Projects

Advancing the Clinical Imaging and Treatment of Neuroblastoma Tumours through the use of Conjugated Polymer Nanoparticles.

Neuroblastoma (NB) is a childhood cancer of the autonomic nervous system which regulates involuntary physiologic processes including heart rate, blood pressure and respiration. Children are typically diagnosed with NB at 17 months of age with tumours occurring in the neck, chest, abdomen, or pelvis. In the UK, NB accounts for 6% of childhood cancers (100 annual diagnoses) and 10% of all childhood cancers that result in mortality. More than 50% of NB are diagnosed as being ‘high risk’ where the cancer has spread from its original site to other organ systems. Only 50% of these cases are curable.

Surgery plays an important role in the treatment of NB. However, the surgical removal of tumour tissue is challenging due to its close association with the nervous system. Currently there is no technology that could help direct surgical decision making in real time by selectively and reliably differentiating NB tumours from healthy tissue. Such technology could significantly improve surgical accuracy and, therefore, patient outcomes.

This study will assess the potential of Conjugated Polymer Nanoparticles (CPNs) to act as a highly fluorescent, NB specific imaging agent that could be used in combination with tissue penetrating near infrared (NIR) radiation to guide surgical decision making and improve post-operative patient outcomes. 

Further, we have shown that CPNs act as NIR responsive reactive oxygen species (ROS) generators. CPNs could therefore also become the light activated payload for a novel antibody drug conjugate (ADC) aimed at the selective, ROS based destruction of tumour cells. 

This dual functionality could offer a significant advance in the imaging and treatment of NB and other complex tumours.

Assaying end-of-treatment bone marrows for minimal residual disease in high-risk neuroblastoma

Neuroblastoma is a common cancer in children under five. It is a cancer of the nerve cells that most often grows in the abdomen and spreads early to many sites, including the bone marrow.

Neuroblastomas can be divided into those that are high-risk and those that are low-risk. Less than half of children with a high-risk neuroblastoma survive [1]. High-risk neuroblastomas are treated with intensive chemotherapy to try to kill all the cancer cells that have spread, and the primary tumour is then surgically removed. Children typically then have more chemotherapy, and some may have radiotherapy or other specialised therapies. Despite this intensive treatment, the cancer comes back in 60% of children with high-risk disease [2]. These children are very unlikely to survive.

The aim of our project is to test if we can use cutting-edge DNA-sequencing methods to detect any neuroblastoma cells that have managed to survive the treatment and linger in the bone marrow. To do this study, we will use the tumour and bone marrow samples that are already taken from children as part of routine clinical management. We will find DNA mutations in the tumour and use sensitive sequencing methods to see if we can find the tumour’s mutations in the bone marrow from the end of treatment: if the cancer’s mutations are there, it suggests that some of the cancer cells have managed to survive. If we can detect low-level residual cancer in a child after treatment, we could focus surveillance and further treatment on these children, and relax it for those in whom we cannot detect residual cancer.

Deciphering relapse in B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia is the most common pediatric cancer. Twenty percent of all children relapse, and in these cases, the 10-year survival rate is below 35%. Thus, there is an urgent need to identify the children that will relapse and to develop better treatments for them. In this project we will use the most recent methodological breakthroughs to study both cancer cells (at diagnose and relapse) to better understand relapse. These will allow us to compare relapsed versus non-relapsed patients and discover new biomarkers to predict relapse. In addition, we will characterize for the first time the alterations (e.g., mutations) at regulatory elements that drive B-cell acute lymphoblastic leukemia
development and relapse to discover new therapeutic targets. In summary, this project will provide novel understanding into how cancer cells survive treatment and will enable us to design therapeutic strategies needed for this subset of children with dismal outcomes.

Determining an effective drug combination in paediatric AML

Current treatments for paediatric AML (pAML) are very toxic and result in long-term side effects which significantly affect the quality of life of pAML survivors. Therefore, there is an unmet need for effective, but less toxic treatments. This project has used a drug screening technique to identify a combination of three drugs which are effective at killing pAML cell lines grown in the lab. We are currently expanding our experiments to test the drug combination on pAML cells which are more representative of those within the body.

