Prostate cancer is the most common non-skin cancer in America, and the most commonly diagnosed cancer among males. When metastatic, the disease can ultimately be incurable. Consequently, alternative strategies to current treatments are sought, especially in the area of immunotherapy. Vaccine immunotherapy using a specific antigen, such as prostate specific antigen (PSA) seeks to stimulate both the innate and adaptive immune system to destroy tumor cells in the body. PSA is an ideal target antigen given that it has a narrow distribution in tissues and is expressed in virtually all prostate cancer cases. An adenovirus encoding for PSA (Ad-PSA) can be used to deliver the genomic data encoding for PSA production and secretion to the target cell. This type of viral gene delivery system has already been shown to have the potential to stimulate anti-tumor activity. To enhance this activity and increase transfection efficiency, we proposed the combination of a viral system with a non-viral system, in the form of a cationic polymer such as poly(ethyl)enimine (PEI) or chitosan. Cationic polymers complex with the negatively charged adenovirus to form nanoparticles that can be used in gene delivery. Delivery in nanoparticle form can give enhanced uptake by the antigen-presenting cells necessary to initiate the targeted immune response. To further augment this response, previous research has shown that CpG sequences act as an adjuvant to enhance the efficacy of the Ad-PSA vaccines' tumor protection. CpG delivered in particulate form has also been shown to be more effective than delivery in solution. The objective of this proposal was to test the hypothesis that co-delivery of this targeted viral/non-viral gene delivery system will enhance tumor protection in a mouse model of prostate cancer. Using the OVA model antigen system, we found that the adenovirus encoding OVA (AdOVA), coupled with the polymer PEI, enhanced tumor protection in vivo compared to AdOVA alone. To move towards our therapeutic model, these experiments were repeated using chitosan as the cationic polymer carrier, delivering AdOVA, and incorporating CpG into some particles. In this set of experiments, we found that AdOVA + CpG gave the best tumor protection in challenge studies. AdOVA + chitosan + CpG showed a decrease in protective levels and numbers of antigen-specific immune cells. Further experiments focused on elucidating the mechanisms by which chitosan and CpG modulate the immune response. Using the therapeutic AdPSA model, chitosan was not found to enhance tumor protection or numbers of antigen-specific immune cells. Additional experiments found that this depression was not due to problems with viral infectivity or secretion due to chitosan complexation. A series of kinetics studies were performed which showed that peak levels of effector T cells were present 14 days later in AdPSA + CpG immunized mice than in AdPSA alone. This delayed effect may explain the increased levels of protection in AdPSA + CpG mice, and be useful in future vaccine design concerning the timing of peak response.

The interplay between cancer cells and the extracellular matrix (ECM) remains a critical regulator of both normal tissue organization and cancer cell invasion. Proteins that function as ECM receptors function to link the cell with the ECM. Abberations in either the structure of the ECM or the expression of ECM receptors leads to disrupted interaction and downstream signaling effects. Dystroglyan (DG) is an ECM receptor that is expressed in a variety of tissue types and functions to mediate sarcolemma stability, epithelial polarity, and is critical in the early formation of basement membranes. However, DG has primarily been studied in muscle where loss of its function is linked to a host of muscular dystrophies. In the epithelium, the role of DG remains enigmatic. While DG has repeatedly been shown to lose function during cancer development and progression, the mechanism and functional consequence of its loss are currently unknown. In order to increase our understanding of DG in cancer development, we analyzed its expression and glycosylation, a functional requirement for DG, in a range of prostate cancer cell lines. Previous work has shown DG to be downregulated in prostate cancer, but the mechanism by which this occurs has remained largely unclear. We found that DG expression is maintained while its glycosylation was heterogeneous in the cell lines. Further investigation revealed that lines with hypoglycosylated DG strongly associated with the loss of expression of the glycosyltransferase LARGE2. Further this enzyme is frequently downregulated in human cancers and appears to serve as a required enzyme in DG glycosylation within prostate epithelium. This is the first work to demonstrate the functional requirement of LARGE2 for DG, and the only work to implicate loss-of-function of LARGE2 in cancer progression. To determine whether loss of LARGE2 is found in other tumor types, we analyzed human clear cell renal cell carcinoma (ccRCC) samples by immunohistochemistry and via in silico analysis with the Cancer Genome Atlas (TCGA). Our work demonstrated a frequent and significant downregulation of LARGE2 expression and its association with DG hypoglycosylation. Additionally, we found the loss of LARGE2 strongly associated with increased mortality. Thus, we again demonstrated a functional requirement of LARGE2 but also found a clinical correlate with increased mortality. Finally, we examined the functional outcome of DG hypoglycosylation or loss of expression in both a mouse model of prostate cancer and a variety of cell lines models. We found that while loss of DG expression does not increase prostate cancer growth or metastasis in one model of cancer, loss of its glycosylation does seem to mediate downstream metabolic changes within cells. The mechanism for this change remains unclear. In summary, these studies have contributed to our understanding of DG glycosylation and function in both prostate and renal carcinoma. Additionally, we have shown a novel mechanism by which DG glycosylation is lost with downregulation of LARGE2 expression. Finally, while we were unable to demonstrate a clear mechanism by which signaling changes arose, we were able to demonstrate a strong correlation between DG hypoglycosylation and increased mortality in ccRCC. These insights could be used to improve treatment of multiple cancer types as our understanding of DG function continues to improve.

