Pathology - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
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.
-
ItemUsing DNA methylation to study benign breast and breast tumours( 2011)Breast cancer is a heterogeneous disease comprising of numerous distinct entities that have different biological and clinical behaviours. Clinical diagnosis and accurate prognosis are not infrequently difficult to make, leading to over or under treatment. Although improvements in survival have been observed over the last 10 years, a substantial number of women die from the disease. A better understanding of the molecular mechanisms and signalling pathways that are involved in breast cancer development (e.g. the hypoxia pathway) as well as a deeper appreciation of the role of epigenetic alterations through DNA methylation, will greatly improve our combative efforts against breast cancer. The aim of this thesis is to assess DNA methylation of candidate genes in breast cancer with the goal of identifying biologically important pathways of hypoxia-inducible factor 1 (HIF-1) and to find novel diagnostic and prognostic markers. HIF-1 is the master regulator of cellular oxygen homeostasis that is induced by hypoxia. DNA methylation assessment was performed on the genes that regulate HIF-1 to understand the link between DNA methylation changes in cancer and the altered response to hypoxia. However, promoter methylation of a panel of HIF-1α regulating genes (VHL, CUL2, TCEB1, TCEB2, RBX1, PHD1, PHD2, PHD3, FIH, SDHA, SDHB, SDHC, SDHD, FH and SIAH2) was not present in breast cancers. This showed that promoter methylation is unlikely to be an important mechanism in stabilising HIF-1 in breast cancers through the down-regulation of the expression of these genes. Nevertheless, the study has demonstrated that the loss of expression of CITED4, a negative regulator of HIF-1, in breast cancer is in some cases due to DNA methylation that leads to increased HIF-1α activity and its downstream genes. Demethylation and histone modification was able to reactivate CITED4 gene expression in the methylated cell lines and lead to changes in tumour behaviour. The data of the study also suggests that histone modification may be the alternative epigenetic mechanism that regulates CITED4 expression. The distinction between fibroadenomas and benign phyllodes, which are both fibroepithelial tumours of the breast, can be difficult with no specific features that can reliably distinguish these neoplasms. I have demonstrated that the strong methylation of RASSF1A and/or TWIST1 was associated with phyllodes tumours. TWIST1 methylation is also correlated with increasing malignancy in phyllodes tumours. Hence, RASSF1A and TWIST1 methylation, and potentially other markers such as APC and WIF1, may be useful clinically for distinguishing phyllodes tumours from fibroadenomas. Additionally, the absence of frequent methylation in fibroadenomas supports a non-neoplastic origin. DNA methylation profiling and array comparative genomic hybridisation have been demonstrated as a valuable diagnostic tool in differentiating newly arisen tumours for ‘true’ relapses from new independent tumours. DNA methylation profiling results showed that ipsilateral tumours have a higher chance to be a recurrent tumour, whereas contralateral tumours were mostly a de novo tumour. Overall, this thesis have demonstrated that DNA methylation is an important mechanism in understanding the depth of hypoxia in breast cancer and has the potential to be a useful diagnostic tool in helping to answer some clinical questions in breast cancer.
-
ItemEvaluation of high resolution melting methodology for biomarker studies in non-small cell lung cancer( 2011)The adoption of molecularly targeted therapies in non-small cell lung cancer (NSCLC) has demonstrated the clinical efficacy of treating individual patients based on tumour molecular defects, emphasising the necessity of accurate and reliable methodology for molecular biomarker studies. High resolution melting (HRM) is a fast, sensitive, and cost-effective methodology that can screen both genetic and epigenetic biomarkers. Using HRM-based assays developed as part of this thesis, testing for a range of genetic and epigenetic biomarkers that have a predictive role for various therapies in NSCLC has been studied with a focus on the problems associated with diagnostic material. Mutational profiling of seven selected genes in the EGFR signalling pathway (EGFR, HER2, KRAS, BRAF, PIK3CA, and AKT1/3) was performed in a panel of 200 NSCLC samples that had been sent for diagnostic EGFR testing. Gain-of-function mutations were detected in 54% of the NSCLC samples. All 75 EGFR mutations that were previously identified by Sanger sequencing were detected by HRM analysis. Forty nine per cent of the NSCLC tumours harboured either EGFR exon 18-21 mutations (36.5%) or KRAS codon 12 and 13 mutations (12.5%). Somatic PIK3CA (3.5%), BRAF (3%), and AKT1 (0.5%) mutations were less frequently detected. EGFR, KRAS, and BRAF mutations were mutually exclusive whereas concomitant PIK3CA and EGFR mutations were detected in four NSCLC tumours. Limited copy number (LCN)-HRM was developed for screening and detection of low levels of mutations present below the sensitivity of Sanger sequencing as the limited sensitivity of this method is a particular problem in lung cancer diagnostics. The analytic sensitivity of HRM (5%) was shown to be greater than of Sanger sequencing (10%), demonstrating that low levels of mutations (5-10%) was detectable by HRM only. When clinical samples showing the discordant results between Sanger sequencing and HRM were tested by LCN-HRM, both low levels of true mutations and PCR artefacts were detected. Template-mediated PCR artefacts, mainly transitional changes (G>A and C>T), were prevalent in DNA extracted from formalin-fixed paraffin-embedded tissues. STK11 mutations were screened by HRM in a panel of tumours from 195 NSCLC patients who were surgically treated in combination with platinum-based chemotherapies. A total of 27 non-synonymous STK11 mutations (14%) were detected, including 9 nonsense, 5 frame-shift, 6 missense, and 7 splice site variants. The majority of STK11 mutations (93%) were detected in the kinase domain. STK11 mutations were significantly associated with worse progression free survival and overall survival of the NSCLC patients. The frequency of promoter region methylation in two sets of genes, DNA repair genes and selected genes previously associated with recurrence after surgery, was examined in 56 N1 tumours using methylation sensitive (MS)-HRM. DNA methylation was not detected in BRCA1, MLH1, and XPC, contrary to previous reports in the literature. Methylation was detected at a low frequency in ERCC1 (2%) and RAD23B (2%). Modest levels of MGMT methylation was seen in more individuals (12.5%) and was significantly associated with the ‘T’ allele of the MGMT promoter rs16906252 SNP (P<0.0001). A high frequency of methylation in the APC, CDH13, RASSF1A, and p16 genes was confirmed in NSCLC tumours, giving an overall methylation frequency of 25%, 50%, 32%, and 25% respectively. A strong association between KRAS mutation and CDH13 methylation was also detected. In conclusion, HRM is a sensitive and accurate methodology that can be used in a range of molecular diagnostic tests for genetic and epigenetic biomarkers. When used in combination with sequencing, it is suitable for routine diagnostic testing for molecularly tailored therapies for NSCLC patients.