Clinico-pathological correlation of serial measurement of circulating tumor cells in 24 metastatic colorectal cancer patients receiving chemotherapy reveals interpatient heterogeneity correlated with CEA levels but independent of KRAS and BRAF mutation.

BACKGROUND: The Veridex CellSearch is an FDA-approved technology for enumerating circulating tumor cells in blood samples of metastatic colorectal cancer mCRC) patients and has prognostic value. It is important to understand how counts of circulating tumor cells (CTCs), which are advocated to be tools for "liquid biopsy" of tumors, correlate with clinical and pathologic variables of significance in these patients. In this study, we have attempted to make such correlations along with evaluating how CTC counts change during the course of chemotherapy. PATIENTS AND METHODS: Following an IRB-approved protocol, blood samples were collected from 24 patients with mCRC along with relevant clinico-pathological data. Blood was collected at defined time-points both prior to as well as during the course of treatment with combination chemotherapy, and CTC counts were enumerated from 7.5 ml of blood. RESULTS: Seventeen out of 24 patients with mCRC showed a CTC count of 2 or less cells in 7.5 ml of blood at base-line assessment before chemotherapy while 7 patients showed 3 or more cells in 7.5 ml of blood at that point. A correlation was found between high carcino-embryonic antigen (CEA) levels and high CTC counts (P = 0.018) although it was also found that some patients had elevated CTCs without an elevated CEA. No correlation with the time interval between detection of primary tumor and appearance of secondary (metastatic) tumor(s) was found. CTC counts did not correlate with the presence of lung or liver metastases, i.e. a number of mCRC patients with lung or liver metastases had a count of zero CTCs at baseline. We also noted no correlation between CTC number and the status of KRAS or BRAF mutation. CTC counts dropped immediately after the start of chemotherapy in 11 out of 21 patients, and also reduced from the baseline at the end of chemotherapy in 5 out of 10 patients. Six of 7 patients who started with 3 or more CTCs in 7.5 ml at baseline also showed a final CTC reduction at the end of the therapy assessment. CONCLUSIONS: 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.

Circulating tumor cell levels are elevated in colorectal cancer patients with high tumor burden in the liver.

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 EpCAM+ CTCs were analyzed with the FDA-approved CellSearch® 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® 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.

Circulating tumor cell isolation during resection of colorectal cancer lung and liver metastases: a prospective trial with different detection techniques.

BACKGROUND: Colorectal cancer (CRC) metastasectomy improves survival, however most patient develop recurrences. Circulating tumor cells (CTCs) are an independent prognostic marker in stage IV CRC. We hypothesized that CTCs can be enriched during metastasectomy applying different isolation techniques. METHODS: 25 CRC patients undergoing liver (16 (64%)) or lung (9 (36%)) metastasectomy were prospectively enrolled (clinicaltrial.gov identifier: NCT01722903). Central venous (liver) or radial artery (lung) tumor outflow blood (7.5 ml) was collected at incision, during resection, 30 min after resection, and on postoperative day (POD) 1. CTCs were quantified with 1. EpCAM-based CellSearch® system and 2. size-based isolation with a novel filter device (FMSA). CTCs were immunohistochemically identified using CellSearch®'s criteria (cytokeratin 8/18/19+, CD45- cells containing a nucleus (DAPI+)). CTCs were also enriched with a centrifugation technique (OncoQuick®). RESULTS: CTC numbers peaked during the resection with the FMSA in contrast to CellSearch® (mean CTC number during resection: FMSA: 22.56 (SEM 7.48) (p = 0.0281), CellSearch®: 0.87 (SEM ± 0.44) (p = 0.3018)). Comparing the 2 techniques, CTC quantity was significantly higher with the FMSA device (range 0-101) than CellSearch® (range 0-9) at each of the 4 time points examined (P < 0.05). Immunofluorescence staining of cultured CTCs revealed that CTCs have a combined epithelial (CK8/18/19) and macrophage (CD45/CD14) phenotype. CONCLUSIONS: 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.

Apoptotic circulating tumor cells (CTCs) in the peripheral blood of metastatic colorectal cancer patients are associated with liver metastasis but not CTCs.

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.

Flexible micro spring array device for high-throughput enrichment of viable circulating tumor cells.

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 10(4) enrichment, and better than 80% viability from 7.5-mL whole blood samples in <10 min on a 0.5-cm(2) 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.

Novel antineoplastics targeting genetic changes in colorectal cancer.

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.