Surgery (Austin & Northern Health) - Theses
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ItemThe role of zinc in prostate cancer( 2018)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.
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ItemCharacterisation of tumour-infiltrating lymphocytes in benign and malignant prostate tissues( 2018)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.
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ItemSelection and management of men for active surveillance in low risk prostate cancer( 2016)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.