C-Terminal glycine-extended prohormones are enzymatically converted to á-amidated peptides, by peptidylglycine á-amidating monooxygenase (PAM). PAM is a bifunctional enzyme with two catalytic domains: peptidylglycine á-hydroxylating monooxygenase (PHM) and peptidylglycine peptidylglycineaminoglycolate lyase (PAL). PAM has a significant role in the proliferation of androgen-independent prostate cancer. Thus, the inhibition of PAM could halt cancer growth. Hippurate and hippurate analogs were used as lead compounds for developing inhibitors for PAM. The hippurate analogs exhibiting the highest affinity to PAM (lowest inhibition constant) did inhibit the growth of human androgen-independent prostate cancer DU 145 cells.

External beam radiation therapy for prostate cancer can result in urinary, sexual, and rectal side effects, often impairing quality of life. A polyethylene glycol-based product, SpaceOAR hydrogel (SOH), implanted into the connective tissue between prostate gland and rectum can significantly reduce the dose received by the rectum and hence risk of rectal toxicity. The optimal way to manage the hydrogel and rectal structures for plan optimization is therefore of interest. A retrospective planning study was completed with 13 patients to examine optimal planning and treatment methods. Computerized tomography (CT) scans were taken pre- and post-SOH implant. Six hypofractionated (60 Gy in 20 fractions) treatment plans were produced per patient using either a structure of rectum plus the hydrogel, termed composite rectum wall (CRW), or rectal wall (RW) as the inverse optimization structure and intensity modulated radiotherapy (IMRT) or volumetric modulated arc therapy (VMAT) as the treatment technique. Dose-volume histogram metrics were compared between plans to determine which optimization structure and treatment technique offered the maximum rectal dose sparing. RW structures offered a statistically significant decrease in rectal dose over CRW structures, whereas the treatment technique (IMRT vs VMAT) did not significantly affect the rectal dose. However, there was improvement seen in bladder and penile bulb dose when VMAT was used as a treatment technique over IMRT. Overall, treatment plans using the RW optimization structure offered the lowest rectal dose while VMAT treatment technique offered the lowest bladder and penile bulb dose. These treatment techniques and optimization structures have now been implemented at BC Cancer - Victoria based on this retrospective study. SOH implant has been shown not to be equally effective in all patients. Determining a priori patients in which the implant will offer most benefit allows for effective management of SOH resources. Several factors have been shown to be correlated to reduction in rectal dose including distance between rectum and planning treatment volume (PTV), volume of rectum in the PTV and change in rectum volume pre- to post-SOH. Several of these factors along with other pre-SOH CT metrics were found via multiple linear regression models to predict reduction of rectal dose using data from 21 patients who received SOH implant. Two high rectal dose metrics were modeled, change in the relative volume receiving 55 Gy and change in the partial high dose integral, integrating over the dose-volume histogram (DVH) from 55 Gy to 60 Gy. Models were also produced to predict pre-SOH RV55Gy. These models offered R-squared between 0.57 and 0.87 with statistical significance in each model. Applying a 3.5% lower limit on pre-SOH RV55Gy removed one third of patients as implant candidates. This may offer a clinically useful tool in deciding which patients should receive SOH implant given limited resources. Predictive models, nomograms and a workflow diagram were produced for clinical management of SOH implant. / Graduate

