The long-term natural history of ovarian reserve after adjuvant chemotherapy for breast cancer has been poorly described. We recruited 52 breast cancer survivors treated with adjuvant chemotherapy before 40 years of age who remained premenopausal after chemotherapy treatment. Twenty (38.5%) were more than five years out from treatment. Ovarian reserve estimates were compared with a control group. Anti-Müllerian hormone (AMH), follicle stimulating hormone and luteinizing hormone demonstrated significant differences consistent with reduced ovarian reserve in breast cancer survivors. Mean AMH was 6.65 pmol/l in survivors compared to 17.43 in controls (p < 0.001). Attained age and age at the time of treatment were correlated with AMH levels in breast cancer survivors. Conclusion: Ovarian reserve is significantly reduced in young breast cancer survivors. Age is the major predictor of AMH level in survivors. A 35 year old breast cancer survivor has an AMH level similar to a 45 year old control.

The aggressive nature of pancreatic cancer, characterized by invasiveness, resistance to treatment, rapid progression, and its high prevalence in the population urges the need for developing more effective treatments. Many studies have attributed resistance to therapeutics of pancreatic cancer to activity of the transcription factor nuclear factor kappa B (NF-kB). NF-kB is regulated by the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). GSK-3 is a key mediator of pathways such as insulin, wnt, and PI3K/Akt and has roles in proliferation, glucose metabolism, apoptosis, motility and neuroprotection. Depending on the cellular context, GSK-3 activity can promote or inhibit cell survival. GSK-3 inhibition was recently reported to have anti-cancer effects against pancreatic cancer cells. This effect was in part attributed to suppression of NF-kB. In this thesis, I showed that while blocking GSK-3 disrupts NF-kB, and has anti-survival effects on pancreatic cancer cells, it does not sensitize to the chemotherapeutic drug gemcitabine. NF-kB inhibition by curcumin also resulted in similar effects. These results questions previous reports that NF-kB activation plays a major role in chemo-resistance of pancreatic cancer. The inhibition of NF-kB by genetic disruption of GSK-3 was previously reported to sensitize mouse embryonic fibroblasts and hepatocytes to TNF-alpha cytotoxicity. I therefore tested whether GSK-3 inhibition could sensitize pancreatic cancer cells to apoptosis induced by the clinically applicable member of the TNF-alpha family, TNF-alpha related apoptosis inducing ligand (TRAIL). In contrast to the results obtained with gemcitabine, the combination of genetic or pharmacological inhibition of GSK-3 and TRAIL was found to be highly synergistic in apoptosis induction. Analysis of the apoptotic mechanisms, point towards effects of GSK-3 inhibition on caspase-8 activation, consistent with inhibition of the death receptor signalling pathway. It was found that not only caspase-8 but also mitochondrial anti-apoptotic proteins such as Bcl-XL and Mcl-1 were mediating the TRAIL sensitization. Furthermore, for the first time the in vivo effects of GSK-3 inhibition in combination with TRAIL treatment was investigated. The results indicate a significant enhancement of apoptosis in pancreatic cancer xenografts with minimal toxic effects. Together, these studies provide a rationale for developing combination treatments based on GSK3 inhibition and TRAIL death receptor activation to treat pancreatic cancer.

Siblings of children with cancer encounter stressors and challenges that can lead to severe distress and a host of psychological difficulties. Factors including age, gender, and disease characteristics of the child with cancer are reported to influence sibling adjustment. The majority of research, however, is dated, inconsistent, and marred by methodological problems. Guided by the disability-stress-coping model, the study examined the: (a) frequency of sibling and parent reported symptoms of anxiety and depression, internalizing and externalizing behavior problems, and somatic problems, (b) influence of sibling, family, and disease factors on sibling adjustment, (c) moderating effects of age on the relationship between sibling factors and sibling adjustment, and (d) mediating effect of primary cognitive appraisal on the relationship between self-esteem and sibling adjustment. One hundred and eight siblings (7-17 years; 51 males; 57 females) participated. Siblings completed the State-Trait Anxiety Inventory for Children and the Children’s Depression Inventory to provide measures of sibling reported symptoms of anxiety and depression. Parents completed the Child Behavior Checklist to provide measures of parent reported internalizing behavior problems, externalizing behavior problems, and somatic problems. The communication and intrapersonal thoughts and feelings subscales of the Sibling Perception Questionnaire, completed by siblings, were used to assess perceived social support and primary cognitive appraisal. Self-esteem was assessed with the global self-worth subscale of the Self Perception Profile for Children/Adolescents, completed by siblings. Hierarchical regression analyses were conducted to assess the direct and indirect effects of sibling, family, and disease factors on psychological outcomes. Siblings and parents reported higher incidents of clinically significant symptoms of anxiety, internalizing behavior problems, and somatic problems than expected in a normative population. Sibling age and gender, diagnosis of the child with cancer, social support, self-esteem, and primary cognitive appraisal were significantly associated with sibling and parent reported psychological adjustment measures. Age moderated the relationship between gender, social support, and primary cognitive appraisal and several adjustment outcomes. Lastly, primary cognitive appraisal partially mediated the relationship between self-esteem and sibling reported anxiety and depression symptoms. These findings highlight the need for sibling psychosocial interventions and provide direction for the development and implementation of such groups.

