Surgery (Austin & Northern Health) - Theses
Now showing items 1-12 of 34
Protection of the kidney against Ischaemia-Reperfusion injury using zinc
Acute kidney injury (AKI) continues to be a major cause of morbidity and mortality worldwide. Septic shock, hypovolaemia, and renal ischaemia related to major surgeries are the primary contributors to AKI in hospitalised patients. AKI is associated with a four-fold increase in mortality in hospitalised patients, and a two-fold increase in the likelihood of discharge to a short- or long-term care facility. As a result, the estimated healthcare costs associated with AKI in hospitalised patients in the US alone exceeds US$10billion per year. Studies have also demonstrated that AKI resulting from ischaemia-reperfusion (IR) is a causative determinant in the development, and progression, of chronic kidney disease (CKD). Despite major medical advances to the current day, short of supportive measures there is still no definitive therapeutic option available to prevent AKI in these settings. Preconditioning (PC) against renal IR injury has been heralded as a promising solution to abrogate this major healthcare problem, and an extensive volume of research has amassed in this area. Preconditioning is a phenomenon whereby an innate tissue adaptation occurs in response to a sublethal stimulus, which leads to protection of an organ or tissue against a subsequent insult. PC was first discovered in the context of ischaemic PC (IPC), where brief sublethal periods of ischaemia led to protection against a subsequent more sustained period of ischaemia in the canine heart. Since the discovery of the IPC phenomenon in 1986, tissue protection against ischaemia by means of IPC has been demonstrated by a number of methods in a variety of tissues, including the heart, brain, liver, kidney, and striated smooth muscle. The promise of these findings has also prompted research into the use of alternative methods of tissue PC, and studies have since investigated the use of pharmaceutical agents to promote these tissue adaptations by pharmacological preconditioning (PPC). The race for a pharmaceutical agent capable of eliciting protective adaptations against tissue ischaemia has involved many classes of pharmaceutical compounds, endogenous proteins, and trace elements. Zinc (Zn) is a metal that is essential to many biological functions, including cell growth and survival. The omnipresence of Zn in cellular interactions, and its importance in so many biological processes has led to the investigation of augmenting Zn homeostasis as a means of protection against tissue ischaemia. This is the primary topic of this thesis. The promise of enabling organ protection against IR injury has major clinical implications, spanning many areas of medicine. However, despite the extensive volume of clinical and basic science research in this area, there is still currently no effective method of PC that will protect the human kidney against IR injury. The aims of this thesis are to investigate if parenteral Zn can be used as a therapeutic strategy to protect the kidney against IR injury. The body of research on the topic of tissue PC has highlighted some potential targets for PC agents, however to date a definitive mechanism has not been elucidated, and therefore a further aim of this thesis is to investigate the potential mechanisms through which Zn effects renal tubular and glomerular epithelial cell survival in the setting of renal IR.
The oncological outcomes of dose escalated radiotherapy and its impact on biochemical control and toxicity in men with prostate cancers
Introduction: Radiation therapy (RT) for prostate cancer (PC) has steadily evolved over many years, with improvement in biochemical relapse free survival (bRFS). An association between overall survival and doses greater than or equal to 75.6 Gray in men with intermediate and high-risk PC has been reported in population-based studies. Contemporary RT techniques such as image guided radiotherapy, intensity modulated radiotherapy, and stereotactic body radiotherapy, has facilitate further dose escalation. Brachytherapy is an internal form of RT that also developed substantially and can be delivered in combination with external beam radiation therapy (EBRT). However, dose escalation can come with increased gastrointestinal (GI) toxicity and new devices such as rectum spacers have been developed to spare this critical normal structure. Methods: Our large prospective brachytherapy database, that I created, which included patients treated with low dose rate (LDR) and high dose rate (HDR) brachytherapy was interrogated to determine the long-term oncological outcomes. In addition, I was one of the first radiation oncologists in Australia to use a novel polyethylene glycol hydrogel rectal spacer and its iodinated counterpart. We were able to implement its use as a fiducial marker in the post-prostatectomy setting and its use as a tissue expander in the intact prostate for EBRT with or without high dose rate brachytherapy as well as in the post-prostatectomy setting. Results: I found that the use of LDR and HDR brachytherapy with or without EBRT to be safe and efficacious. The bRFS for LDR brachytherapy alone for low to intermediate risk PC was excellent as was its use in combination with EBRT for men with predominantly unfavorable intermediate risk PC. In addition, the use of HDR brachytherapy in combination with EBRT for men with intermediate and high-risk PC also yielded excellent bRFS comparable to any other series reported in the literature. I successfully introduced the use of hydrogel spacers into our practice with marked reduction in rectal volumes irradiated to high radiation doses which allowed appropriate dose escalation of EBRT with or without HDR brachytherapy. This has translated to a marked reduction in late GI toxicity. In addition, we also successfully used hydrogel spacers in the post-prostatectomy setting both as a spacer to allow for ultra-high dose radiation therapy and as a fiducial marker with hydrogel spacer in its iodinated form. Conclusion: Although the use of brachytherapy has declined in the last few years, our results confirm its outstanding efficacy in PC and as such we will continue to advocate for its use. We will continue to support a brachytherapy unit for the treatment of PC. In addition, my work on hydrogel spacers has resulted in its use as standard practice in all PC patients who require EBRT.
