mRNA expression profiles in circulating tumor cells of metastatic colorectal cancer patients

The molecular characterization of circulating tumor cells (CTCs) is a promising tool for the repeated and non-invasive evaluation of predictive and prognostic factors. Challenges associated with CTC characterization using the only FDA approved method for CTC enumeration, the CellSearch technique, include the presence of an excess of leukocytes in CTC-enriched blood fractions. Here we aimed to identify colorectal tumor-specific gene expression levels in the blood of patients with and without detectable CTCs according to CellSearch criteria. Materials and methods: Blood of 30 healthy donors (HDs) and 142 metastatic colorectal cancer (mCRC) patients was subjected to CellSearch CTC enumeration and isolation. In all samples, 95 mRNAs were measured by reverse transcriptase quantitative PCR (RT-qPCR). HD blood samples and patient samples with three or more CTCs were compared to identify CTC-specific mRNAs. Patient samples without detectable CTCs were separately analyzed. Results: Thirty-four CTC-specific mRNAs were higher expressed in patients with ≥3 CTCs compared with HDs (Mann-Whitney U-test P<0.05). Among patients without detectable CTCs, a HD-unlike subgroup was identified which could be distinguished from HDs by the expression of epithelial genes such as KRT19, KRT20 and AGR2. Also, in an independent patient set, a similar HD-unlike group could be identified among the patients without detectable CTCs according to the CellSearch system. Conclusion: Extensive molecular characterization of colorectal CTCs is feasible and a subgroup of patients without detectable CTCs according to CellSearch criteria bears circulating tumor load, which may have clinical consequences. This CTC-specific gene panel for mCRC patients may enable the exploration of CTC characterization as a novel means to further individualize cancer treatment

Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells

Identification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management. However, to be effective, the method used to identify circulating tumor cells must detect all tumor cell types. We investigated whether the five subtypes of human breast cancer cells that have been defined by global gene expression profiling—normal-like, basal, HER2-positive, and luminal A and B—were identified by CellSearch, a US Food and Drug Administration–approved test that uses antibodies against the cell surface–expressed epithelial cell adhesion molecule (EpCAM) to isolate circulating tumor cells. We used global gene expression profiling to determine the subtypes of a well-defined panel of 34 human breast cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single healthy human donor, and the mixtures were subjected to the CellSearch test to isolate the breast cancer cells. We found that the CellSearch isolation method, which uses EpCAM on the surface of circulating tumor cells for cell isolation, did not recognize, in particular, normal-like breast cancer cells, which in general have aggressive features. New tests that include antibodies that specifically recognize normal-like breast tumor cells but not cells of hematopoietic origin are needed

Detection of circulating tumor cells in breast cancer may improve through enrichment with anti-CD146

Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, "normal-like" cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [J Natl Cancer Inst 101(1):61-66, 2009]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch (TM) technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients

CD49f-based selection of circulating tumor cells (CTCs) improves detection across breast cancer subtypes

Circulating tumor cells (CTCs) can be enumerated using CellSearch, but not all breast cancer subtypes, specifically those with epithelial-mesenchymal transition (EMT) characteristics, sufficiently express the enrichment (EpCAM) and selection (CK8/18/19) markers used in this method. While CD146 can detect EpCAM-negative CTCs, we here evaluated the value of various cytokeratins and CD49f to detect CK8/18/19-negative CTCs. The tested cytokeratins provided no substantial benefit, but adding CD49f to CK8/18/19 as a selection marker resulted in improved recovery of normal-like cell lines. Combined staining of CK8/18/19 and CD49f after CD146/EpCAM enrichment is likely to further improve CTC detection in breast cancer. (c) 2011 Elsevier Ireland Ltd. All rights reserved

Generation of mRNA and miRNA gene expression profiles in circulating tumor cells of metastatic colorectal cancer patients

Abstract Patients with solitary resectable liver metastases might benefit from curative liver resection, but unfortunately, half of the patients relapse within one year after this major surgery. Reliable prognostic factors are urgently needed to identify those patients benefitting from liver metastasis resection. Circulating tumor cells (CTCs) have been identified as a powerful prognostic marker in metastatic colorectal cancer. In addition to enumeration, CTCs can be enriched for subsequent molecular characterization. In this sense, CTCs are a very promising diagnostic and prognostic tool enabling the repeated and non-invasive evaluation of the expression of drug targets, predictive and prognostic factors at the time of metastasis resection. Here, we describe the identification of a CTC-specific mRNA and miRNA profile generated on CTC-enriched fractions isolated from blood drawn from metastatic CRC patients at the time of liver metastasis resection, and their correlation with clinical parameters. In this study we included 161 metastatic colorectal cancer patients who were about to undergo liver metastasis resection and 30 healthy donors (HD). CTCs were isolated from 30 mL blood using the CellSearch Profile Kit (Veridex LLC) after a modified Ficoll enrichment, and HD blood was subjected to the same procedure. Total RNA was isolated from the enriched CTC fraction and a panel of mRNAs and miRNAs with potential clinical relevance was measured in CTCs and in enriched HD blood to identify CTC-specific genes. Gene expression levels of CTC-specific mRNAs and miRNAs were correlated with clinical parameters. From 100 of these patients, the liver resection indeed showed pathology-confirmed metastasis of colorectal origin and both mRNA and miRNA was of sufficient quality. These 100 patients (mean age 63, range 40 - 85) had a median CTC count of 1 (range 0 - 35) per 30 mL blood. Most patients (63%) presented with synchronous metastatic disease and median time between primary tumor resection and metastasis was 313 days (range 0 - 2486). Just over half of patients (52%) had received induction chemotherapy before liver surgery. Out of 96 mRNAs and 45 miRNAs, we were able to identify a panel of CTC-specific mRNAs and miRNAs, of which the transcripts were more abundantly expressed in patients with β2 CTCs compared to 30 healthy donors (Mann-Whitney U-test P&amp;lt;0.05). Among the differentially expressed were, besides epithelial-specific genes, genes involved in cell differentiation, proliferation and inflammation. Associations between CTC gene expression and clinicopathologic characteristics will be presented at the meeting. In this study, we show that it is feasible to molecularly characterize rare CTCs from colorectal cancer patients in a background of leukocytes, a promising application which will greatly increase the amount of information that can be obtained from the CTCs of colorectal cancer patients. 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 3409. doi:1538-7445.AM2012-3409</jats:p