Clinical Research (Excluding Clinical Trials), 2021
Background: Minimal Residual Disease (MRD) detection is vital for therapy monitoring and relapse ... more Background: Minimal Residual Disease (MRD) detection is vital for therapy monitoring and relapse prediction in cancers. While RNA-based MRD assays are shown to be effective at diagnostic time-points, serial response monitoring does not provide additional predictive value.DNA-based assays may be more sensitive and allow longitudinal monitoring of the disease trajectory. This study aimed to develop MRD assays based on whole genome sequencing (WGS) data for high-risk neuroblastoma (HRNB) and Ewing sarcoma (ES) patients, to establish their sensitivity in quantitative PCR (qPCR) anddroplet digital PCR (ddPCR) formats, and to directly compare against RNA-based MRD assays. Methodology: We analyzed WGS data to identify patient-specific chromosomal breakpoints in HRNB (N=6) and ES (N=6) patients, then established qPCR and ddPCR assays. Assay performance was validated using patient-derived cells spiked into bone marrow samples and in serially collected clinical samples as available. The DNA-M...
516 The majority of child cancer arises in embryonal cells which have persisted beyond birth, hav... more 516 The majority of child cancer arises in embryonal cells which have persisted beyond birth, having failed to undergo cell death following normal organogenesis in utero. The mechanism of embryonal cell persistence, and thus, tumour initiation are unknown. Resistance to spontaneous regression by neuroblasts is an important early step in human neuroblastoma tumorigenesis. We have previously shown that perinatal expression of a MYCN transgene in paravertebral murine neuroblasts mediates transient neuroblast proliferation and resistance to apoptotic cell death, as the first step in neuroblastoma tumorigenesis (Hansford et al., PNAS 2004). Here we analysed the mechanism of resistance to apoptotic cell death in primary ganglion cells from MYCN transgenic, compared with normal mice. MYCN-mediated resistance to cell death was seen in ganglion cells following a variety of different death stimuli: nerve growth factor (NGF) withdrawal, hypoxia, nutrient deprivation and doxorubicin. The mRNA e...
Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplificat... more Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplification) of the cancer-causing gene MYCN strongly predict poor outcome. Neuroblastomas are reliant on high levels of cellular components called polyamines for their growth and malignant behavior, and the gene regulating polyamine synthesis is called ODC1. ODC1 is often coamplified with MYCN, and in fact is regulated by MYCN, and like MYCN is prognostic of poor outcome. Here we studied a naturally occurring genetic variant or polymorphism that occurs in the ODC1 gene, and used gene editing to demonstrate the functional importance of this variant in terms of ODC1 levels and growth of neuroblastoma cells. We showed that this variant impacts the ability of MYCN to regulate ODC1, and that it also influences outcome in neuroblastoma, with the rarer variant associated with a better survival. This study addresses the important topic of genetic polymorphisms in cancer. Abstract Ornithine decarboxylas...
The ultimate cause of treatment failure for many forms of malignant and infectious diseases is th... more The ultimate cause of treatment failure for many forms of malignant and infectious diseases is the development of resistance to a broad range of cytotoxic drugs. Hence, despite significant advances in cancer treatment, multidrug resistance (MDR) remains a major clinical problem and leads to limited therapeutic options and poor patient outcome in a number of cancers, including high-risk neuroblastoma, leukaemia and carcinomas of the prostate, breast, lung and ovary. Therefore, gaining a better understanding of the mechanisms that lead to drug resistance is of utmost importance in order to improve the current chemotherapy regimes in cancer treatment. While MDR may develop in response to a specific drug or drug combination, the resultant phenotype typically confers resistance to a variety of agents, often with diverse mechanisms of action. A large body of research devoted to exploring cellular MDR has revealed that resistance most commonly arises through (a) insufficient uptake of wate...
DIPG is an aggressive paediatric brainstem tumour, with a median survival of less than 1 year. Po... more DIPG is an aggressive paediatric brainstem tumour, with a median survival of less than 1 year. Polyamines are intracellular polycations that control important aspects of cell growth and are often upregulated in cancer. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is a key driver of polyamine synthesis. We investigated the efficacy of polyamine pathway inhibitors as a therapeutic strategy against DIPG. We found high expression levels of synthetic enzymes in the polyamine pathway in primary patient samples and cultures. Using cytotoxicity and clonogenic assays, we found that DFMO inhibited the proliferation of DIPG neurospheres. However, DIPG cells compensated for DFMO inhibition by increasing expression of the polyamine transporter SLC3A2. Gene expression analysis showed that the polyamine transporter, SLC3A2, was significantly overexpressed in DIPG compared with all other high-risk childhood cancers. Addition of polya...
