Catherine Alix-Panabières

Circulating Tumor Cells: Liquid Biopsy of Cancer

BACKGROUND 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. CONTENT 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). SUMMARY 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.

Challenges in circulating tumour cell research

During the past ten years, circulating tumour cells (CTCs) have received enormous attention as new biomarkers and the subject of basic research. Although CTCs are already used in numerous clinical trials, their clinical utility is still under investigation. Many issues regarding the detection and characterization of CTCs remain unknown. In this Opinion article, we propose a conceptual framework of CTC assays and point out current challenges of CTC research, which might structure this dynamic field of translational cancer research.

Circulating tumour cells in cancer patients: challenges and perspectives

Ultrasensitive methods have been recently developed to detect circulating tumour cells (CTCs) in the peripheral blood and disseminated tumour cells (DTCs) in the bone marrow (BM) of cancer patients. Studies with these new methods indicate that BM is a common homing organ and a reservoir for DTCs derived from various organ sites including breast, prostate, lung and colon. Peripheral blood analyses, however, are more convenient for patients than invasive BM sampling and many research groups are currently assessing the clinical utility of CTCs for prognosis and monitoring response to systemic therapies. Moreover, molecular analyses of CTCs/DTCs have provided new insights into the biology of metastasis with important implications for the clinical management of cancer patients.

Circulating tumor cells and circulating tumor DNA.

Solid tumors derived from epithelial tissues (carcinomas) are responsible for 90% of all new cancers in Europe, and the main four tumor entities are breast, prostate, lung, and colon cancer. Present tumor staging is mainly based on local tumor extension, metastatic lymph node involvement, and evidence of overt distant metastasis obtained by imaging technologies. However, these staging procedures are not sensitive enough to detect early tumor cell dissemination as a key event in tumor progression. Many teams have therefore focused on the development of sensitive assays that allow the specific detection of single tumor cells or small amounts of cell-free tumor DNA in the peripheral blood of cancer patients. These methods allow the detection and characterization of early metastatic spread and will provide unique insights into the biology of metastatic progression of human tumors, including the effects of therapeutic interventions.

Circulating Tumor Cells and Bone Marrow Micrometastasis

Sensitive immunocytochemical and molecular assays allow the detection of single circulating tumor cells (CTC) in the peripheral blood and disseminated tumor cells (DTC) in the bone marrow as a common and easily accessible homing organ for cells released by epithelial tumors of various origins. The results obtained thus far have provided direct evidence that tumor cell dissemination starts already early during tumor development and progression. Tumor cells are frequently detected in the blood and bone marrow of cancer patients without clinical or even histopathologic signs of metastasis. The detection of DTC and CTC yields important prognostic information and might help to tailor systemic therapies to the individual needs of a cancer patient. In the present review, we provide a critical review of (a) the current methods used for detection of CTC/DTC and (b) data on the molecular characterization of CTC/DTC with a particular emphasis on tumor dormancy, cancer stem cell theory, and novel targets for biological therapies; and we pinpoint to (c) critical issues that need to be addressed to establish CTC/DTC measurements in clinical practice.

Cell-free Tumor DNA in Blood Plasma As a Marker for Circulating Tumor Cells in Prostate Cancer

Purpose: Circulating cell-free DNA in the blood of cancer patients harbors tumor-specific aberrations. Here, we investigated whether this DNA might also reflect the presence of circulating tumor cells (CTC). Experimental Design: To identify the source of cell-free DNA in blood, plasma derived from 81 patients with prostate cancer was examined for CTCs and cell-free DNA. An epithelial immunospot assay was applied for detection of CTCs, and a PCR-based fluorescence microsatellite analysis with a panel of 14 polymorphic markers was used for detection of allelic imbalances (AI). Results: The plasma DNA levels significantly correlated with the diagnosis subgroups of localized (stage M0, n = 69) and metastasized prostate cancer (stage M1, n = 12; P = 0.03) and with the tumor stage of these patients (P < 0.005). AI was found on cell-free DNA in plasma from 45.0% and 58.5% of M0 and M1 patients, respectively. Detection of CTCs showed that 71.0% or 92.0% of the M0 and M1 patients harbored 1 to 40 CTCs in their blood, respectively. The occurrence of CTCs correlated with tumor stage (P < 0.03) and increasing Gleason scores (P = 0.04). Notably, significant associations of the number of CTCs with the AI frequencies at the markers D8S137 (P = 0.03), D9S171 (P = 0.04), and D17S855 (P = 0.02) encoding the cytoskeletal protein dematin, the inhibitor of the cyclin-dependent kinase CDKN2/p16 and BRCA1, respectively, were observed. Conclusions: These findings show, for the first time, a relationship between the occurrence of CTCs and circulating tumor-associated DNA in blood, which, therefore, might become a valuable new source for monitoring metastatic progression in cancer patients.

