Enhanced resistance to radiation could be caused by both chronic hypoxia and acute hypoxic which has been reported in prostate cancer in various studies. Therefore currently used dose prescriptions (70Gy in 35 fractions) for external beam radiation therapy (EBRT) of prostate cancer has been suggested insufficient to provide optimum clinical outcome. In this study, we propose a Biologically Guided Radiation Therapy approach to boost dose in hypoxic prostate tumor regions while sparing the urethra. A previously proposed hypoxia model was modified for prostate cancer and incorporated into treatment plan optimization. The concept of equivalent uniform dose (EUD) was used in the optimization and evaluation of results. CT data from 25 prostate cancer patients who recently received EBRT at the British Columbia Cancer Agency (BCCA) and hypothetical hypoxic regions manually drawn on these CT scans were selected for this study. The results show that our methods could boost dose in target volume to substantially higher levels. EUD of planning target volume increased to more than 80Gy, despite accounting for effects of hypoxia. This increase was achieved with only minor changes in dose in normal tissues, typically less than 5Gy. Notably, urethra sparing was excellent with a EUD around 64Gy. Robustness of the proposed approach is verified against various hypoxic settings. EUD comparison between RT plans in biological guided and conventional approaches using the same RT technique (Volumetric Modulated Arc Therapy) also suggests that biologically guided radiation therapy (BGRT) approach is more suitable for dose painting purposes with the advantage of delivering sufficient dose to hypoxia region in different scenarios and sparing normal tissue better. Furthermore, we also investigated the impact of inter-fraction patient set-up error and intra-fraction organ motion on the high dose gradients achieved with this proposed dose painting method and explored the feasibility of adapting geometrical uncertainties (represented as systematic error and random error) into treatment planning. Image error obtained from EPID images are used to derive systematic uncertainty and random uncertainty. During the geometrical uncertainty adapted optimization, dose matrix in PTV is shifted based on systematic error and convolved with a Gaussian kernel which is pre-calculated using random error. CT sets and organ contours from five patients who enrolled in the previous dose painting i study are selected. For each of them, seven plans are generated using cumulated uncertainty data which was collected after every five fractions. We also present the outcome in terms of equivalent uniform dose (EUD). For four of the patients, EUD history of all seven plans suggests using the proposed optimization method with uncertainty data from the first five fractions, it is possible to achieve the same target coverage of static treatment plans (difference in EUD less than 1Gy). Meanwhile, the elimination of PTV margin also leads to a significant dose reduction (more than 15Gy) in rectum.

Prostate cancer (PC) is the most common visceral cancer amongst men in Australia and elsewhere in the world. The American Cancer Society estimated that in 2006, PC alone would account for about 33% of newly diagnosed cancer in men, and be responsible for 9% of cancer related deaths in men. In men with advanced, metastatic PC, hormone therapy is widely accepted as the treatment of choice and produces good initial responses in most patients. However, many patients will relapse and become resistant to further hormone manipulation. Currently used treatment modalities for these patients are intended to palliate symptoms and therefore improve quality of life; the long term survival benefit of currently used management strategies is marginal at best. The limited treatment modalities with survival benefit for patients with advanced PC results in the need for development of novel therapies. Enhancement of the natural anti-tumour defences of the immune system to recognise and destroy tumour cells appears as a favourable alternative to prior therapies. Use of autologous dendritic cells (DC) as stimulators of an anti-tumour response has shown promise in several phase I, II and III trials. The Mater Medical Research Institute has developed a novel system for the isolation of blood DC (BDC). This system utilizes an antibody selection system based on a human/mouse chimeric CMRF-56 (huCMRF-56) monoclonal antibody (mAb) which has been engineered from the prototype murine IgG1 CMRF-56 mAb. Pre-clinical studies have demonstrated that the huCMRF-56 mAb isolates a CMRF-56+ cell population comparable to that obtained with the murine IgG1 CMRF-56 mAb with a sufficient CMRF-56+ BDC purity and yield to warrant its use as a BDC based immunotherapy. The objective of this research project was to validate the use of the huCMRF-56 mAb isolated BDC preparations in PC immunotherapy. This was achieved by; i) determining the optimal concentration of the huCMRF-56 mAb at which to perform isolations in order to obtain a CMRF-56+ cellular preparation with purity, yield and cellular composition, in terms of DC subsets, B cells, monocytes and contaminating cells, comparable to that obtained with the murine mAb, which has shown efficacy in in vitro studies; ii) Demonstrating that CMRF-56+ preparations obtained from PC patients using the huCMRF-56 BDC mAb are comparable to those from healthy donors (HD) and iii) demonstrating functional capacity of both freshly isolated and cryopreserved CMRF-56+ BDC isolated preparations to induce anti-tumour cytotoxic T lymphocyte (CTL) responses in both HD and PC patients. These studies utilised