Sir Peter MacCallum Department of Oncology - Theses

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    Tracking Disease In Breast Cancer Using Circulating Tumour DNA
    Lo, Louisa Lisa ( 2023-03)
    Plasma circulating tumour DNA (ctDNA) correlates with disease status in breast cancer and can provide more comprehensive genomic information than the heterogeneous nature of a single biopsy site. In metastatic breast cancer it allows the study of underlying cancer driving mutations, characterization of treatment resistance and can potentially guide future treatment adaptation and selection to improve patient survival. In early breast cancer, it has also been explored as a potential minimal residual disease (MRD) biomarker with the promise that it can significantly predict disease relapse and aid in prognostic stratification. This work has utilised ctDNA as a minimally invasive strategy to study molecular information in patients undergoing novel combination and targeted therapies in breast cancer. Using different sequencing technologies, this work has shown the feasibility and ability of ctDNA to predict and profile the molecular subtypes of breast cancer that would respond to different treatments. Additionally, serial analysis of ctDNA has been able to accurately monitor disease during therapy. When breast cancer patients progress on treatment, the molecular changes captured by ctDNA were studied to characterize genomic resistance mechanisms associated with the applied therapies, providing insights into future strategies to circumvent these changes. Nevertheless, the reliance on genetic information alone has limited the sensitivity and specificity of ctDNA as a prognostic biomarker in cancer of low volume disease. This thesis has therefore explored new methodologies to interrogate the epigenetic profile of ctDNA in breast cancer. When coupled with ctDNA genetic information, a breast cancer ctDNA classifier was derived and tested for its sensitivity and specificity for disease detection in patients with oligometastatic disease. As a result, this work provides a platform for future research to refine the use of combined genomic and epigenomic ctDNA test as a sensitive and specific disease monitoring strategy in high risk early and metastatic breast cancer.
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    Clinical implications of genomic driver alterations in hormone receptor-positive, human epidermal growth factor receptor (HER) 2-negative early breast cancer
    Luen, Stephen James ( 2021)
    Global initiatives applying next generation sequencing to thousands of primary and metastatic tumour samples have detailed the genomic landscape of breast cancer. These unique insights have transformed our understanding of the biological processes underlying these tumours. Despite this, the clinical relevance of these findings remains poorly understood. This is because these datasets are frequently heterogeneous, include patients that may not have received contemporary treatments, often have limited clinical annotation, and commonly lack long-term follow-up for patient survival outcomes. In order to better assess the clinical relevance of genomic driver alterations in breast cancer, this research project performed genomic sequencing using primary tumour samples from patients enrolled in two pivotal phase 3 clinical trials – the Breast International Group 1-98 (BIG 1-98) study and the Suppression of Ovarian Function Trial (SOFT). This provided a well-defined population with hormone receptor-positive, human epidermal growth factor receptor (HER) 2-negative early breast cancer in both postmenopausal and premenopausal populations, as well as long-term survival outcome data collection. Crucially, this allowed for further investigation of late distant recurrence, a unique feature of hormone receptor-positive, HER2-negative breast cancers, as well as the ability to focus on distinct high-risk subpopulations, such as breast cancer arising in young women. In the postmenopausal BIG 1-98 study, comprehensive panel sequencing was performed using 538 tumour samples from patients with hormone receptor-positive, HER2-negative early breast cancer. PIK3CA mutations were the most common driver (49%) and were associated with a reduction in the risk of distant recurrence. Contrastingly, TP53 mutations, amplifications on 11q13 and 8p11, and increasing number of driver alterations were associated with an increase in the risk of distant recurrence. Additionally, PIK3CA mutations were predictive of a greater magnitude of benefit from letrozole compared with tamoxifen. Using the same dataset, we further investigated the association of oncogenic drivers with late distant recurrence (5 years or greater from the time of randomisation). PIK3CA mutations were associated with reduced risk of late distant recurrence, whereas amplifications on 8p11 and BRCA2 mutations were associated with increased risk of late distant recurrence. In the premenopausal SOFT study, comprehensive panel sequencing was performed using 1258 tumour samples from the entire study cohort, and whole exome sequencing was performed using 82 tumour samples from very young patients with high-risk, hormone receptor-positive, HER2-negative early breast cancers. Subgroups of tumours with distinct copy number-amplifications had a higher risk of distant recurrence than tumours that were amplification-devoid, validating previously reported data. Importantly, the study further defined unique molecular characteristics that were enriched in very young women and may explain their poor prognosis. This includes subgroups of tumours with homologous recombination deficiency, a high-risk PIK3CA mutated subgroup, amplification-enriched subsets, and a low estrogen receptor expressing subtype with immune infiltration. Taken together, these results highlight the clinical relevance of genomic sequencing of hormone receptor-positive, HER2-negative early breast cancers. They provide a new prognostic framework using genomic stratification, and highlight molecular targets that should be prioritised for future clinical trial research.
