Circulating tumour cells: the evolving concept and the inadequacy of their enrichment by EpCAM-based methodology for basic and clinical cancer research

Annals of Oncology, Aug 2014

Increasing evidence suggests that circulating tumour cells (CTCs) are responsible for metastatic relapse and this has fuelled interest in their detection and quantification. Although numerous methods have been developed for the enrichment and detection of CTCs, none has yet reached the ‘gold’ standard. Since epithelial cell adhesion molecule (EpCAM)-based enrichment of CTCs offers several advantages, it is one of the most commonly used and has been adapted for high-throughput technology. However, emerging evidence suggests that CTCs are highly heterogeneous: they consist of epithelial tumour cells, epithelial-to-mesenchymal transition (EMT) cells, hybrid (epithelial/EMT+) tumour cells, irreversible EMT+ tumour cells, and circulating tumour stem cells (CTSCs). The EpCAM-based approach does not detect CTCs expressing low levels of EpCAM and non-epithelial phenotypes such as CTSCs and those that have undergone EMT and no longer express EpCAM. Thus, the approach may lead to underestimation of the significance of CTCs, in general, and CTSCs and EMT+ tumour cells, in particular, in cancer dissemination. Here, we provide a critical review of research literature on the evolving concept of CTCs and the inadequacy of their enrichment by EpCAM-based technology for basic and clinical cancer research. The review also outlines future perspectives in the field.

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Circulating tumour cells: the evolving concept and the inadequacy of their enrichment by EpCAM-based methodology for basic and clinical cancer research

P. K. Grover 2 A. G. Cummins 1 T. J. Price 0 I. C. Roberts-Thomson 1 J. E. Hardingham 0 0 Haematology-Oncology, The Queen Elizabeth Hospital , Woodville South , Australia 1 Gastroenterology and Hepatology 2 Departments of Surgery Increasing evidence suggests that circulating tumour cells (CTCs) are responsible for metastatic relapse and this has fuelled interest in their detection and quantification. Although numerous methods have been developed for the enrichment and detection of CTCs, none has yet reached the 'gold' standard. Since epithelial cell adhesion molecule (EpCAM)-based enrichment of CTCs offers several advantages, it is one of the most commonly used and has been adapted for high-throughput technology. However, emerging evidence suggests that CTCs are highly heterogeneous: they consist of epithelial tumour cells, epithelial-to-mesenchymal transition (EMT) cells, hybrid (epithelial/EMT+) tumour cells, irreversible EMT+ tumour cells, and circulating tumour stem cells (CTSCs). The EpCAM-based approach does not detect CTCs expressing low levels of EpCAM and non-epithelial phenotypes such as CTSCs and those that have undergone EMT and no longer express EpCAM. Thus, the approach may lead to underestimation of the significance of CTCs, in general, and CTSCs and EMT+ tumour cells, in particular, in cancer dissemination. Here, we provide a critical review of research literature on the evolving concept of CTCs and the inadequacy of their enrichment by EpCAM-based technology for basic and clinical cancer research. The review also outlines future perspectives in the field. - introduction Although the risk of cancer death is linked to the stage of the disease at the time of diagnosis, >90% of cancer deaths result from the development of disseminated metastases [14]. Currently, the mechanism of genesis of metastases from solid tumours is not fully understood and remains one of the most enigmatic aspects of cancer biology. New insights are fast causing paradigm shifts in our understanding of metastatic phenomena. For instance, recent evidence indicates that tumour cells disseminate at a relatively early stage of the natural history of tumour growth [5, 6]. Current tumour staging procedures and high resolution imaging technologies are not sensitive enough to detect micro-metastases or early tumour cell dissemination, the key events in tumour progression. Therefore, detection of disseminated tumour cells (DTCs) might be complementary to the current imaging procedures used for tumour staging and improve the identification of cancer patients at high risk of metastatic relapse [710]. This view has spurred interest in the development of sensitive assays that allow the specific detection of single DTC in cancer patients. Since sampling for the detection of DTCs is an invasive procedure, therefore, the focus in recent years has shifted to circulating tumour cells (CTCs) in the peripheral blood. Over the years, many procedures have been developed for the detection of CTCs in the peripheral blood, yet none has reached the gold standard of sensitivity and more importantly, of specificity. Epithelial cell adhesion molecule (EpCAM) is expressed ubiquitously, albeit at variable levels, in epithelial cells and their cancers, but is absent in blood cells [1113]. This galvanized our group to pioneer the development of first-ever use of EpCAMbased enrichment followed by reverse transcription-polymerase chain reaction (RT-PCR) for detecting CTCs in cancer patients [14]. The use of our anti-EpCAM-labelled microbeads for this purpose was later commercialized as Epithelial Enrich (Dynal Biotech, Norway). Among the current EpCAM-based technologies, the automated CellSearch system (Veridex, Warren, NJ, USA) has gained considerable attention over the past several years [8]. The system has been cleared by the Food and Drug Administration for monitoring CTCs in metastatic breast, colon, and prostate cancers [1517]. Currently, the EpCAMbased approach is one of the most commonly used in exploring CTCs, and a great deal of effort and resources has been invested in adapting the method for modern automated high-throughput The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: . technologies [1824]. Numerous studies from our laboratory [6, 25, 26] and others have shown that the presence of elevated levels of CTCs, as determined by the EpCAM-based approach including the CellSearch system, is negatively correlated with prognosis in patients with various types of cancers. However, the relatively low sensitivity and specificity of the approach is a significant concern [2729]. Furthermore, increasing evidence suggests that CTCs are highly heterogeneous [10, 3036]. They consist of epithelial tumour cells, epithelial-to-mesenchymal transition (EMT) cells, hybrid (epithelial/EMT+) tumour cells, irreversible EMT+ tumour cells, and circulati (...truncated)


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P. K. Grover, A. G. Cummins, T. J. Price, I. C. Roberts-Thomson, J. E. Hardingham. Circulating tumour cells: the evolving concept and the inadequacy of their enrichment by EpCAM-based methodology for basic and clinical cancer research, Annals of Oncology, 2014, pp. 1506-1516, 25/8, DOI: 10.1093/annonc/mdu018