the appropriate immunostaining techniques and flow cytometry to determine the CMRF-56+ cell yield, CMRF-56+ BDC purity and viability of the preparation. To determine functional capacity CMRF-56+ preparations were isolated from HD and PC patients, and loaded with the prostate tumour associated antigens (TAA) and control antigen peptides: prostate specific antigen-3 (PSA-3), prostatic acid phosphatase- 5 (PAP-5), prostatic specific membrane antigen-2 (PSMA-2), flu matrix protein (FMP) and/or melanoma antigen recognised by T cells (MART). Co-culture experiments were set up with autologous peripheral blood mononuclear cells (PBMC) and induction of CTL responses were assessed using pentamer staining and ELISPOT assay. The working concentration of the huCMRF-56 mAb of 1.28mg/ml was determined to immunoselect a cellular preparation with maximal BDC purity, BDC yield and total viable cell number. Phenotyping studies of this preparation demonstrated it to be predominately comprised of antigen presenting cells (APC) and enriched for BDC with several BDC subsets immunoselected. CMRF-56+ preparations immunoselected from HD and PC donors were similar and comparable to previously described preparations immunoselected using the original murine CMRF-56 mAb. From antigen loaded CMRF-56+ preparations, specific major histocompatibility complex (MHC) class 1 restricted CD8 lymphocyte responses were generated against prostate TAAs in 2 of 5 HD and 1of 3 PC donors and against FMP in 1 of 3 PC and 5 of 5 HD. Cryopreserved antigen loaded CMRF-56+ BDC preparations from HD generated antigen specific FMP or MART-1 CTL responses in 3 of 4 HD, however anti-prostate TAA CTL responses were not observed. The humanised CMRF-56 mAb immunoselects a cellular preparation enriched in BDC and capable of effective uptake and presentation of antigen. The CMRF-56+ BDC enriched preparation, loaded with PC TAA is capable of inducing specific anti tumour responses in vitro. While further preclinical studies are required, the preparation, loaded with PC TAA shows promise as a potential immunotherapy for PC.

Prostate cancer is the second leading cause of death from cancer in Australian men. As prostate cancer cells are reliant on androgens for growth and survival, the standard therapy for metastatic disease is androgen ablation therapy (AAT). AAT inhibits androgen signalling by altering androgen synthesis or prevent binding of androgens to their intracellular mediator, the androgen receptor (AR). Although initially effective, virtually all patients relapse, beyond which there are limited treatment options. The failure of AAT is not necessarily due to a decreased requirement for androgen signalling, but rather the AR is able to maintain signalling and tumour growth in an androgen-depleted environment. Therefore novel strategies that directly target the AR may provide a more effective therapeutic approach. We have endeavoured to suppress AR activity in prostate cancer cells by utilising a dominant negative AR. The most effective dominant negative construct developed, ARi41O, lacks amino acids 39-410 in the AR amino terminal transactivation domain. In studies of transcriptional activity, ARi410 has no intrinsic activity but inhibits the activity of wild type AR (wtAR) and also clinically relevant AR variants, by up to 95%. The objective of this thesis was to characterise the mechanisms of action of ARi410 and assess the functional effects of introducing this dominant negative receptor into prostate cancer cells. To investigate the mechanism by which ARi410 suppresses AR activity, a robust and sensitive AR inhibition assay was developed. This assay revealed that ARi410 is a potent inhibitor of AR activity on three independent AR-regulated promoters, regardless of the level of AR expression. Furthermore, while ARi410 can inhibit AR activity, it does not alter AR protein levels. By using ARi410 variants with mutations and/or deletions in regions of functional importance, the AR inhibition assay was also used to identify the critical regions of ARi410 required for its dominant negative activity. These studies demonstrate that the dominant negative activity of ARi41 0 is ligand-dependent, requires dimerisation through the ligand binding domain (LBD) and an intact DNA-binding domain (DBD). Further investigation into the mechanism of dominant negative activity revealed that ARi410 does not alter the subcellular localisation of AR, as both receptors are predominantly cytoplasmic in the absence of ligand and rapidly co-localise to the nucleus in response to androgens. Furthermore, an interaction between AR and ARi410 was observed in the presence and absence of ligand, and electrophoretic mobility shift assays demonstrated that AR and ARi410 form heterodimers on DNA. These studies led to the conclusion that the mechanism of dominant negative activity by ARi4I0 involves the formation of inactive receptor heterodimers that assemble on DNA and suppress AR activity. To determine the functional consequence of expressing the dominant negative androgen receptor in prostate cancer cells, an adenoviral method of gene delivery was developed. Adenoviral expression of ARi410 in LNCaP prostate cancer cells did not allow assessment of cell viability due to cell-specific toxicity of the viral vectors when expressed long-term. However, short-term expression of ARi410 in LNCaP cells resulted in inhibition of AR signalling, as determined by reduced expression of the androgen regulated genes apolipoprotein D and kallikrein 2. Importantly, this finding is consistent with the inhibitory activity of ARi410 observed using synthetic AR-regulated reporter genes in the AR inhibition assay, and demonstrates that ARi410 can effectively suppress endogenous AR signalling. The results of this thesis indicate that heterodimerisation between AR and ARi410 is the most likely mechanism of dominant negative inhibition of AR function by ARi410, and that the DBD and dimerisation through the LBD are required for optimal dominant negative activity. Furthermore, this thesis has demonstrated that ARi410 is an effective inhibitor of AR signalling and provides a basis for further functional studies and evaluation of the dominant negative androgen receptor in vitro and in vivo. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1338478 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

