Hypermethylation of CpG islands is one of the many processes that a developing cancer cell may use for the inactivation of tumour suppressor genes. The Sav/Hippo/Warts pathway was originally identified in Drosophila and shown to be responsible for controlling both growth and apoptosis, implying this is a tumour suppressor pathway. This pathway is both evolutionarily and functionally conserved in mammals. Work presented here shows that apart from FAT1 and YAP other pathway members are not epigenetically silenced in common epithelial or haematological cancers. FAT1 and YAP were frequently methylated in childhood acute lymphoblastic leukaemia (ALL) but unmethylated in epithelial cancers. Childhood ALL is a blood cancer with peak prevalence between the ages of 3-5 years. The epigenetics of this cancer were examined with three separate approaches; the first, a candidate gene approach, second a NotI restriction enzyme based array examining the methylation of genes residing on chromosome 3, and thirdly the methylated-CpG island recovery assay (MIRA) combined with CpG island arrays examining methylation on a genome-wide scale. These approaches identified a large number of novel genes which were frequently methylated in ALL. Many of the identified genes were new methylation targets and were shown to be likely targets for methylation in both common epithelial and haematological cancers. A series of these genes was seen to be specifically methylated in different leukaemia sub types, and to cluster T-ALL and B-ALL samples into high and low methylation clusters. When examined in chronic lymphoblastic leukaemia (CLL) methylation of two of the above genes was associated with disease progression and methylation of another gene was associated with response to clinical treatment.

Iron is central to the aetiology of gastrointestinal disease. Specifically, the toxic effects of excess, unabsorbed "luminal" iron ingested from the diet has been shown to be important in the development of inflammatory bowel disease and intestinal cancer. A platform for therapeutic intervention is likely to involve chelation of this luminal pool of iron. As such, a range of dietary iron chelators have been tested for their iron binding capacity. Natural biopolymers extracted from seaweed (alginates) and a variety of natural polyphenolic compounds were stratified in terms of their iron binding potential. One alginate, Manucol LD, was unique in its iron binding and demonstrated luminal iron chelation properties. With respect to the polyphenols, only one of the tested compounds (quercetin) displayed iron chelation activity in vitro and was able to suppress cellular concentrations of reactive oxygen species acting as an antioxidant. As such, it has been demonstrated that a unique alginate, Manucol LD, is an excellent candidate for sequestering luminal iron present in the gastrointestinal tract. These results underpin the rationale in utilising these types of natural and safe bio-polymers for the prevention and treatment of gastrointestinal disease.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and poses a significant health burden due to its rising incidence. The proto-oncogene PTTG is overexpressed in HNSCC and correlates with poor patient prognosis. A recent unpublished GEO profile eDNA array analysis has further suggested a potential upregulation of its binding partner PBF in HNSCC. PTTG and PBF cause transformation in vitro and tumour formation in vivo, both effects thought to be partly mediated by their interactions with the tumour suppressor protein p53. Dysregulation of the p53 pathway is frequently observed in HNSCC, thus alluding to the importance of functionally active p53 in the suppression of HNSCC initiation and progression. The work presented in this thesis describes the functional relationship between PTTG, PBF and p53 in HNSCC. Initial studies confirmed that PTTG and PBF are overexpressed in HNSCC tumours compared to matched normal tissue. In addition, high tumoural PTTG expression correlated with HPV status, whereas high tumoural PBF expression was associated with a significant gender bias. Further investigations established that PTTG and PBF functionally interact with p53 and cooperate to reduce p53 protein stability in HNSCC cells. Moreover, attenuation of PTTG or PBF expression led to dysregulated expression of p53-related genes involved in DNA repair and apoptosis, indicating that both proto-oncogenes may serve to promote genomic instability and HNSCC cell survival. Functionally, depletion of PTTG or PBF significantly repressed cellular migration and invasion, and impaired colony formation in HNSCC cells. Overall, this research has provided novel insights into the roles of PTTG and PBF in HNSCC tumour initiation and progression, through modulation of p53 activity and function.