Philosophiae Doctor - PhD / 2018, the International Agency for Research on Cancer (IARC) estimated that prostate cancer (PCa) was the second leading cause of death in males worldwide. The number of deaths are expected to raise by 50 % in the next decade. This rise is attributed to the shortcomings of the current diagnostic, prognostic, and therapeutic biomarkers used in the management of the disease. Therefore, research into more sensitive, specific and effective biomarkers is a requirement. The use of biomarkers in PCa diagnosis and management takes advantage of the genetic alterations and abnormalities that characterise the disease. In this regard, a microRNA, hsa-miR-5698 was identified in a previous study as a differentiating biomarker between prostate adenocarcinoma and bone metastasis. Six putative translational targets (CDKN1A, CTNND1, FOXC1, LRP8, ELK1 and BIRC2) of this microRNA were discovered using in silico approaches. The aim of this study was to analyse via expression profiling and characterization, the target genes of hsa-miR-5698 in order to determine their ability to act as putative dynamic network biomarkers for PCa. The study was conducted using a combined in silico and molecular approach. The in silico part of the study investigated the putative transcriptional effects of hsa-miR-5698 on the promotors of its translational targets, the correlation between hsa-miR-5698 and mRNA expression profiles as well as the co-expression analysis, pathway analysis and prognostic ability of the target genes. A number of computational software were employed for these purposes, including, R Studio, Trident algorithm, STRING, KEGG, MEME Suite, SurvExpress and ProGgene. The molecular part of the study involved expression profiling of the genes in two PCa cell line LNCaP and PC3 via qPCR.

This thesis describes experimental work performed (1998-2001) during the author's involvement with the Brachytherapy group at the Peter MacCallum Cancer Centre (PMCC), where he was employed by its Department of Physical Sciences and subsequent modeling and analytical studies. When PMCC added HDR brachytherapy to its radiation therapy practice, an existing operating suite was considered the ideal location for such procedures to be carried out. The integration of brachytherapy into the theatre environment was considered logical due to the relatively invasive nature of brachytherapy techniques and the availability of medical equipment. This thesis contains the detailed study of three key Research Questions involved in clinical aspects relating to quality assurance of an HDR brachytherapy practice. An investigative chapter is dedicated to the pursuit of each of the Research Questions. The first question asked… Is the novel approach to using modular shielding combined with time and distance constraints adequately optimized during HDR brachytherapy? In order to establish optimal clinical practices, this project evaluates the effectiveness of additional shielding added to the modular shielding system without modification of the previously determined time and distance constraints for PMCC staff, other patients, and member of the public. The DOSXYZnrc user code for the EGSnrc Monte Carlo radiation transport code has been used to model exposure pathways to strategic locations used for measurement in and around the operating theatre suite. Modeling allowed exposure pathways to various areas with the facility to be tested without the need to use real sources. The second Research Question asked… How well is dose anisotropy characterized in the near field range of the clinic's HDR 192Ir source? This study experimentally investigated the anisotropy of dose around a 192Ir HDR source in a water phantom using MOSFETs as relative dosimeters. In addition, modeling using the DOSRZnrc user code for the EGSnrc Monte Carlo radiation transport code was performed to provide a complete dose distribution consistent with the MOSFET measurements. Measurements performed for radial distances from 5 to 30 mm extend the range of measurements to 5 mm which has not been previously reported for this source construction. The third Research Question is aimed at the patient level. Is the dose delivered to in vivo dosimeters, located within critical anatomical structures near the prostate, within acceptable clinical tolerance for a large group of HDR prostate patients? An in vivo dosimetry technique employing TLDs to experimentally measure doses delivered to the urethra and rectum during HDR prostate brachytherapy was investigated. Urethral and rectal in vivo measurements for 56 patients have been performed in the initial fraction of four-fraction brachytherapy boost. In the absence of comparable in vivo data, the following local corrective action level was initially proposed: more than 50% of the prostatic urethra receiving a dose 10% beyond the urethral tolerance. The level for investigative action is considered from the analyses of dose differences between measured data and TPS calculation.

