**7. Conclusion**

such as Melan-A for melanoma CTCs, for example. Alternatively, there is a need to distinguish co-purified CECs from CTCs using specific endothelial markers not expressed on cancer cells. The endothelial marker CD34 has been used to distinguish CD146-enriched breast cancer CTCs from CECs [44]. Whether CD34 is the most reliable or specific marker to distinguish true CTCs

Our preliminary data suggest that the accumulation of false positives, most likely endothelial cells, is also an issue when using N-cadherin-based immunomagnetic CTC isolation. While CTC numbers isolated from advanced ovarian cancer patients were approximately four times

positive 'CTCs' in the blood from seven individuals without any history of cancer (data not shown). N-cadherin, EGFR and cytokeratin expression of endothelial cells suggest that targeting these proteins in CTC isolation or identification might lead to similar problems. Moreover, other cells in the circulation, such as monocytes, macrophages and neutrophils, also express the EMT markers EGFR, vimentin and N-cadherin. Further, tumour-associated macrophages of breast cancer and prostate cancer patients were also shown to express cytokeratin and therefore could be confused with CTCs [52–55]. Thus, while non-EpCAMbased CTC isolation techniques appear to produce higher CTC counts and favour isolation of CTCs with EMT features, they also may enrich for false-positive cells, and as long as identifi‐ cation solely relies on CK and CD45 staining of nucleated cells, these cannot be sufficiently well discriminated from CTCs. Advances in identifying CTCs and distinguishing them from false positives, in particular endothelial cells, will refine CTC detection and help avoiding

Circumstantial evidence linking EMT changes to advanced disease and increased metastasis is strong. EMT phenotype in patient tumour tissue is often prognostic and correlates negatively to overall survival and disease progression. Most notably, a switch from the expression of the epithelial cell-cell adhesion molecule E-cadherin to the mesenchymal N-cadherin is thought to be central to EMT, and it is commonly found in association with disease progression in various cancers including melanoma, pancreatic-, bladder- and colorectal cancer (reviewed by Cavallaro et al. [56]). Other studies found elevated expression of a number of E-cadherin transcriptional repressors such as Twist, Slug, ZEB1/2 and Snail1 linked to poorer prognosis in endometrial-, colorectal-, hepatocellular-, bladder-, gastric- and lung cancer [57–63]. Equally, increased expression of vimentin was associated with poorer outcomes for patients with gastric-, colorectal-, bladder- and breast cancer [58, 64–66]. However, the correlation of EMT with poorer overall or disease-free survival is not universal. A recent study, which established a comprehensive EMT gene expression signature in tumour tissue, found that poorer disease-free survival was associated with an EMT gene expression pattern in ovarian and colorectal cancer but not in breast cancer; therefore, it will be important to better define the context in which EMT gives cancer cells a selective advantage [67]. As detailed above, EMT marker gene or protein expression has also been studied in CTCs, and the overall emerging

, CK+

, CD45<sup>−</sup> false-

higher than EpCAM-isolated CTC numbers, we also detected more Hoechst+

diagnostic errors when progressing CTC-based assays into the clinic.

**6. EMT-phenotype CTCs, do they have clinical relevance?**

from false positives still needs to be confirmed.

248 Tumor Metastasis

Despite open questions regarding how EMT contributes to cancer progression and drug resistance, there is strong evidence that EMT changes, per se, are useful prognostic markers. Consequently, EMT-CTC isolation and analysis have the capacity to progress EMT research and importantly allow the development of feasible, non-invasive diagnostic tests to predict and monitor the effectiveness of specific therapies. More reliable identification of these cells will permit the translation of EMT and CTC research into clinically relevant tests to guide therapy.

This work was supported by the Cancer Institute New South Wales through the Centre for Oncology Education and Research Translation (CONCERT). J.P. is recipient of an Australian Rotary Health Gynaecological Oncology PhD Scholarship. D.L. is recipient of an Ingham Research Institute Director's PhD Scholarship. Human ethics approval, HREC/13/LPOOL/158, was obtained and managed by the CONCERT Biobank.