Biochimica et biophysica acta. Gene regulatory mechanisms, 2018
Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tu... more Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy, remarkably refractory to therapeutic treatments. One of the most powerful independent prognostic indicators for this disease is the amplification of the MYCN oncogene, which occurs at high levels in approximately 25% of neuroblastomas. Interestingly, amplification and not just expression of MYCN has a strong prognostic value, although this fact appears quite surprising as MYCN is a transcription factor that requires dimerising with its partner MAX, to exert its function. This observation greatly suggests that the role of MYCN in neuroblastoma should be examined in the context of MAX expression. In this report, we show that, in contrast to what is found in normal cells, MAX expression is significantly different among primary NBs, and that its level appears to correlate with the clinical outcome of the disease. Importantly, controlled modulation of MAX expression in neurob...
DIPG is an aggressive pediatric brainstem tumor, with a median survival below 12 months. Tumor ce... more DIPG is an aggressive pediatric brainstem tumor, with a median survival below 12 months. Tumor cells are dependent upon arginine, a semi-essential amino acid, metabolised by arginase enzymes into ornithine, a pivotal precursor to the polyamine pathway. Polyamines, frequently upregulated in cancer, are intracellular polycations controlling key biological processes – the inhibition of which we have previously shown to be highly efficacious in preclinical DIPG models. Pegylated arginase (BCT-100) has recently been shown to significantly delay tumor development, prolonging survival of neuroblastoma-prone Th-MYCN mice. This study investigated the effects of arginine depletion therapy as a single agent and in combination with polyamine pathway inhibitors in DIPG. We found that ARG2, the gene encoding for arginase II, is expressed significantly more highly in DIPG tumors compared to normal brain. Arginine depletion via BCT-100 reduced DIPG cell proliferation and colony formation in patient...
Approximately 25% of children with acute lymphoblastic leukemia (ALL) relapse after frontline the... more Approximately 25% of children with acute lymphoblastic leukemia (ALL) relapse after frontline therapy. Current stratification methods using clinical/biological criteria fail to identify a significant proportion of these children. Numerically more patients classified as standard/medium risk relapse than those with high-risk ALL. Early identification of this group with intensification of therapy may prevent this. We compare two techniques, multi-parameter flow cytometry (FCM) and real-time polymerase chain reaction (RQ-PCR) to monitor minimal residual disease (MRD) in bone marrow after initial therapy in newly diagnosed patients. We report our initial experience in patients with B-lineage ALL from a single institution (CHW) treated on the ANZ Haematology-Oncology Group Study VIII protocol for ALL. Multiparameter FCM was performed using two different four colour combinations of antibodies: CD19APC/CD45PerCP/CD10FITC/CD20PE and CD19APC/CD45PerCP/CD34FITC/CD9PE. A series of dual paramete...
Clinical Research (Excluding Clinical Trials), 2021
Background: Minimal Residual Disease (MRD) detection is vital for therapy monitoring and relapse ... more Background: Minimal Residual Disease (MRD) detection is vital for therapy monitoring and relapse prediction in cancers. While RNA-based MRD assays are shown to be effective at diagnostic time-points, serial response monitoring does not provide additional predictive value.DNA-based assays may be more sensitive and allow longitudinal monitoring of the disease trajectory. This study aimed to develop MRD assays based on whole genome sequencing (WGS) data for high-risk neuroblastoma (HRNB) and Ewing sarcoma (ES) patients, to establish their sensitivity in quantitative PCR (qPCR) anddroplet digital PCR (ddPCR) formats, and to directly compare against RNA-based MRD assays. Methodology: We analyzed WGS data to identify patient-specific chromosomal breakpoints in HRNB (N=6) and ES (N=6) patients, then established qPCR and ddPCR assays. Assay performance was validated using patient-derived cells spiked into bone marrow samples and in serially collected clinical samples as available. The DNA-M...
516 The majority of child cancer arises in embryonal cells which have persisted beyond birth, hav... more 516 The majority of child cancer arises in embryonal cells which have persisted beyond birth, having failed to undergo cell death following normal organogenesis in utero. The mechanism of embryonal cell persistence, and thus, tumour initiation are unknown. Resistance to spontaneous regression by neuroblasts is an important early step in human neuroblastoma tumorigenesis. We have previously shown that perinatal expression of a MYCN transgene in paravertebral murine neuroblasts mediates transient neuroblast proliferation and resistance to apoptotic cell death, as the first step in neuroblastoma tumorigenesis (Hansford et al., PNAS 2004). Here we analysed the mechanism of resistance to apoptotic cell death in primary ganglion cells from MYCN transgenic, compared with normal mice. MYCN-mediated resistance to cell death was seen in ganglion cells following a variety of different death stimuli: nerve growth factor (NGF) withdrawal, hypoxia, nutrient deprivation and doxorubicin. The mRNA e...
Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplificat... more Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplification) of the cancer-causing gene MYCN strongly predict poor outcome. Neuroblastomas are reliant on high levels of cellular components called polyamines for their growth and malignant behavior, and the gene regulating polyamine synthesis is called ODC1. ODC1 is often coamplified with MYCN, and in fact is regulated by MYCN, and like MYCN is prognostic of poor outcome. Here we studied a naturally occurring genetic variant or polymorphism that occurs in the ODC1 gene, and used gene editing to demonstrate the functional importance of this variant in terms of ODC1 levels and growth of neuroblastoma cells. We showed that this variant impacts the ability of MYCN to regulate ODC1, and that it also influences outcome in neuroblastoma, with the rarer variant associated with a better survival. This study addresses the important topic of genetic polymorphisms in cancer. Abstract Ornithine decarboxylas...