Development of a cell-free DNA methodology to assess organ rejection after liver transplantation
Background: Liver transplantation has revolutionised the prognosis of patients with fulminant liver failure, chronic liver disease, and liver cancer. Although liver transplantation is safe, organ rejection is a common complication after such a procedure. The gold-standard for diagnosing organ rejection after liver transplantation is a tissue biopsy. Liver biopsies are invasive. There is thus an unmet clinical need for accurate blood tests to diagnose the episodes of organ rejection after liver transplantation. Donor-specific cell-free DNA (dscfDNA) is an emerging biomarker of organ rejection. Measuring dscfDNA using current methodologies such as next generation sequencing can be both complex and expensive. Novel tests that overcome these limitations would favour adoption of such methodologies for the quantification of dscfDNA and implementation for the surveillance of organ rejection after transplantation. Objectives: The first objective of this thesis was to develop a cell-free DNA based assay for the accurate quantification of dscfDNA that could overcome some of the limitations of existing methodologies. The second objective of this thesis was to deploy this assay to monitor episodes of organ rejection in a prospective cohort of recipients. Main findings: A probe-free droplet digital PCR-based methodology was developed. The methodology overcame some of the common limitations that were observed in next generation sequencing-based and other PCR-based methodologies. The newly developed approach was accurate, economical, and rapid which facilitated the rapid turnaround of results as well as enabled early clinical decision-making (Chapters 3 and 4). The application of this approach to measure dscfDNA was shown to be feasible for the monitoring of dscfDNA in a prospective cohort of forty recipients after liver transplantation (Chapter 5). The levels of dscfDNA were reflective of organ health. Furthermore, a calculated threshold of 898 copies of dscfDNA per mL of recipient plasma identified majority of the recipients with biopsy-proven acute rejection requiring treatment. The diagnostic performance of dscfDNA, in this cohort, was superior compared to routine liver function tests in identifying organ rejection. Conclusion: This thesis presented the application of a novel cfDNA methodology to measure dscfDNA in a prospective cohort of recipients after liver transplantation. The results demonstrated the promising utility of dscfDNA as a marker of organ rejection after liver transplantation. These pertinent findings warrant further validation with a view towards clinical implementation.
Targeting p21-activated kinases in the treatment of pancreatic cancer
Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies worldwide, with a very poor prognosis and a 5-year survival rate less than 9%. This dismal outcome is largely due to lack of early diagnosis, quick disease progression, high rate of post-surgery recurrence and resistance to conventional therapies. Oncogenic Kras mutation is a well-defined hallmark of pancreatic cancer. It is presented in over 95% cases and leads to constitutively active form of Kras protein. Kras still remains as an undruggable target due to absence of a well-defined drug-binding domain. With the aim to fight against Ras-driven cancers, high priority has been given to the novel therapeutic strategies targeting Ras-dependent signalling. P21-activated kinases (PAKs) are a family of serine/threonine kinases that are important down-stream effectors of multiple small GTPases including Ras, Rac1 and Cdc42. Based on the difference in the structure and sequence, all the six members of PAK family are divided into two groups: group I (PAK1-3) and group II (PAK4-6). PAK1 and PAK4 are the most widely studied members and have been reported to be up-regulated in PDA. PAK1 is situated at the convergence of multiple signalling pathways that are associated with cell proliferation, survival/apoptosis, migration/invasion and cytoskeletal regulation. Immunotherapy is now emerging as a promising treatment in the era of personalised anti-cancer therapeutics. However, it can only bring limited clinical benefits for PDA patients, which is largely attributed to the immunosuppressive tumour microenvironment (TME). The role of PAK1 has not been fully elucidated in pancreatic cancer, especially its involvement in re-programming TME. The work presented in this thesis investigated the role of PAK1 in tumour biology and therapeutic regimens, with a focus on its linkage to stroma modulation and anti-tumour immune response. Firstly, the anti-tumour effect of a potent PAK inhibitor (PF-3758309) was determined on a panel of clinical patient-derived PDA cell lines (TKCC 2.1, TKCC15, TKCC18, TKCC22, TKCC23, TKCC26). PF-3758309 treatment inhibited cell proliferation and sensitized PDA cells to different chemotherapies (fluorouracil, gemcitabine and nab-paclitaxel) with a synergistic effect, which was associated with reduction in PAK1 and PAK4 activity and down-regulation of HIF-1α, α-SMA and palladin in vitro. Combination of PF-3758309 and gemcitabine maximally suppressed tumour growth in vivo and had a comparable or even greater therapeutic efficacy compared to combination of gemcitabine and nab-paclitaxel. As mentioned above, the expression of α-SMA was observed in PDA