**5. The pitfalls of non-EpCAM-based CTC isolation**

**EMT markers Main finding/clinical correlation [reference]**

breast cancer [21]

and with poorer OS [30]

HER2, VIM, FN1 Presence of EMT-CTCs correlates to disease stage [23] CK, VIM, FN1 The presence of EMT-CTCs correlates to shorter PFS [32]

**EpCAM-based isolation combined/compared with other methods:**

CK Low CK level CTCs were correlated with receptor negative metastatic breast cancer

Vimentin Vimentin expression in prostate cancer CTCs is associated with decreased OS [31]

Cell-surface vimentin (CSV) EMT-CTC numbers were a more reliable progressive disease marker for breast can‐

CK, EpCAM Most CTCs from metastatic breast cancer patients showed intermittent phenotype

increased EMT-CTC numbers in metastatic breast cancer patients with disease progression [35]

patients with poorer prognostic markers (ie needed neoadjuvant treatment) [36]

EMT-CTCs were more common in primary breast cancer

CK, EpCAM, CDH1, FN1, CDH2 Combined EpCAM/EGFR/Her2-based CTC isolation was linked to

Cell-surface vimentin (CSV) >5 >5 EMT-CTCs more common in progressive colorectal cancer [26]

Vimentin, twist, CK EMT -CTCs more prevalent in CTCs from metastatic breast cancer

EpCAM, CK, VIM CTCs of 'CTC-negative' NSCLC patient by EpCAM based isolation were tumorigenic in mice [29].

Vimentin, CK Pancreatic cancer CTCs without CK or vimentin are more commonly in patients with lymphnode metastasis [38]. CK, EpCAM,VIM, TWIST Proportion of EMT-CTCS is linked to response to therapy in gastric cancer [39] EpCAM, CK, VIM, TWIST Intermittent phenotype and EMT CTCs were predominant in hepatocellular carcinoma and correlated with metastasis [40] OS: overall survival, PFS: progression-free survival. Dependent on their nature, EMT markers follow protein or gene

patients [37]

cer patients when isolated targeting CSV versus EpCAM [27]

portal vein tumor thrombus in liver hepatocellular carcinoma [33].

The expression of both twist and vimentin in CTCs was significantly correlated with

while 16% of patients had EpCAM- only CTCs and 33% EpCAM-null CTCs [34]

Gene expression of EMT markers shown in CTCs [22]

CTCs with intermittent EMT phenotype are common in advanced prostate- and

**EpCAM-based immunomagnetic isolation:**

EpCAM, CK, E-cadherin, vimentin, N-cadherin, CD133

TGFß1, FOXC1, CXCR4, NFKB1,

Vimentin, twist, ZEB1, ZEB2, snail, slug and E-cadherin

TWIST1, SLUG, SNAIL1, ZEB1,

**Isolation by cell size:**

nomenclature

**Targeted immunomagnetic isolation (non-EpCAM):**

**CD45-based immunomagnetic blood cell depletion:**

**Table 1.** Detection of EMT biomarkers in CTCs.

FOXC2

VIM, ZEB2

246 Tumor Metastasis

The clear advantage of EpCAM-based CTC isolation is the observation that EpCAM is only rarely found on cells circulating in the blood stream of healthy individuals, resulting in a limited number of false-positive 'CTCs'. In our hands, using the IsoFlux CTC platform and EpCAM-based enrichment, the average Hoechst+ , CK+ , CD45<sup>−</sup> -false positive 'CTCs', obtained from 10 healthy blood donors is 1.8 per 9 ml of blood with a range of 0–5 cells. By contrast, the greatest problem with the use of EMT markers for CTC isolation or CTC identification, or with CTC isolation techniques relying on physical cell properties such as size and plasticity, is the increased risk of detecting false-positive 'CTCs'. This is the case because some rare cells found in normal blood can express a number of epithelial and mesenchymal markers. For instance, circulating endothelial cells (CECs) can be found at varying numbers in blood samples of healthy individuals (0–29/ml blood) [41] and increased numbers in cancer patients [42]. CECs do not only express cytokeratin, but typical EMT markers such as N-cadherin, EGFR, vimentin and fibronectin [43–48]. Moreover, circulating endothelial cells tend to be above 10 μm in diameter [49], and some endothelial cells might therefore not be excluded from size-based CTC enrichment. There are currently limited data evaluating potential CEC contamination in either filter-enriched CTC samples or samples enriched by positive or negative immunotargeting. However, it is likely that the inclusion of CECs in CTC counts in the literature (i.e. false-positive CTCs) has inadvertently led to overestimation.

A particularly interesting approach to avoid the issues surrounding EpCAM is the use of CD146 (MCAM)-based immunomagnetic CTC isolation. Elevated expression of CD146 has been reported for melanoma, breast-, ovarian- and prostate cancer [50], and CD146-based immunomagnetic CTC isolation was reported for breast cancer and melanoma patients [44, 51]. However, CD146 is also an endothelial marker used to define and target CECs [41]. Thus, CD146-based CTC isolation needs to be complemented by cancer-specific CTC identification, 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 from false positives still needs to be confirmed.

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 higher than EpCAM-isolated CTC numbers, we also detected more Hoechst+ , CK+ , CD45<sup>−</sup> falsepositive '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 diagnostic errors when progressing CTC-based assays into the clinic.
