Avisnata Das

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.

Identifying Circulating Tumor Stem Cells That Matter: The Key to Prognostication and Therapeutic Targeting

TO THEEDITOR: We read with interest the article by Iinuma et al 1 that describes their study examining the clinical significance of cancer stem cells in the peripheral blood of patients with colorectal cancer (CRC). The defining characteristics of a circulating cancer stem cell (CTSC) are its capacity for self-renewal and for initiation of distant metastases; some of these cells are also resistant to traditional chemo- therapy. 2,3 The concept that circulating tumor stem cells can be iden- tified and targeted is attractive and has major diagnostic, prognostic, and therapeutic implications for patients with metastatic cancer. However, the identification of authentic CTSCs continues to be a challenge because of the lack of a clear understanding of the biologic heterogeneity of tumor stem-cell populations and also because of the lack of characterization of surface markers that predict clinically im- portant subsets of CTSCs, such as those with aggressive malignant potential or drug resistance.

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.

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 from7.5 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.

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.

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 Fishers 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 Fishers 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.

Abstract 5102: Analysis of CTCs enriched from whole blood samples of Breast, Lung and Colorectal cancer patients with a flexible microspring array device.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The process by which metastatic cancers release cells that detach from a primary tumor, spread through the circulatory system and invade distant organs accounts for over 90% of cancer related deaths. A fundamental challenge with analysis of these circulating tumor cells (CTCs) in blood samples is the fact that they are so rare, with only a few tumors cells occurring among billions of blood cells. We have developed a flexible micro spring array (FMSA) that employs mechanical separation to effectively enrich CTCs in a nondestructive manner that does not compromise cell viability. The FMSA device geometric design and applied filtration pressures have been optimized to maximize capture efficiency, enrichment against leukocytes, and tumor cell viability. We have successfully enriched and detected varying numbers of CTCs (min=1, max=101) in 7.5mL whole blood samples obtained from Breast, Lung and Colorectal cancer patients. CTCs were analyzed qualitatively and quantitatively based on immunocytochemical determination of phenotype and cell morphological characteristics. Aggregate clusters of cells were observed in all assayed cancer types that comprised of multiple attached CTCs (min=2, max=20) and occasionally leukocytes as well. Citation Format: Ramdane Harouaka, Ming-Da Zhou, Yin-Ting Yeh, Cristina Truica, Avisnata Das, Jussuf Kaifi, Wafik El-Deiry, Xin Liu, Chandra Belani, Tara Baney, Jeffrey Allerton, Si-Yang Zheng. Analysis of CTCs enriched from whole blood samples of Breast, Lung and Colorectal cancer patients with a flexible microspring array device. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5102. doi:10.1158/1538-7445.AM2013-5102

Abstract 2393: Isolation and characterization of circulating tumor cells in a mouse model of colorectal cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Circulating tumor cells (CTCs) are indicative of disease burden in metastatic colorectal cancer (mCRC) and high counts are believed to adversely affect outcome. However there has been a lack of suitable mouse models to study CTCs in mCRC. Here we report the development of an orthotopic model of mCRC in athymic nude mice from which we have successfully isolated and enumerated CTCs using the FDA-approved Veridex Cellsearch technology (Table 1). So far we have injected 5 mice with a million cells each of the human CRC cell line DLD-1 within the layers of the cecal wall for tumor xenografts to grow. 200 microL of blood has been collected every 2 weeks, till the endpoint of 16 weeks or a humane endpoint, from the medial saphenous vein of the mouse for CTC detection. In our protocol, human leucocyte filters are used as a RBC source. The blood is centrifuged at 800 G for 10 minutes in a CellSave tube, the serum removed and replaced by an equal volume of PBS. Mouse blood is transferred to this tube after collection. Efforts are on to show CTCs from mice injected with tumor cells labeled with a suitable cellular dye like Cellvue. At the endpoint, after humanely euthanizing a mouse, the cecum and organs are analyzed for possible secondary spread (liver, lungs, mesenteric lymph nodes) following resection and preservation. H/E staining is performed for evidence of primary or metastatic tumor tissue. Positive tissue samples are further analysed using immunostaining for stem cell markers and prognostic markers CD133, CD26, ALDH1, Mcl-1, EFGR, Ki67. We are analyzing isolated CTCs for expression of these markers and note the extent of concordance. The mouse model should help to understand the role of CTCs in evaluating metastasis as well as to validate the use of CTCs as tools for “liquid biopsy” of tumors. Table 1. CTC counts in the mice at different time points from injection of cells. Mouse 4 is the negative control mouse of the cohort which received injection of normal saline instead of cancer cell suspension. * denotes clotted sample. ![Figure][1] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2393. doi:1538-7445.AM2012-2393 [1]: pending:yes

Abstract 3818: CTC profiling: Integration of biomarker analysis with current Veridex CellSearch technology

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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