Isolation and Enumeration of CTC in Colorectal Cancer Patients: Introduction of a Novel Cell Imaging Approach and Comparison to Cellular and Molecular Detection Techniques

SIMPLE SUMMARY: Despite significant progress in screening and treatment regimens, colorectal cancer (CRC) still is a major health burden lacking profound liquid biomarkers for identifying patients at risk. Circulating tumour cells (CTC) have the potential to non-invasively improve the diagnosis. We have already established a sensitive semi-quantitative RT-qPCR against CK20 for CTC quantification in CRC patients. For clinical translation, this study aims to validate our molecular detection method by terms of cytological approaches, and implement a novel semi-automated microscopic detection after immunofluorescence labelling of CTC. Additionally, we aim to compare our PCR-based approach to a marker-independent, but size-dependent, enrichment process. We have successfully applied the validation techniques and proved their feasibility. Enumeration by size yielded the highest numbers of CTC and demonstrated to be the most reliable strategy for CTC detection in CRC patients. Future studies with larger patient cohorts will have to investigate the clinical significance and prognostic value of this approach. ABSTRACT: Circulating tumour cells (CTC) were proven to be prognostically relevant in cancer treatment, e.g., in colorectal cancer (CRC). This study validates a molecular detection technique through using a novel cell imaging approach for CTC detection and enumeration, in comparison to a size-based cellular and correlated the data to clinico-pathological characteristics. Overall, 57 CRC patients were recruited for this prospective study. Blood samples were analysed for CTCs by three methods: (1) Epithelial marker immunofluorescence staining combined with automated microscopy using the NYONE(®) cell imager; (2) isolation by size using membrane filtration with the ScreenCell(®) Cyto IS device and immunofluorescence staining; (3) detection by semi-quantitative Cytokeratin-20 RT-qPCR. Enumeration data were compared and correlated with clinic-pathological parameters. CTC were detected by either approach; however, with varying positivity rates: NYONE(®) 36.4%, ScreenCell(®) 100%, and PCR 80.5%. All methods revealed a positive correlation of CTC presence and higher tumour burden, which was most striking using the ScreenCell(®) device. Generally, no intercorrelation of CTC presence emerged amongst the applied techniques. Overall, enumeration of CTC after isolation by size demonstrated to be the most reliable strategy for the detection of CTC in CRC patients. Ongoing studies will have to unravel the prognostic value of this finding, and validate this approach in a larger cohort.