Hypoxia has emerged as a crucial part of the aetiology of tumours. It is a negative prognostic factor which is associated to chemoresistance, invasiveness and metastasis. There is a strong association between hypoxia and metabolic transformation in breast cancer due to the alterations of multiple metabolic pathways. However, the current understanding of the nature of metabolic alterations in hypoxia is insufficient. This thesis uses NMR as a tool to investigate both the static metabolome by measuring metabolite concentrations, as well as to determine \(^1\)\(^3\)C metabolic fluxes using stable isotope tracers to reveal metabolic pathway alterations by hypoxia in vitro and by tumour growth in vivo. Firstly, we developed the \(^1\)\(^3\)C isotopomer distribution (CID) analysis to quantify metabolic fluxes by following the evolution of specific isotopomers of specific pathways of interests. MCF7 breast cancer cells were analysed in hypoxia using an integrated approach using gene expression, steady-state metabolite levels and \(^1\)\(^3\)C metabolic flux analysis to pinpoint hypoxia induced metabolic alterations. These most significant alterations were an up-regulation of the pentose phosphate pathway and a down-regulation of mitochondrial oxidative metabolism by lowering the PDH flux. The latter was partially compensated by carbon entry into the mitochondria by increasing flux through pyruvate carboxylase (PC). Further attention was focused towards identifying the shifts in metabolic activity in PC altered cells using [1,2-\(^1\)\(^3\)C]glucose and [3-\(^1\)\(^3\)C]glutamine as precursor nutrients correlated to cellular transformation potential accessed by cell viability. Finally, the \(^1\)\(^3\)C labelled glucose strategy was applied to a cancer model in mice model by infusing mice with [1,2-\(^1\)\(^3\)C]glucose. \(^1\)\(^3\)C glucose administration protocol was optimised in order to enable an investigation of \(^1\)\(^3\)C metabolic fluxes in tumour tissue to identify metabolic pathway differences between earlier stage and advanced stage of mammary gland tumours. In conclusion, an NMR based metabolomics analysis is suitable for discovering metabolic pathway alterations using both in vitro and in vivo models.

by Chan Chi Wai, Angus. / "Submitted in Jan 1999, revised in Jan 2000." / Thesis (M.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 262-294). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

by Anthony Tak Cheung Chan. / "March 1998." / Thesis (M.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 200-242). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

Automated computer-aided systems and approaches are widely required to investigate and analyze histology images for improving the accuracy of cancer diagnosis and effective treatment decision making. Quantitative analysis has immense potential to investigate and analyze the tissue and cellular characteristics of histology images in cancer research. It is based on accurate cellular, morphological, and tissue features. Automated approaches not only make the feature extraction and analysis more objective and more reproducible, but they can also help pathologists look for useful potential clues from a vast amount of hidden information in cancer tissues, whose clinical value may not be fully realized and visualized. This entails the automated computer algorithms with a key role of quantitative analysis of histology images for different cancers. In this thesis, I concentrate on bone marrow cancers and develop automated computer algorithms to extract and realize cellular and texture characteristics of bone marrow biopsies for efficiently characterizing different types of bone marrow cancers in further investigation and analysis. We focus on the development of automated algorithms for identifying various types of cells in bone marrow trephine biopsies, which are tiny cores of bone marrow tissues. All the algorithms are specifically designed for histological sections stained by a standard hematoxylin and eosin (H&E) stain. Firstly, we propose an automated framework with a novel segmentation model for delineating and segmenting megakaryocytes. Secondly, we create a novel deep learning network that processes the nuclear detection with irregular shape for various types of bone marrow stem cells. Then we construct another synchronized deep learning approach to simultaneously do detection and classification. We demonstrate the effectiveness of the network of detection and classification at same time and the training time consumed in this synchronized network.

