Sir Peter MacCallum Department of Oncology - Theses
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ItemPrediction and assessment of pathological complete response following neoadjuvant chemoradiotherapy for locally advanced rectal cancer( 2017)Introduction The management of patients with rectal cancer who develop a pathological complete response (pCR) following neoadjuvant chemoradiotherapy (nCRT) presents an ongoing challenge to clinicians. Some authors have suggested that the presence of a clinical complete response may allow patients to be spared the morbidity and potential risks of surgery through adoption of a ‘watch and wait’ policy with surgery only in the setting of clinical failure. However clinical response and pathological response are not always wellcorrelated and widespread adoption of this regimen is limited by accurate methods to assess and confirm the presence of a pCR without a surgical specimen. Method A systematic review of the literature has been performed to identify methods to predict and assess a pathological complete response to nCRT. The first part of the literature review focuses on factors predictive of a pCR, specifically clinical features, radiological features and histological and molecular markers. The second part of the literature review aims to determine methods to accurately assess the presence of a pCR following neoadjuvant treatment. Following this an institutional database was interrogated to determine clinical and radiological features associated with prediction and assessment of a pCR and a multimodal predictive model has been developed. Molecular analysis has been performed to identify genetic influences on pCR. The role of radiological imaging in the assessment of pCR will be explored and the prognostic and clinical significance of metabolic response assessment by 18F FDG PET CT has been investigated in detail. Results Assessment Histology and clinical assessment remain the most effective methods of assessment of pCR with histology considered the gold standard. Clinical assessment is limited to low rectal tumours and is open to significant inter-rater variability while histological examination requires a surgical specimen for accurate assessment. Radiological assessment of pCR demonstrates the greatest potential for assessment of pCR without the need for surgery with diffusion weighted MRI and 18F FDG PET CT providing the greatest accuracy. It is likely that improved accuracy will be achieved with multimodal assessment of response combining the benefits of clinical, serological, endoscopic and radiological methods of response assessment. Prediction Clear methods to predict pCR prior to the commencement of therapy have not been defined. Clinical and radiological features of the primary cancer have limited ability to predict response. Molecular signatures hold the greatest potential to predict response however adoption of this technology is limited by poor concordance of biomarkers between cohorts. Conclusion Accurate prediction and determination of a complete pathological response is paramount if a non-operative approach is to be undertaken with confidence in oncological outcomes. A variety of methods are available but currently they lack sufficient sensitivity and specificity to define management. Despite a large volume of research the ability to predict which patients are likely to sustain a pCR and accurately assess those patient who have a pCR remains elusive. Further research into models incorporating both prediction and assessment into decision-making is required.
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ItemInvestigating the role of polarity protein, SCRIB, in haematopoiesis( 2017)The evolutionarily conserved scaffolding protein, Scribble (SCRIB), acts as a tumour suppressor in multiple epithelial cancers. However, SCRIB’s role in haematopoiesis and haematopoietic malignancies is largely unknown. As SCRIB knockout mice are embryonically lethal, we utilised conditional knockout mouse models driven by the Mx1 or hScl promoter in conjunction with extensive phenotypic analyses to elucidate SCRIB’s role in haematopoiesis. Our comprehensive analyses revealed loss of SCRIB produced subtle but significant defects in lymphoid and myeloid progenitor fractions although the specific fraction affected varied between the conditional knockout mouse models. As these two models have different kinetics we utilised a reverse transplant assay, with similar kinetics to the Mx1-Cre model, to examine the cell intrinsic effect of SCRIB loss in haematopoiesis. Similar to our previous results, this assay revealed a significant role for SCRIB in early lymphoid and myeloid development. The subtlety of phenotype produced by deletion of SCRIB is most likely due to compensatory mechanisms so we induced stress haematopoiesis through irradiation to mitigate the effect of compensatory mechanisms. Lethally irradiated recipients receiving SCRIB knockout bone marrow revealed a more severe phenotype compared to previous assays with defects in erythropoiesis, myelopoiesis and lymphopoiesis. Taken together, our data reveals a role for SCRIB in multiple haematopoietic lineages during steady-state haematopoiesis thereby suggesting SCRIB may be important during leukemogenesis. Studies are currently underway to investigate how SCRIB impacts on the onset and severity of acute myeloid leukaemia and acute lymphocytic leukaemia.
