Abstract 767: Comparison of different methods for the detection of clusters of circulating tumor cells
Authors: Reduzzi Carolina , Zhang Youbin , Daidone Maria Grazia , Cappelletti Vera , Cristofanilli Massimo 
Published: September 2020

Abstract
BACKGROUND: In the last few years, there has been an increase in research interest and number of studies investigating clusters of circulating tumor cells (CTC clusters) due to their putative higher metastatic potential compared to individual CTCs. Although the majority of studies suggest a link between CTC clusters and worse clinical outcome, their clinical significance and biological role is still not fully demonstrated. A hurdle to this aim is the heterogeneity of the data produced using various technologies and detection strategies. Here, we compare three of the most used approaches to detect CTC clusters in clinical samples.
METHODS: Blood samples (30 ml) were collected from patients with metastatic breast cancer, divided in 3 aliquots and processed in parallel with 3 approaches for CTC cluster enrichment and detection: 1. PAR: Microfluidic chip enrichment (Parsortix®), staining for CD45 and CK (CellSearch CTC kit), detection by fluorescent microscope analysis 2. FIL: Enrichment by filtration (CellSieve™), staining for CD45, CK and CD31, detection by fluorescent microscope 3. CS: CellSearch™ platform for enrichment (EPCAM expression), staining (CD45 and CK) and enumeration.
RESULTS: Blood samples (n=6) were collected from 6 patients with metastatic breast cancer. CTC clusters were defined as groups of ≥2 cells expressing CK and lacking CD45, accompanied or not by leukocytes (CD45+/CK-). The counts of CTC clusters for each sample/approach were compared. By FIL, CTC clusters were detected in all samples, with some clusters formed by &gt;20 CTCs (not observed using PAR and CS). PAR and CS had similar recovery rates for CK+ clusters, but in all the samples processed with PAR, clusters formed by CK-/CD45- cells (possibly mesenchymal CTC clusters) were observed. This type of cluster, being CK-, is not detectable by CS identification criteria. FIL also detected CK- clusters, but it was difficult to distinguish between real clusters and artifacts, due to the high number of contaminating cells and to the fluorescent background given by the filtration membrane. By using PAR, instead, contaminating cells were less and it was possible to better evaluate the cell morphology. Moreover, PAR is a more flexible methodology, allowing downstream applications not feasible with FIL and CS (RNA analysis and cell culture).
CONCLUSIONS: By FIL it is possible to detect CK+ CTC clusters also in samples that would be otherwise considered negative by CS and PAR analysis. Moreover, we could detect clusters formed by CK-/CD45- cells that were undetectable with CS based on the lack of epithelial markers expression. The significance of these clusters is not clear yet and should be further studied. Therefore, in order to completely understand the role of CTC clusters in disease progression, it is instrumental to use technologies able to detect all types of clusters, whose analysis will potentially reveal new features of cluster-mediated dissemination.
Citation Format: Carolina Reduzzi, Youbin Zhang, Maria Grazia Daidone, Vera Cappelletti, Massimo Cristofanilli. Comparison of different methods for the detection of clusters of circulating tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 767.