Medicine (Austin & Northern Health) - Theses
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ItemInvestigating the role of the EHF transcription factor in the regulation of differentiation in colorectal cancer( 2017)Colorectal cancer is characterized by aberrant cell proliferation, resistance to apoptosis and loss of differentiation. While the pathways which drive cell proliferation and resistance to apoptosis in this disease are relatively well characterized, the mechanisms which underpin the loss of differentiation are not well understood. The central aim for this thesis was to identify novel transcription factors which mediate differentiation loss in colorectal cancer. Analysis of gene expression differences between moderately and undifferentiated colorectal cancers identified coordinate downregulation of multiple transcription factors in undifferentiated cell lines. This included the EHF transcription factor (Ets homologous factor) which was highly expressed in well to moderately differentiated colorectal cancer cell lines and downregulated in undifferentiated lines. EHF was also found to be highly expressed in the normal colonic epithelium. Loss of EHF expression also correlated with reduced expression of colonic epithelial differentiation markers in primary colorectal tumours. To assess the direct role of EHF in colorectal cancer cell differentiation, EHF expression was downregulated in moderately differentiated cell lines and stably reexpressed in undifferentiated cell lines. These experiments demonstrated that knockdown or re-expression of EHF alone was not sufficient to alter the differentiation status of colorectal cancer cell lines. Alternatively, EHF was found to play a key role in the suppression of migration and invasion of colorectal cancer cells. As multiple transcription factors are downregulated in undifferentiated colorectal cancers, we next determined whether EHF may impact differentiation status by working in combination with other transcription factors. To address this, systematic knockdown of EHF with other downregulated transcription factors in undifferentiated colorectal cancer cell lines was performed in moderately differentiated colorectal cancer cell lines. This analysis revealed that knockdown of EHF in combination with CDX1 significantly downregulates the expression of differentiation markers and disrupts the glandular architecture of moderately differentiated colorectal cancer cell lines. Conversely, the re-expression of EHF and CDX1 in undifferentiated colorectal cancer cells significantly induced differentiation marker expression and induced some features of gland formation. Furthermore, the re-induction of EHF and CDX1 significantly reduced cell proliferation and migration of colorectal cancer cell lines. These findings demonstrate that EHF in combination with CDX1 regulates the differentiation of colorectal cancer cells, and that the coordinate loss of these transcription factors contributes to the loss of differentiation in colorectal cancer.
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ItemNovel approaches to the endoscopic management of gastrointestinal disorders( 2017)Endoscopy has evolved since its humble beginnings. Over the past decade advances include the rapid evolution in endoscopic visual assessment of the bowel and its role in the management and prevention of colorectal cancer. Endoscopy has developed key diverse technologies that include capsule endoscopy and endoscopic ultrasound. Innovation and advances have not been limited to the endoscopes themselves but increasingly the devices that are utilized during endoscopy help to extend the boundaries of both diagnostic and therapeutic interventions. The aim of this thesis is to assess new and novel endoscopic techniques and technologies in the management of gastrointestinal disorders. The first area of study is to assess novel endoscopic techniques and technologies in the area of colonoscopy. This procedure is of key importance in Australia where despite having one of the highest rates per capita of colonoscopy worldwide, colorectal cancer remains the second most commonly diagnosed malignancy. Given that there is no contemporary local data on colonoscopy and polypectomy practices, we first undertook a national survey of practising endoscopists to assess whether significant variations in colonoscopy and polypectomy practice exist across Australia. The second area of study is whether prediction of colonoscopy surveillance intervals based on real time endoscopic assessment of polyp histology is safe, feasible, accurate and cost effective. A prospective cohort study was performed across public and private centres, 94 patients who underwent colonoscopy and polypectomy of diminutive (<5mm) polyps were recruited, yielding a total of 159 polyps. These polyps are interrogated with narrow band imaging (NBI) which is an optical assessment technology and classified as adenomatous or non-adenomatous according to the Sano-Emura classification system. The endoscopic assessment of histology is used to predict appropriate surveillance intervals. Accuracy of optical diagnosis of diminutive colonic