cells. All-trans retinoid acid (ATRA), a well-known compound that induced quiescence of pancreatic stellate cell (PSC) by decreasing the expression of α-SMA, was utilized to investigate its anti-tumour effect and association with PAK protein and α-SMA in PDA cells. Inhibitory effect of ATRA on PDA cell growth and migration and its synergism with gemcitabine was observed in both wildtype and gemcitabine-resistant PDA cell lines. Expression of PAK1, PAK2, PAK4 and α-SMA was down-regulated by ATRA. Inhibition of PAK1 by shRNA knockdown or PF-3758309 sensitized PDA cells to ATRA. This was the first study to demonstrate the role of PAK proteins in ATRA treatment. The role of PAK1 in tumour immune response was evaluated using an orthotopic mouse model of pancreatic cancer. Inhibition of PAK1 by PF-3758309 or genetic knockout up-regulated intra-tumoural infiltration of CD3+ lymphocytes and splenic CD3+ or CD8+ lymphocytes. Combination of PF-3758309 and gemcitabine synergistically inhibited PDA cell growth in vitro and in vivo. This study not only confirmed the anti-tumour effect of PF-3758309 and its synergism with gemcitabine, but also revealed the potential role of PAK1 in anti-tumour immunity. Finally, the underlying mechanisms of PAK1 in regulating anti-tumour immunity was investigated. Immunohistochemistry was performed on human tissue microarray and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mice samples. PAK1 was identified to be a negative prognostic marker and positively correlated with α-SMA expression. Depletion of PAK1 decreased PSC activity, reduced PSC-stimulated PDA cell proliferation and migration and increased intra-tumoural infiltration of CD4+ or CD8+ lymphocytes. Inhibition of PAK1 decreased both intrinsic and PSC-stimulated PD-L1 expression in PDA cells, which could enhance lymphocyte-induced PDA cell death. This was the first study to demonstrate the important role of PAK1 in regulating PSC activity and PD-L1 expression in PDA. In a summary, these studies revealed the importance of PAK signalling in PDA development, the therapeutic value of PAK inhibitors and its synergism with gemcitabine in PDA treatment. Most importantly, PAK1 is emerging as a potential target to enhance anti-tumour immunity and to facilitate the development of novel immunotherapies.
The effects of cannabinoid derivatives on pancreatic cancer
Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant solid tumours and its treatment has not been efficiently improved during the last decade. The overall 5-year survival rate of pancreatic cancer is 9%, which is expected to become the second major cause of cancer-related mortality by 2030. Chemotherapy has been used as the main treatment of choice in the majority of patients bearing either locally advanced or metastatic tumours. Gemcitabine alone or gemcitabine-based combination chemotherapy is commonly used worldwide for the treatment of pancreatic cancer. The poor treatment response to chemotherapy might be related to a dense stroma that is associated with these cancers. However, more effective treatment techniques and novel targets are urgently needed to improve the treatment response. Plant-derived cannabinoids have been used for medical purposes for over a thousand years. The main active cannabinoid components are tetrahydrocannabinol (THC) and cannabidiol (CBD) that are obtained from Cannabis sativa and Cannabis indica. THC exerts its effects via a receptor dependent mechanism, while CBD can act through a receptor and receptor independent mechanisms. CBD can modulate the adverse effects of THC such as its psychoactive effects. To date, three cannabinoids receptors have been identified, CB1, CB2, and GPR55, which are located on the cell membrane and members of the G-protein coupled receptor (GPCR) family. Cannabinoid receptors play an important role in biological and pathological activities such as inflammation, oxidative stress, metabolism, fibrosis, appetite control, memory and emotion. The main aims of this study were to look at the expression of cannabinoid receptors in pancreatic cancer cell lines and pancreatic cancer specimens, detect the correlation of cannabinoid receptors expression with patient’s outcome, determine the effect of cannabinoids alone or in combination with chemotherapy on pancreatic cancer cells in vivo and in vitro. Methods: Western blot assay, SRB-assay, immunohistochemistry staining, wound healing assay and an animal model were the main methods used in this study. Results: The expression of CB1 and CB2 receptors were shown in all the human and murine pancreatic cancer cell lines, stellate cells, and the gemcitabine resistant cell lines. The significant correlation between high expression of GPR55 and worsened PDAC patients’ outcome were demonstrated. In addition, the positive surgical margin and moderate to dense stromal status are associated with a poorer and prolonged survival in patients, respectively. Both CBD and THC dose-dependently inhibited cell proliferation of pancreatic cancer cells, although CBD was more effective than THC. All the different combination ratios of CBD to THC showed significant effects on the inhibition of cancer cell proliferation compared with a single treatment of drugs, although a 1:1 ratio of THC to CBD demonstrated the maximal inhibitory effect on cell proliferation and migration. Synergistic inhibition was observed for combined THC and CBD treatments, and