Abstract CN3-2: Enumeration and characterization of CTCs
Authors: Sleijfer Stefan 
Published: January 2011

Abstract
Despite many challenges, several assays have recently become available for CTC detection. Enumeration of CTC by several of these techniques has been shown to yield prognostic information in several tumor types. Moreover, CTC enumeration can serve as an early marker to evaluate response to anti-tumor drugs and currently, clinical trials are ongoing to assess whether systemic therapy in cancer patients can be tailored according to CTC counts instead of conventional radiological techniques.
However, despite these successes, it should be realised that there is clear room for improvement. For example, in many breast cancer patients CTC are not detectable despite the presence of metastatic disease. Many CTC assays rely on EpCAM expression by CTC, while it is already known that EpCAM is not expressed in approximately 10% of the primary breast tumors, EpCAM expression can be highly heterogeneous and that EpCAM expression can disappear during dissemination. In our well-characterized breast cancer cell line panel, we have shown that in particular cell lines belonging to the normal-like breast cancer subtype lack EpCAM expression and therefore, when spiked in healthy blood, cannot be detected using the CellSearch System™, which applies anti-EpCAM ferrofluids as enrichment step (JNCI 2009; 101: 61–6). Using these cell lines as a model for EpCAM-negative CTC and by gene expression data and immunohistochemistry, we identified CD146 as a potential alternative marker. By using anti-CD146 ferrofluids instead of anti-EpCAM and by adding CD34 to exclude circulating endothelial cells, EpCAM-negative breast cancer cell lines can be revealed when spiked in blood from healthy donors. Furthermore, CD146-positive CTC can be found in metastatic breast cancer patients and not in healthy female volunteers. Studies have been initiated to establish the clinical value of these CTC.
In addition to improving CTC enumeration, characterisation of CTC holds great promise to improve prognostic and predictive models, to reveal novel targets of treatment, and to get better insight into tumor biology and treatment resistance. Samples enriched for CTC by the CellSearch System™ yield only a few CTC and are contaminated by leucocytes. This clearly hampers CTC characterisation by PCR as positive signals can also be from leucocytes. By using a set of genes with no or only minor expression by leucocytes, we are able to perform quantitative mRNA expression in as little as 1 CTC spiked in healthy donor blood. Additionally, epithelial-specific PCR signals could only be found in patients with detectable CTCs and not in healthy controls (BCRT 2009; 118: 455–68). Recently, we have developed a similar platform enabling the detection of several epithelial-specific miRNAs. Studies are ongoing to assess whether CTC characterization for mRNA or miRNA has indeed clinical value and gives more insight into tumor biology.
The field of CTC enumeration and characterization is rapidly evolving and it is likely that CTC enumeration and characterization will get a place in the standard patient management of several tumor types shortly.
Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN3-2