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ItemInvestigating prognostic and predictive tissue biomarkers in head and neck squamous cell carcinoma( 2017)Head and neck squamous cell carcinoma (HNSCC) comprises a diverse group of cancers that arise from a number of subsites of the upper aerodigestive tract, including the oropharynx, oral cavity, larynx and hypopharynx. These cancers have traditionally been linked to tobacco and alcohol use (Gillison et al 2008; Goon et al 2009) and despite improvements in treatment, the outcome for patients with locally advanced HNSCC remains poor. Current treatments are associated with significant acute and late toxicity which can have a significant long term negative impact on function and quality of life. Current tumor staging methods and biomarkers are limited in their capacity to consistently distinguish groups with different outcomes, and to permit tailoring of therapy based on prognosis and biological features. Furthermore, it is recognised that there are significant clinical differences between the different subsites of head and neck cancer, warranting molecular studies focused on individual sites. Thus, to improve patient outcomes, it is becoming increasing important to identify novel biomarkers of prognosis and response to therapy which will empower clinicians with better patient selection strategies for risk-adapted treatments and emerging molecular therapies. It is now accepted that a significant proportion of oropharyngeal cancers, are caused by infection with human papilloma virus (HPV) (Fakhry et al 2008; Ang et al 2010). HPV positive oropharyngeal cancer is known to differ from HPV negative epidemiologically, clinically and molecularly and most importantly, HPV positive status is associated with a greatly improved prognosis (Fakhry et al 2008; Ang et al 2010). The involvement and importance of HPV in non-oropharyngeal HNSCC sites however remains unclear. Other than HPV, which is often determined diagnostically via p16INK4A immunohistochemical staining (an accepted surrogate biomarker in oropharyngeal cancer), there are no clinically useful prognostic or predictive biomarkers in HNSCC. The identification of relevant biomarkers, which can be easily assayed in a diagnostic setting, utilising readily accessible formalin-fixed, paraffin-embedded (FFPE) tissue blocks, is critical to improving patient outcomes. Such assays include immunohistochemistry (IHC) to look at protein expression and fluorescence in situ hybridisation (FISH) for the study of gene copy number. This thesis comprises five published peer reviewed journal articles containing research investigating the role of HPV/p16INK4A in head and neck cancer; both establishing the role of HPV/p16INK4A in oropharyngeal cancer, as well as work demonstrating that the role of HPV in non-oropharyngeal HNSCC subsites including the oral tongue and larynx is not as clear or important as oropharyngeal cancer. Further, I present work investigating several putative predictive biomarkers and their potential roles in providing clinically useful information for potential patient stratification. All of these studies utilised several large unique HNSCC patient cohorts comprising FFPE tissue blocks and comprehensively annotated clinicopathological and outcome data.
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ItemThe role of Pim-1 in breast cancer metastasis( 2017)Breast cancer is the most common cancer in women. Despite advances in treatment options, the spread of breast cancer to distant organs (metastasis) remains the major cause of morbidity and mortality in breast cancer patients. This is attributed primarily to the impairment of function in affected organs. Thus, there remains a vital need for better-targeted treatments that more effectively inhibit the development or progression of metastases. Pim-1 is a serine/threonine survival kinase that has been implicated in the development of metastasis in several haematological and solid cancers. However, little is known about its role in breast cancer. In our laboratory, we previously identified Pim-1 as upregulated in brain metastatic 4T1Br4 syngeneic mouse cells and tumours compared to parental 4T1 cells. This led us to propose that Pim-1 may play a role in mediating breast cancer brain metastasis. Therefore, the overall objective of this project was to examine the expression and functional role of Pim-1 in breast cancer metastasis, with a focus on organ-specific metastasis. We interrogated public databases to show that Pim-1 expression is low to absent in normal breast tissue and increased in breast tumour tissue. Furthermore we show that the murine (4T1Br4) and human (MDA-MB-231Br) brain metastatic breast cancer cell lines and tumours demonstrate the highest expression of Pim-1 mRNA and protein. To investigate the function of Pim-1 in breast cancer metastasis we tested the impact of inhibiting Pim-1, either by gene knock down using short hairpin RNAs or the pharmacological inhibitor SGI-1776, on the ability of 4T1Br4 and MDA-MB-231Br cells to migrate and invade in vitro. 4T1Br4 cells displayed increased migration and invasion propensity after Pim-1 knock down and this was coupled with a decrease in β4 integrin expression. Conversely, MDA-MB-231Br cells showed a decreased ability to migrate and invade after Pim-1 KD, as well as decreased cell surface expression of β1 and β3 integrins. Treatment with SGI-1776 dose-dependently decreased the ability of both 4T1Br4 and MDA-MB-231Br cells to migrate and invade, decreased cell surface expression of β3 integrin in 4T1Br4 cells, and both β1 and β3 integrins in MDA-MB-231Br cells. To examine the effect of Pim-1 inhibition in vivo, we assessed the metastatic spread of Pim-1 knock down MDA-MB-231Br cells in an experimental metastasis assay. After intracardiac injection of Pim-1 knock down cells, we observed a reduction in the number of circulating tumour cells and decreased bone metastasis, indicating a functional role for Pim-1 in breast cancer metastasis to the bone. Data from brain metastasis in this model were inconclusive. In summary, results from this project highlight the importance of Pim-1 in breast cancer metastasis and provide evidence that Pim-1 contributes to the migration and invasion of breast cancer cells both in vitro and in vivo, possibly via regulation of integrin expression, and indicate that Pim-1 is a relevant therapeutic target for the treatment of metastatic breast cancer.
