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. 2015 Jul 17:5:12270.
doi: 10.1038/srep12270.

The detection of EpCAM(+) and EpCAM(-) circulating tumor cells

Sanne de Wit  1 , Guus van Dalum  1 , Aufried T M Lenferink  1 , Arjan G J Tibbe  2 , T Jeroen N Hiltermann  3 , Harry J M Groen  3 , Cees J M van Rijn  4 , Leon W M M Terstappen  1
Affiliations

The detection of EpCAM(+) and EpCAM(-) circulating tumor cells

Sanne de Wit et al. Sci Rep. .

Abstract

EpCAM expressing circulating tumor cells, detected by CellSearch, are predictive of short survival in several cancers and may serve as a liquid biopsy to guide therapy. Here we investigate the presence of EpCAM(+) CTC detected by CellSearch and EpCAM(-) CTC discarded by CellSearch, after EpCAM based enrichment. EpCAM(-) CTC were identified by filtration and fluorescent labelling. This approach was validated using different cell lines spiked into blood and evaluated on blood samples of 27 metastatic lung cancer patients. The majority of spiked EpCAM(+) cells could be detected with CellSearch, whereas most spiked cells with EpCAM(low) or EpCAM(-) expression were detected using filtration. Five or more CTC were detected in 15% of the patient samples, this increased to 41% when adding the CTC detected in the discarded blood. The number of patients with CTC and the number of CTC detected were doubled by the presence of EpCAM(-) CTC. In this pilot study, the presence of EpCAM(+) CTC was associated with poor outcome, whereas the EpCAM(-) CTC were not. This observation will need to be confirmed in larger studies and molecular characterization needs to be conducted to elucidate differences between EpCAM(-) and EpCAM(+) CTC.

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Conflict of interest statement

Sanne de Wit, Guus van Dalum, Aufried Lenferink, Jeroen Hiltermann and Harry Groen have no conflict of interests to declare. Arjan Tibbe, Cees van Rijn and Leon Terstappen are shareholders of VyCAP BV. Leon Terstappen is a consultant of Janssen Diagnostics and his department MCBP of the University of Twente receives research funding from Janssen Diagnostics.

Figures

Figure 1
Figure 1. A schematic representation of the waste collection and filtration, followed by analysis of the microsieve with CTC.
The Automatic Sample Collection Device collects the blood discarded after immunomagnetic enrichment of EpCAM+ cells by CellSearch Autoprep (A). A pump with disposable filtration unit containing a slide with a microsieve filters the discarded blood (B). The staining of the cells is performed directly on the filter (C) by adding a staining cocktail (1), incubating (2) and removing the liquid by bringing the sieve in contact with an absorbing body (3 and 4). The microsieve is analyzed using fluorescence microscopy for detection of CTC (5).
Figure 2
Figure 2. Thumbnail gallery of CTC and leukocytes in a lung cancer patient identified by CellSearch and in the blood discarded by CellSearch after filtration.
Figure 3
Figure 3. Kaplan-Meier curves for overall survival for CTC subpopulations with a cut-off of 1 CTC or more.
Panel A; EpCAM+, CK 8,18+ or 19+ CTC detected by CellSearch. Panel B; EpCAM+, panCK+ CTC detected by CellSearch. Panel C; EpCAM–, panCK+ CTC after filtration of blood discarded by CellSearch (CS). Panel D; all populations of EpCAM+, panCK+ CTC and EpCAM–, panCK+ CTC.
See this image and copyright information in PMC

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