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ItemDNA methylation and breast cancer risk factors: insights from twin and family studies( 2018)DNA methylation, the most studied epigenetic mechanism regulating gene expression, has been thought to play a critical role in the aetiology of complex diseases and traits, including breast cancer. Twin and family studies have investigated the unmeasured causes of DNA methylation variation; there are, however, several research gaps, such as interpreting the average heritability across sites as the heritability of genome-wide DNA methylation level and assuming DNA methylation variation across sites to have the same causes. Two DNA methylation features, genome-wide average DNA methylation (GWAM) and epigenetic age acceleration, are putatively associated with breast cancer risk. The causes of variation remain unknown for GWAM and have not been well investigated for epigenetic age acceleration. Understanding the potential causality between DNA methylation and conventional breast cancer risk factors, which has been rarely investigated for body mass index (BMI), mammographic density and smoking, might bring a better understanding of breast cancer aetiology. To address current research gaps, my thesis used DNA methylation data from multiple twin and family studies to investigate the causes of variation in GWAM, in site-specific DNA methylation and in epigenetic age acceleration, and to investigate the potential causality between DNA methylation and BMI, mammographic density and smoking with a novel analytic method using data for related individuals - Inference about Causation through Examination of FAmiliaL CONfounding (ICE FALCON). My thesis found that: a) genome-wide methylation level, measured as GWAM, is determined by prenatal environmental factors acting in utero, the effects of which last into old age, and by environmental factors shared by cohabitating family members, including spouse pairs (Chapter 4); b) site-specific variation DNA methylation has specific causes, and substantial variation is explained by measurement error and environmental factors (Chapter 5); c) evidence consistent with twin birth changing the intrauterine environment such that sibling pairs both born after a twin birth are correlated in DNA methylation while sibling pairs both born before a twin birth are not (Chapter 6); d) variation in epigenetic age acceleration is caused by shared environmental factors as well as genetic factors (Chapter 7); and e) BMI, mammographic dense area and smoking are associated with DNA methylation at several genetic loci, and these associations are likely due to the causal effects of the three factors on DNA methylation, the same conclusion to those made by Mendelian randomisation analysis (Chapters 8 to 10). The findings of my thesis suggest that DNA methylation appears to be fundamentally about the way the environment influences the way genes work. Although there might be methylation sites at which the variation has a genetic basis, these are rare. The effects of the environment can start from the time of conception, or at least while in the womb, and continue into adulthood. Some of those environmental effects are shared by family members, even spouse pairs, and these effects can potentially influence breast cancer risk in adulthood. Conventional breast cancer risk factors can cause changes to DNA methylation, indicating that DNA methylation might explain in part why these factors modifying risk. Most of the novel results of this thesis could not have come about without the use of data from twin pairs, or of data from other pairs of relatives including spouse pairs. The thesis has also shown the value of ICE FALCON in making inference about observational epigenetic association based on considering familial confounding - ICE FALCON gives the same conclusion as those being found by Mendelian randomisation. More importantly, ICE FALCON does not require extensive genome knowledge and data that are required by Mendelian randomisation.
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ItemInvestigating epithelial-mesenchymal plasticity in breast cancer using circulating tumour cells and circulating tumour DNA( 2017)Breast cancer is the most frequent invasive cancer among women worldwide with mortality primarily caused by metastasis. One of the key proposed processes underlying metastasis, including the escape to the bloodstream, is epithelial- mesenchymal plasticity (EMP). This refers to the dynamic transition between epithelial and mesenchymal phenotypes of cells within the primary tumour mass. Within the bloodstream, tumour cells and tumour DNA, which are referred to as circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA), respectively, have showcased their potential use as liquid biopsy in cancer management. The presence of CTCs has been shown to associate with poor prognosis in metastatic cancers, which becomes worse with higher CTC numbers. By virtue of carrying genetic and epigenetic features of primary tumours, ctDNA has demonstrated its utility in detecting and monitoring cancer progression. Various studies conducted on the molecular characterisation of CTCs have generated data supporting the role of EMP in generating CTCs. However, the dynamic changes in expression, especially of genes associated with EMP, between primary tumours, CTCs and metastases remain far from conclusive. In keeping with this paradigm, as cancer cells in the primary tumours shift from the epithelial to the mesenchymal phenotype, any released ctDNA may have epithelial and/or mesenchymal features depending on its cellular origin. To date, research on the utility of EMP-associated methylation markers to detect ctDNA is lacking. In light of the suggested role of EMP in different key steps of cancer progression and metastasis, this thesis has aimed to study EMP reflected in CTCs and ctDNA to provide further insights into the CTC molecular characteristics and assist in the detection of ctDNA. This thesis is comprised of two principal areas of study: (1) the gene expression profiling of CTCs in two human breast cancer xenograft models, and (2) the locus- specific methylation profiling of breast cancer cell lines and breast tumours. Firstly, the