A novel method for downstream characterization of breast cancer circulating tumor cells following CellSearch isolation

BACKGROUND: Enumeration of circulating tumor cells (CTCs) obtained from minimally invasive blood samples has been well established as a valuable monitoring tool in metastatic and early breast cancer, as well as in several other cancer types. The gold standard technology for detecting CTCs in blood against a backdrop of millions of leukocytes is the FDA-approved CellSearch system (Janssen Diagnostics), which relies on EpCAM-based immunomagnetic separation. Secondary characterization of these cells could enable treatment selection based on specific targets in these cells, as well as providing a real time window into the metastatic process and offering unique insights into tumor heterogeneity. The objective of this study was to develop a method for downstream characterization of CTCs following isolation with the CellSearch system. METHODS: An in vitro CTC model system focusing on clinically useful treatment predictive biomarkers in breast cancer, specifically the estrogen receptor α (ERα) and the human epidermal growth factor receptor 2 (HER2), was established using healthy donor blood spiked with breast cancer cell lines MCF7 (ERα(+)/HER2(−)) and SKBr3 (ERα(−)/HER2(+)). Following CTC isolation by CellSearch, the captured CTCs were further enriched and fixed on a microscope slide using the in-house-developed CTC-DropMount technique. RESULTS: The recovery rate of CTCs after CellSearch Profile analysis and CTC-DropMount was 87%. A selective and consistent triple-immunostaining protocol was optimized. Cells positive for DAPI, cytokeratin (CK) 8, 18 and 19, but negative for the leukocyte-specific marker CD45, were classified as CTCs and subsequently analyzed for ERα and HER2 expression. The method was verified in breast cancer patient samples, thus demonstrating its clinical relevance. CONCLUSIONS: Our results show that it is possible to ascertain the status of important predictive biomarkers expressed in breast cancer CTCs using the newly developed CTC-DropMount technique. Downstream characterization of multiple biomarkers using a standard fluorescence microscope demonstrates that important clinical and biological information may be obtained from a single patient blood sample following either CellSearch epithelial or profile analyses. TRIAL REGISTRATION: Clinical Trials NCT01322893