Le cancer de la prostate est la seconde cause de mortalité par cancer chez l'homme. Actuellement, les traitements hormonaux visent à diminuer le taux d'androgènes actifs. Malheureusement, avec le temps, les patients développent un cancer androgènes dont l'issue est fatale. Le calcium (Ca2+), second messager ubiquitaire, est impliqué dans de nombreux processus tels que l'apoptose ou la prolifération. Le Réticulum Endoplasmique (RE) est un acteur essentiel de la signalisation calcique. Ainsi, l'étude de canaux calciques réticulaires est fondamentale dans le développement de nouvelles stratégies thérapeutiques. Les travaux effectués ont permis d'identifier deux nouvelles protéines sur le RE : le translocon et le canal TRPM8 (Transient Receptor Potential Melastatin 8). Ces deux protéines seraient des éléments majeurs de la signalisation calcique en régulant la concentration de Ca2+ du RE. Par ailleurs, l'étude de l'évolution de la signature calcique au cours de la cancérogenèse prostatique a permis de mettre en évidence qu'Orai1 (identifié ici comme étant la protéine responsable de l'Entrée Capacitive de Ca2+) est moins exprimée dans les cancers les plus agressifs. A l'opposé, TRPV6 (Transient receptor potential Vanilloid 6), canal calcique impliqué dans l'entrée constitutive de Ca2+, est surexprimé dans des stades avancés de cancer. Ainsi, les variations d'expression de ces protéines seraient responsables respectivement d'un défaut d'apoptose ou d'une augmentation de la prolifération des cellules cancéreuses prostatiques androgéno-indépendantes. Ceci permettrait d'expliquer l'évolution du cancer de la prostate vers des stades plus agressifs.

Le principal objectif de cette thèse est la conception et la production d'outils à destination des radiologues pour la délinéation des organes à risque dans le cadre du traitement par radiothérapie du cancer de la prostate. Les images passées entrées sont des images CT. Elles sont d'abord placées dans un repère commun à l'aide d'un recalage log-euclidien concentré sur les structures osseuses du pubis. Une suite progressive de traitements est ensuite appliquée: dans un premier temps, la vessie est segmentée, puis la prostate est ensuite localisée paralellement à la vessie, pour finir avec l'intégration de la délinéation du rectum. Compte tenu de l'hétérogénéité des images de la base de données sur laquelle nous avons travaillé, notre contribution principale est la flexibilité. La vessie est une structure à forte variabilité en termes de forme et d'intensité, notamment à cause du degré de remplissage et la présence ou l'absence d'un produit de contraste. La méthode proposée s'adapte non seulement aux formes très différentes des vessies de notre base de donnée, mais aussi au degré de replissage donnant lieu, dans le cas ou un produit de contraste a été administré, une h'etérogénéité notable dans la structure à segmenter. Le contraste de la prostate avec les tissus environnants est quasi-nul; son interface avec la vessie est souvent très difficile à distinguer, même par les experts médicaux. L'incorporation d'informations anatomiques sur la forme et d'informations images, couplée à une nouvelle contrainte d'interaction entre deux maillages, permet d'obtenir une bonne segmentation de la prostate et d'éliminer les ambiguités au niveau de l'interface entre les deux structures. L'incorportation du rectum est l'étape la plus délicate: les différences entre les protocoles d'acquisition de la base de données utilisée interdisent toute modélisation de l'intérieur du rectum: présence de matières fécales, insuflation d'air, présence d'un produit de contraste, présence d'une sonde etc. Les hypotheses faites sur les tissus connexes au rectum ainsi qu'une nouvelle contrainte tubulaire couplée a une pré-segmentation du squelette du rectum permettent d'obtenir un résultat probant. La chaîne de traitement qui a conduit a l'élaboration de cette thèse est en cours d'incorporation dans le logiciel Isogray produit par DOSIsoft, ce qui permet une validation plus approfondie dans des conditions cliniques.

