BACKGROUND The dissemination of circulating tumor cells (CTCs) that cause metastases in distant organs accounts for the majority of cancer-related deaths. CTCs have been established as a cancer biomarker of known prognostic value. The enrichment of viable CTCs for ex vivo analysis could further improve cancer diagnosis and guide treatment selection. We designed a new flexible micro spring array (FMSA) device for the enrichment of viable CTCs independent of antigen expression. METHODS Unlike previous microfiltration devices, flexible structures at the micro scale minimize cell damage to preserve viability, while maximizing throughput to allow rapid enrichment directly from whole blood with no need for sample preprocessing. Device performance with respect to capture efficiency, enrichment against leukocytes, viability, and proliferability was characterized. CTCs and CTC microclusters were enriched from clinical samples obtained from breast, lung, and colorectal cancer patients. RESULTS The FMSA device enriched tumor cells with 90% capture efficiency, higher than 104 enrichment, and better than 80% viability from 7.5-mL whole blood samples in <10 min on a 0.5-cm2 device. The FMSA detected at least 1 CTC in 16 out of 21 clinical samples (approximately 76%) compared to 4 out of 18 (approximately 22%) detected with the commercial CellSearch® system. There was no incidence of clogging in over 100 tested fresh whole blood samples. CONCLUSIONS The FMSA device provides a versatile platform capable of viable enrichment and analysis of CTCs from clinically relevant volumes of whole blood.

Blood sampling during CRC metastasis resection is an opportunity to increase CTC capture efficiency. CTC isolation with the FMSA yields more CTCs than the CellSearch® system. Future studies should focus on characterization of single CTCs to identify targets for molecular therapy and immune escape mechanisms of cancer cells.

Enumeration of circulating tumor cells (CTCs) by the CellSearch system provides prognostic information in metastatic colorectal cancer, regardless of metastatic site. We found that CTCs generally represent <1% of observed events with CellSearch analysis and adapted scoring criteria to classify other peripheral blood events. Examination of twenty two metastatic colorectal cancer patients' blood revealed that patients with high CEA or liver metastases, but not lung or distant lymph node metastases, possessed significant numbers of apoptotic CTCs prior to treatment initiation by Fischer's exact test. Six out of eleven patients with liver metastasis possessed apoptotic CTCs whereas one of nine patients with other metastases had measurable apoptotic CTCs. An elevated CTC number was not necessarily associated with apoptotic CTCs or CTC debris by Spearman's correlation, suggesting the metastatic site rather than CTCs per se as contributing to the origin of these events.

Background: Metastatic spread is the most common cause of cancer-related death in colorectal cancer (CRC) patients, with the liver being the mostly affected organ. Circulating tumor cells (CTCs) are a prognostic marker in stage IV CRC. We hypothesized that tumor burden in the liver correlates with CTC quantity. Methods: Blood (7.5 ml) was prospectively collected from 24 patients with novel stage IV CRC diagnosis. Baseline EpCAMC CTCs were analyzed with the FDA-approved CellSearch Ò system. Clinicopathological data were collected, and hepatic tumor burden was determined by radiographic liver volumetry with contrast-enhanced CT scans. CRC primary tumors were immunohistochemically stained for EpCAM expression with BerEP4 monoclonal antibody. Statistical analyses were performed using 2-sample T-test, non-parametric Wilcoxon Rank-Sum test, and Fisher's exact test. Results: CTCs were detected n 17 (71%) of 24 patients. The overall mean CTC number as determined by EpCAM-based CellSearch Ò detection was 6.3 (SEM 2.9). High baseline CTC numbers (3) correlated significantly with a high tumor/liver ratio (30%), and with high serum CEA levels, as determined by two-sample T-test on log-transformed data and by Fisher's Exact test on categorical data analysis (P < 0.05). The CRC primary tumors were consistently expressing EpCAM by immunostaining. Conclusions: High tumor burden in the liver and high baseline serum CEA levels are associated with high number of baseline CTCs in stage IV CRC patients. Future studies should further investigate the biological role and expression patterns of single CTCs in cancer patients to further improve personalized treatment strategies.

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Analysis of circulating tumor cells may be of use in monitoring response to therapy in mCRC, either in combination with CEA monitoring or alone when CTCs are elevated but CEA level is not.

Cytotoxic chemotherapy remains the mainstay of the medical -management of colorectal cancer (CRC). Research over the last two decades has led to a molecular understanding of the oncogenic mechanisms involved in CRC and has contributed to the rational development of antineoplastics that target these mechanisms. During carcinogenesis, genetic changes often occur in molecules that play key functional roles in cancer such as cell proliferation, angiogenesis, apoptosis, cell death and immune-mediated destruction of cancer cells. Here, we review novel antineoplastics that are approved or in development for CRC that target molecules associated with genetic aberrations in CRC. Some of these targeted antineoplastics have proven effective against other solid tumors and hold promise in treating CRC whereas others are now routinely used in combination with cytotoxic agents. This article reviews antineoplastics that target genetic changes in CRC, their antitumor mechanisms, and their stage of development.

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