Abstract 1443: Improved molecular characterization of circulating tumor cells.
Authors: Rao Chandra , Foulk Brad , Nielsen Karl , Vaidya Bhavesh , Patel Jaymala , Gaffney Dana , Smirnov Denis , Karkera Jayaprakash , Connelly Mark 
Published: August 2014

Abstract
Circulating tumor cells (CTCs) of epithelial origin are present in the blood of carcinoma patients at very low frequency (&lt; 10 / ml blood). In order to characterize CTCs, they need to be enriched from large volumes of blood (7.5 mLs). The CELLSEARCH® Circulating Tumor Cell (Epithelial) assay uses magnetic particles conjugated to anti-epithelial cell adhesion molecule (EpCAM) to selectively capture CTCs. After enrichment, the sample contains CTCs and a number (1000 - 5000) of white blood cells (WBCs). The presence of WBCs in the CTC enriched sample may interfere with the molecular analysis of CTC biomarkers that may be co-expressed by WBCs. We have improved the molecular characterization of CTCs by selectively depleting WBCs following the enrichment of CTCs. Normal donor blood was spiked with tumor cell lines and the CTCs enriched using the CELLSEARCH® Profile Kit. Contaminating WBCs were labeled with antibody to the leukocyte marker CD45 conjugated to fluorescein isothiocyanate (FITC). Samples were then incubated for 1 hour in microtiter plates coated with anti-FITC antibody. The WBCs bound to the anti-FITC on the microtiter plates and the unbound cells, containing the CTCs, were removed with the supernatant fluids. The unbound cells were counted and analyzed by flow cytometry and molecular methods. Flow cytometric results show greater than 85% of the CTCs were recovered in the supernatant fluids, while greater than 80% of WBCs had been depleted; demonstrating a substantial improvement in CTC purity. For molecular analysis, normal donor blood was spiked with a range (0, 10, 50, or 150) of Vertebral-Cancer of the Prostate (VCaP) cells and enriched as above using the CELLSEARCH® Profile Kit. Enriched cells from parallel samples were either lysed immediately for RNA isolation (non-depleted sample) or subjected to the CD45 depletion method (depleted samples) and then lysed for RNA isolation. To measure efficacy of depletion, RNA was also isolated from the cells that adhered to the microtiter plate during depletion (adherent fraction). RT-PCR results of the WBC specific markers PTPRC (CD45) and BST1 in WBC depleted samples show differences in cycle threshold (Δ Ct) values of 2.5 and 2.4 PCR cycles, respectively, compared to non-depleted samples. These measurements correspond to ∼80% reduction in WBC specific RNA load and are consistent with the flow cytometric observations. VCaP cell specific markers TMPRSS2:ERG and AR show Δ Ct values of only 0.04 and 0.02 cycles (or ∼ 3% reduction in VCaP-specific RNA load). This demonstrates that the cell population following WBC removal is significantly depleted of WBC with minimal loss of cancer cells. The method thus has a potential to improve detection of tumor-specific markers in clinical samples enriched for the presence of circulating tumor cells.
Citation Format: Chandra Rao, Brad Foulk, Karl Nielsen, Bhavesh Vaidya, Jaymala Patel, Dana Gaffney, Denis Smirnov, Jayaprakash Karkera, Mark Connelly. Improved molecular characterization of circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1443. doi:10.1158/1538-7445.AM2013-1443