Evaluation of a novel in vitro model of sonic hedgehog medulloblastoma by single cell transcriptomics

Medulloblastoma (MB) is the commonest malignant brain cancer in children that arises from a structure called the cerebellum. Four subtypes are recognised which have different biology and vary in their outlook. One subtype termed sonic hedgehog (SHH) medulloblastoma (SHH-MB) includes a lethal form that has a mutation in the gene, TP53. In these tumours, primitive so-called cancer stem cells are responsible for tumour initiation and relapse. To understand their biology and find targets for therapy it is essential to grow tumour cells in the laboratory. In general however, these cells are hard to grow with exceptions including a cell line that harbours a mutation in TP53. Unfortunately, these laboratory-grown cells poorly recapitulate patient tumours. Moreover, medulloblastoma mouse models also differ in their properties from patient tumours. For these reasons, therapies that seem promising in the laboratory usually fail in the clinic. Accumulating evidence points to the lack of a human environment for laboratory-grown medulloblastoma cells as a reason for these tumour model deficiencies. To address this issue, we have attempted to recapitulate a cerebellar microenvironment for these lab grown tumour cells by producing ‘mini’ human cerebellum organoids in vitro from a type of human stem cell which can make any cell type in the body. By growing medulloblastoma cell lines in these human cerebellar organoids, we were able to sample thousands of healthy and tumour cells to determine their properties in a technique termed single cell sequencing. Surprisingly, a subgroup of SHH-MB cells adopts a cancer stem cell identity that is not observed in cell lines grown in typical laboratory conditions. We now wish to investigate whether, in patient SHH-MB tumours with and without mutations in TP53, we can detect cells with the same or a closely similar cancer stem cell profile. If so, that would help to validate our laboratory tumour model and could lead to the testing of novel therapies using our model as a platform. Moreover, if our in vitro findings are confirmed in patient SHH-MB this would represent a major advance in the understanding of cancer stem cell biology.

Germline Cancer Predisposition Associated Tumourigenesis

Everyone has a chance of developing cancer throughout their lifetime. However, some people are born with changes in their body’s instructions (known as ‘genes’) that increase this chance. This means that they are more likely to develop cancer, and to develop it earlier in their lifetime. In this study, we want to understand better why this is the case and the impact these changes have on their bodies, as well as on the cancers they develop. In this study we are particularly interested in a rare cancer called adrenocortical carcinoma, which affects the gland on top of the kidneys known as the adrenal gland.

Not everyone has the same gene change that increase their chance of cancer. However, a person with a change in gene A and another person with a change in gene B can develop the same cancer.

We want to understand how the different gene changes lead to the same cancer type. Moreover, we want to know if the same cancer type behaves the same for person A and person B. This will improve patient care as we will better understand how each individual cancer develops and behaves which will allow for personalised treatments for patients.

Genes are contained in a molecule called DNA which is changed to another molecule called RNA in order to carry out its role in the body. To better understand how the DNA and RNA has changed in these tumours, we will cut into the tumours with a laser to obtain DNA and RNA. This will allow us to find changes in different part of the tumour and understand their effects. By doing so we will be able to see differences in the way different tumours behave and function. Through this understanding, we will

be able to hopefully identify key differences which can later be used to treat individual patients in the clinic. Indeed, we might identify differences that suggest cancer A caused by gene change A should not be treated the same as cancer B when gene change B is present which would revolutionise care for patients with a very high chance of developing cancer.

High-dimensional resolution of cell heterogeneity in PRC2 mutated T-cell acute lymphoblastic leukemias (T-ALL)

T-cell acute lymphoblastic leukemia, or “T-ALL”, is a type of blood cancer that affects

children and adults worldwide, with approximately 30 new cases per million children and 10 new cases per million adults diagnosed each year. While current treatments cure about 80% of children, only about 40% of adults are as fortunate. Even for those who are cured the chemotherapy they have to go through makes them very sick and can cause other, long term health problems.

In T-ALL, several cases carry mutations that inappropriately activate a gene called EZH2. This gene is of particular interest, when it comes to cancer, because it is known to help tumor cells in evading chemotherapy and driving the disease relapse. Dr. Giambra and his collaborators have recently generated and validated an innovative method to genetically modify EZH2 in T-ALL cells. Using this experimental approach, they have identified new markers specifically expressed in leukemia cell subsets of T-ALL patients with EZH2 mutations. Based on these findings, the next step of this research activity will be 1) to better understand the biological mechanisms underlying the maintenance and progression of these aggressive cancer cells before and after drug treatments and 2) to optimize a sort of “targeted” therapy against EZH2 mutations which, in combination with conventional chemotherapy, should be more effective in curing patients with T-cell leukemia, at the same time providing lead and means for the treatment of other cancers as well.

