Department of Pathology and Molecular Medicine Graduate Theses

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    Enhancing Anti-Tumour Cytotoxic Lymphocyte Responses by Targeting FES in Antigen Presenting Cells
    Dmytryk, Natasha; Pathology and Molecular Medicine; Greer, Peter
    The non-receptor tyrosine kinase FES has been implicated in the suppression of inflammatory signaling pathways in antigen-presenting cells (APCs) proposing that it might be dampening APC production of inflammatory cytokines in the tumour niche. These cytokines constitute the third and final signal of cytotoxic lymphocyte (CTL) activation – one of the main anti-tumour immune cells. This thesis explores the effect of FES disruption in APCs, specifically, its effect on inflammatory signaling, Signal 3 cytokine production and CTL priming. Immunoblotting analysis of the inflammatory signaling cascade showed that lipopolysaccharide (LPS)-stimulated fes-/- bone marrow-derived macrophages (BMDMs) display enhanced induction of inflammatory signaling cascades, compared to fes+/+ BMDMs. Additionally, RNA sequencing analysis revealed that in response to LPS-stimulation, certain key Signal 3 cytokine mRNA transcripts show a trend of higher production by fes-/- BMDMs. While a multiplexed cytokine detection assay did not reveal differences between fes+/+ and fes-/- BMDM secretion of these cytokines into culture media, flow cytometry analysis revealed a significantly higher frequency of fes-/- BMDMs with detectable IL-12 levels at the cell surface. This provides a possible rationale for fes-/- BMDMs being more effective at presenting antigen to and activating CTLs; and this was experimentally observed using co-cultures of OT-I CTLs with BMDMs presenting chicken ovalbumin (OVA). Collectively, these observations suggest that the apparent immunosuppressive function of FES makes it an actionable target for improving the efficacy of immunotherapies aimed at enhancing anti-cancer adaptive immune responses, including immune checkpoint inhibitors.
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    Using Culturomics to Characterize the Gastrointestinal Microbiota of Donors for Patients with Bipolar Disorder Experiencing Depression
    Saleem, April; Pathology and Molecular Medicine; Prameet, Sheth
    Depressive disorders such as bipolar disorder (BD) affect more than 310 million people worldwide, reduce quality of life, co-occur with other physical disorders, and increase risks of premature death. This high prevalence reflects the challenges in the tolerability and efficacy of current treatments. Bacteria residing in the human gastrointestinal tract communicate with the central nervous system through a bi-directional network, referred to as the gut-brain axis, which can influence mood and behaviour. Fecal microbiota transplantation (FMT) has been shown to alter the gut bacterial composition of recipients. In a phase 2/3 double-blinded randomized controlled trial (RCT) completed in 2022, participants diagnosed with BD were randomized 1:1 to receive either rigorously screened and processed donor stool (allogenic FMT) or their own stool (autologous FMT) via colonoscopy. Fecal samples were collected from donors and participants at baseline, 3, and 6 months. This thesis will provide a review of the current literature on the role of the gut microbiota, depressive disorders, and methods of altering gut microbiota composition and i) Results of cultured and isolated bacteria from the donor stool with ii) Species and/or strain level identification; and iii) Compare the metagenomic community profiles of the donors to the bacteria isolated from culturing. Using a culturomics approach, donor fecal samples were cultured on 10 different selective and differential media. The taxonomy of bacterial isolates was identified using MALDI-TOF mass spectrometry, 16S rRNA gene sequencing, and Sanger sequencing. Bacterial community profiles from fecal samples were characterized using shotgun metagenomics. From the donor stool, 78 different species of bacteria were isolated across 43 genera including Bacteroides, Bifidobacterium, Collinsella, Streptococcus, Dorea, and Coprococcus. Patients diagnosed with BD, experiencing severe depression who have undergone FMT treatment, have shown reduction in depressive symptoms and different gut bacterial composition. Isolated bacteria from donor stool provides a collection of viable species of interest for future use. These findings add valuable insights into FMT as a method to modify the gut microbiota and analyze the restorative potential to inform the development of further studies examining the relationship between the gut-brain axis and depressive disorders.
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    Comparison of Gene Enrichment between Prostate Tissue and Urine: Epithelial and Stromal Signature Genes And their Implications to Urine Biomarker Development
    Xing, Yuandong; Pathology and Molecular Medicine; Berman, David; Gooding, Robert
    Background: For prostate cancer (PCa), early diagnosis and patient stratification based on biomarkers dramatically improve prognosis after treatment. PCa biomarkers are best credentialled in tissue after comprehensive histopathologic review. However, urinary biomarkers are obtainable non-invasively and thus safter to obtain. It is warranted to understand how to develop urinary biomarkers based on gene expression in tissue. As a critical step, comparison of gene enrichment in prostate tissue versus urine may help in selecting transcripts highly enriched in urine as biomarker candidates. Here, we focus on prostate epithelial and stromal signature genes for gene set enrichment analysis. Methods: Epithelial and stromal genes were identified, filtered, and validated based on public data from Human Protein Atlas (HPA) and Gene Expression Omnibus (GEO). Next, we profiled 367 transcripts in prostate tissue (n=9) and urine (n=9) samples from patients undergoing cancer surgery. We developed a Tissue-Urine Comparability Index (TUCI) to evaluate the efficacy of different data processing methods in aligning gene expression profiles from tissue and urine. Processed by the best method, tissue and urine data were employed to compare the enrichment of epithelial and stromal gene sets, as well as gene sets available in the Molecular Signature Database (MSigDB). Results: Epithelial and stromal gene sets, including nineteen and thirteen genes respectively, reliably represented epithelial and stromal signatures and had potential diagnostic value. Correcting batch effect followed by normalizing to GNAS and TAF6L transcripts achieved the highest (best) TUCI. Both epithelial and stromal gene sets were significantly enriched in tissue compared to urine, while the stromal gene set was enriched to a larger extent in tissue than epithelial gene set. Moreover, compared to tissue, immune gene sets were more enriched in urine. Tissue-enriched gene sets include gene sets targeted by miRNA (e.g., miR-148/152 family, miR-302 family, miR-520 family, and miR-372/373 cluster), gene sets targeted by transcription factors (e.g., STAT5), and gene sets involved in biological processes (e.g., cell growth and organ development). Conclusion: Prostate tissue-enriched gene sets differed from urine-enriched ones. These differences provide new insights into prostate tissue and urine contents and direct future work on developing urine based PCa biomarkers.
