Subset Of Circulating Tumor Cells Research Articles

Subclassification of prostate cancer circulating tumor cells by nuclear size reveals very small nuclear circulating tumor cells in patients with visceral metastases.

Although enumeration of circulating tumor cells (CTCs) has shown some clinical value, the pool of CTCs contains a mixture of cells that contains additional information that can be extracted. The authors subclassified CTCs by shape features focusing on nuclear size and related this with clinical information. A total of 148 blood samples were obtained from 57 patients with prostate cancer across the spectrum of metastatic states: no metastasis, nonvisceral metastasis, and visceral metastasis. CTCs captured and enumerated on NanoVelcro Chips (CytoLumina, Los Angeles, Calif) were subjected to pathologic review including nuclear size. The distribution of nuclear size was analyzed using a Gaussian mixture model. Correlations were made between CTC subpopulations and metastatic status. Statistical modeling of nuclear size distribution revealed 3 distinct subpopulations: large nuclear CTCs, small nuclear CTCs, and very small nuclear CTCs (vsnCTCs). Small nuclear CTCs and vsnCTC identified those patients with metastatic disease. However, vsnCTC counts alone were found to be elevated in patients with visceral metastases when compared with those without (0.36 ± 0.69 vs 1.95 ± 3.77 cells/mL blood; P<.001). Serial enumeration studies suggested the emergence of vsnCTCs occurred before the detection of visceral metastases. There are morphologic subsets of CTCs that can be identified by fundamental pathologic approaches, such as nuclear size measurement. The results of this observational study strongly suggest that CTCs contain relevant information regarding disease status. In particular, the detection of vsnCTCs was found to be correlated with the presence of visceral metastases and should be formally explored as a putative blood-borne biomarker to identify patients at risk of developing this clinical evolution of prostate cancer.

Morphological Subsets of Circulating Tumor Cells in Advanced Prostate Cancers: A Potential Biomarker for Patients with Visceral Metastases

Circulating tumor cells (CTCs) are commonly identified in men with advanced prostate cancer (PC) but their significance beyond enumeration is unknown. We subclassified CTCs using a standard pathology approach and focused on the correlation between CTC subsets and visceral metastasis (VM) in advanced PC. We obtained blood samples from 50 PC patients across the spectrum of metastatic states. The patients were divided into training and validation cohorts. CTCs were captured and enumerated on NanoVelcro Chips and were subjected to pathologic review for cellular morphology and nuclear size. The nuclear size distribution was analyzed using a Gaussian Mixture Model, which revealed 3 distinct subpopulations in the training cohort: large-nuclear CTCs, small-nuclear CTCs, and very-small-nuclear CTCs (vsnCTCs). Subsequent correlational analyses revealed elevated vsnCTC counts in PCs with VM when compared to those without (p=0.005). This difference remained statistically significant in the validation cohort (p=0.007). Serial enumerations showed emergence of vsnCTCs prior to detection of newly developed VM. From these data we concluded that CTC subsets contain relevant information on PC disease status when analyzed with pathologic approaches such as nuclear size assessment. In particular, the detection of vsnCTCs correlated with the presence of VM and should be explored as a potential biomarker to identify patients at risk for developing this more aggressive form of PC.

Cytometric characterization of circulating tumor cells captured by microfiltration and their correlation to the CellSearch(®) CTC test.

Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch(®) CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45-/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45- cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45- cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45-/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non-EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc.