polyps is measured against the gold standard histological assessment. The final study examines the utility of colonoscopy through a prospective multi-centre observational study of right-sided colonic retroflexion and its impact on polyp detection in 1351 consecutive adult patients undergoing elective colonoscopy. Withdrawal from the caecum is performed in the forward view initially and identified polyps removed. Once the hepatic flexure is reached the caecum is re-intubated and the right colon assessed in the retroflexed view to the hepatic flexure. Adenoma detection (ADR) and polyp rate (PDR) in the retroflexed view is compared with forward view examination of the right colon to assess if there is a significant improvement. The second line of investigation of this thesis examines the role of novel endoscopic techniques and technologies in the management of oesophageal and gastric cancers. The first study assesses the feasibility of endoscopic ultrasound (EUS) guided fiducial placement for the multidisciplinary management of gastric cancer. A prospective phase II feasibility study is undertaken in consecutive adult patients with primary gastric adenocarcinoma. Gold fiducial markers are inserted under EUS guidance into the margins of the gastric cancer primary tumour. The main outcome is the successful insertion of the fiducial without complications for response assessment and anatomic localization. The second study sought to address the limitations identified with EUS guided fiducial insertion and assess the utility of a novel marker inserted via a gastroscope for the multidisciplinary management of oesophagogastric cancer. The third and final area to be assessed in this thesis is the use of novel endoscopic devices in the management of upper gastrointestinal disorders. The first study examines the ability for risk stratification of upper gastrointestinal bleeding with an esophageal capsule. A diagnostic, nonrandomized, single-blind (investigator) study is undertaken across three tertiary centres. Eighty-three consecutive adult patients referred for management of upper gastrointestinal bleeding (UGIB) undergo a capsule endoscopy (CE) prior to gastroscopy for the investigation and management of UGIB. The main outcome measures are the detection rates of UGIB source and identification of a low-risk group of patients who may be suitable for outpatient gastroscopy based on CE findings. The second study assesses the management of pancreatic fluid collections (PFCs) with a novel endoscopically placed fully covered self-expandable metal stent (FCSEMS). A retrospective case series is undertaken across thirteen tertiary and private health care centres. Forty-seven patients undergo endoscopic management of PFCs with a recently developed lumen-opposing, fully covered self-expandable metal stent (FCSEMS). The main outcome measures are the technical and clinical success rate, adverse event rate. The aim of this thesis is to assess novel endoscopic innovations and their impact on gastrointestinal disease. Through the studies that will be presented we aim to demonstrate current techniques that can be rapidly incorporated into current clinical practice with significant impact, niche techniques with tremendous potential and novel devices, which exist in current clinical practice without adequate evaluation. Innovation is a key element to the evolution of endoscopy, however it must be evaluated within a formal framework to ensure optimal patient outcomes whilst remaining cost effective to our health system.
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ItemNovel antibodies for treatment of solid tumours( 2015)This thesis describes three different strategies aimed to optimize the use of novel targeted antibodies for the treatment of metastatic solid tumours, including colorectal cancer and renal cell carcinoma. The ability of recombinant antibodies to adequately penetrate into tumours is a key factor in achieving therapeutic effect; however, the behaviour of antibodies at a cellular level in tumours is poorly understood. The purpose of this first study was to investigate those factors that influence the macroscopic and microscopic intratumoural distribution of an anti-GPA33 IgG1-humanised antibody, huA33, in colorectal tumours. Twelve patients were infused with radiolabelled huA33 7 days prior to elective surgery for colorectal carcinoma. Macroscopic huA33 uptake was determined by both gamma well counter and autoradiography measurements of the resected tumour specimens. Microscopic uptake was then quantitated at a cellular level and compared to vascular penetrance. The impact of variation in GPA33 tumour antigen expression, tumour size, specimen type (primary vs metastatic), presence of macroscopic necrosis, and tumour vasculature on huA33 uptake were examined. The mean ± SD [131]I-huA33 uptake in whole tumour sections was 5.13±2.71×10(−3) % injected dose per gram (ID/g). GPA33 was expressed in all viable tumour cells, and huA33 uptake was excellent regardless of tumour size and specimen type. In tumours with macroscopically evident central necrosis (n=5), huA33 uptake in tumour necrotic centres was