Circulating Tumor Cell Isolation: A Marathon Race Worth Running
It took almost 150 years after the first report on the presence of circulating tumor cells (CTCs) 2 by Thomas Ashworth in 1869 (1 ) to reach the point at which their detection and molecular characterization is one of the hottest fields in cancer research (2 ). The clinical significance of CTCs has been evaluated in many types of solid cancers, and the Food and Drug Administration (FDA) cleared the testing of CTCs in metastatic breast, colorectal, and prostate cancer almost a decade ago (3 ) .
... decade ago (3 ) . In contrast to tumor biopsies, peripheral blood samples can be frequently and sequentially obtained for CTC isolation and downstream analysis. The noninvasive isolation of CTCs from peripheral blood and their further downstream molecular characterization at the protein, DNA, and RNA levels could now serve as a "liquid biopsy" approach, eventually offering additional information and, even more, a serious advantage over the conventional and well-established tumor biopsy approach (4, 5 ). Cell-free DNA circulating in plasma or serum of cancer patients has also been recently proposed as an alternative to the CTC liquid biopsy approach (6 ). By the use of extremely powerful and highly sensitive detection techniques, the presence of specific mutations in the plasma of patients with cancer could give valuable information concerning responses to specific molecular targeted therapies (7 ). However, there is a substantial difference between these two approaches; CTCs are viable cells that circulate in blood, and understanding their biology in a holistic way could give valuable information on metastatic spread, clarify their connection to cancer stem cells, and reveal active and possible targetable signaling networks. Cell-free DNA could give specific information as a circulating biomarker for the presence or absence of specific alterations indicating therapy response. The prognostic relevance of CTCs has been evaluated in numerous studies so far, and a recent metaanalysis clearly indicated that the detection of CTCs is a reliable prognostic factor in patients with early-stage and metastatic breast cancer (8 ). Nowadays, CTC molecular characterization has a strong potential to be translated into individualized targeted treatments (9 ) and the number of ongoing trials that evaluate CTCs as markers for early prediction of treatment efficacy is continuously rising (10 ). A tremendous effort is ongoing toward the development of novel state-of-the-art technologies for CTC isolation and molecular characterization (4, 5 ). CTC molecular characterization systems are mainly based on protein and image-based approaches or on molecular assays based on the analysis of the nucleic acids in CTCs. Nowadays, the application of extremely powerful next generation sequencing technologies in the area of CTC molecular characterization in combination with reliable single-CTC isolation opens new frontiers for the management of patients. There is, however, substantial variability in the rates of positive samples determined by using existing CTC isolation and detection techniques. This lack of standardization of technology hampers the implementation of CTC measurement in clinical routine practice. Direct comparison of different methodologies for detecting CTCs in blood samples from patients with breast cancer has revealed substantial variations in the detection rates (11 ). CTC phenotypes have not yet been fully defined. According to the FDA-cleared CellSearch system, which has shown clinical relevance, CTCs are defined as epithelial cell adhesion molecule (EpCAM) ϩ , cytokeratin ϩ , and CD45 Ϫ . However, there is ongoing discussion on the variable expression of cell antigens used for CTC enrichment with immunoaffinity-based isolation methods. Expression of EpCAM is variable across epithelial cancers and, more importantly, clinically relevant subpopulations of CTCs may go through an epithelial-to-mesenchymal transition or exist as cancer stem cells (12 ). We now know that CTCs are highly heterogeneous and that different analytical systems detect different cell populations on the basis of the approach being used for their isolation. The concordance between different analytical CTC detection systems is far below 100%, indicating that each system is actually identifying different and partially overlapping