Abstract 3832: Characterizing breast cancer CTCs for brain metastasis competence

Abstract Mechanisms causing fatal breast cancer brain metastasis (BCBM) remain largely unknown as well as functionalities of brain-colonizing circulating tumor cells (CTCs) in BCBM disease.We have characterized CTCs isolated from peripheral blood mononuclear cells of patients with breast cancer by multi-parametric flow cytometry. In epithelial cell adhesion molecule (EpCAM)-negative CTCs, we identified a “brain metastasis selected marker” (BMSM) profile or HER2+/EGFR+, HPSE+ and Notch1+ CTCs. These CTCs, which are not captured by the CellSearchTM (Veridex, LLC)platform because of their EpCAM negativity, were analyzed for cell invasiveness and metastatic competency in vivo. CTCs expressing the BMSM profile were highly invasive and capable of generating metastases when xenografted in nude mice. Notably, increased brain metastatic capabilities, frequency and quantitation were detected in EpCAM− CTCs overexpressing the BMSM profile. The presence of proteins of this profile was also verified in the metastatic lesions of animals. Further, investigations of viable BMSM CTCs were carried out using the DEPArrayTM, a dielectrophoresis-based platform able to detect and sort pure CTCs which were investigated by whole genome amplification and proteomic content. CTC subsets were derived possessing combinatorial levels of the BMSM profile and distinct abilities to generate BCBM.Collectively, we provide evidence of isolation, culture and characterization of human breast cancer CTCs leading to the description of a BMSM protein profile that is considered necessary for CTC metastatic competency to brain. Studies are presently undergoing to capture multiple CTC subsets and validate the presence of the BMSM profile in longitudinal studies of populations of breast cancer patients with the objective to not only better understand BCBM onset but also to predict disease course for improved and informed clinical treatment. Citation Format: Dario Marchetti. Characterizing breast cancer CTCs for brain metastasis competence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3832. doi:10.1158/1538-7445.AM2014-3832

Prognostic value of circulating tumor cells with podoplanin expression in patients with locally advanced or metastatic head and neck squamous cell carcinoma.

Podoplanin (PDPN) is a prognostic factor for head and neck squamous cell carcinoma (HNSCC). However, PDPN expression in circulating tumor cells (CTCs) and its prognostic value are not clear. The PowerMag system was used to enumerate CTCs from 53 patients with HNSCC prechemotherapy and 61 healthy donors. PDPN expression was determined by immunofluorescence staining. Results were correlated with clinicopathological parameters and clinical outcome, such as patient survival by receiver operating characteristic (ROC) and univariate and multivariate analyses. PDPN was expressed in a subset of CTCs. Both EpCAM-positive CTC and PDPN-positive CTC counts were statistically different between the disease and nondisease groups (p < .0001) with no prognostic value. After a median follow-up of 10.5 months (range, 6.6-18.5 months), the PDPN-positive/EpCAM-positive CTC ratio >20% was a significant prognostic factor for death within 6 months (p = .011) and was correlated with poor progression-free survival (p = .016) and overall survival (p = .015). PDPN-positive/EpCAM-positive CTC ratio is a prognostic factor and defining the ratio in patients with HNSCC might be valuable to clinical management.

Functional analysis of single cells identifies a rare subset of circulating tumor cells with malignant traits

Ample evidence supports genetic and functional heterogeneity in primary tumors, but it remains unclear whether circulating tumor cells (CTCs) also exhibit the same hierarchical organization. We examined the functional diversity of viable, single CTCs using an array of subnanoliter wells (nanowells). The compartmentalization of single cells by nanowells allowed clonal comparison and mapping of heterogeneity of single cells or preformed clusters of cells. By measuring the short-term viability, invasiveness and secretory profiles of individual CTCs, it was evident that only a rare subset of CTCs possessed malignant traits indicative of metastatic potential in late-stage, progressing metastatic castration-resistant prostate cancer (mCRPC) patients. These CTCs were resistant to anoikis after being in the circulation, were invasive in their epithelial state, or secreted proteases capable of cleaving peptide substrates. Every CTC observed, however, did not exhibit such metastatic potential, suggesting that enumeration of CTCs alone may be insufficient to understand metastasis or stratify patients.