lower than in viable peripheries (0.606 ± 0.493 vs 2.98 ± 2.17×10(−3) %ID, p=0.06). However, when corrected for low cell viability in necrotic centres, uptake of huA33 at the cellular level was highly comparable to that in the more viable tumour periphery (7.10 ± 5.10×10(−9) vs 3.82 ± 3.67×10(−9) %ID/cell, p=0.4). In the five patients who exhibited macroscopic necrosis in their tumours, huA33 showed excellent tissue penetration, with a maximum penetration distance of 26 μm in peripheral tumour regions and 118 μm in central regions. No correlation was observed between [131]I-huA33 uptake in tumour on a cellular basis and tumour vascularity. In patients with colorectal carcinoma, monoclonal antibody huA33 effectively targeted viable tumour cells in all cellular milieus examined, including effective penetration into necrotic tumour centres, a novel and therapeutically important finding. For the second strategy, a Phase I imaging and pharmacodynamic study of anti-death receptor 5 (DR5) antibody CS-1008 investigated the impact of CS-1008 dose on the biodistribution, quantitative tumour uptake and anti-tumour response in patients with metastatic colorectal cancer. Pretreated patients with metastatic colorectal cancer were infused with weekly CS-1008 in five dose cohorts. Day 1 and day 36 doses were trace-labeled with Indium-111 ([111]In), followed by whole-body planar and regional SPECT imaging at several time points over 10 days. Nineteen patients were enrolled. [111]In-CS-1008 uptake in tumour was observed in only 12 patients (63%). [111]In-CS-1008 uptake and pharmacokinetics were not affected by dose or repeated drug administration. [111]In‐CS‐1008 biodistribution showed gradual blood pool clearance and no abnormal uptake in normal tissue. No anti-CS-1008 antibody response was detected. One patient achieved partial response (PR, 3.7 months duration), 8 patients had stable disease (SD), and 10 patients had progressive disease (PD). Disease control rate (SD + PR) in patients with [111]In-CS-1008 uptake in tumour was 58% vs 28% of patients with no uptake. An analysis of individual lesions showed that lesions with antibody uptake were 1/3 as likely to progress compared to those without antibody uptake (p=0.07). DR5 expression in archived samples did not correlate with [111]In-CS-1008 uptake (p=0.5) or tumour response (p=0.6). In conclusion, DR5 imaging with [111]In-CS-1008 revealed significant inter- and intra-patient heterogeneity of uptake in tumour, is not dose dependent, and is predictive of clinical benefit in the treatment of metastatic colorectal cancer patients. The third strategy examined the safety and tolerability of the combination of cG250 and sunitinib in patients with advanced clear cell renal cell carcinoma. cG250 is an IgG1 kappa chimeric antibody that recognises carbonic anhydrase IX, an antigen present on more than 85% of renal cell carcinoma, but with limited expression in normal tissues. If binding of cG250 to its antigen interrupts signalling, this may lead to synergistic effects with sunitinib. We hypothesized that combination of cG250 and sunitinib would induce beneficial clinical effects by targeting complementary signalling pathways and that sunitinib’s acute effects on tumour vasculature would influence cG250 biodistribution, tumour metabolism and tumour blood flow. Up to 2 cycles of treatment were given to eligible patients who had measurable metastatic or unresectable clear cell renal cell carcinoma with at least one lesion >2 cm assessable by FDG-PET. cG250 was administered at 10 mg/m2/week for 5 weeks (1st and 5th doses tracelabeled with [124]I). Sunitinib was commenced on day 8 of the 1st cycle at 50 mg/day for 4 weeks for sunitinib-naive participants or continued at the same dose for those already receiving sunitinib. Six patients were enrolled and evaluable for toxicity, biodistribution, pharmacokinetics and tumour uptake; 4 patients were evaluable for tumour response. The study was terminated early because dose limiting toxicities (DLTs) occurred in 2 patients: Grade (G) 3 fatigue and G5 cardiogenic shock. These events were considered possibly related to sunitinib. No other G3/4 adverse events (AEs) were reported. No AEs were attributable to cG250. Three patients had SD on CT and stable metabolic disease on FDG-PET, 1 patient had PR on CT and complete metabolic response on FDG-PET. There was a significant decrease in tumour blood flow ranging from 38.5% to 73.2%.[124]I-cG250 showed excellent tumour targeting on PET. No patients developed HACA responses. No differences in biodistribution, tumour uptake or pharmacokinetics were observed between the first and fifth infusion. As expected, the antiangiogenic activity of sunitinib resulted in a significant decrease in quantitative tumour blood flow which was clearly seen as soon as two weeks after its initiation. Despite sunitinib’s acute effects on tumour vasculature, [124]I-cG250 uptake was not affected by tyrosine kinase inhibitor (TKI) treatment in clear cell renal cell carcinoma lesions. These findings may ultimately have implications for the design of studies combining both TKIs and antibody-based treatments.