when THC/CBD was combined. with gemcitabine (combination index < 1; using the Chou-Talalay method). In this study, cannabis oil, which contained a 1:1 ratio of CBD to THC and other cannabinoid components has been used for in vitro and in vivo experiments. The effects of this oil on pancreatic cancer cell proliferation and migration was less than either the pure CBD or THC alone. Moreover, cannabis oil alone or in combination with gemcitabine did not significantly inhibit tumour growth in a xenograft model. Bioavailability and absorption patterns of this oil are unknown and further studies are required. Conclusion: Cannabinoid receptors are highly expressed in pancreatic cancer cell lines. The high intensity of GPR55 was correlated with poor patients’ outcome. The 1:1 ratio of THC to CBD provided a significant inhibitory effect on pancreatic cancer proliferation and migration. Combination of cannabinoids with gemcitabine had a synergistic effect on pancreatic cancer inhibition in in vitro study. The cannabis oil used in our in vitro study showed a lesser effect on blocking proliferation and migration of cancer cells than purified THC and CBD. We could not determine the clinically significant effect on pancreatic cancer in our in vivo study using cannabis oil. No absorption characteristics of studied cannabis oil have been reported, therefore more in vivo bioavailability studies are required for the future work.
Communication, technology and instruments in the modern contemporary setting of urology
The 2017 Sensis Social Media Report demonstrated an increase in Australians being involved in Social Media Platform. About 84% of Australians access the Internet on a daily basis with higher usage among the age group under 30 year old and less common among people age group of 40 and over. Delivering accurate medical information to patients or caregivers is essential in any medical practice. Having free access healthcare information with the unknown potential of having inaccurate medical information or unmoderated website for quality control while online consumers with health information-seeking behaviour could lead to false health information. Technological innovation has aided the growth in the healthcare industry. The wide expansion of modes of communication and surgical equipments, especially in urology has definitely changed the practice in the past few decades. It is one of the surgical specialty that has increasingly become a technology-driven specialty. With new innovative surgical instruments aiming to improve the outcomes for patients and the ease of accessibility of communication technology in medical practice has certainly improved in various ways within the healthcare setting. Technology has been part of medical and surgical practice and this continue to change the way medicine would be practice in the future. With regards to surgical instruments, the advancement of endoscopic procedures have shown to be a huge advantage in the surgical practice. Although it does not involve all types of surgery but it has been a turning point in some surgical cases. For example, open surgery was the conventional method of treating benign prostatic hyperplasia (BPH) but new treatments have been developed over the years without going through major surgery. BPH is a common condition for men as they get older which causes prostate enlargement which leads lower urinary tract symptoms (LUTS). The BPH prevalence examined in several autopsy studies showed that 20% for men stated to develop in their 40s, reaching between 50% to 60% for men in their 60s, and from 80% to 90% for men in their 70s and 80s. BPH If left untreated, this will eventually lead to blockage of the urinary tract, causing problems to bladder, urinary tract or kidney. The current "gold standard" of treating BPH surgically is transurethral resection of the prostate (TURP) using endoscopic method to remove prostate tissue. Although this method is an effective treatment clearing obstructive prostate tissue, the main side effects include bleeding. Laser surgical therapy for BPH was introduced for more than 20 years. To this date, there are various types of laser therapy that are readily available for surgical treatment of BPH with the unique properties of each laser to allow accurate treatment of BPH. The GOLIATH study demonstrated the GreenLightTM laser was noninferior to TURP in terms of the outcomes and had better results in length of catheterisation and length of hospital stay. The impact of technology in any surgical setting with the aim to improve outcomes in treating any disease while minimizing the risk of complication, or improving has changed significantly. With the rapid evolution of this of technology in the surgical field, however,needs thorough research to ensure high standards of care is delivered when utilizing latest surgical instruments. Essentially, the background outlined here not only highlights the core aim of this thesis: to observe the trend and understand the impact of technology in the current surgical practice. Given that this is will be a broad perspective, this thesis will be focusing on two parts, that is, one related to modes of communication using online technology and how it benefits the users and observing the risks involved. The other part of the thesis will be related to advancement of surgical instruments in urological surgery and the outcomes from the new equipments.
