The field of targeted cancer therapy has progressed in recent years with the approval of new oncology drugs. Coupled with the benefits that these agents provide, has come an appreciation for challenges that occur when attempting to translate successful experiments from the laboratory into effective clinical trials. One such challenge has been predicting the optimal dose and schedule to take into clinical evaluation, given the possibility that certain targeted therapeutics may exhibit maximal anti-tumour efficacy well below maximum tolerated doses. Recent work with a targeted antibody to the mouse vascular endothelial growth factor receptor-2 demonstrated that detection of increased levels of its endogenous ligand in the plasma, namely VEGF, could address this issue, as maximal increases in VEGF paralleled optimal drug activity. The VEGF result has become recognized as a potential class effect for this family of inhibitors. This thesis summarizes experiments designed to build upon this discovery by investigating whether the utility of ligand measurement might also apply to drugs that inhibit the epidermal growth factor receptor (EGFR), which have also received recent regulatory approval, and inhibit angiogenesis as one of their mechanisms of action. In addition, we investigated the potential application of EGFR inhibitors to influence other markers of tumour angiogenesis, specifically their effects on levels of circulating endothelial progenitor cells (CEPs). Finally, we evaluated combination treatment of EGFR inhibition with anti-angiogenic scheduling of chemotherapy. The EGFR ligand TGF-alpha increased in a dose dependent fashion following treatment with cetuximab, and levels in the circulation paralleled anti-tumour activity. This was a host-dependent effect that was not observed with the lower affinity antibody nimotuzumab. Inhibition of host EGFR also reduced plasma CEPs, but at higher doses these drugs increased off target growth factors VEGF and G-CSF, as well as CEPs. In a model of advanced triple negative breast cancer, the combination of nimotuzumab and metronomic cyclophosphamide was efficacious and well tolerated, leading to a potential new treatment strategy for this aggressive disease. Taken together, these studies identify new and useful applications for EGFR-targeted antibodies, and shed further light on their contributions within the field of tumour angiogenesis and antiangiogenic therapy.

Background LHRH agonists decrease tumour size/activity by suppressing testosterone in prostate cancer; however, initial injection causes testosterone surge that triggers flare symptoms. Anti-androgen given with agonist may reduce/avoid flare symptoms. When LHRH antagonist/blocker is introduced, testosterone suppression is immediate, but there is uncertainty about significance of flare symptoms without anti-androgen. Objective Systematic review compared significance of flare symptoms avoided and cost utility analysis using modelling comparing incremental value of blocker (degarelix) OR agonist (goserelin)+anti-androgen (bicalutamide) VERSUS agonist alone in prostate cancer patients. Outcome Incremental cost/QALY of bone pain as flare symptom between treatments Results Thirteen studies were reviewed. There was no standard definition for flare symptoms or data on LHRH antagonist versus other treatments on flare. From societal perspective, goserelin+bicalutamide was dominated over goserelin alone and similarly, from public perspective, goserelin+bicalutamide had favourable cost effectiveness profile against goserelin. Conclusion With bone pain as clinical endpoint, LHRH agonist+anti-androgen had favourable cost-effectiveness profile compared to goserelin.

Background LHRH agonists decrease tumour size/activity by suppressing testosterone in prostate cancer; however, initial injection causes testosterone surge that triggers flare symptoms. Anti-androgen given with agonist may reduce/avoid flare symptoms. When LHRH antagonist/blocker is introduced, testosterone suppression is immediate, but there is uncertainty about significance of flare symptoms without anti-androgen. Objective Systematic review compared significance of flare symptoms avoided and cost utility analysis using modelling comparing incremental value of blocker (degarelix) OR agonist (goserelin)+anti-androgen (bicalutamide) VERSUS agonist alone in prostate cancer patients. Outcome Incremental cost/QALY of bone pain as flare symptom between treatments Results Thirteen studies were reviewed. There was no standard definition for flare symptoms or data on LHRH antagonist versus other treatments on flare. From societal perspective, goserelin+bicalutamide was dominated over goserelin alone and similarly, from public perspective, goserelin+bicalutamide had favourable cost effectiveness profile against goserelin. Conclusion With bone pain as clinical endpoint, LHRH agonist+anti-androgen had favourable cost-effectiveness profile compared to goserelin.