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ItemThe renin angiotensin system and macrophages in regulation of colorectal liver metastases( 2012)Metastasis to the liver is the leading cause of death for colorectal cancer (CRC) patients. The systemic treatment of CRC liver metastases is suboptimal and with limited response rates. Targeting of the renin angiotensin system (RAS) may be a potential adjunct therapeutic strategy in this disease. Blockade of the RAS can inhibit tumour growth in a mouse model of CRC liver metastases. However, the underlying mechanisms remain unclear. Participation of the RAS in inflammatory diseases and in malignancy suggests that macrophages may be a novel mediator of RAS-induced effects. This thesis addressed the role of the RAS in regulating macrophage biology and its consequent impact on the growth of CRC liver metastases. Macrophage depletion studies using an orthotopic murine model of CRC liver metastases demonstrated the bimodal role of macrophages in determining tumour growth. They exhibit an early inhibitory and a later stimulatory effect. Alterations in iNOS- and VEGF- expressing cells, and T-cell responses may be responsible for the observed reduction in tumour burden following depletion of pro-tumour macrophages at the late stage of metastatic growth. Using combined in-vitro with in-vivo experiments the potential of the RAS to alter macrophage function was demonstrated. In-vivo, the anti-tumour affects of ACE inhibition (captopril) on CRC liver metastases was mediated by changes in macrophage biology that inhibited initial tumour seeding and proliferation, as well as promoting macrophage migration. In-vitro, both key RAS peptides, Ang II and Ang-(1–7) were capable of altering tumour-regulatory factors, including iNOS, MMP-9, VEGF and TNF-α in murine macrophages. These factors are equally important in directing macrophage polarisation (M1 or M2 macrophages). Conditioned media from macrophages stimulated with Ang-(1–7) reduced the proliferation and viability of both human and mouse CRC cells, but increased cell migration in-vitro. Supernatant of Ang II-treated macrophages also altered the kinetics of mouse, but not human CRC cells. Interestingly, Ang II and its receptor inhibition did not induce distinct macrophage polarisation. It is clear the RAS has important immunomodulatory roles that can regulate tumour progression and its metastasis. Further understanding of these physiological mechanisms will enable agents targeting the RAS to reach their full therapeutic potential in the treatment of CRC liver metastasis.
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ItemMolecular determinants of therapeutic response to targeted therapies in metastatic colorectal cancer( 2011)Given the high cost, optimizing survival of patients with colorectal cancer requires research into two important questions. Firstly, can there be improved efficacy of these agents by using novel combinations with existing or other new drugs? Secondly, what are the mechanisms of both innate and acquired resistance to these drugs? Restricting use from those patients who have factors that govern immediate innate resistance will enrich and maximise initial response rates (RR). Identification of mechanisms governing acquired resistance may allow novel future strategies to avoid these mechanisms and allow ongoing durable responses to therapy. This thesis aims to explore methods of optimizing the targeted treatment of the EGFR pathway and the VEGF related pathway in colorectal cancer. The biology of each pathway will be reviewed. Therapeutic options for inhibiting these pathways in colorectal cancer will be discussed, focusing on targeted agents. The concept of maximising the effect of targeting the EGFR pathway by using two drugs that target the same pathway will be reviewed. Additionally, biological characteristics of the tumour that may predict resistance and response to targeting each individual pathway will also be reviewed.