The role of zinc in prostate cancer
Prostate cancer (PCa) is the most common cancer amongst Australian men. Zinc is an essential metal and is vital for normal function of the prostate gland. Castrate-resistant prostate cancer (CRPC) is becoming increasingly resistant and treatment options are limited in number and often associated with poor clinical outcomes. Therefore a pertinent clinical issue is to develop more effective treatment regimes. Zinc appears to play a role in PCa, but a true understanding leading to therapeutic developments is yet to be achieved. In particular evidence regarding cell proliferation and zinc uptake and levels in PCa cells is conflicting. HIF1α is a well-known prognostic marker in PCa associated with poor prognosis, resistance to treatment and development of metastatic disease, however the cause over-expression in CRPC remains a mystery. Therefore the ability of PCa cells to uptake and store zinc, and the role of zinc in PCa cell proliferation, tumour growth and HIF1α mediated survival was investigated in this thesis. CRPC-like human PC3 cells are significantly resistant to docetaxel chemotherapy and overexpress HIF1α protein, compared to normal prostate epithelial control cells (PNT1A). Cell proliferation assays (MTT) demonstrated that physiological zinc administered to PC3 cells significantly slowed growth compared to normal PNT1A and androgen-sensitive PCa (LNCaP) cells. The effect of zinc supplementation or zinc chelation (by TPEN) does not affect macroscopic growth in PC3 xenograft tumours. There is no significant difference in baseline total zinc concentration (measured by ICP-MS) between cell lines normal (PNT1A), androgen sensitive (LNCaP) and CRPC (DU145 and PC3) cells. However, CRPC-like PC3 cells contain significantly higher unbound free Zn2+ and Immunofluorescence Microscopy (IFM) subcellular distribution of Zn2+ in PC3 cells is unlike that seen in normal prostate epithelial cells. PC3 cells are resistant to oxidative stress injury. Zinc strongly induces HIF1α protein expression in these cells in a time and dose dependent manner. Zinc mediated oxidative protection in PC3 cells is a HIF1α dependent as demonstrated in a PC3 HIF1α-KD model. No such zinc protection was seen PNT1A cells. Zinc could be essential in the resistant nature of CRPC cells as Zn2+ ions rescue HIF1α protein expression and are implicated in the normoxic stabilisation of the HIF1α protein by competing with Fe2+ ions at the PHD binding sites. Therefore zinc dysregulation in CRPC cells is an important factor in the development of resistance, as well as potentially progression to metastatic disease and poor prognosis in PCa.
Characterisation of tumour-infiltrating lymphocytes in benign and malignant prostate tissues
Over the past decade, we have seen a significant advancement in the treatment of metastatic castrate-resistant prostate cancer (PC). Cancer immunotherapy is now a reality, but the information on tumour infiltrating lymphocytes (TILs) in human prostate tissues is still limited. This project aims to evaluate and compare T cell characteristics in blood and tissues from patients with benign and malignant prostate conditions using flow-cytometry. Patients with benign prostatic hyperplasia (BPH) and normal prostate (NP) were used as controls and compared with patients with PC. We also examined the effects of androgen deprivation therapy (ADT) on systemic immune responses and immune regulation in patients with advanced/metastatic PC. The T cell characteristics evaluated were: (1) The proportion of regulatory T cell (Treg) and CD8/Treg ratio; (2) The percentage of activated cytotoxic lymphocytes (CTLs); and (3) The T cell maturation status. We found that the proportion of Treg in PC tissue was 5.6%, consistent with other studies. We did not find any differences in the proportion of Treg or CD8/Treg ratio in tissues for the three patient groups. When evaluating PC involved vs PC non-involved tissues, as an internal control comparison, PC involved tissues were found to have a higher percentage of Treg (6.89% vs 4.15%, p-value of 0.045) and a lower CD8+/Treg ratio (54 vs 119, p-value of 0.0032) than PC non-involved tissues. Despite a small number of 6 cases, the differences were statistically significant. These findings support the hypothesis of local immunosuppression in PC-involved tissues, and that such immunosuppression might be compartmentalised even to the level of specific lobes of the prostate. In this compartmentalised environment, PC cells can evade and survive the immune system’s defences. This study did not find any correlation between Gleason scores and the percentage of Treg in the CD4+ T cell subset or the CD8/Treg ratio either in PBMC or PC tissue. In the PC group, the same percentages of CD8+ T cells were found in both tissues and peripheral blood for majority of the patients. This showed that CD8+ T cells were not expelled or excluded from the tumour micro-environments, therefore the PC tissues were of the T-inflamed phenotype. When comparing tissue with PBMC, the levels of Treg are two times higher in tissues (both benign and malignant) than in peripheral blood. This could be a phenomenon across all tissue types and not exclusive to the prostate. Their role presumably is to maintain peripheral tolerance. We also found that the percentage of activated CD8+ T cells was ~20 times higher in tissues than in blood, regardless of the tissue type. Again, it is possible that this is not exclusive to the prostate. Profiling the immune infiltrates of other tissue types will help to clarify this. Using PCR, we have assessed the cytokine profiles in benign and malignant prostate tissues, and there were no significant differences in the levels of IFNγ, TNFα, TGFβ and IL-10 expressions. Our assays did not detect IL-4, IL-5 and IL-13 in any sample. To assess the impact of ADT on T cells, we evaluated the T cell characteristics in peripheral blood of 15 patients before ADT, at 3 months and then at 9-12 months after ADT. There was a significant reduction in the percentage of Treg by 15% after 3 months of ADT. This suggested there was an initial reduction in immune suppression during the first year of ADT. There was a significant decreased in the proportion of naïve T after 9-12 months of ADT. This suggested naïve T cells might have developed into other more mature T cell subsets after ADT.