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ItemNo Preview AvailableBioimaging in colorectal cancer - prediction of response to neoadjuvant treatment( 2015)Over the last decade the management of colorectal cancer has changed significantly with the benefits of neoadjuvant therapies and new adjuvant treatments becoming apparent. Surgical strategies have also evolved with initial evidence that some patients can be successfully managed with local excision or omission of any surgery at all, resulting in a shift towards the individualisation of cancer management. The management of rectal cancer is based on primary staging assessment which relies on imaging techniques such as CT, MRI and ERUS. Recent advances in technology have improved the accuracy and widened the applications of these techniques. With the progress in medical and surgical treatments for rectal cancer, the optimal management of rectal cancer has become more complex. The evolving ability to tailor optimal treatment to the individual has created new roles for imaging such as prediction of response to treatment, restaging with assessment of response to treatment and prediction of prognosis. Consequently, prediction of response will become an important component of modern pre-operative assessment of rectal cancer to optimise individualisation of medical and surgical treatment. Beyond the established role of primary staging of malignancies, the role of conventional imaging techniques in re-staging following neoadjuvant treatment may be of increasing importance. Novel functional imaging techniques such as FDG-PET, DW (diffusion weighted) MRI are also emerging, the roles of which are yet to be determined. This thesis will examine the current status of bio-imaging and explore new imaging techniques in rectal cancer. At the Peter MacCallum Cancer Centre, we have been routinely performing staging and restaging imaging with CT, MRI and PET for the last 5 years which has resulted in a cohort of patients in whom these imaging techniques can be evaluated. This thesis also aims to evaluate a recent and evolving functional imaging technique- DW-MRI, in the prediction of response of rectal cancer to chemo-radiation.
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ItemInvestigating mechanisms of metastasis in melanoma( 2016)Metastasis is a complex process that is responsible for most human cancer deaths. Autopsy findings across multiple cancers suggest that metastatic dissemination from the primary site of disease to distant organs is not a random process; cancers can have different predilections to metastasize to particular organs. These observations raise the general question of whether determinants of metastasis, including organ-specificity, are driven by cancer cell-intrinsic or cell-extrinsic factors, or a combination of both. A fundamental question in cancer biology is whether metastasis capability in human tumours is acquired in a heritable manner. Work pertaining to this thesis tested whether organ-specificity during melanoma metastasis is a biologically heritable trait, using PDX modelling. Collectively, these studies suggest that while metastatic potential per se is a biologically heritable trait, organ-specificity of metastasis is not. To examine a candidate cell-intrinsic regulator of melanoma metastasis, desmoglein 2 (DSG2), expression and correlative studies were also performed on patient melanomas. DSG2, a member of the cadherin class of proteins that mediate cell-cell contacts, has been previously but inconsistently detected in human melanoma, however its role and function remain poorly defined. Herein, DSG2 expression was investigated as a potential prognostic factor in melanoma linked to the development of vasculogenic mimicry and poor patient outcomes. These studies confirm that DSG2 is expressed in primary and metastatic melanomas, but not in normal cutaneous melanocytes, and is associated with poor survival in melanoma. Although DSG2-positive melanomas consistently contained more features of VM than DSG2-negative, the differences were not statistically significant.
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ItemCooperative tumourigenesis : analysis of novel tumour suppressors in ras oncogene driven epithelial tumours( 2015)Cancer is a cooperative process, involving mutations in multiple genes. Activation of a cancer-driving gene, the Ras small GTPase, via a mutation that locks Ras in the GTP-bound active form (RasV12), occurs in ~30% of human cancers. However, alone it is not sufficient for tumour formation. A loss of function screen previously performed in the vinegar fly, Drosophila melanogaster, identified 947 genes that potentiate RasV12-mediated tumourigenesis and metastasis (Zoranovic, et al. in prep.). This list has been narrowed down to 234 genes that 1) show increased tumourigenicity with RasV12 in vivo, 2) are in the top 100 genes down-regulated in human cancer, and 3) are known to regulate the cytoskeleton, polarity, adhesion or cell motility. This study has successfully confirmed involvement of autophagy-related genes Atg8a, Atg7 in regulating RasV12-mediated proliferation in the Drosophila eye epithelial tissue using the UAS/GAL4 system. The study identified the autophagy-related genes Atg1, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg12 and Atg101 that when knocked down cooperate with RasV12 and lead to increased tissue overgrowth in the Drosophila eye epithelium. Atg8a was chosen as the representative target gene to investigate this cooperation. It was observed that Atg8a cooperates with RasV12 through the Raf pathway. The role of p62 in this Ras-mediated cooperation with Atg8a was also examined and it was found that p62 levels increase in RasV12+ Atg8aRNAi expressing tissue in comparison with control. Investigations were also carried out to ascertain if knockdown of Atg genes cooperate with Ras through the JNK pathway. It was discovered that in the presence of oncogenic Ras, knock down of Atg8a increases the expression of the JNK target MMP1. The finding of this work could lead to use of this autophagy related genes as prognostic markers in Ras-driven oncogenesis and might reveal effective therapeutic targets to combat this deadly disease.