Abstract 1929: A sensitive cell-based assay for the evaluation of prostate cancer cells in urine.
Authors: Nickens Kristen , Tan Shyh-Han , Ravindranth Lakshmi , Ali Amina , McLeod David G. , Srivastava Shiv , Petrovics Gyorgy 
Published: August 2014

Abstract
Introduction: The current prostate cancer (CaP) screening in the United States includes the prostate specific antigen (PSA) test and the digital rectal exam (DRE). Abnormal results lead to a prostate biopsy, pathologic evaluation and diagnosis. The PSA test has high sensitivity, but low specificity, leading to a high percentage of unnecessary biopsies. Therefore, there remains an urgent need for a specific and sensitive, non-invasive assay for CaP detection. Our objective was to develop an assay for the detection of CaP cells in post-DRE urine.
Methods: Urine CaP (UCaP) assay development was performed by spiking CaP cell lines (VCaP, LNCaP, and NCI-H660) into normal urine and optimizing cell stability, recovery, and biomarker detection by immunohistochemistry (IHC). Cells were recovered from the urine by translucent membrane filtration. Using the filter as the assay platform, retained cells were assessed for the detection of selected protein markers (ETS related gene [ERG]-CaP marker; alpha-methylacyl-CoA racemase [AMACR]-CaP marker; PSA-normal prostate marker). As a clinical assessment, post-DRE urine from patients undergoing diagnostic biopsy was analyzed for the presence of CaP cells.
Results: Assay sensitivity was enhanced by maximizing the percentage of cells recovered following filtration. To assess enhanced cell recovery of the UCaP assay over currently reported methods (cytospin, centrifugation), low numbers of cultured prostate cancer cells (10 or 100 cells) were spiked into centrifuged urine samples. Cells captured on the filter membrane were stained with DAPI nuclear dye and counted. Between 70% and 85% of cells were recovered after spiking VCaP, LNCaP and NCI-H660 CaP cells. Cells were recovered 100% of the time, even when spiking in as little as a single cell. This is in contrast to published cell recovery studies that require at least 1,000 cells spiked into urine for consistent detection. Furthermore, detection of ERG, AMACR, and PSA protein expression was demonstrated by IHC in cells recovered from post-DRE urine. A feasibility test of ten patients was conducted. Our assay confirmed the presence of CaP marker positive cells in all biopsy positive patients (3 of 3). We also detected the presence of CaP cells in two patients whose biopsy results were negative.
Conclusions: This novel yet simple cell capture platform for CaP cell detection and characterization yielded enhanced assay sensitivity. In a clinical feasibility test the assay confirmed positive biopsy results, and detected CaP cells in some urine specimens potentially missed by biopsy. A larger study is in progress for further evaluations of the UCaP assay.
Citation Format: Kristen Nickens, Shyh-Han Tan, Lakshmi Ravindranth, Amina Ali, David G. McLeod, Shiv Srivastava, Gyorgy Petrovics. A sensitive cell-based assay for the evaluation of prostate cancer cells in urine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1929. doi:10.1158/1538-7445.AM2013-1929