In chronic illnesses, such as prostate cancer, multiple health outcomes need to be considered. This project focused on two types of health outcomes: health-related quality of life (HRQoL) and cognitive function. The first aim was to investigate cognitive effects of pharmacological androgen-suppressing treatment. Numerous studies have shown cognitive performance to be associated with sex hormones. One of the main groups of drugs used for hormonal ablation in men with prostate cancer, the luteinising hormone releasing hormone (LHRH) agonists, has been associated with adverse cognitive effects in controlled studies in women and in case reports of female and male patients. However, there have been no published studies on the effect of LHRH agonists and other androgen-suppressing treatments on cognitive functioning in male patients. The second aim was to investigate the effect of treatments on HRQoL in men with prostate cancer. There are few randomised treatment studies of HRQoL in these patients. The third aim was to study additional predictors of HRQoL, examining stress and coping theory as a theoretical basis for understanding individual differences in HRQoL. The fourth aim was to examine patients’ subjective experiences of prostate cancer. To investigate these questions, 82 men with non-localised prostate cancer were randomly assigned to receive leuprorelin (LHRH agonist), goserelin (LHRH agonist), cyproterone (steroidal antiandrogen), or close clinical monitoring. These patients and 20 community volunteers matched for age, marital status, and general health undertook medical, psychosocial, and cognitive assessments before treatment and after 6 and 12 months of treatment. The main question for statistical analysis was whether dependent variables would show Group x Time interactions in the predicted directions. Compared with baseline assessments, men administered androgen suppression monotherapy performed worse in 3/12 tests of attention, memory, and executive function. Twenty-six percent of men randomised to active treatment demonstrated clinically significant decline in one or more cognitive tests at 6 months compared with baseline performance. By contrast, no community volunteers or patients randomised to close monitoring showed decline in test performance. Men on hormonal treatments reported impaired sexual function on treatment compared with baseline assessments. Men assigned to close monitoring and cyproterone treatment reported increased emotional distress over time. Groups did not differ in change in existential satisfaction, subjective cognitive function, physical symptoms, or social and role functioning. For individuals, hormonal treatments were more frequently associated with decreased physical, sexual, social and role functioning, but were also associated with improved HRQoL for some individuals. In hierarchical regression analysis, HRQoL was lower for men who had more comorbid illnesses, a history of neurological dysfunction, higher threat appraisals, or higher use of emotion-focused coping strategies. Coping strategies also showed some longitudinal associations with HRQoL, even when earlier levels of HRQoL had been taken into account. Subjective reports demonstrated that many patients viewed prostate cancer as a relatively manageable problem. Several patients said that other health problems affected them more than prostate cancer, whereas no patient said that prostate cancer was worse than other problems. Comments about the seriousness of prostate cancer were equally divided between patients who reported it as very serious (14.3% of patients) and those who saw it as a relatively minor problem (14.3%). Other patient observations were grouped into categories of personal responses to prostate cancer, health, and health research; life circumstances that were not directly associated with health; attributions about medication; and function prior to the study. The results demonstrated that pharmacological androgen suppression therapy was associated with impaired memory, attention, and executive function in male patients. Hormonal treatments and close monitoring had differential effects on patients’ HRQoL, particularly in terms of sexual function and emotional distress. HRQoL was also associated with appraisal and coping, to a greater extent than it was associated with medical variables, supporting the applicability of stress and coping theory for these patients. Observations from participants placed these findings in the context of participants’ concerns, demonstrating that issues such as cognitive and sexual function were relevant for these patients. These findings suggest that cognitive function should be given increased attention as a health outcome, not only in “neurological” disorders but also in other “non-neurological” conditions such as prostate cancer. They also support continued efforts to understand both beneficial and adverse effects of treatments for chronic illness on HRQoL and individual factors that affect health outcomes.