The field of targeted cancer therapy has progressed in recent years with the approval of new oncology drugs. Coupled with the benefits that these agents provide, has come an appreciation for challenges that occur when attempting to translate successful experiments from the laboratory into effective clinical trials. One such challenge has been predicting the optimal dose and schedule to take into clinical evaluation, given the possibility that certain targeted therapeutics may exhibit maximal anti-tumour efficacy well below maximum tolerated doses. Recent work with a targeted antibody to the mouse vascular endothelial growth factor receptor-2 demonstrated that detection of increased levels of its endogenous ligand in the plasma, namely VEGF, could address this issue, as maximal increases in VEGF paralleled optimal drug activity. The VEGF result has become recognized as a potential class effect for this family of inhibitors. This thesis summarizes experiments designed to build upon this discovery by investigating whether the utility of ligand measurement might also apply to drugs that inhibit the epidermal growth factor receptor (EGFR), which have also received recent regulatory approval, and inhibit angiogenesis as one of their mechanisms of action. In addition, we investigated the potential application of EGFR inhibitors to influence other markers of tumour angiogenesis, specifically their effects on levels of circulating endothelial progenitor cells (CEPs). Finally, we evaluated combination treatment of EGFR inhibition with anti-angiogenic scheduling of chemotherapy. The EGFR ligand TGF-alpha increased in a dose dependent fashion following treatment with cetuximab, and levels in the circulation paralleled anti-tumour activity. This was a host-dependent effect that was not observed with the lower affinity antibody nimotuzumab. Inhibition of host EGFR also reduced plasma CEPs, but at higher doses these drugs increased off target growth factors VEGF and G-CSF, as well as CEPs. In a model of advanced triple negative breast cancer, the combination of nimotuzumab and metronomic cyclophosphamide was efficacious and well tolerated, leading to a potential new treatment strategy for this aggressive disease. Taken together, these studies identify new and useful applications for EGFR-targeted antibodies, and shed further light on their contributions within the field of tumour angiogenesis and antiangiogenic therapy.

Despite the combination of chemotherapy and surgery in osteosarcoma treatment, the survival of patients remains low. The lack of treatment improvement prompted me to investigate deregulated Hedgehog pathway as a potential target for intervention. During development, the Hedgehog pathway is tightly regulated to control organ and tissue development. Specifically, the Indian Hedgehog pathway (IHH) is important for bone development. Aberrantly activated Hedgehog pathway through ligand-dependent or ligand-independent mechanisms has been reported in numerous cancers. Several small molecule antagonists of the Hedgehog pathway are being explored clinically to improve patient outcome. I examined the expression of IHH pathway components, IHH, SMO, PTCH1 and GLI1, in 43 high-grade primary osteosarcoma tumors and 6 cell lines and found various transcript levels of IHH pathway components in osteosarcoma samples. The high levels of co-expressed IHH and targets, PTCH1 and GLI1, in a subset of osteosarcoma samples are indicative of ligand-dependent activation. Trends toward worse survival for patients with higher IHH (positive regulator) levels and toward better survival for patients with higher PTCH1 (negative regulator) levels were observed. Through genetic analyses, PTCH1 inactivating mutations and GLI1 amplification were found not to be responsible for ligand-independent activation in osteosarcoma. Characterization of Hedgehog signaling in osteosarcoma cell lines showed that cells with high IHH, PTCH1, and GLI1 levels were sensitive to small molecule modulators of both SMO and GLI, which supported the ligand-dependent activation observed in the clinical samples. The inverse correlation of endogenous GLI2 levels and Hedgehog pathway induction levels, and the sensitivity of high-GLI2 cells to GLI inhibition, but not SMO inhibition, in cell lines suggest that GLI2 overexpression may be a mechanism of ligand-independent activation in osteosarcoma. Furthermore, in patient-derived osteosarcoma xenograft models, I observed autocrine and possibly paracrine ligand-dependent Hedgehog signaling in the tumor and stromal compartments. I also showed that a clinically relevant SMO antagonist, IPI-926, was effective at specifically inhibiting all ligand-dependent Hedgehog signaling interactions. A trend toward decreased proliferation and increased apoptosis in treated tumors was observed warranting additional research and demonstrating the potential of Hedgehog pathway inhibitors as novel targeted therapeutics for osteosarcoma treatment.