Cellules tumorales circulantes : biopsie liquide du cancer

The detection and molecular characterization of circulating tumor cells (CTCs) are one of the most active areas of translational cancer research, with more than 400 clinical studies having included CTCs as a biomarker. The aims of research on CTCs include: a) estimation of the risk for metastatic relapse or metastatic progression (prognostic information); b) stratification and real-time monitoring of therapies; c) identification of therapeutic targets and resistance mechanisms; and d) understanding metastasis development in cancer patients. This review focuses on the technologies used for the enrichment and detection of CTCs. We outline and discuss the current technologies that are based on exploiting the physical and biological properties of CTCs. A number of innovative technologies to improve methods for CTC detection have recently been developed, including CTC microchips, filtration devices, quantitative reverse-transcription PCR assays, and automated microscopy systems. Molecular characterization studies have indicated, however, that CTCs are very heterogeneous, a finding that underscores the need for multiplex approaches to capture all of the relevant CTC subsets. We therefore emphasize the current challenges of increasing the yield and detection of CTCs that have undergone an epithelial-mesenchymal transition. Increasing assay analytical sensitivity may lead, however, to a decrease in analytical specificity (e.g., through the detection of circulating normal epithelial cells). A considerable number of promising CTC detection techniques have been developed in recent years. The analytical specificity and clinical utility of these methods must be demonstrated in large prospective multicenter studies to reach the high level of evidence required for their introduction into clinical practice.

Abstract B51: ApoStream™ isolated circulating tumor cells from primary breast cancer patients reveals heterogeneous phenotypes related to epithelial-mesenchymal transition and stem cell markers.

Abstract Background: Detection of circulating tumor cells (CTCs) is an indicator of poor prognosis in patients with metastatic breast cancer and not in primary breast cancer (PBC). The classical phenotypic definition of a CTC is a nucleated cell that is cytokeratin (CK) positive but CD45 negative. Several reports have shown that EpCAM based capture methods detect only a fraction of CTCs and not the heterogeneous subpopulations of CTCs. Moreover, subsets of CTCs may acquire a more aggressive phenotype with features of invasiveness and motility by undergoing an epithelial to mesenchymal transition (EMT) and down regulate the epithelial cell adhesion molecule, EpCAM. EMT is a hallmark of cellular invasion and metastasis and CTCs undergoing EMT may express the putative cancer stem cell like phenotype, CD24lowCD44+. CTCs undergoing EMT (CTC-EMT) are not readily detected by current CTC detection technologies. Thus, it is desirable to isolate CTCs using capture methods independent of EpCAM to recover a heterogeneous CTC population for more extensive characterization. Here we use ApoStream™, a novel antibody-free CTC isolation device that does not rely on EpCAM to capture circulating rare cells, to evaluate the molecular heterogeneity of CTCs. Methods: Baseline blood samples from 14 newly diagnosed PBC patients were collected and processed using ApoStream™. Isolated cells were stained with anti-CK and anti-CD45, and DAPI. In addition, a multiplexed immunofluorescence assay and laser scanning cytometry analysis were applied to identify multiple combinations of CK+CD45− cells for the expression and distribution of EpCAM, vimentin, CD44, CD24, β-catenin and E-cadherin. Results: ApoStream™ recovered both EpCAM+ and EpCAM− cells. CK+CD45− cells were detected in 9 out of 14 PBC patients. The expression of EpCAM− vimentin+ in the CK+CD45− population was heterogeneous across the patient population. E-cadherin and β-catenin were detected in 0-94% (Mean 52 %) and 0-37% (Mean 8 %), respectively of the CK+CD45− population. All patients with CK+CD45− cells had a subset of cells with the putative phenotype of CD44+CD24low cells. Conclusions: Heterogeneous CTC phenotypes with CD44+CD24lowin both EpCAM+ and EpCAM− cells were observed in patients with PBC. Our aim is to correlate ApoStream™ EMT-CTC counts in patients with PBC with the pathological clinical response (pCR). This study will continue to enroll PBC patients and test the hypothesis that low EMT-CTC count patients have higher pCR rates compared to high EMT-CTC count patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B51. Citation Format: Kenna L. Anderes, Insiya Jafferji, Vladislava O. Melnikova, Jackson A. Summer, Darren W. Davis, James M. Reuben, Naoto T. Ueno. ApoStream™ isolated circulating tumor cells from primary breast cancer patients reveals heterogeneous phenotypes related to epithelial-mesenchymal transition and stem cell markers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B51.

