Department of Pathology and Molecular Medicine Graduate Theses

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https://hdl.handle.net/1974/759

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    Elucidating functional interactions of the RET receptor tyrosine kinase in cancer using a synthetic dosage lethal screen
    (2024-09-25) Hussain, Montdher; Pathology and Molecular Medicine; Mulligan, Lois
    RET (REarranged during Transfection) is a receptor tyrosine kinase that plays essential roles in the early development of the kidneys and neural crest-derived lineages (e.g., enteric nervous system). Its ability to activate pro-growth signaling pathways has implicated RET in several malignancies. While a clear oncogenic role for RET has been established, the precise molecular mechanisms that contribute to RET-mediated oncogenic growth are less well known. To address this gap, we performed an unbiased, genome-wide synthetic dosage lethal (SDL) screen in collaboration with Dr. Franco Vizeacoumar (University of Saskatchewan) to uncover genes that are functionally linked to RET-mediated growth of SH-SY5Y neuroblastoma cells. This project used an integrative bioinformatics approach that combined statistical analysis with machine learning to identify and prioritize candidate genes from the screen results. Two genes, ASCL1 and NCAM1, were selected for experimental validation of our approach. The work presented in this thesis provides the first known evidence of a role for ASCL1 in transcription of RET in a neuroblastoma cell line. Furthermore, we show that NCAM1 may play a role in transdifferentiation of SH-SY5Y cells which may modulate cell growth. Together, this project serves as an important proof-of-principle study for assessing the utility of the SDL screen in uncovering genes that contribute to RET-mediated processes.
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    Assessing the Clinical Relevance of BRCA1 BRCT Domain Variants of Uncertain Significance
    (2024-09-18) Torretto, Gabriella; Pathology and Molecular Medicine; Davey, Scott; Feilotter, Harriet
    Breast and ovarian cancer are among the most common cancers in Canadian women. Approximately 5-10% of breast and 20-25% of ovarian cancers are inherited, with pathogenic germline BRCA1 and BRCA2 variants causing the majority of hereditary cases. While genetic testing is used to identify pathogenic BRCA variant carriers who would subsequently benefit from personalized screening, prophylactic and treatment steps, its widespread use has resulted in the discovery of thousands of variants of uncertain significance (VUS). VUSs pose a critical clinical challenge as they are unable to be effectively interpreted, limiting clinicians’ ability to accurately assess cancer risk and recommend appropriate management steps. We sought to build a computational classifier specific to BRCA1’s BRCT domain to accurately predict missense VUS pathogenicity and stratify VUSs to prioritize for functional analyses like phosphopeptide binding assays. All BRCA1 BRCT missense variants were collected from the ClinVar database and were analyzed using 50 different in silico tools. Molecular Feature Selection Tool (MFeaST) ranked tools based on their ability to discriminate pathogenic and benign variants. Supervised classifiers were then trained using combinations of the most discriminative in silico tools, with the most accurate classifier being used to score all VUSs. Phosphopeptide binding assays were conducted on select suspected pathogenic and benign VUSs to assess their impact on binding activity and folding. Our results show that an Ensemble Subspace kNN classifier trained with 9 in silico tools (CADD hg19, MetaRNN, ClinPred, VEST4, BayesDel AF, EVE, Eigen PC, gMVP and PolyPhen2) demonstrated the best performance out of all trained supervised models, with 91.1% and 87.9% accuracy on the training and validation sets, respectively. Compared to individual in silico and AI protein language models, our model demonstrated the highest accuracy on the training set and comparable accuracy on the validation set of BRCA1 BRCT variants. Results from the phosphopeptide binding assays show that suspected pathogenic VUSs demonstrated either reduced BRCT binding ability, reduced BRCT protein levels, or a combination of both. Suspected benign VUSs demonstrated retained BRCT binding ability and BRCT protein levels. The computational and functional evidence obtained through this study will contribute to the reclassification of BRCT VUSs to pathogenic or benign, strengthening and broadening variant classification databases essential for clinicians to make decisive management recommendations for BRCA1 variant carriers. Additionally, this study highlights the potential of domain-specific computational approaches for characterizing missense variants in other multi-domain cancer susceptibility genes.