Prostate cancer is a frequently diagnosed malignancy which ranges from an indolent asymptomatic tumour to an aggressive, rapidly lethal systemic disease. Determination of chromosomal, genetic and epigenetic alterations associated with prostate carcinogenesis have led to the characterisation of functional consequences of these alterations, thereby elucidating pathways contributing to malignant growth that can be utilised clinically and therapeutically. FLJ22318 is a novel hypothetical protein that was identified by yeast two-hybrid analysis to interact with the prostatic homeodomain protein, NKX3-1. Expression of NKX3-1 is largely restricted to epithelial cells of the adult prostate where it is involved in maintaining the prostatic phenotype, while NKX3-1 expression is reduced or absent in prostate tumours. In contrast, FLJ22318 expression is documented in cDNA libraries derived from a variety of human adult and foetal tissues including the prostate, suggesting that it may be ubiquitously expressed and that it potentially interacts with a number of proteins in addition to NKX3-1. FLJ22318 expression is undocumented in human prostate tumours. Bioinformatic analyses have postulated multiple FLJ22318 mRNA transcripts however the proposed open reading frames encoded by these transcripts predict the FLJ22318 protein to contain three strong protein-protein interaction domains, a Lissencephaly type-1-like homology (LisH), a C-terminal to LisH (CTLH) and a CT11-RanBPM (CRA) domain. Of the 44 single nucleotide polymorphisms identified within the FLJ22318 gene, none are located within the protein coding region suggesting that FLJ22318 may be critical for cell survival and/or function. Comparison of the amino acid sequence between human FLJ22318 and its orthologues in a diverse range of mammalian species identified >97% sequence homology, providing further strong evidence of the critical cellular function of FLJ22318. To characterise the biological activity of FLJ22318 in prostate cancer cells, the FLJ22318 coding region was amplified by polymerase chain reaction (PCR) and ligated into mammalian and bacterial expression vectors to encode V5-, myc-, GFP-, HA-, and GST-FLJ22318 fusion proteins. Interaction between FLJ22318 and NKX3-1 was confirmed using (reverse) yeast two-hybrid, GST pull-down and co-immunoprecipitation assays. These data were supported by confocal microscopy which demonstrated the perinuclear and nuclear co-localisation of FLJ22318 and NKX3-1 in prostate cancer cells. Northern blotting identified expression of ~2Kb and ~4Kb FLJ22318 mRNA’s in prostate cancer cell lines, which was consistent with bioinformatic analyses of mRNA species. Transfection of prostate cancer cells to overexpress FLJ22318 did not alter endogenous NKX3-1 levels, however FLJ22318 exhibited transcriptional repressor function on an NKX3-1 responsive element and increased NKX3-1 transcriptional repressor activity on this element. To further investigate FLJ22318 function, additional yeast two-hybrid analyses were performed in prostate cancer cells to identify potential FLJ22318 binding proteins. These studies isolated cDNA’s encoding 33 different proteins involved in cell metabolism and apoptosis as well as transcriptional regulators associated with control of cellular proliferation. One of the candidate FLJ22318 interactors, protein kinase, interferon-inducible double stranded RNA dependent activator (PRKRA/PACT) was shown using confocal microscopy to extensively co-localise with FLJ22318 in the cytoplasm and perinuclear regions of prostate cancer cells. Preliminary co-immunoprecipitation and GST pull-down assays have provided additional evidence of the interaction of PRKRA and FLJ22318. Results of this thesis have generated important information characterising the structure of the FLJ22318 gene and protein, the interaction between FLJ22318 and NKX3-1 and the potential functions of FLJ22318 in prostate cancer cells. As the FLJ22318 gene is located on 5q35, a chromosomal region frequently disrupted in a variety of tumours, future studies of the biological activity of FLJ22318 will clarify its normal cellular functions and its contribution to tumorigenesis or malignant progression in the prostate and in other tissues.

Thesis (Ph.D.)--Edith Cowan University, 2006. / Submitted to the Faculty of Communications, Health and Science. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M.S.)--University of Delaware, 2008. / Principal faculty advisor: Carlton R. Cooper, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph.D.)--University of Western Australia, 2005.

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. Includes bibliographical references: leaf 239-252.