Clinical application of circulating tumor cells in breast cancer: overview of the current interventional trials

In 2004, circulating tumor cells (CTC) enumeration by the CellSearch® technique at baseline and during treatment was reported to be associated with prognosis in metastatic breast cancer patients. In 2008, the first evidence of the impact of CTC detection by this technique on survival of cM0(i+) patients were reported. These findings were confirmed by other non-interventional studies, whereas CTC were also investigated as a surrogate for tumor biology, mainly for HER2 expression/amplification. The aim of this report is to present the current prospective large interventional studies that have been specifically designed to demonstrate that CTC enumeration/characterization may improve the management of breast cancer patients: STIC CTC METABREAST (France) and Endocrine Therapy Index (USA) assess the CTC-guided hormone therapy vs chemotherapy decision in M1 patients; SWOG0500 (USA) and CirCe01 (France) assess the CTC count changes during treatment in metastatic patients; DETECT III (M1 patients, Germany) and Treat CTC (cM0(i+) patients, European Organization for Research and Treatment of Cancer/Breast International Group) assess the use of anti-HER2 treatments in HER2-negative breast cancer patients selected on the basis of CTC detection/characterization. These trials have different designs in various patient populations but are expected to be the pivotal trials for CTC implementation in the routine management of breast cancer patients.

Plasticity of disseminating cancer cells in patients with epithelial malignancies

Current models suggest that at a certain but yet undefined time point of tumour development malignant cells with an aggressive phenotype start to disseminate via the blood stream into distant organs. This invasive phenotype appears to be associated with an epithelial–mesenchymal transition (EMT), which enables detachment of tumour cells from a primary site and migration. The reverse process of mesenchymal–epithelial transition (MET) might play a crucial role in the further steps of metastasis when circulating tumour cells (CTCs) settle down in distant organs and establish (micro-)metastasis. Nevertheless, the exact mechanisms and interplay of EMT and MET are only partially understood and their relevance in cancer patients is unclear. Research groups have just started to apply EMT-related markers in their studies on CTCs in cancer patients. In the present review, we summarize and discuss the current state of investigations on CTCs in the context of research on EMT/MET.

Establishment and Characterization of a Cell Line from Human Circulating Colon Cancer Cells

Circulating tumor cells (CTC) in blood are promising new biomarkers potentially useful for prognostic prediction and monitoring of therapies in patients with solid tumors including colon cancer. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in patients with cancer. However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of patients with colorectal cancer. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. Here, we describe, for the first time, the establishment of cell cultures and a permanent cell line from CTCs of one patient with colon cancer. The cell line designated CTC-MCC-41 has been cultured for more than one year, and the cells have been characterized at the genome, transcriptome, proteome, and secretome levels. This thorough analysis showed that CTC-MCC-41 cells resemble characteristics of the original tumor cells in the patient with colon cancer and display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem cell-like properties, and an osteomimetic signature, indicating a bone marrow origin. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. The establishment of this first colon cancer CTC line allows now a wealth of functional studies on the biology of CTCs as well as in vitro and in vivo drug testing.

Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer.

IntroductionWe evaluated whether CK19, one of the main cytoskeleton proteins of epithelial cells, is released as full-length protein from viable tumor cells and whether this property is relevant for metastatic progression in breast cancer patients.MethodsEPISPOT (EPithelial ImmunoSPOT) assays were performed to analyze the release of full-length CK19 by carcinoma cells of various origins, and the sequence of CK19 was analyzed with mass spectrometry. Additional functional experiments with cycloheximide, Brefeldin A, or vincristine were done to analyze the biology of the CK19-release. CK19-EPISPOT was used to detect disseminated tumor cells in bone marrow (BM) of 45 breast cancer patients who were then followed up over a median of 6 years.ResultsCK19 was expressed and released by colorectal (HT-29, HCT116, Caco-2) and breast (MCF-7, SKBR3, and MDA-MB-231) cancer cell lines. The CK19-EPISPOT was more sensitive than the CK19-ELISA. Dual fluorescent EPISPOT with antibodies against different CK19 epitopes showed the release of the full-length CK19, which was confirmed by mass spectrometry. Functional experiments indicated that CK19 release was an active process and not simply the consequence of cell death. CK19-releasing cells (RCs) were detectable in BM of 44% to 70% of breast cancer patients. This incidence and the number of CK19-RCs were correlated to the presence of overt metastases, and patients with CK19-RCs had a reduced survival as compared with patients without these cells (P = 0.025, log-rank test; P = 0.0019, hazard ratio, 4.7; multivariate analysis).ConclusionsFull-length CK19 is released by viable epithelial tumor cells, and CK19-RCs might constitute a biologically active subset of breast cancer cells with high metastatic properties.