Abstract 1584: Analytical and clinical validation of the Epic Circulating Tumor Cell (CTC) Platform: enrichment-free CTC detection and biomarker characterization
Authors: Graf Ryon P. , Valenta David T. , Landers Mark , Taggart Bryan , Greene Stephanie , Werner Shannon L. , Dittamore Ryan , Marrinucci Dena 
Published: August 2015

Abstract
Introduction:
CTCs hold promise as prognostic and predictive biomarkers for cancer management. Many CTC technologies enrich CTCs based on cell surface markers or physical parameters such as cell size. However, genomic and phenotypic intra-tumor heterogeneity is well documented and similarly observed in CTCs. We sought to validate the Epic Platform, an enrichment-free approach, for CTC identification and biomarker characterization. In addition, we sought to enumerate and molecularly characterize CTCs in Metastatic Castrate Resistant Prostate Cancer (mCRPC) patients.
Experimental Procedures:
To assess intra/inter-assay and -operator reproducibility, cell line cells (CLCs) were spiked into healthy donor (HD) blood and processed onto slides (∼0.5mL/slide) in dilutions ranging from 6-300 CLCs/slide and assessed by%CV of CLC counts in replicate samples. Accuracy and linearity were assessed via linear regression and slope of all dilutions. CLCs were also molecularly characterized by immunofluorescence (IF), FISH, and NGS.
To test clinical sensitivity, ∼1mL of blood from 28 mCRPC patients was processed. CTCs (CK+, CD45-, DAPI+) and CTC subtypes including apoptotic (CK+, CD45-, fragmented DAPI), and CK- (CK-, CD45-, DAPI+) were enumerated. Androgen receptor (AR) expression was quantitated on each CTC. A subset of samples were further analyzed for ERG rearrangements via FISH and whole genome copy number variation (CNV) by NGS. In addition, 18 HDs were tested to assess clinical specificity.
Results:
Intra/inter-assay and operator reproducibility across 3 runs each produced ≤ 20% CV at 300 CLC/slide and ≤ 30% CV at 25 CLC/slide. The CTC enumeration assay demonstrated accuracy, linearity and sensitivity throughout all dilutions tested, 6-300 CLC/mL, (x = 1.15, r2 = 0.9987,%CV ≤ 30%). CLCs exhibited expected biomarker expression (IF) and genetic aberrations (FISH and NGS).
No CTCs or CTC subtypes were detected in HD samples. At least 1 CTC/mL was detected in 82% of mCRPC patient samples. Including CK- and apoptotic CTCs, 100% of mCRPC patients had at least 1 CTC/mL. Analysis of AR expression/localization, ERG rearrangements by FISH, and whole genome CNV analyses were consistent with malignant origin.
Conclusions:
We established the reproducibility, linearity, accuracy, and sensitivity of the Epic Platform. CTCs and CTC subtypes were absent in HDs and present in all mCRPC samples tested. We also demonstrated integrated IF, FISH, and NGS characterization of single CTCs, which has powerful implications for improved patient selection for targeted therapy and could enable a better understanding of intra-patient heterogeneity, clonal evolution, and drug resistance and response over time.
Citation Format: Ryon P. Graf, David T. Valenta, Mark Landers, Bryan Taggart, Stephanie Greene, Shannon L. Werner, Ryan Dittamore, Dena Marrinucci. Analytical and clinical validation of the Epic Circulating Tumor Cell (CTC) Platform: enrichment-free CTC detection and biomarker characterization. [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 1584. doi:10.1158/1538-7445.AM2015-1584