Michael Anglesio

High-grade serous ovarian cancer (HGSOC) represents approximately 70% of cases of ovarian carcinoma. Investigation into its molecular features led to the discovery of four subtypes of HGSOC, which differ from each other in their gene expression, cellular composition and survival outcomes. However, methods of whole-genome expression clustering have limited the reproducibility of existing subtype-specific research findings as well as being unsuitable for use in individual cases, such as in a clinical setting. PrOTYPE has been developed as a clinical-grade tool for determining the subtype of HGSOC tumours according to expression of a select panel of genes. To investigate the potential utility of determining HGSOC subtype using PrOTYPE in the clinical decision-making process, this thesis has two aims: 1) explore subtype-specific differences in survival outcomes of HGSOC patients treated with the anti-angiogenic drug bevacizumab, to determine whether subtype could be used to inform treatment decisions, and 2) evaluate the subtypes of paired samples collected before and after treatment to platinum and taxane chemotherapy to determine whether exposure to chemotherapy induces changes in subtype. In Aim 1, I found that cases belonging to the immunoreactive C2.IMM subtype generally had improved overall and progression-free survival when treated with bevacizumab. Results also suggested that the C5.PRO subtype may be associated with poorer survival when treated with bevacizumab compared to if treated with platinum and taxane chemotherapy alone. However, these findings could not be validated in independent cohorts; stratification of patients by subtype-specific treatment response in a clinical setting would require further investigation before being implemented. Additionally, a search for novel genes associated with differential treatment outcomes with bevacizumab identified several genes that may be useful in future research on targeted approaches to bevacizumab treatment. In Aim 2, I found that change in subtype from before to after exposure to neoadjuvant chemotherapy occurred in the majority (11/16) of the cases studied; this indicates that pre-chemotherapy subtype cannot be determined from post-chemotherapy samples. Clinical and prognostic significance of HGSOC subtype after chemotherapy remains an area for future investigation. Subtyping using PrOTYPE could be used in the clinical context to guide treatment decisions and evaluate patient prognosis.

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Endometriosis is a gynaecological condition that affects 10-15% of people with a uterus and is defined when endometrial-like tissue forms lesions in ectopic environments, most commonly within the pelvic cavity [1, 2]. The reflux of eutopic endometrium into the pelvic cavity, via retrograde menstruation, is the most common explanation for endometriosis development; however, more factors (such as gene expression alterations and immune dysregulation) are likely to contribute to the development of endometriotic lesions [3, 4]. It has recently been discovered that patients with deep infiltrating endometriosis, an invasive subtype of endometriosis, tend to have a mutation in KRAS codon 12 confined to the epithelium of the endometriosis lesions and within the eutopic endometrium [5]. To achieve a deeper understanding of the roles of the KRAS mutation in the development of endometriosis, this thesis has two aims: 1) compare the morphology and expression profiles between Kras-mutant and Kras-wildtype eutopic endometrium and 2) develop a novel mouse model for endometriosis with epithelium-restricted Kras mutation. In Aim 1, I found significant downregulation of genes related to the extracellular matrix and upregulation of genes involved in inflammation in Kras-mutant compared to Kras-wildtype uterine tissue. Furthermore, differential regulation of genes involved in epithelial-mesenchymal transition, decidualization, and ferroptosis were also identified. The differential expression of various genes between Kras-mutant and wildtype uterine tissue may partially explain the development of endometriosis and associated symptoms. Aim 2 investigates this potential role of Kras mutations in the development of endometriosis; however, lesion establishment was unsuccessful. Nonetheless, this model provided a good framework to inform the development of future endometriosis mouse models. The characterization of differentially regulated genes between mutated and wildtype endometrial tissue could have large implications in future preclinical studies that focus on the role of somatic mutations in the proliferation and growth of the invasive tissue, with the end goal of developing novel personalized treatments suitable for patients affected by endometriosis.

