Strategies for the enrichment of circulating tumour DNA in clinical samples

Current drive for personalised medicine approaches is demonstrating an increasing need for reliable biomarkers and robust detection methods with less invasive and low-cost technologies. Despite of the limitations, early detection of genetic and genomic alterations shows a great potential to contribute to cancer patient management at many different levels, including diagnosis, treatment choice and monitoring, and identification of drug resistance. However, current molecular methods still lack the analytical sensitivity to detect low abundance mutations in high wild type DNA background. Recently developed minor-allele enrichment assays are practical and cost-effective, and allow detection of very low abundance mutations in liquid biopsy samples, that could have relevance in clinical management. Thus this work aimed to develop mutation specific nuclease based enrichment for most common oncogenic driver PIK3CA hotspot mutations. Enrichment was validated using cutting-edge technology, such as digital PCR, as well as combined with a novel, in-house, SYBR Green real-time quantitative PCR detection method. This advancement enabled detection of PIK3CA mutations in a variety of clinical breast cancer samples (tissue biopsy and blood) with a low mutant allele frequency. Moreover, this work led to a novel prediction model to accurately calculate initial mutant allele frequency in clinical samples. Finally, addressing a lack of standardisation in liquid biopsy sample preparation, in this thesis a microfluidic component application for circulating cell-free DNA preparation from whole blood to enriched circulating tumour DNA was developed and tested. This versatile workflow has the potential to be applied for routine diagnostic testing at the point of care.