This study presents an integrated analysis of pancreatic ductal adenocarcinomas (PDACs) for identification of putative cancer driver genes in somatic copy number gains (SCNGs). SCNG data on 60 PDAC genomes was extracted to identify 756 genes, mapping to 20 genomic loci that are recurrently gained. Through copy number and gene expression analysis on a panel of 29 human pancreatic cancer cell lines, this gene catalogue was refined to 34 PDAC high-confidence candidate genes. The performance of these genes was assessed in pooled shRNA screens and only ECT2 showed significant essentiality to cell viability in specific PDAC cell lines with genomic gains at the 3q26.3 locus that harbor this gene. Targeted shRNA-mediated interference of ECT2, as well as pharmacological inhibition, are supportive of the pooled shRNA screen findings. These results favor ECT2 as a candidate target gene for further evaluation in the subset of PDACs presenting with 3q26 somatic copy number gains.

APLF (Aprataxin and Polynucleotide kinase/phosphatase-Like Factor) is an FHA (forkhead-associated)-domain-containing nuclear protein that facilitates the repair of single-strand and double-strand breaks (SSBs and DSBs). Specifically, the APLF-FHA domain mediates interactions with XRCC1 and XRCC4, factors involved in SSB and DSB repair, respectively. A novel substitution was identified in pancreatic cancer patients, where a conserved histidine was substituted to leucine (H42L) in the APLF-FHA domain. The functional and biological characterization of this “variant of unknown significance” was investigated. This thesis shows that the H42L substitution affects APLF phosphorylation, impairs APLF retention at laser-induced DNA breaks, disrupts protein-binding to XRCC1 but not to XRCC4, and reduces cell survival following irradiation and etoposide exposure. Collectively, these data suggest that the H42L substitution impacts APLF participation in global DNA damage repair. The findings from this work could provide insight into the role of APLF in genomic integrity and, moreover, in cancer predisposition.

DKK1, SFRP1, WIF-1, and Wnt5a encode Wnt pathway genes that are frequently silenced by promoter hypermethylation in colorectal cancer. Despite attractive biological consequences of these events, it is unclear whether they contribute to patient prognostication or may influence tumour cell biology within distinct patient subsets. I sought to determine the prognostic roles of these methylation events in a large cohort of colorectal carcinomas from Ontario and Newfoundland. Methylation was quantified and associated with patient clinicopathlogical features. Methylation was present in cancer tissue. DKK1, Wnt5a, and SFRP1 were strongly and independently associated with tumour subtype in a manner that suggested subtype-specific activity of Wnt signaling. Methylation of DKK1 was a borderline prognosticator of favourable outcome. These results offer intriguing insight into subtype-specific biology and lead to a proposed model whereby methylation-induced Wnt bias may contribute to patient outcome.

Colorectal cancer (CRC) is common in Ontario. This study described the processes of care following CRC resection, and identified CRC relapse from administrative data. Methods: CRC patients aged 18-80 from 1996-2001 with a colorectal resection were identified from the Ontario Cancer Registry. Linked discharge abstracts and physician billings were examined for physician visits, body imaging and endoscopy over the 5 year follow-up period. Administrative codes suggesting disease relapse were compared with patient charts. Results: Overall, 12,804 patients were identified and 8,804 had no evidence of relapse. Most (96.2%) patients had general practitioner follow-up, while 49.3% had medical oncology and 80.4% had general surgery follow-up. Greater than 90% of patients received endoscopy, while only 68.7% of patients received body imaging. Detecting disease relapse was 87.5% sensitive and 93.0% specific. Conclusions: There is potential for improving post-resectional follow-up in CRC patients. It is possible to detect relapse through administrative databases.

Intensity modulated radiotherapy (IMRT) can potentially increase the dose delivered to liver tumours while sparing normal tissues from dose. More complex IMRT, with more modulation of the radiation beam is more susceptible to geometric and dosimetric uncertainties than simpler radiotherapy plans. Simple breath-hold liver IMRT using few radiation beam segments (<30) was investigated in 27 patients to determine the quality of treatment in terms of tumour dose coverage and normal tissue sparing as compared to index IMRT using >30 segments. In all 27 plans number of segments was reduced to <30 without compromising tumour coverage or normal tissue dose constraints, at the expense of dose conformity. Delivered tumour and normal tissue dose did not differ statistically between IMRT plans when accounting for treatment residual geometric error. This research supports considering the use of simple IMRT for treatment of liver cancer, except when loss of dose conformation is undesirable (i.e. very high doses).