Identification of specific and safe immunotherapeutic targets for T-cell ALL using integrative multilayer OMICs

T-Cell Acute lymphoblastic leukemia (T-ALL)is diagnosed at any time throughout lifetime. Despite improved outcomes with intensive chemotherapy, near to 70% ofT-ALL patients end up relapsing or not responding. These refractory/relapsing T-ALL patients become high-risk patients and their clinical outcome is usually unfavourable. The use of the patient's oWD immune cells to eradicate cancer is already a reality for many tumors. Development of manipulated T-cells expressing 2 Chimeric Antigen Receptor (CAR) molecule to specifically recognize and attack the patient's tumoral cells is very effective. Unfortunately, however, T-ALL tumoral cells and normal T-cells share the same cell surface molecules so it has not been possible to develop CAR T-cell therapies for this disease. We here propose to discover a new target to develop and preclinically validate a safe and universal CART-cell treatment in T-ALL.

Investigating the inflammatory immune microenvironment of Burkitt Lymphoma

Burkitt Lymphoma (BL) is a cancer of B lymphocytes that affects children all over the World, is often fatal in low- middle income countries (LMIC), and in high income countries is treated aggressively with chemotherapy leaving children with both short- and long-term side-effects. Overall, the quality of life for children with Burkitt lymphoma is poor and better treatments are urgently needed. When this cancer occurs in areas where malaria is dominant it is called endemic Burkitt lymphoma whereas in other regions it is considered to develop as a sporadic disease. In both cases, the cancer doubles in size every 24 hours and therefore needs rapid and aggressive treatment. The past few decades have witnessed a tremendous interest in and understanding of the role of immune cells in cancers, and how these can be ‘reprogrammed’ to kill cancer cells. This field is known as cancer immunotherapy. Although significant progress has been made in studying tumour-immune cell interactions and identifying/administering immunotherapies to adults with solid tumours, progress on these fronts has been restricted for paediatric malignancies, particularly the rarer (and most fatal) ones such as Burkitt Lymphoma. This study serves to bridge this gap by comprehensively studying the immune microenvironment of Burkitt Lymphoma to understand the roles played by specific immune cell types in governing disease progression/therapeutic response. The over-arching aim is to identify novel targets for the immunotherapy of this cancer to improve the lives of children with this disease.

Investigating tumour heterogeneity and genetic and non-genetic determinants of evolution in paediatric solid tumours, through the analysis of post mortem samples, with a focus on neuroblastoma and sarcomas

Neuroblastoma and sarcomas cause a large proportion of cancer deaths in children and young people. In order to find better treatments, we need to understand how cancer changes over time. We also know that cancer is heterogeneous, so different areas present different characteristics. However we still need to understand in depth tumour heterogeneity, in order to design treatments which can target the whole cancer. We are already collecting tissue and blood from many patients, through two research studies. With this proposal, we intend to study the tissue taken post mortem, when parents have consented to the donation, to understand how cancer evolves and how cancer is different in different areas

Investigating whether chemotherapy-induced senescence affects the behaviour of cancer stem cells in Wilms Tumour

Wilms tumour (WT) is the most common kidney cancer in children and is diagnosed in around 85-90 children every year in the UK. WT forms because cells that are important for the formation of the kidneys during human development, remain after birth, become abnormal, multiply and form a cancer. The treatment of children with WT includes chemotherapy and surgical removal of the cancer which is generally successful. However, in about 10-15% of affected children the cancer grows back (relapses) and may be fatal.

We believe that a main reason why cancers can relapse is because specific cancer cells are not removed by chemotherapy, and/or not fully removed by surgery. It is thought that during chemotherapy, cells within the cancer communicate with each other, allowing specific cancer cells to remain and potentially grow back or migrate to other organs. Instead of being destroyed, these cells adapt to escape the effects of chemotherapy. One method of adaptation is for cancer cells to become inactive or ‘senescent’, which normally means the cells will stop growing. Unfortunately, senescent cells may re-activate, probably because they communicate with neighbouring cells some time following the end of chemotherapy.

