published_or_final_version / Pathology / Master / Master of Philosophy

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It is known that many types of leukemias and lymphomas are of viral origin. A new strain of immunologically deficient mice, the BALB/c x C57B1/6 beige nude mice, has been observed to develop spontaneous lymphomas of unknown origin at a high frequency. It is possible the tumors originate from a retroviral infection, which we attempted to show by detection of viral reverse transcriptase (RT) activity. We measured the (RT) activity in the supernatants of cocultures from the spleen and lymph node tissues of the beige nude animals by two methods, tritiated thymidine triphosphate incorporation in a standard RT assay, and the commercially available RT-DetectTM (DuPont) method. Of all supernatants tested, none showed a significant amount of RT activity compared with a cell line that was known to be actively producing the retrovirus MuLV. Upon electron microscopic analysis of the tumor-like cells grown in coculture, no viral particles were observed. Flow cytometric analysis of the tumor-like cells showed two general phenotypes; one predominately of a helper T cell type, and the other of a less differentiated immature thymocyte type. / Department of Biology

Microarray technology allows researchers to monitor the mRNA transcription levels of thousands of genes in parallel which opens the door for more advanced cancer research. This thesis focuses on a case study of papillary thyroid carcinoma data. Fourteen publicly available Affymetrix microarray data sets were used where seven samples were collected from normal thyroid tissue and the remaining seven were collected from papillary thyroid carcinoma tissue. The present study compared the results obtained from three different normalization processes: MAS5.0, RMA and GCRMA in detecting differentially expressed genes under two conditions. Internal consistencies within the methods as well as the results across three methods were compared. Statistical packages 82.5.1 and Bioconductor 2.08 are used to perform the data analysis. Each step of normalization with MAS5.0 and RMA is described. Statistical package Limma is used to fit a linear model. Finally an empirical Bayes method is used to detect the significantly differentially expressed genes. First, considering all genes a comparison is made among the three normalization methods where RMA and GCRMA showed the maximum agreement in detecting differentially expressed genes. Then using unspecified filtering process a set of genes was selected and the whole process was replicated where the top fifty differentially expressed genes did not show any overlap with each other. / Department of Mathematical Sciences

Small cell lung cancer (SCLC) is an aggressive form of lung cancer that frequently develops multidrug resistance (MDR) during chemotherapy. Vault RNA1 (vRNA1), a non-structural component of the MDR-associated vault organelle, is believed to act as a microRNA (miRNA) and may contribute to MDR by regulating the expression of genes involved in apoptosis, inflammation, and/or drug metabolism. Since vaults function to aid cells in survival, we hypothesized that vRNA1 might be free in the cytoplasm and able to inhibit expression of pro-survival mRNAs when vaults are open in drug-sensitive GLC4/S cells but not in the MDR GLC4/ADR cells where vaults might be closed with the miRNA sequestered within. In order to establish the role of vRNA1 as a regulator of survival in SCLC cells, siRNA-mediated down-regulation of vRNA1 was employed in GLC4/S and GLC4/ADR SCLC cells. Fluorescence microscopy using a green fluorescent 3’ AlexaFluor-488 negative siRNA control was used to estimate transfection efficiency, yielding 56% for GLC4/S and 89% for GLC4/ADR. However, these values and the level of apoptosis before and after transfection, as judged by trypan blue hemacytometer cell counts, were not entirely reliable due to cell clumping. The latter counts indicated a 2-fold decrease in viability in GLC4/S cell following transfection but no decrease in GLC4/ADR cells (p< 0.05). RT-PCR revealed that transfection significantly (p<0.05) decreased vRNA1 expression in GLC4/S cells but not in GLC4/ADR cells, confirming our hypothesis concerning the availability of vRNA1 in the two cell types. Caspase activity measurements showed vRNA1 down-regulation in the GLC4/ADR cells significantly (p≤0.05) increased survival via a 6.1-fold reduction in caspase 3/7 activity, further supporting our hypothesis. However, GLC4/S cells showed a similar loss of apoptosis when transfected with either sivRNA1 or the negative control siRNA. vRNA1 down-regulation did not significantly (p≤0.05) affect the expression of major pro-survival (Bcl-2, Bcl-xL), pro-apoptotic (Bad), or pro-inflammatory (IL-6, NFĸB p65) factors in either GLC4/S or GLC4/ADR cells. However, the drug metabolism protein CYP3A (previously shown by Persson et al., 2009 to be regulated by vRNA1) was significantly (p≤0.05) lowered (~16%) following vRNA1 down-regulation in the GLC4/S cells. In conclusion, we were successful in down-regulating vRNA1 which enhanced cell survival as hypothesized, but we were not able to identify new proteins regulated by vRNA1. / Department of Biology