Large intergenic RNAs (lincRNA) are involved in numerous cellular processes, including many relevant to normal development and cancer progression. In my doctoral research, we hypothesized that lncRNAs are functionally important to human endothelial biology, more specifically, to the process of human blood vessel formation or angiogenesis. To detect lincRNAs that are functionally important to human angiogenesis, a custom microarray was used to profile long noncoding transcripts in human vascular endothelium in two-dimensional versus three-dimensional pro-angiogenic cultures, with or without VEGF-A165. We identified a VEGF-A-responsive lincRNA near the VEGFR1 gene, which we termed lincRNA-VEGFR1 (LIVE1). Unbiased mRNA microarrays defined a number of potential target genes when LIVE1 was functionally disrupted using RNA interference. Importantly, knockdown and over-expression studies indicated that LIVE1 exerts transcriptional control over VEGFR1 as well as other VEGF receptors and direct angiogenesis in vitro. Furthermore, we found that LIVE1 is highly expressed in glioblastoma, and is enriched in glioma stem cell (GSC) fractions and neoplastic endothelial progenitor populations. In vivo knockdown of LIVE1 in a glioblastoma xenograft model decreased microvascular density, vascular perfusion, pericyte coverage, tumor volume and slowed tumour progression. Our results establish LIVE1 as a key mediator of angiogenesis and demonstrate the potential of lincRNA-based therapeutics.

Large intergenic RNAs (lincRNA) are involved in numerous cellular processes, including many relevant to normal development and cancer progression. In my doctoral research, we hypothesized that lncRNAs are functionally important to human endothelial biology, more specifically, to the process of human blood vessel formation or angiogenesis. To detect lincRNAs that are functionally important to human angiogenesis, a custom microarray was used to profile long noncoding transcripts in human vascular endothelium in two-dimensional versus three-dimensional pro-angiogenic cultures, with or without VEGF-A165. We identified a VEGF-A-responsive lincRNA near the VEGFR1 gene, which we termed lincRNA-VEGFR1 (LIVE1). Unbiased mRNA microarrays defined a number of potential target genes when LIVE1 was functionally disrupted using RNA interference. Importantly, knockdown and over-expression studies indicated that LIVE1 exerts transcriptional control over VEGFR1 as well as other VEGF receptors and direct angiogenesis in vitro. Furthermore, we found that LIVE1 is highly expressed in glioblastoma, and is enriched in glioma stem cell (GSC) fractions and neoplastic endothelial progenitor populations. In vivo knockdown of LIVE1 in a glioblastoma xenograft model decreased microvascular density, vascular perfusion, pericyte coverage, tumor volume and slowed tumour progression. Our results establish LIVE1 as a key mediator of angiogenesis and demonstrate the potential of lincRNA-based therapeutics.

Despite the combination of chemotherapy and surgery in osteosarcoma treatment, the survival of patients remains low. The lack of treatment improvement prompted me to investigate deregulated Hedgehog pathway as a potential target for intervention. During development, the Hedgehog pathway is tightly regulated to control organ and tissue development. Specifically, the Indian Hedgehog pathway (IHH) is important for bone development. Aberrantly activated Hedgehog pathway through ligand-dependent or ligand-independent mechanisms has been reported in numerous cancers. Several small molecule antagonists of the Hedgehog pathway are being explored clinically to improve patient outcome. I examined the expression of IHH pathway components, IHH, SMO, PTCH1 and GLI1, in 43 high-grade primary osteosarcoma tumors and 6 cell lines and found various transcript levels of IHH pathway components in osteosarcoma samples. The high levels of co-expressed IHH and targets, PTCH1 and GLI1, in a subset of osteosarcoma samples are indicative of ligand-dependent activation. Trends toward worse survival for patients with higher IHH (positive regulator) levels and toward better survival for patients with higher PTCH1 (negative regulator) levels were observed. Through genetic analyses, PTCH1 inactivating mutations and GLI1 amplification were found not to be responsible for ligand-independent activation in osteosarcoma. Characterization of Hedgehog signaling in osteosarcoma cell lines showed that cells with high IHH, PTCH1, and GLI1 levels were sensitive to small molecule modulators of both SMO and GLI, which supported the ligand-dependent activation observed in the clinical samples. The inverse correlation of endogenous GLI2 levels and Hedgehog pathway induction levels, and the sensitivity of high-GLI2 cells to GLI inhibition, but not SMO inhibition, in cell lines suggest that GLI2 overexpression may be a mechanism of ligand-independent activation in osteosarcoma. Furthermore, in patient-derived osteosarcoma xenograft models, I observed autocrine and possibly paracrine ligand-dependent Hedgehog signaling in the tumor and stromal compartments. I also showed that a clinically relevant SMO antagonist, IPI-926, was effective at specifically inhibiting all ligand-dependent Hedgehog signaling interactions. A trend toward decreased proliferation and increased apoptosis in treated tumors was observed warranting additional research and demonstrating the potential of Hedgehog pathway inhibitors as novel targeted therapeutics for osteosarcoma treatment.