<p> In the work of this doctoral dissertation a new resonator sensor technique, first presented in 1989, has been further developed and evaluated with focus on technical characteristics and applications within the medical field.</p><p> In a first part a catheter-type tactile sensor using the resonator sensor technique was evaluated in a silicone model and applied to human prostate in vitro. The main finding was that different histological compositions of prostate tissue correlated with the frequency shift, .fS, of the resonator sensor and that the common property was the hardness of the tissue. The results indicated that hardness of the prostate tissue, and maybe hardness of human tissue in general, can be expressed according to a cone penetration standard (DIN ISO 2137) and that the hardness can be measured with this tactile sensor system. The tissue hardness application for the resonator sensor technique has to be further developed and evaluated in a larger study. The study also produced results that has led to the basic understanding of the resonator sensor system. One important result was that .fS of the sensor system was related to the contact area between sensor and sample. This indicated that the resonance sensor could be used for contact area measurement.</p><p> In a second part, containing three studies, the area-sensing capability from the first study was utilised in the development and evaluation of the applanation resonator sensor (ARS) for measurement of intraocular pressure (IOP). For the purpose of evaluating IOP-tonometers, an in vitro pig-eye model was developed, and it was shown that a saline column connected to the vitreous chamber could be used successfully to induce variations in IOP.</p><p> A ARS sensor with a flat contact surface was applied onto the cornea with constant force and .fS was measured. A mathematical model based on the Imbert-Fick law and the assumption that .fS was linearly related to contact area was proposed and verified with a convincing result. IOP measured with the ARS correlated well (r=0.92, n=360) with the IOP elicited by a saline column.</p><p> The ARS in a constant-force arrangement was evaluated on healthy human subjects in vivo. The results verified the sensor principle but revealed a nonnegligible source of error in off-centre positioning between the sensor and cornea. The sensor probe was redesigned and evaluated in the in vitro model. The new probe, with a spherical contact surface against the eye reduced the sensitivity to off-centre positioning. It was also shown that a .fS normalisation procedure could reduce the between-eye differences.</p><p> The ARS method for IOP measurement was further developed using combined continuous force and area measurement during the dynamic phase when the sensor initially contacts the cornea. A force sensor was included with the resonator sensor in one probe. Evaluation was performed with the in vitro pig-eye model. The hypothesis was that the IOP could be deduced from the differential change of force and area during that phase. The study showed good accuracy and good reproducibility with a correlation of r=0.994 (n=414) between measured pressure in the vitreous chamber and IOP according to the ARS. Measurement time was short, 77 ms after initial contact. Problems with inter-eye differences and low resolution at high pressures were reduced. The ARS method is the first to combine simultaneous, continuous sampling of both parameters included in the applanation principle. Consequently, there is a potential for reducing errors in the clinical IOP tonometry. </p>

<p>The aims of the thesis were to</p><p>• explore whether radical prostatectomy is beneficial compared with watchful waiting in survival and disease progression</p><p>• find possible effect modifiers</p><p>• evaluate a protocol of multiple biopsies and investigate if men with previous benign prostate biopsies are a group at risk for later prostate cancer</p><p>• inquire into patients’ and clinicians’ experiences of randomization in order to find out what made this study possible to conduct, and thereby contribute to improve randomization in the future</p><p>The background material was a large randomized clinical trial, the Scandinavian Prostatic Cancer Group Study Number 4, or SPCG-4, which was open for inclusion from February 1989 through December 1999. It comprised 695 men in Sweden, Finland and Iceland who had localized prostate cancer and were randomized to either radical prostatectomy or watchful waiting. </p><p>After a mean follow-up time of 6.2 years the first analyses, according to intention-to-treat, showed that radical prostatectomy reduced disease specific mortality, risk of metastases and risk of local progression but did not statistically significantly reduce overall mortality. </p><p>The second analyses confirmed our earlier findings and furthermore, at ten years, radical prostatectomy also statistically significantly reduced overall mortality. Age appeared as an independent effect modifier that will be further investigated.</p><p>A total of 547 men, with a suspicion of prostate cancer that had undergone multiple biopsies, and whose biopsies had benign histology were later compared with the background population to evaluate whether they were a group at risk of developing prostate cancer. Within six years of follow-up, there was no increased risk of prostate cancer.</p><p>Patients as well as clinicians used individual strategies to cope with the situation. The randomizing clinician has to understand the patient’s strategy and his expectations in order to individualize the information accordingly.</p>