The number of Circulating Tumor Cells (CTCs) that can be isolated from blood of cancer patients is prognostic for the course of the disease. A higher number of CTCs correlates with a worse prognosis. A change from a higher number to a lower number of CTCs indicates a benefit of the current treatment for the patient and vice versa. For this reason the number of CTCs found with the CellSearch test can be used as a tool in the management of patient treatment. Repeated molecular analysis of CTCs enables the detection of changes during the course of the disease. This analysis could unmask details on proliferation stimulating pathways in the cells, which can lead to a change to a more effective treatment. With this information, targeted drugs could effectively be used until new analysis shows resistance and discloses possible new druggable pathways. This thesis covers the whole workflow from blood draw up to the analysis of single CTC genomes. We give an overview of all work done to analyze or unmask the cells captured with CellSearch tests and a review is included of single cell isolation methods suitable for sorting CTCs. In a review of Whole Genome Amplification (WGA) methods we show the technical details of the commercially available kits and the applicability is discussed on upstream target DNA quality and downstream applications. Cancerous origin of the epithelial cells isolated with CellSearch was expected but has been conclusively proven by the development of a Fluorescence In Situ Hybridization (FISH) test. The analysis of a large number of CTC’s from a series of prostate cancer patient samples clearly shows aberrant chromosome numbers. During development of FISH using unique sequence probes a highly efficient method was developed to remove repetitive DNA from these probes. We present the procedure to remove the repetitive DNA and show the quality and efficiency of the derived FISH probes. The possibility of analyzing genomes of single CTCs after CellSearch shows to be informative but labour intensive. The presented method using Fluorescence Assisted Cell Sorting (FACS) is a multi step procedure suitable for research but needs to be optimized and simplified to be able to use in routine diagnostics. The microwell single cell isolation technique has the potential of automation, speed and still a high degree of multi marker confirmation to authenticate the CTCs. It is a promising step forward in handling and separating CTCs.
|Award date||13 Jan 2017|
|Place of Publication||Enschede|
|Publication status||Published - 13 Jan 2017|