Dissertation

Capture of colorectal circulating tumour cells for mutational analysis

Nageswaran, Haritharan

2016

Overview

Colorectal cancer accounts for 13% of all cancers in the UK. Surgical resection offers the best prospects of a cure but 45% of these patients will still develop metastases with haematogenous spread suspected to be the main method of dissemination to distant sites. Circulating tumour cells (CTCs) are intermediaries in this process and whilst their exact role remains unclear, their presence has proven to be an independent prognostic factor in breast, prostate and colorectal cancer. The capture and isolation of intact CTCs for mutational analysis could not only shed light on the process of metastases but could guide therapy. However, CTCs are extremely rare events in the blood stream and their detection and capture have been elusive, particularly the capture of sufficient numbers to allow mutational analysis. Capture of CTCs based on their larger size in comparison to most other cells in the blood, combined with immunohistochemical characterization to identify them as tumour cells has shown promise in the past. This method of capture also causes minimal disruption to the cell structure and therefore enables accurate mutational analysis. In this study a novel filtration based technology was used to capture and isolate CTCs from the peripheral blood of colorectal patients to allow assessment of their KRAS mutational status. An immunohistochemical staining protocol to identify colorectal CTCs captured from whole blood was developed and incorporated into the functioning of the filtration device. Two patient populations were examined to identify and enumerate colorectal CTCs: those undergoing liver surgery for colorectal liver metastases and those undergoing palliative therapy for inoperable disease. Significant numbers of CTCs were only identified in those with inoperable disease. Mutational analysis was performed using a commercially available cast-PCR kit designed to detect KRAS mutations. Preliminary experiments found that DNA extracted directly from captured CTCs was insufficient in quantity and quality for successful analysis. Whole genome amplification of DNA from intact cells was therefore performed prior to mutational analysis. A multi-step protocol of cell capture, whole genome amplification and cast PCR was developed and tested on patient samples to show that KRAS mutational analysis could be performed on CTCs using this filtration based technology. In conclusion, the study shows that capture and isolation of intact CTCs is possible using filtration based technology. However, even in patients with relatively high numbers of CTCs, whole genome amplification needs to be performed prior to performing mutational analysis.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biomedical engineering
• ISBN: 1073967646, 9781073967643