Amongst the neighbouring cells are inflammatory cells which may also be involved in the re-activation. Some re-activated cells can behave as cancer stem cells. These cancer stem cells and other re-activated cells often reside within a specific region of the cancer called ‘blastema’. Increased levels of chemotherapy- resistant blastema content in WT are linked to a higher risk of relapse. The cancer stem cells may either lead to relapse at the site of the kidney (if they have spread locally before the kidney is removed), or potentially spread, leading to relapse elsewhere (most commonly the lungs in WT). Our understanding of these biological processes is very limited.

This research proposal will increase our knowledge of how cancer cells communicate with neighbouring  cells, become senescent, re-activate, and how some of those cells behave as cancer stem cells. This understanding has major implications for chemotherapy resistance and future disease relapse. The knowledge gained in this project will contribute to work on driving new therapies which may block these adaptations thus preventing relapse.

In this study we aim specifically to characterise the cancer stem cells and other re-activated cells within the blastemal areas of tumour together with neighbouring cells in archived tissue from WT patients. To achieve this, we will use novel technologies to determine the full set of molecular markers specific for cancer stem cells and blastema cells and define their individual characteristics and behaviour. Furthermore, we aim to explore the signalling molecules with which the cancer stem and blastema cells and their neighbouring cells communicate with each other and more widely within the cancer. This understanding will allow us to more accurately identify which Wilms tumours contain cancer stem cells and blastemal cells and are potentially at increased risk of relapse. The identified pathways may prove susceptible to targeted therapies providing a novel treatment approach.

Learning from outliers to improve the outcomes of children with rhabdomyosarcoma

We believe that is feasible to deliver immediate benefits to children with rhabdomyosarcoma by targeting existing therapies more precisely. For example, some children with non-high-risk disease will relapse. If we knew who these children were at diagnosis, we could treat them more intensely from the outset. In order to target existing therapies more precisely, we require markers that accurately predict tumour behaviour and that outperform existing established markers of risk (eg, fusion status, site of tumour, age, etc.). The aim of our work is to discover such markers.

Molecular profiling of craniopharyngioma

Craniopharyngioma is a challenging tumour that affects children and adults. These tumours often have solid and liquid/cystic parts. Growth, close to important structures in the brain results in damage to important brain structures leading to blindness, hormone problems, difficulty regulating weight and poor quality of life. Current treatment is surgery with or without radiotherapy, however despite this around one quarter of cases regrow. Our previous study of samples within previous CCLG projects, and other, studies has improved our understanding of the biological processes causing tumours to develop and grow. Through this, we have identified potential new therapies which will now be tested in clinical trials. In this study we want to continue to improve our understanding of this type of tumour by analysing the DNA, RNA, proteins and other processes, e.g. energy processes, called metabolism, inflammation, in solid and fluid parts of the tumours. We will also analyse samples from patients treated with new treatments to better understand how these do, or don’t, work. We will continue to work closely with the wider craniopharyngioma research community around the UK and world.

Molecular profiling of Malignant Rhabdoid Tumours to support translation of molecular biological diagnostics to the clinic.

Malignant Rhabdoid Tumours (MRT) are rare but aggressive tumours of early childhood. Prognosis is relatively poor and current treatments are often ineffective or else frequently toxic and/or have a high risk of damaging long-term effects. For many other childhood cancers, biological profiling is used to discriminate between patients who are likely to respond to therapy and those not. In this way damaging therapies are avoided in low-risk patients and novel therapies attempted in those patients for which current therapies are known to be ineffective. This type of precision medicine has not yet been applied to treating MRTs, in part because of the comparative rarity of the disease and a lack of clinical trials.

By analysing biological samples from MRT patients collected retrospectively, we and others have shown a potential relationship between biology and response to therapy. Using state-of-the-art genomics technology, profiling tens of thousands of genes, we can detect at least three biological subtypes of MRT which appear to impact upon patient survival.

Furthermore, we can use artificial intelligence techniques to extract biological patterns which predict at the point of diagnosis, how likely a patient is to respond to their treatment. Whilst this is a promising development there are limitations, in terms of the size of the studies and the extent to which patients were treated in a uniform way, which prevent us from confidently advancing these findings into everyday practice. DNA from tumours can be detected in the biological fluids (i.e. blood, spinal fluid) of patients. 