Abstract C23: Functional characterization of viable circulating tumor cells using nanowells.

Abstract Current characterizations of circulating tumor cells (CTCs) are limited to enumeration, genotyping, and gene expression. Because these assays each require fixation or lysis, it has not been possible to evaluate several important characteristics of live CTCs at the single-cell level. We have interrogated viable, single CTCs using arrays of subnanoliter wells (nanowells), performing functional measurements of viability, migration, and secretion of soluble factors. We developed a process using nanowells to isolate and characterize viable CTCs from whole blood. Each poly(dimethylsiloxane) (PDMS) array comprises 84,762 cubic wells of 275 pL each. CTCs were enriched from whole blood by negative selection against CD45 and loaded onto the array to settle into the wells by limiting serial dilution. Because CTCs are rare, the loading biased the occupancy of the wells to single CTCs or CTCs in preformed clusters, allowing comparisons among individual CTCs. The defined address of each well allowed spatiotemporal tracking of single CTCs, and subsequent registration of data for functional measures on each CTC. Using this approach, we made three types of measurements on viable CTCs isolated from prostate cancer patients: (1) immediate and short-term viability of CTCs, (2) migration and invasion potential of CTCs, and (3) secretion of soluble factors. We found a rare subset of CTCs to possess malignant traits indicative of metastatic potential in late-stage, progressing prostate cancer patients. These CTCs were resistant to anoikis after being in the circulation as they stained Calcein AM+/Annexin V−, and remained viable for at least a week in culture. They were invasive in their epithelial state when embedded in Matrigel. They could also secrete proteases capable of cleaving peptide substrates. However, we did not observe such metastatic potential in every CTC, pointing to the presence of heterogeneity within CTCs and suggesting that enumeration of CTCs alone may be insufficient to understand metastasis or stratify patients. By interrogating the functional behavior of individual CTCs, we identified a rare subset of CTCs with phenotypes consistent with more efficient metastasis. The nanowell technology holds the potential to further investigate the cellular characteristics of rare tumor cells and design therapies to counter metastasis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C23. Citation Format: Xiaosai Yao, Atish D. Choudhury, Yvonne J. Yamanaka, William C. Hahn, K Dane Wittrup, J Christopher Love. Functional characterization of viable circulating tumor cells using nanowells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C23.

Micrometastasis in gastric cancer

Circulating tumor cells (CTCs) are a crucial contributor and indicator for cancer metastasis. The presence of CTCs and other two forms of occult disease including circulating tumor microembolus (CTMs) as well as disseminated tumor cells (DTCs) as a sign for micrometastasis has been associated with adverse survival of both localized and metastatic cancers. However, their assignments in micrometastasis are still not well understood. This mini-review is aimed to provide an overview of the biological features and the clinical impact of CTCs, DTCs and CTM in gastric cancer (GC), and to summarize the technical limitations of detection methods in addition to the significance of distinct migration modes in epithelial–mesenchymal transition (EMT) and gastric micrometastasis. Although there are controversies over current CTC identification strategies, both single and collective migration modes as indispensable parts of micrometastasis are influential participants in GC progression, diagnosis and prognosis. CTCs, CTM and DTCs are not separated forms of the occult disease; however available techniques fail to detect all subsets of them. Therefore, micrometastatic detection should be combined with conventional pathological examinations in order to make more accurate predictions of tumor outcomes. Novel markers further defining the features of CTC subsets are warranted.