18F-fluorodeoxyglucose positron emission tomography as a biomarker for colorectal cancer liver metastases
Background: Colorectal cancer is the second most common cause of cancer-related death in Australia. The majority of patients with colorectal cancer develop liver metastases but only those amenable for surgical resection have a possibility of long term survival. Recent advances in achieving macroscopic resectability of colorectal liver metastases needs to be balanced urgently, by an ability to assess systemic micrometastatic disease. Tumour staging by 18F-fluorodeoxyglucose positron emission tomography (PET) is a non-invasive tool already in routine use. Aim: To explore metabolic characteristics assessed by PET as biomarkers for colorectal cancer liver metastases. Methods / Results: Four studies were performed, each addressing separate aspects regarding the utility of tumour metabolic assessment. The first three studies were performed on retrospective cohorts while the fourth study was a prospective study. The studies and main novel findings are summarized below: 1) The Prognostic Impact of Tumour Metabolism an a Single PET Scan after Preoperative Chemotherapy Various parameters that characterize and quantify tumour metabolism were assessed for their prognostic ability. These parameters were compared to clinical and pathological features as well as previously verified prognostic scoring systems. The metabolic parameters corresponding to metabolic tumour burden were found to be most prognostic on a single PET scan following preoperative chemotherapy. 2) The Prognostic Impact of Tumour Metabolic Response to Preoperative Chemotherapy The prognostic ability of metabolic response to preoperative chemotherapy was assessed using the serial assessment of various metabolic parameters. In comparison, tumour size shrinkage on computed tomography and pathological response, the current gold standards of chemotherapy response evaluation, were assessed. Metabolic response to preoperative chemotherapy was shown to be the best prognostic indicator. 3) Metabolic Response Correlated to Biological Mechanisms The biological mechanisms underlying the prognostic impact of metabolic response was explored. Immunohistochemical analysis of six tumour biomarkers showed an inverse correlation between metabolic response and the expression of Ki-67, a marker of cellular proliferation; and a direct correlation between metabolic response and the expression of p16, a tumour suppressor. 4) Early Metabolic Response Assessment The use of early tumour metabolic response after only the first cycle of preoperative chemotherapy was assessed for the ability to predict eventual metabolic response. Early tumour metabolic response after one cycle of chemotherapy did not predict eventual metabolic response or clinical outcome. Conclusion: This thesis showed tumour metabolism to be a powerful prognostic indicator for patients with colorectal cancer liver metastases. In particular, it reveals the burden of disease as well as the sensitivity of the metastases to systemic chemotherapy. PET assessment of tumour metabolic response to chemotherapy should be routinely performed, particularly in patients undergoing complex liver surgery.
Role of p21-activated kinases in pancreatic cancer
Pancreatic cancer remains one of the most lethal of all solid tumours with an overall 5-year survival rate of 7%. Management has not improved significantly over the last thirty years and based on current trends, is expected to become the second leading cause of cancer-related mortality by 2030. Treatment options are limited and gemcitabine-based chemotherapy remains the standard of care as a single agent. Furthermore, the presence of the dense stroma, characteristic of pancreatic cancer, contributes to therapeutic resistance and poor therapeutic response. Thus, a better understanding of the underlying genetic and molecular mechanisms is urgently required to find targeted and effective therapies. There is growing evidence that p21-activated kinases (PAKs) are involved in pancreatic carcinogenesis. The PAK family consist of six isoforms, two of which, PAK1 and PAK4, are upregulated and/or hyper-activated in pancreatic cancer. PAK1 can mediate many different cellular processes including the regulation of cytoskeletal dynamics and cell adhesion, the evasion of apoptosis, the promotion of cell survival, proliferation, migration and invasion, the fibrosis that constitutes the stroma, and the interplay between cancer cells and the stroma. PAK1’s role has not been fully elucidated in pancreatic cancer and has not been evaluated as a target for therapeutic intervention. The work presented in this thesis investigates the role of PAK1 in pancreatic cancer and the effect of PAK1 inhibitors, alone and in combination with gemcitabine, on pancreatic cancer growth, metastasis, stroma, and survival. First, we investigated the effect of glaucarubinone, a known inhibitor that reduces the activity of PAK1 and PAK4, on pancreatic cancer growth, migration and murine survival. Using 4 human and 2 murine pancreatic cancer cell lines, PAK1 and PAK4 was expressed in all pancreatic cancer cell lines tested and proliferation and