The p53 tumour suppressor gene is inactivated by mutation in over 50% of all human cancers. The p53 protein is activated and stabilized through several post-translational modifications in response to various stresses and promotes cell cycle arrest and apoptosis. Thus, regulation of p53 is critical for normal cellular function. Pirh2 is a p53-regulated gene recently identified in our laboratory which encodes an E3 RING-finger ubiquitin ligase that binds to p53 and negatively regulates p53 by targeting it for ubiquitin-mediated proteolysis. Pirh2 is similar to another well-characterized E3 RING finger ubiquitin ligase, Mdm2, which also participates in a similar negative feedback loop with p53. At least seven E3 ubiquitin ligases are known to target p53 for degradation and the reason for this functional redundancy is unclear. The purpose of this study is to characterize Pirh2 activity. This study has two aims the first is to identify additional interacting proteins for Pirh2, and the second is to delineate Pirh2 regulation of p53. Using several tandem affinity purification strategies and a GST-pull down approach, we have identified PKC delta as a candidate interacting protein. The second aim is to further characterize Pirh2 regulation of p53. Splenocytes and thymocytes from Pirh2-/- mice demonstrate a subtle increase in total p53 levels after irradiation when compared to wild-type controls. Phosphoserine 15 p53 levels are significantly higher in splenocytes and thymocytes from Pirh2 -/- mice relative to wild-type counterparts. Cells stably transfected with Pirh2 have decreased levels of phosphoserine 15 p53 and decreased induction of p21 relative to vector control and Mdm2 expressing cells. The stability of the p53 protein is primarily regulated through ubiquitin mediated proteolysis, and there are multiple ubiquitin ligases targeting p53 for degradation. Here we are able to address the question of functional redundancy by indicating that Pirh2 can target serine 15 phosphorylated p53 which is reported to not be regulated by Mdm2.

The aggressive nature of pancreatic cancer, characterized by invasiveness, resistance to treatment, rapid progression, and its high prevalence in the population urges the need for developing more effective treatments. Many studies have attributed resistance to therapeutics of pancreatic cancer to activity of the transcription factor nuclear factor kappa B (NF-kB). NF-kB is regulated by the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). GSK-3 is a key mediator of pathways such as insulin, wnt, and PI3K/Akt and has roles in proliferation, glucose metabolism, apoptosis, motility and neuroprotection. Depending on the cellular context, GSK-3 activity can promote or inhibit cell survival. GSK-3 inhibition was recently reported to have anti-cancer effects against pancreatic cancer cells. This effect was in part attributed to suppression of NF-kB. In this thesis, I showed that while blocking GSK-3 disrupts NF-kB, and has anti-survival effects on pancreatic cancer cells, it does not sensitize to the chemotherapeutic drug gemcitabine. NF-kB inhibition by curcumin also resulted in similar effects. These results questions previous reports that NF-kB activation plays a major role in chemo-resistance of pancreatic cancer. The inhibition of NF-kB by genetic disruption of GSK-3 was previously reported to sensitize mouse embryonic fibroblasts and hepatocytes to TNF-alpha cytotoxicity. I therefore tested whether GSK-3 inhibition could sensitize pancreatic cancer cells to apoptosis induced by the clinically applicable member of the TNF-alpha family, TNF-alpha related apoptosis inducing ligand (TRAIL). In contrast to the results obtained with gemcitabine, the combination of genetic or pharmacological inhibition of GSK-3 and TRAIL was found to be highly synergistic in apoptosis induction. Analysis of the apoptotic mechanisms, point towards effects of GSK-3 inhibition on caspase-8 activation, consistent with inhibition of the death receptor signalling pathway. It was found that not only caspase-8 but also mitochondrial anti-apoptotic proteins such as Bcl-XL and Mcl-1 were mediating the TRAIL sensitization. Furthermore, for the first time the in vivo effects of GSK-3 inhibition in combination with TRAIL treatment was investigated. The results indicate a significant enhancement of apoptosis in pancreatic cancer xenografts with minimal toxic effects. Together, these studies provide a rationale for developing combination treatments based on GSK3 inhibition and TRAIL death receptor activation to treat pancreatic cancer.