The prognostic value of circulating tumor cells lacking cytokeratins in metastatic breast cancer patients

In this study, we detected epithelial circulating tumor cells (CTCs) that expressed cytokeratins and potential circulating tumor cells (pCTCs) that had lost expression of cytokeratins in metastatic breast cancer (MBC) patients. The aim of the study was to evaluate the prognostic significance of these two kinds of CTCs in MBC patients. We detected CTCs and pCTCs from 66 MBC patients using MACS immunomagnetic enrichment technology combined with immunocytochemistry (ICC). A cutoff score of 5 CTCs (or pCTCs) was set as a benchmark for prognosis in patients. Progression free survival (PFS) was calculated and analyzed during the following 24 months. We evaluated the sensitivity of this method and recovery rates of the CTCs by spiking experiments. The loss number of tumor cells by our method was 0-15. The population with fewer than 5 CTCs showed significantly higher PFS than the group with 5 or more CTCs. The difference in PFS between the patients with 5 or more pCTCs and those with fewer than 5 pCTCs was statistically significant. The presence of these pCTCs more accurately predicted poor prognosis than the CTCs that express cytokeratins. There is a subset of CTCs that lose epithelial markers such as pCTCs. Due to the heterogeneity of the expression of epithelial antigens in CTCs, different subtypes of CTCs exist. Independently of CTCs, the groups of patients with pCTCs had poorer prognoses.

Circulating Tumor Cells: Liquid Biopsy of Cancer

The detection and molecular characterization of circulating tumor cells (CTCs) are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker. The aims of research on CTCs include (a) estimation of the risk for metastatic relapse or metastatic progression (prognostic information), (b) stratification and real-time monitoring of therapies, (c) identification of therapeutic targets and resistance mechanisms, and (d) understanding metastasis development in cancer patients. This review focuses on the technologies used for the enrichment and detection of CTCs. We outline and discuss the current technologies that are based on exploiting the physical and biological properties of CTCs. A number of innovative technologies to improve methods for CTC detection have recently been developed, including CTC microchips, filtration devices, quantitative reverse-transcription PCR assays, and automated microscopy systems. Molecular-characterization studies have indicated, however, that CTCs are very heterogeneous, a finding that underscores the need for multiplex approaches to capture all of the relevant CTC subsets. We therefore emphasize the current challenges of increasing the yield and detection of CTCs that have undergone an epithelial-mesenchymal transition. Increasing assay analytical sensitivity may lead, however, to a decrease in analytical specificity (e.g., through the detection of circulating normal epithelial cells). A considerable number of promising CTC-detection techniques have been developed in recent years. The analytical specificity and clinical utility of these methods must be demonstrated in large prospective multicenter studies to reach the high level of evidence required for their introduction into clinical practice.

Circulating Tumor Cells in Hepatocellular Carcinoma: Detection Techniques, Clinical Implications, and Future Perspectives

Hepatocellular carcinoma (HCC) is a primary liver cancer with a huge challenge in terms of its complex etiology and its management. The fact that the most common site of early tumor recurrence in liver transplantation for HCC is the transplanted allograft strongly suggests that circulating tumor cells (CTCs) are really an active source of HCC metastasis or recurrence. In the past decade, with the tremendous progress in the technology of CTC detection, there is convincing evidence that CTCs have great potential as a marker for metastatic disease and poor prognosis in patients with a malignancy. Currently some interesting and encouraging results have been achieved in HCC CTC detection, although the knowledge about its clinical relevance in HCC is lagging behind other major tumor types. Here we will review existing and developing methodologies for CTC detection, discuss future perspectives, and describe the potential clinical impact of the identification and molecular characterization of CTC subset or circulating cancer stem cells in HCC patients. Particular attention is given to the results based on the HCC CTC study.

P4-07-08: Subsets and Molecular Signatures of Circulating Tumor Cells in Breast Cancer Brain Metastasis.