Copies of author's previously published articles inserted. / Amendments pasted onto back-end paper. / Bibliography: leaves 255-286. / ix, 286, [15] leaves, [5] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to isolate the gene(s) responsible for Fancomi anaemia and breast cancer using a positional cloning strategy / Thesis (Ph.D.)--University of Adelaide, Dept. of Cytogenetics and Molecular Genetics, 1999

Despite decades of effort, the mechanistic underpinnings of many cancers remain unsolved It has increasingly become appreciated that cancers can be more readily classified by their transcriptional identities rather than by genomics alone. A fuller understanding of the mechanistic connections between the aberrant genomics leading to the transcriptional dysregulation of tumors is key to both improving our knowledge of cancer biology as well as developing more precise and effective therapeutics. This thesis explores the development and application of a network based multi-omic master regulator framework designed to elucidate these pathways. In Chapter 2 we apply this analysis across 20 tumor types from the Cancer Genome Atlas and in doing so identify 407 key master regulators responsible for canalizing a high percentage of the driver genetics present across these samples. Further evaluation of these key regulators revealed a highly modular structure, indicating that the regulators work in coordinated groups to implement a variety of key cancer hallmarks. Genetic and pharmacological validation assays confirmed the predicted interactions and biological phenotypes. Chapter 3 focuses on the application of this analytical framework specifically on gastroesophageal tumors. Using a more fine-grained approach we find 15 distinct subtypes across a cohort of these heterogenous tumors. These subtypes align well with previously identified features of these cancers but also reveal novel genomic associations and key master regulators that can serve as potential avenues for therapeutic treatment.