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    Circulating tumour DNA analysis for personalised care in breast cancer
    Zivanovic, Andjelija ( 2020)
    Phenotypic diversity of breast cancers poses insurmountable challenges in the treatment of this lethal disease. Recent advances in next generation sequencing have led to unprecedented insight into the genomic landscape underlying breast tumours. This has resulted in burgeoning development of targeted treatments and predictive biomarkers, several of which have recently demonstrated clinical activity. However, key challenges hinder optimal application. On the background of extensive molecular heterogeneity, most biomarkers represent minority patient subpopulations, hampering clinical development. Furthermore, considerable genomic evolution of breast tumours impacts accuracy of genomic characterisation that is thus far heavily reliant on the sequencing of non-contemporaneous and invasive tumour tissue biopsies. Finally, stratification to genomically-matched targeted therapies also fails to fulfil the extent of its promise. In many cases relentless tumour growth remains unperturbed, while in others resistance ultimately develops. Crucially, molecular mechanisms underlying resistance remain poorly understood, while follow-on treatment options are often poorly defined. Central to the promise of personalised medicine is the robust and accurate characterisation of the tumour genome. Minimal invasiveness and convenience of circulating tumour DNA (ctDNA) analysis, with ability to detect tumour genomic aberrations from a blood draw, highly recommends this approach. Recent technological advances have paved the way to a range of clinical applications, with evolving potential for ctDNA analysis to address the continuum of challenges posed to precision medicine throughout patient management. Toward this end, extensive clinical development is required, while prevailing technological hurdles need to be addressed. This thesis explores a multi-faceted and rigorous approach towards the integration of ctDNA analysis in the management of breast cancer patients. Firstly, the development and validation of multiple assays (allele-specific and NGS-based) tailored to breast cancers, enabled comprehensive genomic analysis with in-built flexibility to be readily applicable to a variety of clinical scenarios. Subsequent establishment of a prospective ctDNA-based molecular profiling program across a large cohort of metastatic breast cancer (mBC) patients demonstrated feasibility of real-time analysis in the clinical setting across a range of genomic targets of variable abundance. Importantly, integration of longitudinal testing in this program throughout patient management demonstrated capacity for ctDNA analysis to reflect genomic evolution in real-time to optimise precision-guided patient management. Finally, exploratory longitudinal ctDNA analysis for the study of resistance mechanisms to CDK4/6i, constituting a novel class of targeted compounds for breast cancer, highlights established and novel resistance markers. Indeed, this study also serves to demonstrate a workable framework for ctDNA analysis as a highly effective approach for the de novo elucidation of resistance mechanisms to novel targeted agents that is relevant across cancer types.