Abstract Background: Circulating tumor cells (CTCs) represent the “seeds” of intractable brain metastatic breast cancer (BMBC); however, properties of CTCs targeting the brain remain elusive. For example, the FDA-approved CTC platform (CellSearch™, Veridex, LLC) detects only CTCs positive for epithelial cell adhesion molecule (EpCAM) and cytokeratins (CKs), but is unable to capture any other CTC subtypes or analyze biomarkers of brain-homing CTCs. We hypothesized that profiling CTCs from BMBC patients might result in the identification of brain-colonizing CTC signatures. Materials and Methods: We employed CellSearch™ and a novel technology that uses analysis of specific antigenic markers by immunofluorescence, coupled with detecting gene amplification by fluorescence in situ hybridization on the same cells; and quantification of the signal via automated scanning (FICTION; BioView Duet-3™ system). Results: We established that our approach was feasible by performing CTC analyses on peripheral blood mononuclear cells isolated from BMBC patients or patients not possessing overt metastatic disease. We detected a differential gene amplification for human epidermal growth factor receptor1 and 2 (EGFR and HER2, respectively). Second, the number of EpCAM-positive CTCs visualized by the BioView™ platform was at least three orders of magnitude higher than one obtained from CellSearch™ CTC analyses using the same specimen. Third, we identified the presence of CTCs positive for CKs but negative for EpCAM. Conversely, high levels of prometastatic heparanase, in conjunction with the expression of aldehyde dehydrogenase-1 (ALDH1), a known cancer stem-cell marker, were detected in CTCs from BMBC patients; with a correlation between heparanase, ALDH1, and high EGFR amplification. Finally, extensive flow cytometric/FACS analyses validated the presence of CTC subsets negative for EpCAM and CD45, a hematolymphoid marker, however enriched for heparanase/ALDH1 expression. Discussion: These findings indicate that the BioView™ platform not only captures more EpCAM-positive CTCs than CellSearch™ but also allows the detection of novel CTC subtypes possessing varying EpCAM levels. Importantly, they suggest that profiling CTC subtypes in patients with BMBC can be relevant towards the discovery of BMBC founder CTCs. Work is ongoing to further characterize these CTC subtypes, and to assess their abilities to metastasize to brain in xenotransplantation studies using immunodeficient mice. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-08.

Circulating tumor cells in the cerebrospinal fluid: “tapping” into diagnostic and predictive potential

Metastatic spread of breast cancer into the central nervous system (CNS) carries an especially ominous prognosis. Successful treatment depends on early and accurate diagnosis [1]. Therefore, it is of paramount clinical importance to develop sensitive and specific methods to detect circulating tumor cells (CTCs) in the cerebrospinal fluid (CSF). Routine cytological examination of the cerebrospinal fluid for tumor cells has many limitations including low sensitivity necessitating repeated lumbar punctures to increase the diagnostic yield [2]. In the October issue of Oncotarget, Patel et al developed a method to adapt the FDA approved CellSearch platform to isolate CTCs from the CSF after spiking them into blood [3]. This novel approach adds a “molecular dimension” to the detection of malignant epithelial cells in the CSF. It hones in on single cells and begins to characterize their surface markers thus opening the door to a better understanding of their role in the metastatic cascade. The CellSearch system defines CTCs as EpCAM+, Cytokeratin+, CD45- nucleated cells. These cells, when isolated from the CSF, may represent a special subpopulation of CTCs capable of surviving the perilous journey in the blood stream and subsequent invasion of the CNS. The next logical step in understanding the biology of this unique cell population will be to search for markers that would predict special affinity for homing into and invading the CNS, surviving and proliferating (i.e. stem cell markers) or for drug resistance [4]. Studying ER/PR and HER-2 concordance between primary or metastatic tumor sites and CTCs in the CSF could also have profound implications. Further studies are needed to create surface marker and genetic “profiles” of this important subset of CTCs. This study also demonstrated correlation between CTC counts in the CSF and the administration of chemotherapeutic agents, a pattern that mirrors that observed with CTCs in the peripheral blood of patients with metastatic breast cancer. Previous studies have confirmed the validity of circulating tumor cell counts in predicting progression free survival (PFS) and overall survival (OS) in patients with metastatic breast cancer. For instance, in one study of metastatic breast cancer, patients with more than 5 CTCs had lower PFS (2.7 months vs. 7.0 months, P 18 months, P<0.001). These differences persisted at the first follow-up visit after the initiation of therapy [5]. It would be interesting to see if larger studies confirm similar prognostic and predictive correlation between CTC counts in the CSF and survival or response to therapy. Our understanding of the role of CTCs in the biology of metastases continues to evolve. This valuable study sheds the light on a subset of CTCs capable of invading the blood brain barrier to reach the CNS. Further characterization and profiling of CTCs in the CSF is likely to yield valuable insights into the biology and the behavior of this important subset of cells and to improve our understanding of how CNS metastases occur. Correlation between CTCs counts, their biological characteristics and clinical outcomes will continue to make this area both promising and clinically relevant.