Breast cancer is a major health issue, affecting annually approximately 1.4 million women worldwide. It is a highly heterogeneous disease with the different subtypes having distinct clinical outcomes and different sensitivity to various treatment modalities. The focus of the present dissertation was the identification of novel proteomic prognostic markers for patients with early stage breast cancer. Three different approaches to identify potential prognostic markers were undertaken. First, we hypothesized that since different breast cancer subtypes have distinct clinical outcomes, breast cancer subtype-specific proteins may retain prognostic potential. Second, given the central role of estrogen signaling in breast epithelial cell biology, we hypothesized that estrogen-regulated proteins may be useful in predicting patient outcome. Finally, we hypothesized that genes related to survival based on meta-analyses of publicly available breast cancer tissue microarray data, may also demonstrate prognostic potential at the proteome level. As such, a variety of mass spectrometry-based approaches and biological samples were utilized for the discovery of these potential prognostic protein markers resulting in twenty-four candidates. Upon the identification of candidate biomarkers, a mass spectrometry-based assay for the simultaneous quantification of these proteins in breast cancer tissue samples was established. The developed assay was used for measuring the relative expression levels of the potential biomarkers in a cohort of 96 breast cancer tissue samples from untreated patients with early stage breast cancer. This exercise uncovered two proteins that showed the potential to discriminate between ER-positive patients at high and low risk of disease recurrence, namely KPNA2 and CDK1. In conclusion, the present dissertation describes the development of a preclinical exploratory study, from the discovery to the preliminary verification of potential prognostic biomarkers for breast cancer patients.

This thesis employed methods used in health care decision making to evaluate strategies for prevention and treatment of febrile neutropenia (FN) in non-Hodgkin lymphoma (NHL) patients. The objectives of this thesis were to quantify the cost-effectiveness of filgrastim and pegfilgrastim as primary prophylaxis against FN in NHL patients, to develop an algorithm for converting health-related quality of life data collected in non-Hodgkin lymphoma patients into preference-based health utility values, and to evaluate NHL patients’ preferences for outpatient treatment of FN. The cost-effectiveness analysis demonstrated that neither filgrastim, nor pegfilgrastim are cost-effective, with respective incremental cost-effectiveness ratios [95% confidence interval] of $4,599,000/QALY [$597,045, dominated] and $6,272,000/QALY [$730,692, dominated], well above the normally accepted threshold of $50,000/QALY. The algorithm for deriving health utility values was based on a regression model that used health utility values obtained from the EQ-5D instrument as the outcome variable and the four subscales of the Functional Assessment of Cancer Therapy – General (FACT-G) questionnaire as the predictor variables. The model final model included three of the FACT-G subscales, and had an R-squared value of 0.502 and a mean squared error of 0.013. A discrete choice experiment was used to examine patients’ preferences for out patient treatment of FN, and demonstrated that out-of-pocket costs, unpaid caregiver time required daily, and probability of return to hospital are all significant attributes when considering outpatient therapy for FN. Adjusted odds ratios [95% confidence intervals] of accepting outpatient treatment for FN were 0.84 [0.75 to 0.95] for each $10 increase in out-of-pocket cost; 0.82 [0.68 to 0.99] for each 1 hour increase in daily unpaid caregiver time; and 0.53 [0.50 to 0.57] for each 5% increase in probability of return to hospital. These results provide important information for clinicians and health care decision makers involved in implementing programs for NHL patients with FN.