Ovarian cancer is the fifth most common cause of cancer death in females and the most lethal gynecologic cancer. Globally, an estimated 240,000 people are diagnosed with ovarian cancer each year, with 22,530 new cases in the United States in 2019. Parity, oral contraceptive use, and lactation are protective, while early menarche, late menopause, and nulliparity have opposite effects. The “incessant ovulation” theory has thus emerged, in which a higher number of ovulations may be a cause of epithelial ovarian cancer (EOC). However, the mechanisms of this theory are unknown; one possibility is that the chance of acquiring a cancer-initiating pathogenic variant increases with each ovulatory cycle because of a microenvironment that promotes DNA damage. In this dissertation, we aimed to leverage genetic epidemiologic data to test this potential mechanism by evaluating the presence of gene-environment interaction between DNA repair capacity (measured through the presence of pathogenic variants in DNA repair genes) and lifetime ovulatory years (LOY). In the first aim of this dissertation, we conducted a systematic review and meta-analysis, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, to formally evaluate the strength of evidence and to generate summary point estimates for the association between LOY and EOC. We then executed two analytic aims to evaluate if the presence of pathogenic variants in DNA repair genes exacerbated the increase in ovarian cancer risk associated with LOY. In Aim 2, we evaluated interaction on the additive scale in the United Kingdom (UK) Biobank through use of a novel DNA repair capacity score developed in this dissertation, measured by quantifying the number of pathogenic variants present per individual from a list of 163 DNA repair genes, using whole exome sequencing (WES) data. In Aim 3, we evaluated the presence of interaction between pathogenic BRCA1/2 status and LOY in the Breast Cancer Family Registry (BCFR), a cohort enriched for familial risk. In both empirical aims, we assessed the presence of interaction on the additive scale using the relative excess risk due to interaction (RERI) formula. We compared results across the two empirical aims. We found the relationship between lifetime ovulations and ovarian cancer risk to be consistent and replicable in the published literature. In pooled estimates from 22 published studies, a one-year increase in LOYs was associated with a 4% (3-6%) increased risk of ovarian cancer and those with a high number of ovulations (compared to low LOYs) had a 2.15-fold (95% CI 1.82, 2.54) increased risk of ovarian cancer. We also confirmed the positive association between increasing LOYs and ovarian cancer risk in the UK Biobank and the BCFR cohorts. Although interaction on the additive scale was not detected, there were strong positive associations between pathogenic variants in DNA repair genes and ovarian cancer risk. In the UK Biobank, the presence of at least one pathogenic variant in a DNA repair gene was associated with a significant 27% increased risk of epithelial ovarian cancer (EOC) (95% CI 5-55%). Among women at high risk of ovarian cancer due to family history of breast and/or ovarian cancer, there was a strong relationship between BRCA1/2 pathogenic variants and ovarian cancer, regardless of the number of ovulations experienced. The association between LOY and ovarian cancer was found to be consistent and replicable, despite differences in study design, covariates, and measurement. We also detected robust evidence that increasing lifetime ovulations and pathogenic DNA repair variants were associated with ovarian cancer risk. Such variants were exceedingly rare in both cohorts, which limited power to detect interaction in an already rare cancer. Despite such associations, there was no evidence of synergy between LOY and impaired DNA repair capacity, but rather, high LOY and impaired DNA repair capacity may be independent risk factors of ovarian cancer. Each exposure may describe a separate class of women at increased risk of ovarian cancer that should be targeted for future prevention and screening strategies.

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

This dissertation examines the production and management of genetic risk for breast and ovarian cancer in the United States in the new era of multi-gene panel testing. Drawing on three years of ethnographic fieldwork and in-depth interviews with genetics health professionals and women with mutations, this project is the first social science study to examine how breast and ovarian cancer genetic risk is constructed and managed among women with variants of uncertain significance or moderate-risk mutations. Moving beyond an individual-level focus on women’s risk management decisions, this project instead explores how the structures, practices, and organization of genetic medicine constrain and enable those decisions. There are four key findings from this study. First, the adoption of panel testing has shifted the boundaries of risk, disease, and patienthood and contributed to a spectrum of medicalization of breast and ovarian cancer risk. Women with high-risk breast and ovarian cancer mutations are now typically viewed and treated like full patients with a "disease," while women with moderate-risk mutations occupy a liminal space of qualified patienthood. Second, the structures and organization of genetic medicine in the United States point women with breast and ovarian cancer mutations toward risk-reducing mastectomy and breast reconstruction and encourage choosing those surgical responses over breast surveillance or staying flat. Mastectomy has become the standard “treatment” for the “disease” of genetic risk for breast cancer, regardless of whether women have high- or moderate-risk mutations and despite more conservative recommendations in clinical guidelines. Third, the structures of genetic medicine and the contemporary gender order in the United States are mutually constituted and co-produced. Breast reconstruction and gynecologic surgery practices both emerge from and reinforce gendered social and cultural norms that prioritize women's appearance and their reproductive capacity over their embodied experiences and daily quality of life. Finally, the discourses and practices of genetic medicine leave many women un- or under-prepared for the duration and severity of the side effects and consequences associated with breast reconstruction and risk-reducing salpingo-oophorectomy. By closely examining the social and structural dimensions of how cancer genetic risk is produced and managed in the United States, this project illuminates how clinical practices that magnify and focus on reducing certain risks simultaneously obscure and generate exposure to others.