Abstract B51: ApoStream™ isolated circulating tumor cells from primary breast cancer patients reveals heterogeneous phenotypes related to epithelial-mesenchymal transition and stem cell markers.
Authors: Anderes Kenna L. , Jafferji Insiya , Melnikova Vladislava O. , Summer Jackson A. , Davis Darren W. , Reuben James M. , Ueno Naoto T. 
Published: August 2014

Abstract
Background: Detection of circulating tumor cells (CTCs) is an indicator of poor prognosis in patients with metastatic breast cancer and not in primary breast cancer (PBC). The classical phenotypic definition of a CTC is a nucleated cell that is cytokeratin (CK) positive but CD45 negative. Several reports have shown that EpCAM based capture methods detect only a fraction of CTCs and not the heterogeneous subpopulations of CTCs. Moreover, subsets of CTCs may acquire a more aggressive phenotype with features of invasiveness and motility by undergoing an epithelial to mesenchymal transition (EMT) and down regulate the epithelial cell adhesion molecule, EpCAM. EMT is a hallmark of cellular invasion and metastasis and CTCs undergoing EMT may express the putative cancer stem cell like phenotype, CD24lowCD44+. CTCs undergoing EMT (CTC-EMT) are not readily detected by current CTC detection technologies. Thus, it is desirable to isolate CTCs using capture methods independent of EpCAM to recover a heterogeneous CTC population for more extensive characterization. Here we use ApoStream™, a novel antibody-free CTC isolation device that does not rely on EpCAM to capture circulating rare cells, to evaluate the molecular heterogeneity of CTCs.
Methods: Baseline blood samples from 14 newly diagnosed PBC patients were collected and processed using ApoStream™. Isolated cells were stained with anti-CK and anti-CD45, and DAPI. In addition, a multiplexed immunofluorescence assay and laser scanning cytometry analysis were applied to identify multiple combinations of CK+CD45− cells for the expression and distribution of EpCAM, vimentin, CD44, CD24, β-catenin and E-cadherin.
Results: ApoStream™ recovered both EpCAM+ and EpCAM− cells. CK+CD45− cells were detected in 9 out of 14 PBC patients. The expression of EpCAM− vimentin+ in the CK+CD45− population was heterogeneous across the patient population. E-cadherin and β-catenin were detected in 0-94% (Mean 52 %) and 0-37% (Mean 8 %), respectively of the CK+CD45− population. All patients with CK+CD45− cells had a subset of cells with the putative phenotype of CD44+CD24low cells.
Conclusions: Heterogeneous CTC phenotypes with CD44+CD24lowin both EpCAM+ and EpCAM− cells were observed in patients with PBC. Our aim is to correlate ApoStream™ EMT-CTC counts in patients with PBC with the pathological clinical response (pCR). This study will continue to enroll PBC patients and test the hypothesis that low EMT-CTC count patients have higher pCR rates compared to high EMT-CTC count patients.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B51.
Citation Format: Kenna L. Anderes, Insiya Jafferji, Vladislava O. Melnikova, Jackson A. Summer, Darren W. Davis, James M. Reuben, Naoto T. Ueno. ApoStream™ isolated circulating tumor cells from primary breast cancer patients reveals heterogeneous phenotypes related to epithelial-mesenchymal transition and stem cell markers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B51.