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Representing approximately 5% of all epithelial ovarian cancers, low-grade serous ovarian carcinoma (LGSC) is a rare histotype with unique genetic, molecular, and pathological features. LGSC patients are often detected at an advanced stage and respond poorly to platinum-based chemotherapy. Therefore, targeted therapies focused on exploiting oncogenic dependencies have homed in on the RAS/RAF/MEK/ERK (MAPK) signaling pathway, due to frequent activating MAPK mutations in LGSC tumours. Trials using MEK inhibitors (MEKi) have seen a modest increase in patient response, however, no cellular markers that distinguish MEKi response have been identified. This research aimed to discover and target protein markers of MEKi response by analyzing the global proteome of in-vitro cell lines previously established from patient samples through mass spectrometric analyses. 95 proteins were identified as being significantly differentially expressed between four MEKi sensitive and three MEKi resistant LGSC cell lines. Nine of these proteins were selected for further analysis, with the differentially protein expression of five markers being successfully validated. One of these confirmed markers, EGFR (a common driver of oncogenesis), was selected for paired inhibition with MEK using a combination of small molecule inhibitors specific for MEK and EGFR to induce cellular apoptosis in MEKi resistant LGSC lines. Four de novo and three acquired MEKi resistant LGSC cell lines were treated with a combination of erlotinib (EGFRi) and either selumetinib or trametinib (MEKi’s). Cellular response was analyzed by live-cell imaging, cell proliferation, and viability assays. A linear mixed effects model and drug synergism analysis was implemented to evaluate the efficacy of MEKi and EGFRi in combination. In the four de novo resistant LGSC lines, the drug combination induced complete cell death in two lines and demonstrated a degree of drug synergism in another. In the acquired MEKi resistance setting however, none of the cell lines responded to dual EGFR and MEK inhibition. Therefore, the combined inhibition of EGFR and MEK may represent a potential therapy for overcoming de novo MEKi resistance in LGSC, but not as a strategy for combatting acquired MEKi resistance.

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Introduction: Endometriosis is a chronic, inflammatory gynecological disease characterized by the ectopic growth of endometrial-like tissue. Previous studies have established endometriosis as the precursor to clear cell and endometrioid ovarian carcinomas. The presence of somatic driver mutations in endometriosis is believed to represent early events in transformation, however our group has recently described the presence of such mutations in nearly one-quarter of cases of deep infiltrating endometriosis (DE) – a form of endometriosis that rarely progresses to malignancy. These mutations may play a fundamental role in the pathogenesis of endometriosis outside of the context of cancer, however it is unclear whether they occur in other forms of endometriosis or the eutopic endometrium – the likely tissue of origin for endometriosis. The purpose of my study is to: 1) analyze and compare the mutational profiles of DE and incisional (iatrogenic; IE) endometriosis and 2) characterize somatic cancer-drivers that exist in the eutopic endometrium and determine whether the presence of such mutations reflect the aging of this tissue.Methods: I macrodissected endometriosis tissue from women with IE or DE. Extracted DNA was analyzed by targeted sequencing and mutations were orthogonally validated by droplet digital PCR. PTEN and ARID1A immunohistochemistry was also performed for each specimen. Using the same protocol, I also analyzed hysterectomy and endometrial biopsy specimens obtained from cancer-free women.Results: Overall, we detected the presence of somatic alterations in 27.5% and 36.1% of IE and DE cases respectively. These events affected canonical components of RAS/MAPK or PI3K-Akt signaling pathways. Furthermore, over 50% of cancer-free women also harboured similar somatic alterations in their eutopic endometrial tissue. The presence of somatic cancer-drivers in the eutopic endometrium are likely regional and are correlated with age (p = 0.048).Conclusions: My findings are consistent with a uterine origin of endometriosis. Somatic cancer-driver alterations are commonly found in both endometriosis and the eutopic endometrium of cancer-free women and may reflect the accumulation of DNA damage over time. These somatic alterations alone are insufficient for malignant transformation and should be interpreted with caution in the early diagnosis of gynecologic malignancies given their common occurrence in cancer-free women.

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