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    Investigating microRNA-375 as a Tissue and Circulating Marker for Neuroendocrine Cells and Neoplasms
    Mayotte, Harrison R.; Pathology and Molecular Medicine; Renwick, Neil
    Neuroendocrine neoplasia is an enigmatic disease that is currently without reliable diagnostic markers. Normal neuroendocrine cells are difficult to recognize upon routine H&E staining, requiring several IHC markers to confirm their cell-type. A lack of a single, specific marker has led to difficulties in successfully diagnosing neuroendocrine neoplasms (NENs), leading to poor patient outcomes. This has contributed to nearly 50% of cases becoming metastatic in the disease course. microRNAs (miRNA) are small non-coding RNA that negatively regulate gene expression through messenger RNA destabilization. Due to their disease specificity, miRNAs are being increasingly researched as potential candidate diagnostic markers. miRNA-375 (miR-375) was previously shown to be more highly expressed in NEN tissue compared to site matched controls using RNA sequencing. In this study, we investigate the potential for miR-375 to be used as a marker for neuroendocrine cell differentiation as well as neuroendocrine neoplasia. We first visualized miR-375 expression in healthy tissues from varying anatomical sites using chromogenic in situ hybridization. We found that miR-375 was only specific to candidate neuroendocrine cells, scattered throughout many tissues. The presence of miR-375 was then compared in neuroendocrine tumor tissue in organs of the gastroenteropancreatic system with non-neuroendocrine cancer from the same region. miR-375 was present in NEN tissue and absent in control tissues. Finally, we compared the plasma abundance of miR-375 in the circulation of lung-NEN patients to patients with non-NEN lung cancer and non-neoplastic lung disease. miR-375 abundance was found to be higher in the plasma of lung NEN patients compared to controls. With the results from this study, miR-375 has the potential to a be a promising candidate marker for future NEN diagnostics.
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    Investigating the Tumour Microenvironment of Non-Small Cell Lung Carcinoma for Anti-Pd-1 Biomarker Discovery
    Espinosa, Nicole; Pathology and Molecular Medicine; Cottrell, Tricia
    Background: Most advanced non-small cell lung cancers (NSCLCs) lack targetable driver mutations and are now treated with PD-1/PD-L1 blockade as standard therapy. Patients with high tumor cell PD-L1 expression (≥50%, PD-L1high) receive anti-PD-1 monotherapy, but the prognosis for advanced NSCLC is poor. Novel biomarkers to identify this treatment-resistant subset of PD-L1high NSCLCs are needed, specifically within the tumour microenvironment. Aim 1: Assess features of the TME in PD-L1high lung adenocarcinomas that may predict resistance to anti-PD-1 Aim 2: Assess the association of tumour-associated neutrophils (TANs) with other clinical and pathologic features Aim 3: Optimize and validate a multiplex panel to characterize features of the TME associated with TANs Methods: Archival diagnostic lung adenocarcinoma specimens collected at Kingston Health Sciences Centre (KHSC) (2010-2021) were assessed for high PD-L1 expression (≥50%), receipt of anti-PD-1 monotherapy, and clinical annotation. Routine clinical H&E/HPS stained slides were visually evaluated for neutrophils by morphology. TAN (+) tumors were defined as those with a minimum of 5 neutrophils per HPF in at least 3 HPFs. Neutrophils within intact tumor epithelium or intra-tumoral stroma were included. Results: TAN(+) specimens were associated with significantly worse overall survival (OS) in the discovery (n=27) and validation (n= 28) cohorts (p=0.0469 and p=0.0246, respectively), By univariate cox regression analysis, TAN(+) was associated with poor OS (HR 3.26: 95% CI 1.47 to 7.23, p=0.004). Other clinicopathological features such as patient age, gender, KRAS status, necrosis, NLR, and smoking history were not significantly associated with OS. Conclusion: Pathologist assessment for TAN on routine H&E/HPS-stained slides is a readily available biomarker that may identify a subset of PD-L1high lung adenocarcinomas with poor outcomes in need of alternative treatment options. Exploration of the import of TAN in other settings (e.g., PD-L1low, squamous histology, and alternative treatment regimens) is warranted.