According to the cancer stem cell hypothesis a subpopulation of cells within a tumour has the capacity to sustain its growth. These cells are termed cancer stem cells, and are most simply defined as the cells within a primary tumour that can self-renew, differentiate and regenerate a phenocopy of that cancer when transplanted in vivo. Cancer stem cells have now been prospectively identified from numerous human tumours and are actively sought in many cancer types, both clinical and experimental. The cancer stem cell hypothesis remains controversial, with evidence both supporting and challenging its existence in human tumours and in animal models of disease. Here we prospectively identify and study brain cancer stem cells in clinically representative mouse models of the medulloblastoma and glioma. Cancer stem cells from both mouse brain tumour types are prospectively enriched by fluorescent activated cell sorting freshly dissociated cells for the surface antigen CD15, display a neural precursor phenotype, exhibit the hallmark stem cell characteristics of self-renewal and multilineage differentiation, and regenerate a phenocopy of the original tumour after orthotopic transplantation. Additionally, novel mouse medulloblastoma and glioma cancer stem cell lines were established and studied in vitro as adherent cultures in the same serum-free media conditions that support the growth of normal neural stem cells. When mouse and human glioma stem cell lines were compared, many novel molecular mediators of the tumour phenotype were identified, as were chemical compounds that selectively inhibit their growth. Our results have important implications regarding the cancer stem cell hypothesis, the mechanisms that drive brain tumour stem cell growth and the therapeutic strategies that may prove effective for the treatment of glioma and medulloblastoma.

The colony formation assay (CFA) is the gold standard for measuring cytotoxic effects on cells. To increase efficiency, the CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation (IR) on the FaDu and A549 cancer cell lines. Its ability to evaluate combination therapies was investigated using cisplatin and IR. The 96-well CFA was transferred to a robotic platform for evaluation as a high-throughput screen (HTS) readout for the discovery of novel anti-cancer compounds, and radiosensitizers. Screening yielded eight putative anti-cancer hits, and five putative radiosensitizing hits. Secondary screening confirmed 6/8 anti-cancer compounds, and 0/5 radiosensitizing compounds. Thus, the 96-well CFA can be adopted as an alternative assay to the 6-well CFA in the evaluation of cytotoxicity in vitro, providing a possible readout to be utilized in HTS for discovering anti-cancer compounds, but with limited applicability in discovering radiosensitizers.

Ovarian high-grade serous cancer (HGSC) is the most lethal gynecologic malignancy and well-characterized models may improve patient outcomes. Patient-derived xenografts (PDXs) recapitulate disease heterogeneity; however, to be useful in predicting response to novel chemotherapeutics, they must reflect the response of the donor tissue to standard chemotherapy. The objectives of this study were: first, to evaluate the response of PDXs’ to platinum therapy and compare this response to that of the donor; and second, to determine whether treatment with chemotherapy enriches for tumourigenic cells. Eighteen samples formed tumours in the mammary fat pads of NOD-Scid-IL2Rγnull mice and were treated with Carboplatin. There was a 100% concordance between sample status and PDXs response to chemotherapy. HGS histology was confirmed for all cases. A conclusion regarding post-chemotherapy tumourigenicity could not be made due to inadequate statistical power. PDXs represent useful tools for evaluation of novel therapies and identification of patients who are platinum-resistant/sensitive.

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

Pancreatic adenocarcinoma (PDAC) putatively arises from the pancreatic duct, thus usage of the normal human pancreatic duct epithelial (HPDE) cell line is an ideal model to examine the successive accumulation of genetic alterations involved in carcinogenesis. KRAS mutations have been reported in 90% of PDACs. Oncogenic KRAS elicits activation of downstream pathways involved in survival, motility, and cell cycle progression. KRASG12V introduction in the HPDE cell line upregulates Lipocalin-2 (LCN2) expression. LCN2 has been identified in numerous carcinomas and is associated with survival, tumorigenicity, and invasion. In this work, LCN2 was found to be commonly expressed in high grade pancreatic duct neoplastic precursor lesions and PDAC illustrating its potential as a biomarker. Moreover, in vitro and in vivo studies demonstrate that high LCN2 expression promotes gemcitabine resistance, MMP-9 activity, angiogenesis, and tumorigenicity. Loss of Smad4 function is found in 55% of PDAC cases. Smad4 is a critical component in the TGF-β signaling which mediates the transcription of genes involved in processes such as cell cycle arrest, apoptosis, and invasion. This work examined the consequences of KRASG12V expression and Smad4 loss in the HPDE model. Cellular invasion was promoted by KRASG12V expression or knocking down Smad4 by 80% in the HPDE model. A TGF-β resistant HPDE cell line, TβR, was shown to lack Smad4 expression due to deletion, promoter methylation, and nonsense mutation. KRASG12V expression in the TβR model (TβR KRAS) promoted neoplastic transformation and tumour formation in immunodeficient mice with complete penetrance. Smad4 expression in the TβR KRAS cell line reinstated TGF-β signaling, delayed tumour formation, and decreased metastatic spread. This study provides evidence that Smad4 acts as a restriction point in the transformation of HPDE cells. Overall, this work examines the contribution of genes involved in transformation, and identifies a potential therapeutic and diagnostic biomarker in PDAC.