Abstract
Abstract The presence and number of circulating tumor cells (CTCs) in the blood of cancer patients is predictive of clinical outcome. To date only the CellSearch system by Veridex has been FDA approved for use in breast, prostate and colon cancer (cc). However, a limited number of biomarkers are examined in the CellSearch system. In the case of colorectal cancer, cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) expression is used to identify cells as CTCs. Additional analysis of other biomarkers by Cheshire technology is provided as fee for service and only uses the current fluorescent channels utilized by the CellSearch Analyzer. Our aim is to greatly enhance the number of biomarkers that can be tested in an economic and efficient manner and integrating the analysis into the existing CellSearch isolation and analytic platform. We combined Nuance multispectral imaging with quantum dot technology to expand the biomarkers analysis. Quantum dots (QD) have the advantage that they can be excited in the UV wavelengths while emitting across the visible spectrum. Only DAPI in the original CellSearch panel of reagents is excited in this range and its emission does not overlap with the QDs. This allows us to visualize additional biomarkers without interfering with CellSearch CTC analysis. Antibodies against a panel of known biomarkers were labeled with quantum dots. Each antibody was verified by standard immunofluorescence in appropriate tissue culture lines before quantum dot labeling. Antibody labeling and titration was confirmed on tissue culture cell lines before adding it to the panel of CTC staining reagents. To model multiplexing assays, blood is spiked with a known number of cells from appropriate cancer cell lines. Following standard CTC isolation and enumeration, cells are retained in the MagNest and allowed to dry. Cells are then stained with our biomarker panel. Currently 8 quantum dots are commercially available for simultaneous analysis. Our panel of biomarkers is under revision, but includes phospho-AKT, Ki-67, CD26, CD133 and EGFR. We are expanding the panel to include markers for death pathway signaling including Mcl-1, Bcl-XL, Flip, TRAIL, and death receptors. Separate panels are also being developed for breast cancer. Additional panels can include markers for sensitivity to chemotherapeutic reagents (TS, DPD, PTHFR, GSTP1, ERCC1, ERCC2, UGT1A1, BRAF), cell proliferation, known tumor susceptibility genes or tumor markers (APC, p53, HER2, PSA, IGF1-R) or probes for specific signal transduction pathways (NFκB, Wnt). Analysis of samples from patients undergoing chemotherapy -/+ targeted therapy is underway to evaluate the status of the stem cell, prognostic markers and cell death determinants in individual CTCs as a function of time. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3818. doi:10.1158/1538-7445.AM2011-3818
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