Abstract B070: Epithelial-marker absent and disease-marker specific circulating and disseminated tumor cells are rarely detected in men with localized prostate cancer
Authors: Glavaris Stephanie , Chalfin Heather , Lu Changxue , Chen Yan , Caruso Emily , Valkenburg Kenneth , Luo Jun , Pienta Kenneth 
Published: February 2019

Abstract
Introduction and Objectives: Epithelial-marker negative circulating tumor cells (CTCs) have recently been discovered in metastatic prostate cancer (PCa). Many popular CTC detection methods, including the AdnaTest, utilize a selection-based first step of bead pull down against an epithelial marker such as EpCAM. It has previously been shown that disseminated tumor cells (DTCs) are rare at the time of localized PCa using the AdnaTest. Here we queried the matched epithelial-marker depleted buffy coat (DBC) fraction of the AdnaTest for the presence of DTCs and CTCs in a cohort of localized patients undergoing radical prostatectomy (RP).
Methods: 8 mL of bone marrow (BM) were collected from 28 patients (5 controls, 2 metastatic, and 21 localized) along with 8 mL of matched peripheral blood (PB) from 11 patients (3 controls, 2 metastatic, 6 localized). 5 mL of BM and PB were evaluated with the AdnaTest ProstateCancer Select kit (Qiagen) to enrich for EpCAM positive cells using immunomagnetic beads. The DBCs having the remaining cells after AdnaTest enrichment were isolated and queried for the presence of DTCs and CTCs. Cells were lysed and reverse transcribed into cDNA followed by real-time qPCR for the expression of the prostate-specific markers, NKX3.1, androgen receptor (AR), prostate specific antigen (PSA), and HOXB13, as well as the epithelial marker EpCAM. The buffy coats from the remaining 3 mL of nonenriched BM and PB were isolated and stored in -80°C for future evaluation.
Results: EpCAM, NKX3.1, and AR were expressed in localized, control, and metastatic BM and PB and thus were nonspecific to identifying prostate CTCs and DTCs. PSA and HOXB13 positive cells were only identified in the metastatic setting. With the AdnaTest, PSA and HOXB13 positive DTCs were detected in 0/21 (0%) localized patients, but were identified in 2/2 (100%) metastatic samples. Positive PSA and HOXB13 matched in all cases. No controls expressed PSA or HOXB13. The DBC fraction was contaminated with EpCAM positive cells in 27/28 (96%) BM samples in addition to 8/11 (73%) PB specimens. PSA or HOXB13 expression were not detected in any DBC, demonstrating the absence of epithelial-marker negative DTCs. In two instances, EpCAM was detected in the PB depleted fraction but not with the AdnaTest.
Conclusion: EpCAM, NKX3.1, and AR are nonspecific, while PSA and HOXB13 are prostate-specific markers in the PB and BM. PSA and HOXB13 positive prostate CTCs and DTCs are rarely identified in localized men undergoing RP. Epithelial-marker negative DTCs and CTCs were not detected in localized men. The AdnaTest bead selection step did not capture all epithelial cells, and ongoing investigation into a selection-free CTC/DTC detection platform is necessary.
Citation Format: Stephanie Glavaris, Heather Chalfin, Changxue Lu, Yan Chen, Emily Caruso, Kenneth Valkenburg, Jun Luo, Kenneth Pienta. Epithelial-marker absent and disease-marker specific circulating and disseminated tumor cells are rarely detected in men with localized prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B070.