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    The role of inflammatory and prothrombotic biomarkers in severe COVID-19-associated endothelial dysfunction
    (2024-09-05) Hinds, Megan; Pathology and Molecular Medicine; James, Paula
    Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December of 2019, coronavirus disease 2019 (COVID-19) associated endothelial cell (EC) dysfunction has been well documented in critically ill patients. Studies have identified elevated hemostatic factors as indicators of acute and sustained EC activation, including the large, highly adhesive clotting protein, von Willebrand factor (VWF). Reduced activity of the VWF cleaving protease ADAMTS-13 has also been described and is associated with poor disease outcome. These observations have been coupled with serious clinical effects, including deep vein thrombosis (DVT) and acute pulmonary embolism (PE). This suggests that severe COVID-19-driven EC injury is fulminant and sustained, and presents a major risk for thrombotic complications. However, the role of inflammatory and prothrombotic biomarkers in EC activation and injury requires further investigation. Plasma samples were collected from 61 severe COVID-19 patients at various timepoints throughout their ICU stay. Clinical outcomes were recorded, and relevant plasma biomarkers were measured. ECs from healthy donors were treated with severe COVID-19 plasma and the effects of these biomarkers on EC activation, VWF secretion, and monolayer disruption were analyzed via flow cytometry, ELISA, and confocal immunofluorescence microscopy. Clinical outcomes included DVT, PE, stroke, and death. Prothrombotic biomarkers were consistently elevated, with mean values peaking at days 11 to 15 of their ICU stay. Mean ADAMTS13 levels were below normal range, with some patients experiencing a reduction to ≤10% of normal. Cytokine profiles were diverse but represented a significant increase in biomarkers associated with innate immunity coupled with a mean reduction in those associated with adaptive immune system. In addition, ECs exposed to severe COVID-19 plasma displayed significant monolayer disruption, activation, and VWF secretion compared to cells treated with normal plasma. This study confirms the association of biomarkers with significant clinical outcomes and highlights their use as a predictive tool. Severe COVID-19 induces a prothrombotic and hyperinflammatory state that may sustain EC activation and disrupt monolayer integrity independent of direct viral infection by SARS-CoV-2. Results of this study could provide new insights into disease mechanisms, uncover therapeutic targets, and validate cellular models for future studies on EC dysfunction.
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    Enhancing Anti-Cancer Immunotherapy by Disruption of the non-Receptor Tyrosine Kinase Fes
    (2024-09-05) Laight, Brian; Pathology and Molecular Medicine; Greer, Peter
    Cancer immunotherapies are based on the principle that a patient’s own immune system can be engaged to fight their disease. While effective activation of the immune system is crucial for the success of cancer immunotherapies, native checkpoint mechanisms exist to limit immune activation and maintain homeostasis. In the setting of cancer, these checkpoints act as barriers to anti-cancer immunity, and therefore represent important targets for cancer immunotherapy. Here, we demonstrate a novel role of the Fes tyrosine kinase, which is abundantly expressed in macrophages, dendritic cells, NK cells and B cells, as an innate intracellular immune checkpoint. In syngeneic engraftment models of breast cancer and melanoma, FES genetic disruption in the host was associated with delayed tumour growth, improved survival, enhanced response to therapy with doxorubicin, and sensitization of tumours to anti-PD-1 immune checkpoint blockade. These effects were associated with enhanced in vivo ratios of M1/M2 tumour associated macrophages, as well as activation of and PD-1 expression on tumour associated T cells. In vitro, Fes-deficient bone marrow derived macrophages demonstrated an increase in Toll-like receptor signaling in antigen presenting cells, which was associated with an increase in proinflammatory cytokine production and T cell activation capabilities. Furthermore, we demonstrate a novel role for Fes in regulating the retention of cytokines on antigen presenting cell surfaces that may elicit greater downstream signaling in T cells. Our results highlight Fes as a novel innate immune checkpoint with potential as a predictive biomarker for effective immune checkpoint blockade treatment, and a potential therapeutic target to improve this form of anti-cancer immunotherapy.
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    Opportunities in Breast Cancer Patient Care in Southeast Ontario: Evidence-based Recommendations to Implement from Cancer Diagnosis to Treatment
    (2024-09-05) Nasute Fauerbach, Paola Veronica; Pathology and Molecular Medicine; Berman, David
    Despite advances in breast cancer biology and targeted treatments, surgery and early cancer detection are crucial for the best outcomes. In Ontario, the five-year breast cancer-specific mortality rates vary significantly, with Southeastern Ontario (SEO) being among the worst regions, especially in women aged 40-49. The purpose of this thesis was to determine feasible approaches to improve surgical outcomes and investigate potential clinicopathological determinants of poor survival rates in women 40-49. My first manuscript focused on assessing margin status at breast-conserving surgery (BCS) of women with breast cancer in 2016-2017 as inadequate margins (IM) often lead to reoperations and increase healthcare costs. Clinicopathological and imaging data were reviewed for all consecutive 360 patients. Statistical analysis revealed invasive cancers >20 mm, ductal carcinoma in situ, and, most importantly, the lack of a definitive presurgical diagnosis were associated with IM at BCS, which were 30.00%. My second manuscript focused on assessing clinicopathological, imaging, and genetic data collected for 395 patients aged 40-49 from 2009 to 2018. The hereditary cancer rate was within the previously reported range (8.10%), but metastatic rates were high (20.51%) regardless of genetic status and completion of intensive treatment. Moreover, my study showed that 88.35% of patients were symptomatic and only 4.56% had undergone annual screening. Cox analysis demonstrated that cancer dimension >20 mm was the strongest predictor of metastasis. Altogether, my research reveals the importance of a definitive presurgical diagnosis, which can be achieved with biopsy, and underscores the impact of early cancer detection through screening as cancer size is a major factor in both IM at BCS and high metastatic rates. Therefore, I propose practical, evidence-based approaches to improve breast cancer patient outcomes not only in SEO, but also in other regions that face similar challenges.