Abstract 1383: Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis
Authors: Dong Liang , Zhang Zhongyuan , Lu Changxue , Reyes Diane , Groot Amber de , Amend Sarah R. , Luo Jun , Pienta Kenneth J. 
Published: August 2019

Abstract
Purpose: The presence of circulating tumor cells (CTC) in blood is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. For non-metastatic PCa patients and patients with biochemical recurrence (BCR), it is extremely difficult to find CTCs in 5-10 ml of peripheral blood (PB). The purpose of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by other enrichment technologies to increase the CTC yield by using a large screening blood volume.
Methods: Blood samples were collected from one healthy donor and one mPCa patient. During LP procedure, different blood elements were separated to different layers based on their densities. The mononuclear cell layer (that also contains CTCs) was collected. To assess recovery rate of different CTC isolation and detection methods, the healthy donor LP was spiked with a known number of PCa cell line LNCaP cells. Three CTC isolation and detection methods were used, including FAST (Clinomics, Ulsan, South Korea), a size-selection platform followed by immunofluorescence (IF); the AccuCyte-CyteFinder system (RareCyte, Inc., Seattle, WA), a selection-free method that utilizes IF; the AlereTM CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method followed by RT-qPCR. The criteria for defining a CTC are DAPI+, CK/EpCAM+, and CD45-. The Ct value thresholds for positive CTC signal by RT-qPCR were EpCAM 36.4, PSA 36.3, HOXB13 40, AR 35.4.
Results: For the healthy donor LP with spiked LNCaP cells, the CTC recovery rate from FAST was 53.7%~67.2%. For Adna test, 0, 44, 95 and 1000 LNCaP cells were spiked in 5 ml of LP respectively. The Ct values of EpCAM were 36.28, 29.32, 28.22 and 24.76. For the mPCa patient, two tubes of 7.5 ml of PB were collected for FAST and RareCyte. The FAST found 3 CTCs while the RareCyte found 1 CTC. An LP sample was also collected from the same mPCa patient. The LP sample volume was 162 ml (WBC concentration was 83×109/L). Triplicates of 5 ml of samples were sent through AdnaTest and CTCs were identified in all samples (Ct: EpCAM 34.21±0.77, PSA 33.86±1.81, HOXB13 36.25±0.34, AR 35.29±0.22). Then the remainder of the mPCa LP was subjected to Ficoll-Paque density centrifugation to further concentrate mononuclear cells to a 90 ml volume. 1 ml samples were then applied to FAST in triplicate. The CTC numbers were 22 and 15. The density of cells on one of the filters was too high to be successfully scanned.
Conclusion: Finding CTCs in LP samples by size-based technology or AdnaTest is feasible and offers a promising method to do liquid biopsy for mPCa patients. Screening a larger volume of PB by LP has significantly increased the CTC yield compared to using the same capture technology on 7.5 ml of PB.
Citation Format: Liang Dong, Zhongyuan Zhang, Changxue Lu, Diane Reyes, Amber de Groot, Sarah R. Amend, Jun Luo, Kenneth J. Pienta. Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1383.