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

CNS-PNETs are rare, aggressive, paediatric embryonal brain tumours that are poorly studied. We recently identified an aggressive sub-group of CNS-PNETs characterized by the amplification of the C19MC microRNA cluster, however, little is known regarding the features of other CNS-PNET tumours. This study was designed to define additional molecular sub-groups of CNS-PNET by interrogating a large cohort of CNS-PNETs. To elucidate the features of CNS-PNET, we examined transcriptional and copy number profiles from primary hemispheric CNS-PNETs. We then validated and examined the clinical significance of novel sub-group markers in 123 primary CNS-PNETs. This thesis demonstrates that CNS-PNET can be categorized into three molecular sub-groups that are distinguished by distinct primitive neural, oligo-neural and mesenchymal lineage gene expression signatures and also correlated with distinct clinical features. Data from my thesis has generated a substantial body of knowledge to fuel both biological and clinical investigations of childhood CNS-PNETs.

CNS-PNETs are rare, aggressive, paediatric embryonal brain tumours that are poorly studied. We recently identified an aggressive sub-group of CNS-PNETs characterized by the amplification of the C19MC microRNA cluster, however, little is known regarding the features of other CNS-PNET tumours. This study was designed to define additional molecular sub-groups of CNS-PNET by interrogating a large cohort of CNS-PNETs. To elucidate the features of CNS-PNET, we examined transcriptional and copy number profiles from primary hemispheric CNS-PNETs. We then validated and examined the clinical significance of novel sub-group markers in 123 primary CNS-PNETs. This thesis demonstrates that CNS-PNET can be categorized into three molecular sub-groups that are distinguished by distinct primitive neural, oligo-neural and mesenchymal lineage gene expression signatures and also correlated with distinct clinical features. Data from my thesis has generated a substantial body of knowledge to fuel both biological and clinical investigations of childhood CNS-PNETs.

Monoclonal antibodies (mAbs) conjugated to nuclear localization sequences (NLS) and labeled with Auger electron-emitters have great potential for targeted radiotherapy of cancer. This approach may be especially appropriate for the 25-30% of patients with breast cancer whose tumors display overexpression of HER2. Trastuzumab (Herceptin) is a humanized anti-HER2 mAb approved for immunotherapy of HER2-amplified breast cancer. The goal of this research was to radiolabel trastuzumab with [111]In, and to modify it with peptides harboring the NLS (CGYGPKKKRKVGG) of the simian virus 40 large-T antigen for targeted radiotherapy of breast cancer. It was hypothesized that the NLS-peptides would mediate the translocation of covalently linked [111]In-trastuzumab molecules into the nuclei of HER2-overexpressing breast cancer cells where subcellular-range Auger electrons are most damaging to DNA and lethal to cells. Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate for reaction with NLS-peptides and labeled with [111]In using diethylenetriaminepentaacetic acid. The dissociation constant for binding of [111]In-NLS-trastuzumab to HER2-overexpressing SK-BR-3 human breast cancer cells was reduced < 3-fold compared to [111]In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The NLS-peptides did not affect the biodistribution of [111]In-trastuzumab, but promoted its nuclear uptake in HER2-overexpressing MDA-MB-361 xenografts. The cytotoxicity of [111]In-NLS-trastuzumab on breast cancer cells correlated with their HER2 expression. Moreover, [111]In-NLS-trastuzumab was 2-fold and 5-fold more potent at killing MDA-MB-361 and SK-BR-3 cells compared to [111]In-trastuzumab, and nearly 3-fold and 6-fold more effective than unlabeled trastuzumab, respectively. Methotrexate is a known radiosensitizer that can amplify the lethal effects of ionizing radiation on tumor cells. Non-cytotoxic, but radiosensitizing doses of methotrexate were therefore combined with [111]In-NLS-trastuzumab; this enhanced the sensitivity of HER2-overexpressing breast cancer cells to [111]In-NLS-trastuzumab. The blood t1/2 of [111]In-NLS-trastuzumab in non-tumor bearing BALB/c mice was 23-34 h when administered intravenously or intraperitoneally. The maximum tolerated dose was 9.2-18.5 MBq; doses >18.5 MBq caused decreased leukocyte and platelet counts. [111]In-NLS-trastuzumab exhibited strong anti-tumor effects against HER2-overexpressing MDA-MB-361 xenografts, reducing their growth rate 2-fold and 3-fold compared to mice administered [111]In-trastuzumab or unlabeled trastuzumab, respectively. These promising results suggest that [111]In-NLS-trastuzumab may be a useful Auger electron radioimmunotherapeutic agent for HER2-positive breast cancer in humans.