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Title: Investigation of the tissue origins of cell-free DNA and candidate biomarkers for liquid biopsy
Author: Laurent, Danny
ISNI:       0000 0004 8498 0148
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Liquid biopsy is the sampling and analysis of analytes from various biological fluids to detect and diagnose cancer, for which it is an alternative to solid tissue biopsy for the detection of cancer. Compared to solid tissue biopsy, liquid biopsy is minimally invasive. Further, its ability to capture intra-tumoural and inter-metastatic heterogeneity in cancer makes it an attractive platform to complement solid tissue biopsy. Molecules of free circulating DNA in the bloodstream, termed cell-free DNA (cfDNA), can be used as an analyte for liquid biopsy. Various types of tumour-specific alterations can be detected in the cfDNA of cancer patients. To date, liquid biopsy assays based on the analysis of cfDNA are still lacking in consistency and precision. Clinical validity and utility have not been shown for the majority of assays. The composition of cfDNA in health and under particular physiological and pathological conditions still need to be better understood. Further improvements to diagnostic tools are required to enable detection of small numbers of molecules for liquid biopsy. Animal models may be useful as a platform to study cfDNA and cancer biology in liquid biopsy. The utilisation of an animal model may spare ethically sensitive clinical materials from patients and serve as an alternative when serial sampling from patients is challenging. For cancer biology, experiments in animal models are required to understand the mechanisms underlying the onset of malignancies and to identify methods to prevent, diagnose and treat diseases. In this PhD thesis, the tissue origins of cfDNA were investigated using tissue-specific reporter mouse models. Liver injury was induced in mice to investigate the effect of tissue injury on the composition of cfDNA. In addition, the potential of the analyses of cfDNA as a biomarker of tissue injury in paracetamol (APAP) overdose patients was investigated. Absolute measurement of the tissue origins of cfDNA using ddPCR showed that myeloid, lymphoid and erythroid cells were major tissue contributors to cfDNA, adding up to ~78% of cfDNA pool in healthy tissue-specific reporter mice. A minor contribution was observed from hepatocytes (4.2%). No contribution was detected from muscle cells. Up to ~17.6% of contribution was unaccounted, indicating contribution of other tissues not studied in this project. Following APAP overdose in mice, total concentration of cfDNA increased by ~100-fold and the contribution of hepatocytes increased by ~20-fold. Similarly, a substantial increase in total cfDNA and liver-derived cfDNA was also observed in clinical samples from APAP overdose patients, highlighting the potential of the analyses of cfDNA as a biomarker of tissue injury in APAP overdose patients. In separate experiments, genomic analysis was performed on the cancer genome of the Pirc rat, a mutant rat of colorectal cancer (CRC), to screen for potential candidate biomarkers for liquid biopsy. Genomic analysis in the cancer genome of the Pirc rat showed candidate LOH biomarkers in chromosome 18, along with other types of somatic alterations in relevant cancer genes. These are potential markers of cancer development in future cfDNA studies in the Pirc rat. To conclude, successful analyses in rodent models showed the tissue origins of cfDNA in the healthy state and following APAP overdose, as well as potential candidate biomarkers of CRC for liquid biopsy.
Supervisor: Aitman, Timothy ; Arends, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: liquid biopsy ; cfDNA ; mouse model ; liver injury ; ddPCR