A feasibility study of gene expression analysis from enriched circulating tumor cells (CTC) in metastatic breast cancer (MBC).

e21156 Background: The most common methods for CTC isolation from peripheral blood depend on positive selection based on tumor cells’ surface EpCAM expression. However, EpCAM expression may vary between CTCs and may be downregulated in cells undergoing epithelial-to-mesenchymal transition, potentially resulting in the failure to capture CTC subsets having a critical role as intermediates in cancer progression. Methods: We previously reported on a feasibility study using an unbiased approach with three different enrichment methods, involving both positive and negative selection, and obtained consistently high recovery of small numbers of MDA-MB-231 cancer cells spiked into donor human peripheral blood using the RosetteSep Human Circulating Epithelial Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Vancouver, BC Canada). Cytospins prepared from recovered cells were used for identification and enumeration by immunocytochemistry using fluorescently laballed anti-CD45 and anti-CK19 antibodies. We optimized RNA amplification from the equivalent of one MDA-MB-231 cell (~10 pg) and used the resulting amplified RNA in microarray experiments. Peripheral blood from MBC patients was enriched for CTC, RNA was isolated and amplified, and microarray and qPCR analysis was used to investigate global and gene-specific transcription changes, respectively. Results: Numbers of CTC enriched from eight MBC patients ranged from 1 to 125 per 5 mL blood. EpCAM, CK19, vimentin, and CD45 expression by CTC was determined by qPCR. Preliminary results from microarray analysis of enriched CTC from four of six initial patients showed ~ 130 common genes upregulated more than twofold, including genes involved in tumorigenesis. Two of six patients had insufficiently amplified RNA. Gene enrichment analysis showed an abundance of genes involved in translation. Conclusions: Although preliminary, these methods may lead to a more thorough characterization of micrometastases in advanced breast cancer and potentially early stage disease. Further microarray analysis and optimization methods are ongoing in MBC patients.

Circulating tumor cells: detection, molecular profiling and future prospects

Disseminated malignancy is responsible for the vast majority of cancer-related deaths. During this process, circulating tumor cells (CTC) are generated, spread from the primary tumor, colonize distant organs and lead to overt metastatic disease. CTC are essential for establishing metastasis; however, they are not sufficient as this process is highly inefficient and most will fail to grow in target sites. Several CTC die during migration while others remain dormant for several years and very few grow into macrometastases. CTC have been well documented in the bloodstream of cancer patients; however, the clinical relevance of this detection is still the subject of controversies and their biology is poorly understood. Indeed, available markers fail to distinguish between subgroups of CTC, and several current methods lack sensitivity, specificity or reproducibility in CTC characterization and detection. The advent of more precise technologies is renewing the interest in CTC biology. We will review herein recent findings on CTC biology, on the role of host–tumor interactions in CTC shedding and implantation, available methods of CTC detection and future perspectives for the molecular characterization of the CTC subset(s) responsible for the development of metastasis. Ultimately, understanding CTC biology and host–tumor ‘complementarities’ will help define metastasis-related biomarkers providing formidable and tailored novel therapeutic targets.