Feasibility of multimodal molecular characterization of the androgen receptor (AR) in circulating tumor cells (CTCs) from patients with castrate-resistant prostate cancer (CRPC).

Abstract

133 Background: Despite the wealth of agents targeting the androgen receptor (AR) signaling pathway in castrate-resistant prostate cancer (CRPC), emergence of resistance is common. Mechanisms for resistance are diverse and likely include quantitative alterations in AR as well as alterations in nuclear localization. Clinical interrogation of such events is challenged by difficulties in biopsying metastatic sites and limitations in available circulating tumor cell (CTC) platforms. We set out to explore different methodologies of AR interrogation within CTCs. Methods: CTCs and cultured prostate cancer cells were isolated from nucleated blood cells using fluorescence associated cell sorting (FACS) targeting the Epithelial Cell Adhesion Molecule (EpCAM) with CD45 negative selection. In addition to standard immunofluorescence imaging, ImageStreamX spectral flow analysis was used to analyze AR protein expression and nuclear localization. Targeted DNA sequencing and TaqMan copy number evaluation were used for downstream analysis of AR alterations. Patients with highly refractory disease were enrolled for these assay development studies. Results: Thirteen of 15 (87%) patients in an initial feasibility cohort had CTCs isolated for downstream evaluation. Targeted DNA sequencing of as few as 10 C4-2 cells isolated from nucleated blood cells confirmed specificity for prostate cells by identifying a known AR mutation. TaqMan copy number evaluation revealed AR amplification in two out of four evaluable patients. Fluorescence microscopy demonstrated feasibility of AR staining, with 10 of 10 patients exhibiting AR staining above negative control and 50% staining above that of AR+ LAPC4 cells. Though the numeric variability in AR expression using ImageJ software was high, AR protein interrogation with ImageStreamX allowed for more precise evaluation of nuclear localization of the AR revealing that in CRPC patients there can be distinct CTC populations with respect to AR localization. Conclusions: Identifying mechanisms of AR derangement is crucial for the precision management of advanced prostate cancer. CTCs are a promising way to evaluate molecular characteristics of AR in patients with late stage disease.