migration/invasion inhibited by treatment of glaucarubinone with reduction in PAK1 and PAK4 activity in vitro. Synergistic inhibition was observed when combined with gemcitabine with decrease in pancreatic cancer proliferation in vitro, decrease in pancreatic cancer growth in human xenograft tumours in vivo, and increase in murine survival in an orthotopic immunocompetent model in vivo. This was one of the first studies that showed clinical benefit of targeting and reducing PAK1 in pancreatic cancer. Using more direct methods of reducing PAK1 activity, shRNA knockdown systems, and a PAK1 selective inhibitor, FRAX597, were utilised. shRNA knockdown of PAK1 resulted in a reduction in pancreatic cancer cell proliferation and survival and sensitised cells to gemcitabine in vitro. PAK1 was also found to be key regulator of signalling pathways such as PI3K and HIF1α. FRAX597 treatment decreased pancreatic cancer cell proliferation and migration/invasion and synergised with gemcitabine to decrease cell proliferation in vitro. FRAX597, combined with gemcitabine, reduced pancreatic tumour volume and increased murine survival in preclinical orthotopic immunocompetent murine models in vivo. Although, further clinical validation is required, it illustrates the clinical potential of a PAK1 inhibitor, FRAX597, combined with gemcitabine to improve pancreatic cancer patient outcomes. PAK1’s role was investigated in pancreatic stellate cells (PSCs), which are primarily responsible for the fibrosis that constitutes the pancreatic cancer stroma. This was the first study to show the presence of PAK1 activity in isolated human PSCs. The treatment of the selective PAK1 inhibitor, FRAX597, on PSCs resulted in a reduction in their activation, proliferation, and increase in apoptosis in vitro. PAK1 knockout mice tumours had decreased expression and activity of PAK1, associated with increased murine survival, showing the effect of depleting host PAK1 in an orthotopic immunocompetent murine model in vivo. These results implicate PAK1 as a regulator of PSC activation, proliferation and apoptosis and targeting stromal PAK1 could increase therapeutic response and survival of patients with pancreatic cancer. Together, these results illustrate the importance of PAK1 signalling in pancreatic cancer and the possible therapeutic benefit of targeting PAK1 with gemcitabine on pancreatic cancer growth and the stroma to increase the survival of pancreatic cancer patients.
Selection and management of men for active surveillance in low risk prostate cancer
Aims: To investigate: 1. Selection of men for active surveillance of prostate cancer a. Validation of risk calculators b. Suitability for inclusion of Gleason 3+4 disease. 2. Performance of prostate biopsy during AS a. Differences in quality of diagnostic biopsy between academic and referral centres. b. Optimization of biopsy templates c. Examination of prognostic indicators for disease progression Methods: Data were obtained from several difference sources: • Men suitable for AS on prostate biopsy but undergoing upfront radical prostatectomy were pooled from 3 international academic institutions in Cambridge (UK), Toronto (Canada) and Melbourne (Australia). • Prospectively maintained AS prostate cancer database at Princess Margaret Cancer Centre (PMCC) (1997-2012). Analyses performed: • Four risk calculators were assessed for their ability to predict different definitions of insignificant prostate cancer by area under the curve (AUC) of receiver operating characteristic curves and Brier scores for discrimination, calibration curves and decision curve analysis. • Men with biopsy Gleason 3+4 disease, suitable according to modified Royal Marsden, Sunnybrook Toronto and PRIAS selection criteria, were assessed for presence of adverse pathology at upfront radical prostatectomy. • Patients on AS at a tertiary referral centre (PMCC) were dichotomized depending on where their diagnostic biopsy was performed (interval versus external). Multivariate logistic regression was performed to examine for predictors of re-classification at the second, or confirmatory, biopsy. • Mapping of all patients with pathological progression at PMCC for location of disease progression enabled comparison of hypothetical biopsy templates (sextant and standard extended) to the institutional template used. • Men on AS at PMCC were evaluated for presence of disease progression at serial biopsy in the prostate transition zone (TZ). Multivariate Cox proportional hazards regression evaluated predictors of TZ progression. • At PMCC, men were dichotomized based on presence of cancer at their confirmatory biopsy. Pathological progression was investigated using a Cox proportional hazards regression model. Results: • All 4 models predicting presence of insignificant prostate cancer had weak discrimination at best (AUC 0.618-0.664). • Presence of Gleason 3+4 at biopsy, compared to 3+3 disease, increases risk of adverse pathology at radical prostatectomy if modified Sunnybrook Toronto criteria are used (19% versus 33%, p≤0.001). Using a stricter protocol such as PRIAS, there was no statistical difference between the groups. • External biopsy predicted both grade related