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Title: Investigation of mutant DNA in plasma of patients with colorectal neoplasia
Author: Clark, Andrew J. E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2012
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Introduction: Colorectal cancer is a major clinical and public health problem. Early stage colorectal cancer is amenable to surgical intervention with a high cure rate. Screening for the disease has now been rolled out throughout the UK, although current screening modality of faecal occult blood testing (FOBT) is suboptimal. Specific genetic alterations found in primary tumours, including mutations in proto-oncogenes, microsatellite instability and loss of heterozygosity (LOH), have been shown to be detectable in the host plasma. Colorectal cancer is an excellent paradigm in which to investigate the application of assays to detect tumour-related plasma DNA. There is substantial data regarding specific mutations and their frequency in colorectal cancer tissue and adenomas. The ability to detect early stage colorectal neoplasia using DNA plasma assays holds considerable promise as a non-invasive screening modality. Materials and Methods: Assay performance was assessed using colorectal cancer cell lines and archived material from genetic studies. Assays were then applied to a prospective cohort of 124 colorectal neoplasia cases and controls. Plasma DNA was quantified using a sensitive DNA binding fluorescent dye and detection platform. A well-characterised tumour-specific mutation was used as the target for the first assay. This mutation was in a poly A tract of the transforming growth factor beta receptor II (TGFpRII) gene and was assessed using a restriction fragment length polymorphism (RFLP) assay. Microsatellite analysis was performed using 3 polymorphic markers relevant to colorectal neoplasia in matched tumour, normal and plasma DNA samples. A novel assay was developed exploiting real-time fluorescent PCR amplification of single nucleotide polymorphisms in the adenomatous polyposis coli (APC) gene as a means to detect tumour-specific allelic imbalance. Assay performance was determined in spiking experiments, and performance assessed in clinical samples. A further iteration of the assay was developed to quantify tumour specific alleles in plasma by counting individual alleles PCR amplified from plasma DNA of cases and controls. Results: Quantification of total plasma DNA revealed a significant difference between cases and controls (area under the receiver operator curve: 0.7). Despite intensive efforts to overcome the problem, the TGFpRII RFLP assay was affected by technical difficulties when applied to plasma DNA. The required high number of PCR cycles introduced artefact and limited its applicability. Microsatellite analysis demonstrated LOH in matched tumour and plasma samples with maximal sensitivity of 67% but specificity was only 32%. The quantitative PCR assay to detect APC LOH was able to detect 3-5ng of homozygous DNA introduced into 1 ml of heterozygous plasma, and accurately quantified LOH in tumour tissue. However, it was not able to discriminate cases from controls by assessment of plasma DNA. Counting of alleles in plasma DNA from a test case demonstrated matching LOH to that seen in the primary tumour. Allele counting of a further cohort suggested that this approach has a low false positive rate. Discussion: Analysis of plasma DNA is technically challenging due to a low abundance of partially fragmented mutant DNA sequences admixed with normal DNA in plasma with DNAases and PCR inhibitors. However total quantity of plasma DNA was higher in cancer cases compared to controls with good specificity for cancer at high DNA concentrations. Fluorescent microsatellite analysis of plasma DNA demonstrated encouraging overall sensitivity but poor specificity that waslikely at least partly a reflection of low DNA abundance and hence sampling error within aliquots of plasma DNA. A quantitative PCR approach was developed and validated with relevant levels of in vitro sensitivity, and improved discrimination of LOH in some clinical samples. Adaptation of this approach to count alleles individually is labour intensive and expensive but appears to address issues of sampling error due to abundance and hence improves specificity. These data, whilst highlighting some of the technical challenges in the field, demonstrate associations of plasma DNA in colorectal neoplasia that warrant further investigation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available