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    Effectiveness and cost-effectiveness of programs for BRCA pathogenic variant carrier cancer risk management
    Petelin, Lara ( 2019)
    Background Women who inherit a germline pathogenic variant in the BRCA1 or BRCA2 genes have a significantly elevated lifetime risk of breast and ovarian cancer. Women who are aware of their BRCA carrier status can mitigate their increased risk by undergoing intensive breast cancer screening from a young age for early detection, and risk-reducing surgery for prevention of breast and/or ovarian cancer. The effectiveness of these interventions is dependent on BRCA carriers taking up these risk management strategies at an appropriate time, considering factors such as their age, personal preferences, and life stage. The most effective approach to ensuring carriers adhere to risk management recommendations is unknown. This project evaluates the lifetime health outcomes and cost-effectiveness of long-term clinical management of BRCA carriers in the context of structured clinical programs, using real-world data. Methods This thesis describes the development and outcomes of a discrete-time state-based microsimulation model. The model, named miBRovaCare, simulates the gene-specific natural histories for breast and ovarian cancer in BRCA carriers. Cost-effectiveness and cost-utility analyses were performed to evaluate the lifetime outcomes of different approaches to clinical management of carriers from the perspective of the Australian public healthcare system. The comparator for the base case analysis was the natural history (no cancer risk management). The interventions included: (i) a structured familial cancer service with a multidisciplinary high-risk clinic, and (ii) a formal annual carrier review program. For the intervention arms, BRCA carriers could undergo annual breast imaging, risk-reducing bilateral or contralateral mastectomy, and risk-reducing bilateral salpingo-oophorectomy. Uptake of and adherence to these strategies (patient behaviour) was based on an analysis of 983 BRCA carriers seen through a clinic in Melbourne, Australia. Additional model inputs were obtained from a local hospital database, the literature, government reports, and expert opinion. Costs and health outcomes were discounted by 5%. Results Long-term management of BRCA carriers through a familial cancer service is likely to be cost-effective, with or without an annual review program. A familial cancer service was the preferred strategy if the willingness-to-pay was at least $29,000 per quality-adjusted life-year (QALY) for BRCA1 carriers and $57,000 per QALY for BRCA2 carriers. Inclusion of an annual review program for BRCA1 carriers had a 75% probability of being cost-effective at a willingness-to-pay threshold of $50,000 per QALY. For BRCA2 carriers, a familial cancer service with or without an annual review program had only a 37% probability of being cost-effective at a $50,000 per QALY willingness-to-pay threshold. Discounting of health outcomes had, by far, the greatest impact on cost-effectiveness outcomes. Conclusions This thesis describes a novel microsimulation model for optimising clinical management of BRCA carriers. BRCA carriers are likely to benefit from access to structured clinical programs and regular review, due to fewer cancer diagnoses, improved life expectancy and an increase in QALYs. Genetic testing for hereditary breast and ovarian cancer predisposition syndromes is steadily expanding, and may even be available at a population-based level in the near future. Maximising adherence to evidence-based risk management guidelines along with access to appropriate follow-up services will therefore be increasingly important. The model developed for this thesis can enable faster evaluation of emerging risk management strategies and behavioural interventions, and can be easily adapted to alternative settings and healthcare systems.