re-classification (OR 4.14, C.I. 2.01-8.54, p<0.001) and volume related re-classification (OR 3.43, C.I. 1.87-6.25, p<0.001). • Sextant and standard extended biopsy templates were inferior to the institutional biopsy template in detecting presence of cancer (84% and 99% versus 100%), and pathological progression (47.9% and 81.9% versus 100%). • At each subsequent biopsy during AS, 2.7-6.7% of men had disease progression only in the TZ which would not have been detected if TZ biopsy was not performed. Predictors of TZ progression were maximum % single core (HR 1.99, C.I. 1.30-3.04, p=0.002), and MRI reporting cancer (HR 3.19, C.I. 1.23-8.27, p=0.02). • Men with no cancer at confirmatory biopsy were less likely to have pathological progression (HR 0.47, CI 0.29-0.77, p=0.003). Sub-analysis showed this was predictive of volume-related progression (HR=0.36, CI 0.20-0.62, p=0.0006) and not grade-related progression. Conclusions: • Utilization of models predicting suitability for AS should be used with caution as external validation in our cohort was weak. • If considering biopsy Gleason 3+4 disease for AS, a stricter protocol such as PRIAS must be utilized. • At PMCC, patients who had their initial diagnostic prostate biopsy for AS done externally, were more likely to have worse pathological features and re-classify on the second biopsy. • For men on AS, sextant and standard extended biopsy are less likely to detect prostate cancer or disease progression than the template used at PMCC. • TZ biopsy should be considered for all men having serial biopsy on AS, in particular those with high % core involvement or positive MRI findings. • Absence of cancer on B2 is associated with a significantly decreased risk of volume-related but not grade-related progression.
The role of hypoxia inducible factor 1 alpha (HIF1α) in prostate cancer
Prostate cancer (PC) is one of the most prevalent cancers in men. Although many PCs are indolent, a significant proportion will metastasize and develop resistance to therapy. Contemporary screening tests lack the finesse to accurately differentiate aggressive PCs from indolent tumours, potentially leading to over-diagnosis and over-treatment. While cellular hypoxia often plays an integral role in carcinogenesis and tumour progression, this connection has been difficult to demonstrate in PC. However, a downstream marker of hypoxia, Hypoxia inducible factor 1α (HIF1α), which is a transcription factor that protects cells against noxious stimuli, is frequently over expressed in PC. Therefore, the role of HIF1α in PC was investigated in this thesis. The Castrate resistant PC (CRPC)-like human PC cell lines PC3 and DU145 were found to over-express HIF1α protein compared to an androgen-sensitive cell line LNCaP under normoxic conditions. Using HIF1α 5’UTR-luciferase constructs in PC3 cells, further experiments revealed that increased translation of HIF1α mRNA regulated by a 70bp GC-rich, secondary structure in the 5’UTR of the HIF1α promoter may be responsible for normoxic HIF1α overexpression. Cell proliferation assays revealed that PC3 cells over-expressing HIF1α were more resistant to destruction by cytotoxic agents (H2O2 and 5-fluorouracil) than androgen-dependent LNCaP cells. Reduction of HIF1α expression in PC3 cells using RNA interference decreased both the resistance towards cytotoxic agents and cell migration. Conversely, in the androgen-dependent LNCaP cells overexpression of HIF1α increased the resistance to cytotoxic agents. One hundred prostate tumours were then immune-stained for HIF1α and outcomes measured. On multivariate analysis HIF1α was an independent risk factor for progression to metastatic PC (Hazard ratio (HR) 9.8, p = 0.017) and development of CRPC (HR 10.0, p = 0.021) in patients on androgen-deprivation therapy (ADT). Notably the tumours that did not express HIF1α did not metastasise or develop CRPC. Next, the effects of non-specific HIF1α inhibitors (digoxin, metformin and angiotensin-2 receptor blockers) were investigated in ninety-eight patients who had continuous ADT as first line therapy and developed CRPC. The median CRPC-free survival was longer in men using HIF1α inhibitors compared to those not on inhibitors (6.7 yrs vs. 2.7yrs, p=0.01) and there was a 71% reduction in the risk of developing CRPC (p=0.02) and an 81% reduction in the risk of developing metastases (p=0.02) after adjustment for Gleason score, age and PSA. Finally, the effects of metformin were investigated in 2055 men treated for PC with external beam radiotherapy. Surprisingly, metformin did not result in any improvement in time to biochemical failure, time to metastases or overall survival in men undergoing radiotherapy, but there was an 1.5 fold increase in PC-specific deaths (p<0.05) in men on metformin who received ADT when adjusted for cancer risk and co-morbidities. In conclusion, the results presented in this thesis indicate that HIF1α is a promising marker in PC, which may be used for early identification of cancers that potentially will progress to metastases and develop resistance to ADT. HIF1α is likely to contribute to metastasis and chemo-resistance of CRPC, targeted reduction of HIF1α may improve outcomes of aggressive PC.