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Clinical Research

A novel method for detection of exfoliated prostate cancer cells in urine by RNA in situ hybridization

Prostate Cancer and Prostatic Diseases volume 24, pages 220–232 (2021)Cite this article

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Abstract

Background

In the current study, we explore the feasibility of detecting exfoliated prostate cancer cells in urine using an RNA in situ hybridization (RISH) assay. We hypothesized that robust and specific labeling of prostate cancer cells could be achieved in post-digital rectal examination (DRE) urine samples using RISH.

Methods

We focused on method development, optimization, and analytical evaluation of RISH-based detection of prostate cancer in urine. We optimized a sample collection, processing, and target detection workflow for urine cytology specimens in conjunction with RNA target detection by RISH. We screened a panel of 11 prostate-specific RNA targets, and selected NKX3-1 and PRAC1 as markers for cells of prostate origin and PCA3 as a marker of prostate malignancy. Following analytical validation of a multiplexed NKX3-1/PRAC1/PCA3 assay, we evaluated whether prostate cancer cells can be detected in a pilot cohort of 19 post-DRE specimens obtained from men diagnosed with prostate cancer.

Results

Using cytology specimens prepared from spiked urine samples, we established the analytical validity of the RISH assay for detection and visualization of prostate cells in urine. Cells of prostate origin could be readily and specifically identified and separated into benign and malignant cell populations based on the multiplex test that consisted of markers specific for prostate cells (NKX3-1, PRAC1) and prostate cancer cells (PCA3). Upon evaluation of post-DRE urine from a pilot cohort of prostate cancer patients, we identified 11 samples in which prostate cells were present, 6 of which were also positive for prostate cancer cells.

Conclusions

Multiplex RISH enables the direct visualization and molecular characterization of individual exfoliated prostate cells in urine. This proof-of-principle study provides evidence supporting the application of RISH as a potential noninvasive tool for prostate cancer detection.

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Fig. 1: RISH assay development and optimization.
Fig. 2: Visualization of RISH targets in urine spiked with LNCaP cells.
Fig. 3: Validation of specific detection of prostate cells by RISH.
Fig. 4: Detection of prostate cells in post-DRE urine.

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Authors and Affiliations

  1. Department of Urology, The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Jillian N. Eskra, Daniel Rabizadeh, Leslie Mangold, Elizabeth Fabian, W. Nathaniel Brennen, David B. Yeater, Kenneth J. Pienta, Alan W. Partin, William B. Isaacs, Christian P. Pavlovich & Jun Luo

  2. Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    W. Nathaniel Brennen

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  1. Jillian N. Eskra
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Correspondence to Jun Luo.

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The Johns Hopkins University has filed a patent application in which 4 co-authors were listed as inventors (JNE, WBI, CPP, JL).

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Eskra, J.N., Rabizadeh, D., Mangold, L. et al. A novel method for detection of exfoliated prostate cancer cells in urine by RNA in situ hybridization. Prostate Cancer Prostatic Dis 24, 220–232 (2021). https://doi.org/10.1038/s41391-020-00272-6

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