expression profiles of EMP-associated genes were characterised in CTCs at the population level and the single cell level, and were compared with the expression profiles of primary tumours and (where possible) metastases, for two xenograft models, the MDA-MB-468 and ED-03. In pooled CTCs relative to primary tumours from both models, a significant increase in expression of mesenchymal markers (SNAI1, VIM, SERPINE1 and NOTCH1), and surprisingly, of a typical epithelial marker CDH1, were observed. A decrease/loss of EPCAM was reproducibly observed in CTCs of both models, while decreased CD24 and EGFR in CTCs were only seen in the MDA-MB-468 model. Genes associating with hypoxia (HIF1A, BNIP3 and APLN) and cellular metabolism (PPARGC1A) were also significantly elevated in CTCs of both models. In additional studies, a direct lysis method was successfully optimised to assist the gene expression study in single cells. The subsequent analysis of single CTCs revealed heterogeneity of CTCs, with co-expression of epithelial and mesenchymal markers, and high expression levels of epithelial markers in individual CTCs. The results reinforced the complex gene expression profiles and alterations seen in pooled CTCs. Secondly, a panel of DNA methylation markers, including those associated with EMP, was developed and tested in breast cancer cell lines, primary tumours and whole blood of normal controls to identify suitable markers for ctDNA detection. In a panel of breast cancer cell lines spanning the epithelial-mesenchymal spectrum, the majority of epithelial cell lines were methylated for cancer-associated markers (i.e., RASSF1A, RARß) and epithelial methylation-based markers (i.e. VIM, DKK3 and CRABP1). Mesenchymal cell lines were exclusively methylated for mesenchymal methylation-based markers (GRHL2, MIR200C and CDH1); however, the level of methylation was quite low. The methylation profiles of the studied genes classified primary tumours into intermediate phenotypes while few tumours were mesenchymal. In addition, MIR200C, RASSF1A, AKR1B1 and TWIST1 were methylated at high frequency in our cohort. Among these, RASSF1A and AKR1B1 showed no methylation in whole blood of normal controls, suggesting their potential use as markers for ctDNA detection from plasma of the breast cancer patients in our cohort. Preliminary experiments established ddPCR assays for these two markers, allowing further testing on patient cell-free DNA samples for the detection of ctDNA. The results of this thesis challenge the conventional model of EMT, where cells in epithelial tumours become mesenchymal, with associated migratory properties, and later re-epithelialise (MET) at a distant metastasis. Firstly, the complex and consistent alterations in the epithelial and mesenchymal markers in CTCs across the two models is suggestive of a ‘hybrid’ phenotype. The overall findings from the CTC work that CTCs were not as mesenchymal as expected also suggest that other processes than EMP directly influence the generation and survival of CTCs. Secondly, nearly all the examined breast tumours exhibited an intermediate rather than a strong epithelial phenotype based on the methylation profiles. This suggested that a plasticity is already present at the solid tumour state. These findings provide an alternative view of EMP in both primary breast cancer and the disseminated forms, and provide an important platform for further research in this field.
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ItemBiomarkers in ductal carcinoma in situ( 2016)Ductal carcinoma in situ (DCIS), a non-invasive form of breast cancer and a non-obligate precursor of invasive carcinoma of the breast, displays heterogeneous behaviour. Most DCIS are adequately managed by local surgical excision alone, but in 20-30% of cases, disease recurrence occurs after local surgical excision either as DCIS or invasive carcinoma. Accurate identification of these two clinical outcome groups at the time of diagnosis is desirable to allow appropriate treatment allocation. In this thesis, genomic and epigenetic alterations in DCIS epithelium, including copy number aberrations, somatic mutations, and DNA methylation were investigated as markers of DCIS biology and outcome. In addition, the expression and significance of LRH-1, a nuclear receptor which acts as a transcription factor, was investigated in both invasive carcinoma and DCIS. Copy number analysis of DCIS of known clinical outcome identified amplification of 20q13 to be associated with disease recurrence, but this was unable to be validated on an independent cohort. Targeted next generation sequencing of a panel of breast cancer-relevant genes revealed that the mutational profile of DCIS was similar to that reported for invasive carcinomas, with the most frequently mutated genes being GATA3, PIK3CA, and TP53. A high prevalence of GATA3 mutations in DCIS was observed and TP53-mutant DCIS was associated with high stromal tumour infiltrating lymphocytes. Mutations of RUNX1 were a novel finding, not previously reported in DCIS. Promoter methylation of a candidate gene panel, consisting predominantly of known tumour suppressor genes, was associated with adverse tumour features in DCIS. Methylation load varied among DCIS cases, suggesting that methylation differs in importance in the tumorigenesis of DCIS, and that assessment of methylation may be useful as a biological classifier of DCIS. Finally, LRH-1 mRNA expression patterns in breast cancers was similar to that reported for breast cancer cell lines and distinct LRH-1 immunohistochemical staining patterns were associated with tumour phenotype in both invasive breast carcinoma and DCIS. The results of this thesis demonstrate that copy number alterations, somatic mutations, DNA methylation, and LRH-1 expression are indicative of DCIS phenotype and hence biology. These markers showed promise as prognostic biomarkers, although validation of their utility was hampered by the small number of pure DCIS cases with both adequate genomic material and long-term clinical outcome data. Nonetheless, the findings of this thesis indicate that assessment of these biomarkers can be performed in routine diagnostic tissue material and provide several avenues for future research.