Almost every malignant rhabdoid tumour has a mutation of one of two genes, SMARCB1 and SMARCA4. This mutation is key to driving the tumour cells aggressive behaviour and we anticipate that tumour DNA bearing these characteristic mutations can be detected and quantified in biological fluids sampled from patients with these tumours. This should mean that the amount of circulating tumour cells present in a patient can be estimated and these measures used to track the progress of a patient during treatment. This should help clinicians to monitor a patient’s response to therapy in real time. These measures could also be used to monitor patients who are in remission and anticipate early if they are likely to relapse again.

We aim to validate and develop further these methods to provide an important tool for clinicians to manage treatment of these patients. If successful we would anticipate these techniques being rolled out to ongoing clinical trials for testing in even larger numbers of patients and to bring these techniques into common clinical practice.

Next Generation Precision Medicine Program: Molecular and Functional Characterization of Paediatric Solid Tumours

We are some of Victoria’s leading research, academic and clinical organisations, working together to make things better for children and adolescents with cancer through world-class medical research and innovation. This work demands that we harness cutting edge technology and methods. Together, the VPCC’s researchers, doctors and families of kids with cancer can deliver research with real-world impact, better medical care, and improved training for the researchers and clinicians of the future. Our partnership organisations are The Royal Children’s Hospital (RCH), Monash Children’s Hospital (MCH), Hudson Institute of Medical Research, Murdoch Children’s Research Institute (MCRI), Monash University, University of Melbourne, Peter MacCallum Cancer Centre, Walter and Eliza Hall Institute of Medical Research (WEHI) and the Children’s Cancer Foundation. Childhood cancer investigators from these organisations have joined forces to drive impactful and interdisciplinary research, improve medical care, and train the childhood cancer leaders of tomorrow.

Current precision medicine programs focus on genomic sequencing to identify discrete mutations that may predict patients’ responses to targeted therapies. Unfortunately, fewer than 20% of cancer patients harbour actionable mutations, and of those who do, only 50% respond to therapy. This underscores a clear need to go beyond genomic sequencing to identify new targeted therapies for paediatric cancers of poorest survival (i.e brain and soft tissue tumours).

The VPCC Next generation precision medicine program aims to identify the next generation of paediatric cancer-targeted therapies through a multi-pronged, paediatric-centric approach that encompasses:

1. Generation of novel models of childhood cancers that faithfully represent the patient’s tumour;

2. Characterisation of models at a multi-omics level (genome, transcriptome, epigenome, proteome);

3. Comprehensive functional genomic screens to identify the genetic drivers and dependencies of low-survival paediatric cancers; and

4. Development of a childhood cell line atlas and data portal to enable cohort-level integrative genomic analyses.

Importantly, paediatric models in VPCC research are coupled to patients whose clinical and molecular data is tracked (in partnership with the Zero Childhood Cancer program). This offers a unique opportunity to correlate patient responses in the clinic/clinical trial setting with both molecular variants and functional dependencies identified in the patient’s avatar model.

To investigate the expression of DLL3 and associated immune populations in neuroblastoma as a potential therapeutic target

Neuroblastoma is one of the most common solid cancers in children. For patients with advanced disease or those where the disease comes back, the chance of cure remains inadequate. Therefore new treatments are desperately needed. In this project we will investigate if a marker called DLL3 is found on neuroblastoma cells, using stains which can be read under the microscope. We will also look to see what immune cells are inside neuroblastoma tumours. By understanding this information we will know whether a new drug that directs the immune system to kill DLL3 positive cancer cells could be used in children with neuroblastoma.

Uncovering mechanisms of transcriptional treatment resistance in T-ALL induction failure

Acute lymphoblastic leukaemia (ALL) is the commonest cancer in children, occurring in approximately 400 children each year in the UK. Although many children can be cured, a significant number still die from the disease. Recent work has shown that children with a subtype of ALL called T-ALL, who fail to respond to the first four weeks of treatment, have a very poor outcome. Recently we carried out a detailed study to see if we could find out more about this group of patients including reasons why their disease is so difficult to treat. 

Often cancer becomes resistant to therapy because of changes (‘mutations’) in the DNA. However we couldn’t find any of these types of changes in the T-ALL group. Given this, we plan to try a new technique called single cell RNA-sequencing. As the name suggests, this allows us to study individual cells so we can find out more about the different programs controlling the cell. We believe this will give us a much greater understanding of why the leukaemia is resistant to chemotherapy and will lead to the development of new more effective treatments.