Abstract 1586: Recovery and characterization of circulating tumor cells (CTCs) in cryopreserved metastatic castrate-resistant prostate cancer (mCRPC) patient samples
Authors: Lu David , Harvey Melissa , Madan Ravi , Heery Christopher , Marte Jennifer , Beasley Sharon , Landers Mark , Krupa Rachel , Louw Jessica , Wahl Justin , Bales Natalee , Marrinucci Dena , Schlom Jeffrey , Gulley James , Dittamore Ryan 
Published: August 2015

Abstract
Introduction: Current CTC detection platforms require the processing of patient blood within 48 hrs after collection to preserve CTC integrity and biomarker expression. Here we demonstrate the recovery of CTCs from banked patient blood samples that have been cryopreserved for up to 7 years. Our results show that cryopreserved CTCs have equivalent morphology and protein expression patterns as CTCs in freshly processed blood and are suitable for genomic analyses. This process enables retrospective studies of banked patient blood samples inclusive of CTC enumeration, biomarker quantification, FISH and genomic analysis via the Epic Platform.
Methods: 30 cryopreserved mCRPC patient blood samples were sent to Epic Sciences alongside 10 matched fresh blood draws (&lt;48 h after draw). All nucleated cells from mCRPC patient samples or cell line-spiked healthy donor (HD) controls were plated onto glass slides and subjected to IF staining, CTC identification by fluorescent scanners and algorithmic analysis. Traditional CTCs (CK+, CD45-, intact DAPI nuclei and morphologically distinct) and CK- CTCs (CK-, CD45-, intact and distinct) were identified. Androgen receptor (AR) expression was assessed by IF staining and PTEN status determined by FISH. Relative CTC enumeration, morphology, protein expression and genomic status were compared between freshly processed and cryopreserved samples.
Results: Initial testing in VCaP (AR+) and PC3 (AR-) cell line-spiked HD blood demonstrated preservation of cell morphology and biomarker expression. In VCaP, CK signal decreased 20% and AR decreased 6% after freezing, but CK and AR expression were 90- and 8-fold higher than negative controls, respectively. In patients, CTCs were detected in 7/10 freshly processed samples and in equal relative abundance in 6/10 matched frozen samples. Cryopreserved CTCs retain CK and AR expression as compared to freshly processed samples: there were no significant differences in mean CK or AR expression among all matched samples compared, and% AR+ CTCs correlated strongly between fresh and frozen samples (r2 = 0.96). Furthermore, PTEN FISH on cryopreserved CTCs resulted in a near-equal percentage of PTEN variant cells as compared to matched fresh draws. After concordance studies, an additional 20 frozen mCRPC patient samples were processed: CTCs were detected in 16/20 samples, with 11/20 harboring AR+ CTCs.
Conclusion: Recovery of CTCs in cryopreserved patient blood samples is feasible using the Epic Platform. These data demonstrate concordance of CTC recovery, protein characterization and genomic analysis in an initial cohort of cryopreserved mCRPC samples. Further studies characterizing cryopreserved CTCs in mCRPC and in cancers of other tissues are warranted, with the goal of enabling retrospective protein and genomic characterization of banked patient samples.
Citation Format: David Lu, Melissa Harvey, Ravi Madan, Christopher Heery, Jennifer Marte, Sharon Beasley, Mark Landers, Rachel Krupa, Jessica Louw, Justin Wahl, Natalee Bales, Dena Marrinucci, Jeffrey Schlom, James Gulley, Ryan Dittamore. Recovery and characterization of circulating tumor cells (CTCs) in cryopreserved metastatic castrate-resistant prostate cancer (mCRPC) patient samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2015-1586