmacodynamic (PD) effect of novel therapeutic compounds in human tissues is used to demonstrate target engagement and to rationalize dosing strategy decisions for emerging targeted therapeutics in clinical trials. Often, human skin biopsies, a readily accessible surrogate tissue, are used for these purposes. Aberrant signaling in the Notch cell surface receptor and the PI3K signal transduction pathways are increasingly implicated in human cancers. In order to test the utility of immunohistochemical (IHC) PD biomarker assays developed in our laboratory for Notch and PI3K inhibitor development programs, we developed ex vivo PD models using respective inhibitors on human skin biopsy explants. Fresh human skin samples were obtained in the Operating-Room from reduction mammoplasty specimens, serially sliced (1-2 mm) and incubated at 37°C in Williams E. media containing 1 µM Notch (gamma-secretase) inhibitor (15 h); and 50 µM PI3K inhibitor (LY294002, 4 h). Specimens were fixed in 10% neutral buffered formalin for 24 hours, and processed into FFPE blocks. Sections were stained for Notch1 intracellular domain (N1ICD, rabbit monoclonal, D3B8) using DAB and for phospho-S6 (S240/S244, rabbit polyclonal) using an alkaline phosphatase-based detection system. N1ICD and phospho-S6 immunoreactivity in cells of the epidermis was analyzed using positive pixel counting. Viability of epidermal cells throughout ex vivo incubation was verified by Ki67 and histomorphologic assessments of the epidermis. Nuclear N1ICD immunoreactivity, a marker of Notch pathway activation, was decreased by 76% when treated with gamma-secretase inhibitor. Similarly, treatment with PI3K inhibitor decreased cytoplasmic phospho-S6 immunoreactivity by 98%. These ex vivo models of human skin provide scientific evidence to support: 1) The N1ICD and phospho-S6 IHC assays developed in our laboratory can demonstrate ex vivo inhibition of these two pathways in human skin; and 2) Feasibility to translate human skin explant findings to patient skin samples from clinical trials. Our explant model systems are closer to real life PD assessments on patient skin samples from clinical trials and can further substantiate the PD data generated in animal models. In some cases, the ex vivo model could replace animal models. Citation Format: Timothy R. Holzer, Leslie A. O'Neill, Angie D. Fulford, Janet M. Grondin, Bradley L. Ackermann, Robert J. Konrad, Kelly M. Credille, Aejaz Nasir. Demonstration of pharmacodynamic effects of Notch and PI3Kinase inhibitors using robust immunohistochemical assays on human skin explant models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4567. doi:10.1158/1538-7445.AM2014-4567