EpCAMlow Circulating Tumor Cells: Gold in the Waste

Hindawi Disease Markers Volume 2019, Article ID 1718920, 5 pages https://doi.org/10.1155/2019/1718920 Review Article EpCAMlow Circulating Tumor Cells: Gold in the Waste Chiara Nicolazzo ,1 Angela Gradilone,1 Flavia Loreni,1 Cristina Raimondi,2 and Paola Gazzaniga1 1 2 Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy Department of Radiological, Oncological and Anatomopathological Sciences, Sapienza University of Rome, 00161 Rome, Italy Correspondence should be addressed to Chiara Nicolazzo; Received 3 May 2019; Revised 6 September 2019; Accepted 11 September 2019; Published 17 September 2019 Academic Editor: Fabrizia Bamonti Copyright © 2019 Chiara Nicolazzo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The CellSearch® system which is still considered the gold standard for the enumeration of circulating tumor cells (CTC) utilizes antibodies against the epithelial cell adhesion molecule (EpCAM) for CTC enrichment. Recently, CTC discarded by the CellSearch® system due to their low EpCAM expression have been isolated and analyzed. We here sought to discuss technical and biological issues concerning the isolation and characterization of EpCAMlow CTC, highlighting the enormous potential of this subpopulation discarded by CellSearch®, which might instead reveal an unexpected clinical significance in tumor types where CTC enumeration has never been validated for prognostic and predictive purpose. 1. Introduction The CellSearch® system (Menarini Silicon Biosystems, Castel Maggiore, BO, Italy) was placed on the market by Veridex Corporation (Warren, NJ) in 2004, and despite 15 years having passed, it still considered the gold standard for the enumeration of circulating tumor cells (CTC), the first and the only one cleared by the US FDA for monitoring of metastatic breast, colorectal, and prostate cancers [1]. The CellSearch® system detects CTC from the whole blood of cancer patients through an immunomagnetic selection using a ferrofluid capture reagent. This is a suspension of magnetic nanoparticles conjugated to a mouse monoclonal antibody recognizing the epithelial cell adhesion molecule (EpCAM) present on the surface of epithelial origin cells. The enriched cells are then labelled with fluorescent dyes for the detection of nucleus; cytokeratins (CK) 8, 18, and 19 (as markers of epithelial origin); and CD45 (expressed on leukocytes), to discriminate the cells of epithelial origin from unwanted blood ones [2]. Therefore, an object is defined as a CTC when having round to oval morphology, a visible nucleus, positive staining for CK, and negative staining for CD45, according to the manufacturer’s definition [3]. The decision to target an epithelial cell antigen for immunomagnetic enrichment of CTC relies on the premise that epithelial cells are absent into bloodstream under physiological conditions [4]. Based on the evidence that monoclonal antibodies directed against EpCAM are broadly reactive with the tissue of epithelial-derived cancers [5], a series of preliminary studies was performed using flow cytometry assay therefore resulting in the choice of EpCAM as the preferential target for CTC immunomagnetic detection [1]. Nevertheless, in the following years, it became clear that higher numbers of CTC can be detected using alternative, EpCAM-independent methods, suggesting that a mixture of EpCAM-positive and EpCAM-negative tumor cells circulates in the blood [6]. In this review, we will argue the unresolved issue of CTC undetected by CellSearch®, with a particular focus on the latest developments reported by the group of Terstappen. In particular, we will discuss technical and biological issues concerning the isolation and characterization of CTC expressing no or low EpCAM, highlighting the enormous potential of this subpopulation discarded by the system, which might instead reveal an unexpected clinical significance in tumor types where CTC enumeration has never been validated for prognostic and predictive purpose. 2 2. EpCAMhigh and EpCAMlow Circulating Tumor Cells The presence of CTC exhibiting different phenotypes in the same patient due to tumor heterogeneity induced Terstappen and Co. to conduct in-depth studies on CTC detection through the CellSearch® system, with a focus on discarded ones expressing no or low EpCAM [7–9]. In 2015, the authors described a method to investigate the presence of two subpopulations of CTC: EpCAMhigh and EpCAMlow CTC. After immunomagnetic depletion of EpCAMhigh cells, the blood sample discarded by CellSearch® was collected through the Automatic Sample Collection Device (ASCS), inserted between the waste tube from CellTracks Autoprep system and the waste container [7]. The discarded blood coming out of the Autoprep was alternatively collected manually by placing a 50 mL conical tube under the outlet [8]. Both ways, the blood sample waste was then passed through the filtration device and the EpCAMlow CTC collected on the microsieve were analyzed by immunofluorescence staining [7–9]. A cocktail of fluorescently labeled antibodies (panCK and CD45) was used to stain cells and to correctly classify them as CTC. The EpCAMlow cells had a nucleus identified by DAPI, expressing CK, but not CD45. Using such proven and relevant testing protocols and tools, three studies were carried out to address how many CTC showing no or low EpCAM expression were discarded during immunomagnetic isolation by CellSearch® and whether their presence was associated with clinical outcome. Results from a pilot study in patients with metastatic lung cancer did not show any significant correlation between the presence of EpCAMlow CTC and overall survival (OS), although the percentage of CTC patients increased when adding the number of CTC found in the blood waste [7]. Similar observations were made in a study involving 97 patients with advanced non-small-cell lung cancer [8]. In 2018, the presence of EpCAMhigh and EpCAMlow CTC was determined in castration-resistant prostate cancer and metastatic breast cancer patients in a multicenter study. Here, again, it was showed that the presence of EpCAMlow CTC was not correlated with poor OS, even though the number of CTC increased when considering both CTC subpopulations [9]. In all these studies, a significant difference was observed for the presence of ≥5 EpCAMhigh CTC in relation to OS whereas no significant difference was observed for ≥5 EpCAMlow CTC, demonstrating that the strong correlation with survival can be solely contributed to EpCAMhigh CTC. 3. EpCAMlow CTC Isolation. Technical and Biological Issues Since CellSearch® was designed for the immunomagnetic enrichment and fluorescent labeling in order to detect circulating epithelial-derived tumor cells [10], it seems obvious that any variation in epithelial antigen expression compr (...truncated)