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    Identification and validation of novel breast cancer predisposition genes
    Li, Na ( 2019)
    The genetic causes of the majority of hereditary breast cancer families remain unresolved (BRCAx families) and lack of this information compromises primary and secondary cancer prevention for the affected women and their family members. Despite intensive efforts, no major new breast cancer predisposition genes with equivalent impact have been discovered since the identification of BRCA1 and BRCA2 in the 1990s. Research from our laboratory provided direct demonstration that the remaining hereditary causes of breast cancer are not due to a few BRCA1-like genes (high penetrance and high carrier frequency) but instead by numerous moderate penetrance genes, each accounting for only a small fraction of families. This finding highlighted the need for very large case-control studies as the only way of resolving the missing breast cancer heritability. In this thesis I aimed to carry out such a study. Using existing germline whole exome and targeted sequencing data, 162 candidate genes were selected for validation in ~8,000 unrelated BRCAx cases and cancer-free controls. Combined with the existing targeted sequencing data of ~4,000 cases and controls, this analysis represents one of the largest targeted sequencing studies of its kind. A significant association with breast cancer predisposition was confirmed for known breast cancer genes PALB2, CHEK2 and ATM. Among the 162 candidate genes, nearly twice as many had more LoF variants in the cases than the controls, compared to those with more variants in the controls than the cases, demonstrating a high enrichment for genuine breast cancer predisposition genes. Most novel candidate genes appeared to convey only low to moderate risks, with a total of 35 genes identified that had an OR>2. Despite the very large sample size, the number of carriers of LoF variants for any candidate gene was still small, further demonstrating the extreme genetic heterogeneity of BRCAx families, and that case-control data is still insufficient on its own to claim the discovery of a specific new breast cancer gene. Nevertheless, this large-scale case-control data will be a valuable guide for future validation. Many of the candidate genes such as NTHL1, WRN, BAP1, PARP2 and CDK9 play essential roles in DNA damage repair, consistent with the function of all the established breast cancer genes. An intriguing exception is CTH which is involved in the trans-sulfuration pathway, and if confirmed, would be the first moderate penetrance breast cancer predisposition not involved in DNA repair. This study also re-evaluated some contentious genes and provided strong evidence to reject RINT1 and RECQL as breast cancer predisposition genes. On the other hand, by combining both case-control data with tumour sequencing data, RAD51C was identified as a triple-negative breast cancer predisposition gene. This study has shown that because of the high genetic heterogeneity of BRCAx families, future validation studies will require either an extremely large sample size (>10,000 subjects), and/or the inclusion of approaches providing independent evidence, such as tumour sequencing to identify bi-allelic inactivation and specific mutational signatures.
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    Genotypic phenotypic correlation in male breast cancer
    Deb, Siddhartha ( 2018)
    Male breast cancers (MBCs) are rare cancers, comprising less than 1% of all breast cancers and less than 1% of all cancers in men. As a result, these cancers have not been well characterized with almost all management extrapolated from the treatment of female breast cancers. More recent studies, however, have demonstrated differences from female breast cancers. This thesis has examined genotypic and phenotypic correlation in a group of 61 familial MBCs (kConFab) and 225 mixed familial and sporadic MBCs (Lund University, Sweden) with robust clinical and pathological data. The hypothesis is that: 1) male and female breast cancer (FBC) is different, 2) familial male and familial FBC is different and 3) familial and sporadic MBCs are different with possible differences within familial MBC subgroups. The penetrance of familial MBCs is different to that of familial FBC, showing an increased proportion of BRCA2 male carriers and underrepresentation of BRCA1 male tumours. Histopathology showed a paucity of lobular and medullary male breast cancers, with less frequent HER2 and Basal phenotypes. An association between BRCA2 mutation carrier status and invasive micropapillary subtype was seen. A BRCA1 associated medullary phenotype was not demonstrated and accordingly TP53 somatic mutations and associated hypoxic drive was not seen in these tumours, highly suggestive of a redundant role for BRCA1 in MBC. The somatic mutation profile in familial MBCs was similar to that seen in luminal A FBCs, with the rare E547K PIK3CA seen several times in MBCs suggesting a possible gender correlation. Methylation of the ERβ/eNOS complex associated tumour suppressor gene, GSTP1, was frequently seen in MBCs, more so in familial than sporadic MBCs. The clinical significance of this may be useful in screening for MBCs in these high-risk populations and may also be a risk modifier as high levels of GSTP1 methylation have also been seen in BRCA2-associated prostate cancers. Similar to familial FBCs, there was observed clustering of tumors by methylation patterns into different BRCA subgroups, albeit with small numbers. Compared to sporadic MBCs, familial MBCs overall showed an earlier median and mean age of onset, with more frequent multifocality or bilateral disease, Familial MBCs also showed a higher proportion of high grade tumours and invasive papillary carcinomas. Familial MBCs also showed a higher amount of gene losses and higher levels of candidate gene methylation. The study demonstrated several differences between male and female breast cancers and sporadic and familial MBCs.