The ultimate cause of treatment failure for many forms of malignant and infectious diseases is th... more The ultimate cause of treatment failure for many forms of malignant and infectious diseases is the development of resistance to a broad range of cytotoxic drugs. Hence, despite significant advances in cancer treatment, multidrug resistance (MDR) remains a major clinical problem and leads to limited therapeutic options and poor patient outcome in a number of cancers, including high-risk neuroblastoma, leukaemia and carcinomas of the prostate, breast, lung and ovary. Therefore, gaining a better understanding of the mechanisms that lead to drug resistance is of utmost importance in order to improve the current chemotherapy regimes in cancer treatment. While MDR may develop in response to a specific drug or drug combination, the resultant phenotype typically confers resistance to a variety of agents, often with diverse mechanisms of action. A large body of research devoted to exploring cellular MDR has revealed that resistance most commonly arises through (a) insufficient uptake of wate...
DIPG is an aggressive paediatric brainstem tumour, with a median survival of less than 1 year. Po... more DIPG is an aggressive paediatric brainstem tumour, with a median survival of less than 1 year. Polyamines are intracellular polycations that control important aspects of cell growth and are often upregulated in cancer. Difluoromethylornithine (DFMO) is an FDA-approved inhibitor of the enzyme ornithine decarboxylase (ODC1) which is a key driver of polyamine synthesis. We investigated the efficacy of polyamine pathway inhibitors as a therapeutic strategy against DIPG. We found high expression levels of synthetic enzymes in the polyamine pathway in primary patient samples and cultures. Using cytotoxicity and clonogenic assays, we found that DFMO inhibited the proliferation of DIPG neurospheres. However, DIPG cells compensated for DFMO inhibition by increasing expression of the polyamine transporter SLC3A2. Gene expression analysis showed that the polyamine transporter, SLC3A2, was significantly overexpressed in DIPG compared with all other high-risk childhood cancers. Addition of polya...
Biochimica et biophysica acta. Gene regulatory mechanisms, 2018
Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tu... more Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy, remarkably refractory to therapeutic treatments. One of the most powerful independent prognostic indicators for this disease is the amplification of the MYCN oncogene, which occurs at high levels in approximately 25% of neuroblastomas. Interestingly, amplification and not just expression of MYCN has a strong prognostic value, although this fact appears quite surprising as MYCN is a transcription factor that requires dimerising with its partner MAX, to exert its function. This observation greatly suggests that the role of MYCN in neuroblastoma should be examined in the context of MAX expression. In this report, we show that, in contrast to what is found in normal cells, MAX expression is significantly different among primary NBs, and that its level appears to correlate with the clinical outcome of the disease. Importantly, controlled modulation of MAX expression in neurob...
DIPG is an aggressive pediatric brainstem tumor, with a median survival below 12 months. Tumor ce... more DIPG is an aggressive pediatric brainstem tumor, with a median survival below 12 months. Tumor cells are dependent upon arginine, a semi-essential amino acid, metabolised by arginase enzymes into ornithine, a pivotal precursor to the polyamine pathway. Polyamines, frequently upregulated in cancer, are intracellular polycations controlling key biological processes – the inhibition of which we have previously shown to be highly efficacious in preclinical DIPG models. Pegylated arginase (BCT-100) has recently been shown to significantly delay tumor development, prolonging survival of neuroblastoma-prone Th-MYCN mice. This study investigated the effects of arginine depletion therapy as a single agent and in combination with polyamine pathway inhibitors in DIPG. We found that ARG2, the gene encoding for arginase II, is expressed significantly more highly in DIPG tumors compared to normal brain. Arginine depletion via BCT-100 reduced DIPG cell proliferation and colony formation in patient...
Approximately 25% of children with acute lymphoblastic leukemia (ALL) relapse after frontline the... more Approximately 25% of children with acute lymphoblastic leukemia (ALL) relapse after frontline therapy. Current stratification methods using clinical/biological criteria fail to identify a significant proportion of these children. Numerically more patients classified as standard/medium risk relapse than those with high-risk ALL. Early identification of this group with intensification of therapy may prevent this. We compare two techniques, multi-parameter flow cytometry (FCM) and real-time polymerase chain reaction (RQ-PCR) to monitor minimal residual disease (MRD) in bone marrow after initial therapy in newly diagnosed patients. We report our initial experience in patients with B-lineage ALL from a single institution (CHW) treated on the ANZ Haematology-Oncology Group Study VIII protocol for ALL. Multiparameter FCM was performed using two different four colour combinations of antibodies: CD19APC/CD45PerCP/CD10FITC/CD20PE and CD19APC/CD45PerCP/CD34FITC/CD9PE. A series of dual paramete...
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