Abstract 651: Development of an RNA in situ hybridization assay for the detection of prostate cancer cells in urine
Authors: Eskra Jillian N. , Rabizadeh Daniel , Pavlovich Christian P. , Mangold Leslie , Fabian Elizabeth , Isaacs William B. , Luo Jun 
Published: August 2018

Abstract
Urine-based detection of prostate cancer represents a promising resource for non-invasive diagnostics. The value of urine as a biospecimen in the management of prostate cancer has already been established, but the utility of urine has yet to be fully explored. The objective of our study was to develop a RNA in situ hybridization (RISH) method for detecting prostate cells in urine and to evaluate the feasibility of using this assay for cancer detection in clinical specimens. We hypothesized that robust and specific labeling of prostate cancer cells could be achieved using this approach, providing benefit over existing urine detection methods, namely the FDA-approved PCA3 test, by allowing for the direct visualization and molecular characterization of individual cells. We optimized the following collection, processing, and preparation procedure for detection of prostate cells in fresh urine specimens collected from patients following digital rectal exam (DRE): the entire urine volume was centrifuged to collect the sediment, which was then formalin fixed, cytocentrifuged onto two slides, and slides were stored at -20°C until proceeding to staining and cytological analysis. Cellularity and sample adequacy was assessed by Papanicolaou staining. For identification of prostate cells in post-DRE urine via RISH, single-probe chromogenic labeling for NKX3.1 or HOXB13 was performed. Pretreatment conditions and staining parameters for RISH were optimized using urine samples spiked with 22Rv1 cells prior to staining patient specimens. We were able to readily identify cells of prostate origin in both spiked urines and patient samples. To further discriminate normal prostate cells from cancer cells, fluorescent multiplex RISH was pursued. We screened a candidate panel of prostate-specific RNA targets that included NKX3.1, HOXB13, KLK3, PRAC1, PRAC2, MALAT1, HOXC6, AMACR, and PCA3. Based on detection specificity, NKX3.1 and PRAC1 were selected as markers to identify cells of prostate origin and PCA3 as a marker of malignancy. Multiplex RISH with these three markers was carried out on 19 patient specimens. Prostate cells, classified as those positive for PRAC1 and/or NKX3.1, were identified in 11 samples, 6 of which were also positive for PCA3. These observations demonstrate that multiplex RISH can be used for the specific detection and visualization of prostate cancer cells in post-DRE urine samples. Although further refinement and validation are necessary, this study provides the first evidence supporting the application of a urinary RISH assay as a potential non-invasive method for prostate cancer detection. Ongoing and future studies will include characterizing performance of additional biomarker panels and validating clinical utility of this technique in a diagnostic setting.
Citation Format: Jillian N. Eskra, Daniel Rabizadeh, Christian P. Pavlovich, Leslie Mangold, Elizabeth Fabian, William B. Isaacs, Jun Luo. Development of an RNA in situ hybridization assay for the detection of prostate cancer cells in urine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 651.