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    An investigation of type-1 interferon and the immune response against breast cancer metastasis
    Rautela, Jai ( 2016)
    Breast cancer is a highly prevalent disease that, like many cancers, lacks effective therapies aimed at treating and preventing metastasis. Harnessing the host immune system to recognise and eliminate malignant cells has recently emerged as an effective therapeutic strategy in many cancers. However, response rates to these approved immunotherapies remain modest in the absence of a more detailed understanding of tumour immunity. The type I interferons are a family of cytokines that have long been understood to enhance the immune response to cancers, though their clinical application has led to underwhelming results in numerous types of cancer. This thesis provides new evidence that proposes the re-visitation of cancer immunotherapies that stimulate the type I interferon pathway. We show that host-derived type interferon is critical for the suppression of breast cancer metastasis through natural- killer cell activation. Induction of a type I IFN response by administering agents that mimic a viral infection (poly(I:C), a double-stranded RNA analog) proved to be powerful anti-metastatic agents in multiple pre-clinical models of triple-negative breast cancer (TNBC). This was linked to widespread immune activation which conferred NK cells with enhanced cytotoxic function to eliminate disseminated tumour cells. The efficacy of this novel immunotherapeutic approach was also found to rely upon the treatment setting in which it was used. Evidence is presented that demonstrates administration prior to primary tumour removal (neo-adjuvant therapy) as the only effective therapeutic regimen. We propose that such immunotherapies are most effective at eliminating circulating and early disseminated cells rather than established metastatic lesions. This provides some explanation to the inefficacy of previous interferon trials that were conducted in patients with late-stage metastatic disease. It also calls into question whether other immunotherapies could be used earlier in cancer treatment to maximise the chances of a clinical response. Finally, we uncover that expression of IRF9, a key transcription factor in the type-I interferon signaling pathway, accurately predicts TNBC patient prognosis. Loss of IRF9 in a patient’s primary tumour predicted significantly poorer overall survival due to metastatic spread. As we show that tumour cells are not directly responsible for the poly(I:C)-induced interferon response, we propose that patients with IRF9-negative TNBC could benefit from neo-adjuvant interferon-based immunotherapy.
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    Investigating MYB in the context of mammary gland biology, transformation and as a therapeutic target in breast and colon cancer
    Cross, Ryan N S ( 2014)
    Breast cancer is the second most common form of malignancy diagnosed in Australian women, imposing an enormous social and economic burden on society. If the cancer spreads to secondary locations, patient survival decreases dramatically. Therapeutic strategies for treatment of metastatic disease are desperately needed in breast cancer. Recently we have shown that when Myb is conditionally deleted from the mammary gland of MMTV-Neu and MMTV-PyMT mice, tumour formation is ablated. To provide further insight into the function of Myb in mammary gland development, cre-mediated conditional knock out of c-myb in the mouse mammary gland was examined. The conditional deletion of c-myb led to a reduction in branching and terminal end bud formation. These data indicate that if MYB could be inhibited for breast cancer therapy, there are potentially few side effects on the normal mammary gland. The ability to inhibit DNA binding transcription factors is a long sort after goal in oncology, as their importance in disease initiation and progression is well documented. To target MYB, we have developed a DNA vaccine. Preclinical studies have largely examined the MYB DNA vaccine in the context of colon cancer models using prophylactic vaccination. However, preliminary data indicate that it may also be effective in reducing the metastatic burden in preclinical breast cancer models. This thesis aims to further investigate the role of MYB during mammary gland development and provide insights into its involvement in tumourigenesis. Furthermore, the MYB DNA vaccine will be assessed for its effectiveness as a therapeutic treatment in clinically relevant surgical models of metastatic breast cancer, as well as further development as a therapeutic in the setting of colon cancer.