Abstract 4959: Multi-level analysis of prostate cancer circulating tumor cells allowing IHC-based identification, 6-parameter fluorescence phenotyping, and individual cell molecular analysis
Authors: Campton Daniel , Needham Rachel , Nordberg Josh , Ramirez Arturo , Drovetto Nick , Clein Alisa , Sabath Daniel E. , Stilwell Jackie , Kaldjian Eric 
Published: July 2016

Abstract
Background. Analysis of circulating tumor cells (CTC) allows non-invasive investigation of prostate cancer biology and response to treatment. The primary level of analysis is the CTC count, which has been demonstrated to be prognostic of outcome. Deeper characterization of CTC phenotype and pertinent biomarkers can confirm cancer lineage and identify drug targets or drug-resistance markers. Single cell analysis of individual CTCs can provide genomic insight into cancer heterogeneity. RareCyte has developed AccuCyte® - CyteFinder® (AC-CF), an integrated technology platform for highly sensitive visual identification and retrieval of rare cells in blood by both immunohistochemistry (IHC) and immunofluorescence (IF) staining. Recently we have developed technology allowing 6-marker assays for broader phenotypic analysis.
Methods. Normal human whole blood samples were spiked with prostate cancer lines as model CTCs (mCTCs). Blood samples from University of Washington patients with advanced prostate cancer were collected under an IRB-approved protocol. Blood was processed using AccuCyte and the nucleated cell fraction was collected and spread onto microscope slides. Slides were stained on an automated stainer using (1) an IHC assay for cytokeratin, (2) a standard 4-wavelength IF assay (DAPI, CD45, cytokeratin and EpCAM) or (3) a novel 6-parameter IF assay using SYTOX-Orange (nuclear stain), cytokeratin, EpCAM, androgen receptor (AR), prostate-specific membrane antigen (PSMA) and CD45. An assay for AR variant 7 (ARv7) was applied to samples with mCTCs with the ARv7 splice variant. Percent recovery of IHC-stained slides (by blinded pathologist review) was compared to IF-stained slides (by CyteFinder image analysis). Individual IHC-stained CTCs were retrieved after on-slide visual identification and re-visualized after dispensing for confirmation. Whole genome amplification (WGA) of retrieved cells was performed, followed by X- and Y-chromosome gene-specific PCR.
Results. There was strong linear correlation between IF and IHC counts of mCTCs over a range of ∼25 - 100 cells/mL (R2 = 0.99). The 6-parameter IF assay was successfully applied to mCTC and clinical samples. AR and PSMA were co-expressed in the majority of epithelial-marker positive clinical CTCs. The ARv7 assay identified mCTCs that express the splice variant. Individual IHC-stained mCTCs spiked into female donor blood were demonstrated to be male after WGA and PCR.
Conclusions. Light microscope identification of IHC cytokeratin-positive mCTCs approximated IF identification. 6-parameter phenotyping of prostate cancer CTCs is feasible and allows identification of lineage-specific markers. IHC-stained cells can be individually retrieved from slides for genome amplification and molecular analysis.
Citation Format: Daniel Campton, Rachel Needham, Josh Nordberg, Arturo Ramirez, Nick Drovetto, Alisa Clein, Daniel E. Sabath, Jackie Stilwell, Eric Kaldjian. Multi-level analysis of prostate cancer circulating tumor cells allowing IHC-based identification, 6-parameter fluorescence phenotyping, and individual cell molecular analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4959.