Clinical Pathology - Theses

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    Evaluating the clinical applicability of tumour mutational signatures in colorectal cancer and related syndromes
    Georgeson, Peter ( 2022)
    Colorectal cancer (CRC) poses a major health burden. It is the second most common cause of cancer death, with the impact of CRC incidence and mortality continuing to grow worldwide. Early detection of CRC substantially improves outcomes, motivating the adoption of screening programs aimed at identifying high-risk individuals for ongoing surveillance. However, efforts to identify individuals predisposed to developing CRC have been hampered by the complexity and heterogeneity of CRC. Recent advances in DNA sequencing technology enable the genome to be studied in high resolution, providing the ability to detect a wide array of somatic mutations and rearrangements in the DNA of cancer cells. Certain mutagenic processes leave identifiable mutational patterns in cancer genomes. The advent of cost-effective large-scale DNA sequencing enables systematic detection of these patterns, known collectively as tumour mutational signatures. To date the main application of mutational signatures has been research focused, where they have been used to determine cancer subtypes and categorise the underlying changes to DNA associated with those subtypes. However, their applicability to clinical contexts have not been sufficiently explored. A limitation to the adoption of mutational signatures clinically is the prevalence of FFPE-preserved tissue in conjunction with whole-exome and panel-sequenced data, in contrast to the use of fresh-frozen whole-genome sequenced data typical in research settings. Formalin is mutagenic, which can result in artefactual variants, while at least an order of magnitude fewer mutations are detected with whole-exome and panel-sequenced data compared to whole-genome. We assess the utility of mutational signatures generated from both whole-exome and panel-sequenced data derived from FFPE-preserved tissue. Specifically, we show that inherited predispositions to CRC, including Lynch syndrome and MUTYH-associated polyposis, can be accurately identified with whole-exome sequenced data from FFPE-preserved tumour tissue, and that, with the correct methodology, biallelic MUTYH carriers can be identified from panel-sequenced FFPE-preserved tumour tissue. Understanding the relationship between environmental exposures and CRC development has implications for both prevention and screening. We consider the ability of mutational signatures to detect mutation patterns arising from exposure to colibactin, the genotoxic compound synthesised by pathogenic E. coli and a potential cause of sporadic (non-inherited) CRC. Demonstrating distinct genomic, clinic-pathological and epidemiological characteristics, we show the potential existence of a distinct subtype of CRC based on the presence of the colibactin-associated mutational signature. The effectiveness of mutational signatures depends on the environment in which they are calculated. We analyse the impact of key analytical parameters and recommend specific filtering settings for variant allele fraction and sequencing depth. We identify situations where mutational signatures are less effective, recommending minimum mutation counts and maximum signature reconstruction error, enabling confidence in mutational signature results to be based on their specific application. The results presented in this thesis have clinical applications. We show that applying mutational signatures to individual tumours provides direct evidence suggesting a particular aetiology. More broadly, mutational signatures provide evidence indicating the likely pathogenicity of co-occurring mutations. Mutational signatures are an important technique for extracting information from sequencing data. This thesis demonstrates clinical applications of mutational signatures in CRC and related syndromes.
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    Characterising tumour and immunological heterogeneity in colorecal cancer
    Shembrey, Carolyn Elizabeth ( 2021)
    Despite improvements in surgical oncology and precision medicine, 5-year survival rates for those with late-stage colorectal cancer (CRC) remain extremely poor and innovative treatment strategies are needed. Although T-cell directed immunotherapies are strikingly effective in many solid cancer types, durable responses are limited to approximately 5% of all CRC patients. Accordingly, there is an urgent need to explore alternate immunotherapeutic strategies which harness other cytotoxic cell types, most notably NK cells. However, the inter-patient variability of NK cell involvement in CRC, and particularly in colorectal liver metastases (CRLMs), is poorly characterised. Moreover, very little is known regarding the influence of tumour heterogeneity on immunotherapy response, mandating the development of novel methodologies which can dissect divergent responses to immuno-, chemo- and targeted therapies at the level of individual tumour cell subpopulations. In this thesis, the scope of tumour and immunological heterogeneity in primary CRC and CRLMs was assessed from transcriptomic, spatial and functional perspectives. A CRC-specific NK cell gene signature which infers the NK cell load of individual tumours from bulk RNAseq data was designed and validated. Differential expression analysis revealed that tumours with high evidence of this NK cell signature were characterised by the upregulation of chemotactic and cytolytic transcriptional programs. Furthermore, amongst patients with primary CRC, those with high NK scores were shown to have better survival outcomes in two independent cohorts. Focussing on metastatic disease, Cell type Identification by Estimating the Relative Subsets of RNA Transcripts (CIBERSORT) analysis revealed significant variance in terms of immune cell composition between CRLMs and adjacent normal (AdjN) liver tissue. Using a novel mIHC panel to quantify the scope of NK cell infiltration and NK cell ligand expression, it was determined that neoadjuvant chemotherapy increased NK cell penetrance of CRLM tissue, restoring NK cell load to levels comparable with the AdjN liver. Furthermore, NK cell densities were comparable in the AdjN liver and CRLM tissue of patients who partially responded to chemotherapy whereas nonresponders showed preferential accumulation in the AdjN liver. Preliminary data is also presented regarding the establishment of a NK cell versus patient-derived organoid (PDO) co-culture killing assay which can be used to assess the functional activity of NK cells against CRLMs. Lastly, a lineage tracing technique termed optical barcoding (OBC) was employed to study the relationship between intra-tumour heterogeneity and treatment response. Stably marking heterogenous populations of CRC cells with unique fluorescent signatures enabled the real-time identification and tracking of different cellular subpopulations under pharmacological selective pressures. Moreover, optimising the fluorescent protein panel allowed the maximum theoretical subpopulation resolution for the OBC technique to be achieved, greatly improving throughput as compared with previously published fluorescent barcoding techniques. Collectively, the results provided herein have deepened our understanding of tumour and immunological heterogeneity in primary CRC and CRLMs, and, by introducing several novel technologies, have laid strong foundations for future studies.