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.

ABSTRACT"Liquid biopsy" focusing on the analysis of circulating tumor cells (CTC) and circulating cell-free tumor DNA (ctDNA) in the blood of patients with cancer has received enormous attention because of its obvious clinical implications for personalized medicine. Analyses of CTCs and ctDNA have paved new diagnostic avenues and are, to date, the cornerstones of liquid biopsy diagnostics. The present review focuses on key areas of clinical applications of CTCs and ctDNA, including detection of cancer, prediction of prognosis in patients with curable disease, monitoring systemic therapies, and stratifi cation of patients based on the detection of therapeutic targets or resistance mechanisms. Signifi cance:The application of CTCs and ctDNA for the early detection of cancer is of high public interest, but it faces serious challenges regarding specifi city and sensitivity of the current assays. Prediction of prognosis in patients with curable disease can already be achieved in several tumor entities, particularly in breast cancer. Monitoring the success or failure of systemic therapies (i.e., chemotherapy, hormonal therapy, or other targeted therapies) by sequential measurements of CTCs or ctDNA is also feasible. Interventional studies on treatment stratifi cation based on the analysis of CTCs and ctDNA are needed to implement liquid biopsy into personalized medicine. Cancer Discov; 6(5); 479-91.

BACKGROUND:The detection and molecular characterization of circulating tumor cells (CTCs) are one of the most active areas of translational cancer research, with Ͼ400 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. Molecularcharacterization 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 epithelialmesenchymal transition. Increasing assay analytical sensitivity may lead, however, to a decrease in analytical specificity (e.g., through the detection of circulating normal epithelial cells).

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Early spread of tumor cells is usually undetected by current imaging technologies. In patients with cancer and no signs of overt metastases sensitive methods have been developed to detect circulating tumor cells (CTCs) in the peripheral blood and disseminated tumor cells (DTCs) in the bone marrow. These technologies can be classified into cytometric and/or immunological and molecular approaches. Interestingly, the bone marrow seems to be a common homing tissue for cells derived from various epithelial tumors, and level 1a data from European and US groups have confirmed the prognostic impact of DTCs in the bone marrow of patients with breast cancer. Sequential peripheral blood analyses, however, are more convenient than bone marrow analyses for patients with solid tumors, and many research groups are currently assessing the clinical use of CTCs for assessment of prognosis and monitoring of systemic therapy. Molecular characterization of DTCs and CTCs opens a new avenue for understanding cancer dormancy, and might contribute to the identification of metastatic stem cells with important implications for future therapies. This Review focuses on the clinical relevance of the latest research results on blood-borne cancer micrometastases in patients with cancer.

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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 Ó2015 AACR.

Distant metastases harbor unique genomic characteristics not detectable in the corresponding primary tumor of the same patient and metastases located at different sites show a considerable intrapatient heterogeneity. Thus, the mere analysis of the resected primary tumor alone (current standard practice in oncology) or, if possible, even reevaluation of tumor characteristics based on the biopsy of the most accessible metastasis may not reveal sufficient information for treatment decisions. Here, we propose that this dilemma can be solved by a new diagnostic concept: liquid biopsy, that is, analysis of therapeutic targets and drug resistance-conferring gene mutations on circulating tumor cells (CTC) and cell-free circulating tumor DNA (ctDNA) released into the peripheral blood from metastatic deposits. We discuss the current challenges and future perspectives of CTCs and ctDNA as biomarkers in clinical oncology. Both CTCs and ctDNA are interesting complementary technologies that can be used in parallel in future trials assessing new drugs or drug combinations. We postulate that the liquid biopsy concept will contribute to a better understanding and clinical management of drug resistance in patients with cancer. Cancer Res; 73(21); 6384-8. Ó2013 AACR.