Multiple myeloma is a cancer of plasma cells. In multiple myeloma, a clone of plasma cells in the bone marrow secretes a unique, monoclonal immunoglobulin (Ig), whose biological properties depend on its type and structure. The monoclonal Ig offers a convenient opportunity for clinicians to monitor the response of the tumour to therapy via the secreted protein, which is readily quantified in a blood sample. Responses to treatment are assigned based on the percentage reduction in monoclonal Ig; however, response criteria do not take into account the different metabolic half-lives of the proteins. 70% of multiple myeloma patients have either monoclonal IgA- or monoclonal IgG-producing clones. IgA and IgG have metabolic half-lives of 6 days and 23 days, at normal concentrations, respectively. The large difference in their metabolic half-lives suggests that they would respond at different rates during therapy. The elimination rate of IgG is concentration-dependent due to saturable recycling by a receptor. This could further impact upon its response during therapy, with the possibility that IgG is eliminated from the body at different rates at the beginning of therapy, when its concentration is high, and at the end of therapy, when its concentration has decreased. In this thesis compartmental models of IgG metabolism from the literature are analysed and parameter values are estimated from available data. A model of IgA metabolism is sourced in the literature. These models are used to predict the responses of monoclonal IgA and IgG during therapy. The simulations are able to replicate typical monoclonal IgA and IgG responses seen in a clinical trial of patients with relapsed and refractory multiple myeloma. Importantly, the plasma cell clone is not directly accessible to measurement and therefore not available to validate model-based predictions. However, monoclonal Ig responses are not evaluated by their ability to predict the tumour burden, but by the strength of their association with patient survival. In this thesis, a prediction is made of how the different metabolic properties of IgA and IgG may influence their association with survival outcomes. Evidence for this effect is then evaluated in data from a clinical trial using the methods of survival analysis.

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.

Background: Lung cancer is the most common cause of cancer death worldwide and is estimated to account for more than 1,380,000 deaths per year. Lung cancer can be separated into two major histological types: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC), accounting for approximately 15% and 85% of cases respectively. Epidermal Growth Factor Receptor (EGFR) is a tyrosine-kinase receptor. In NSCLC EGFR overexpression is found in over 80% of cases, and EGFR copy number gain (CNG) or amplification is found in nearly 60% of them. Tumours with EGFR mutations can be treated using anti-EGFR drugs; however currently genetic analysis has to be performed on tissue which is obtained by biopsy. Aims: This project aims to investigate alternative methods of obtaining tumour DNA for genetic analysis, to potentially improve or support the current diagnostic process. This project will investigate both new testing methods (molecular assays) and new sources of tumour DNA (cell free DNA from plasma). Methods: A number of methods were employed during this project. Initially EGFR and KRAS mutation detection was attempted using a novel Peptide Nucleic Acid Polymerase Chain Reaction (PNA-PCR) assay devised by GeneFirst Ltd (Oxford, UK). The second approach utilised custom designed TaqMan Array 384 well plate assays for the detection of EGFR, KRAS, NRAS and BRAF mutations. 40 clinical EDTA blood samples were obtained for the investigation of the use cfDNA for oncogenic mutation detection. Plasma DNA extracted using two automated platforms (Qiagen EZ1 and Promega Maxwell). The extracted DNA was analysed using the Ion Torrent Next Generation Sequencing (NGS) platform. Results: The GeneFirst novel PNA PCR assays appeared to tolerate low concentration FFPE DNA samples but had a very high false positive rate and the endogenous control assay failed regularly (0- 33.3% failure rate over different assay versions). The TaqMan Array assay was very successful at detecting EGFR, KRAS, NRAS and BRAF mutations from FFPE tissue, displaying 97.62% and 94.74% concordance with previously used diagnostic assays (Qiagen Therascreen EGFR RGQ PCR and Thermo Fisher KRAS castPCR). For the automated isolation of cfDNA, the Promega Maxwell instrument gave consistently superior results to the Qiagen EZ1. CfDNA was successfully used to detect oncogenic mutations using both PCR and NGS assays. Conclusion: This project has utilised a number of approaches in order to investigate new approaches for the detection of clinically actionable oncogenic mutations, both in FFPE tissue (obtained through surgery or biopsy) and the relatively new cfDNA analyte. Two PCR techniques were compared using DNA from FFPE tissue, and the TaqMan Array assay was shown to be vastly superior. The TaqMan Array was subsequently adopted as the primary diagnostic assay in UHCW Pathology. CfDNA (despite the limited number of samples) showed great potential as an alternative for tissue for detection actionable cancer mutations. The Ion Torrent Next Generation Sequencing system proved to be the most sensitive and powerful technique of the ones utilised here, and will prove an invaluable asset for future development of this work.