Radio-Pathomic Approaches in Pediatric Neurooncology: Opportunities and Challenges

With medical software platforms moving to cloud environments with scalable storage and computing, the translation of predictive artificial intelligence (AI) models to aid in clinical decision-making and facilitate personalized medicine for cancer patients is becoming a reality. Medical imaging, namely radiologic and histologic images, has immense analytical potential in neuro-oncology, and models utilizing integrated radiomic and pathomic data may yield a synergistic effect and provide a new modality for precision medicine. At the same time, the ability to harness multi-modal data is met with challenges in aggregating data across medical departments and institutions, as well as significant complexity in modeling the phenotypic and genotypic heterogeneity of pediatric brain tumors. In this paper, we review recent pathomic and integrated pathomic, radiomic, and genomic studies with clinical applications. We discuss current challenges limiting translational research on pediatric brain tumors and outline technical and analytical solutions. Overall, we propose that to empower the potential residing in radio-pathomics, systemic changes in cross-discipline data management and end-to-end software platforms to handle multi-modal data sets are needed, in addition to embracing modern AI-powered approaches. These changes can improve the performance of predictive models, and ultimately the ability to advance brain cancer treatments and patient outcomes through the development of such models

Molecular genetic analysis of paediatric low-grade astrocytoma

The thesis describes the molecular genetic analysis of fifty low-grade paediatric astrocytomas. DNA copy number changes were investigated in paediatric low-grade astrocytomas (WHO grade I and II), using array comparative genomic hybridisation and Affymetrix 250K and 6.0 SNP arrays. A discrete region of DNA copy number gain was identified at chromosome 7q34, primarily although not exclusively in pilocytic astrocytomas of the cerebellum. Further analysis of this region, by PCR and sequencing, demonstrated the presence of gene fusions between KIAA1549 and BRAF. Five KIAA1549- BRAF fusion variants were subsequently identified. A further gene fusion between SRGAP3 and RAF1 was also found in a single tumour with DNA copy number gain at chromosome 3p25. The fusion genes lacked the auto-inhibitory domains of BRAF and RAF1. These were replaced in-frame by N-terminal segments of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. Sequencing confirmed the presence of activating mutations in KRAS and BRAF in three tumours where gene fusions were not identified. Both gene fusions and activating mutations were shown to cause activation of the ERK/MAP kinase pathway by Western blotting. Further sequencing was performed of CDKN2A, PTEN and IDH1/2 to assess the frequency of abnormalities in paediatric low-grade astrocytoma. These genes have previously been found to contain aberrations within adult high-grade astrocytomas. To date, no significant aberrations have been identified in the paediatric astrocytoma samples examined. This confirms previous findings in adult and paediatric astrocytomas, which appear to show distinct molecular changes depending on patient age. Gene fusions or activating mutations were identified in 100% pilocytic astrocytomas studied and were also found in 28% of grade II astrocytomas. These findings highlight the importance of the ERK/MAPK pathway both in the development of paediatric low-grade astrocytomas and as a possible therapeutic target. Gene fusions may provide a means of molecular classification for pilocytic astrocytomas in the future

Radiomics in paediatric neuro-oncology : MRI textural features as diagnostic and prognostic biomarkers

Motivation: Brain and central nervous system tumours form the second most common group of cancers in children in the UK, accounting for 27% of all childhood cancers. Despite current advances in magnetic resonance imaging (MRI), non-invasive characterisation of paediatric brain tumours remains challenging. Radiomics, the high-throughput extraction and analysis of quantitative image features (e.g. texture), offers potential solutions for tumour characterisation and decision support. Aim and Methods: In search for diagnostic and prognostic oncological markers, the aim of this thesis was to study the application of MRI texture analysis (TA) for the characterisation of paediatric brain tumours. To this end, single and multi-centre experiments were carried out, within a supervised classification framework, on clinical MR imaging datasets of common brain tumour types. Results: TA of conventional MRI was successfully used for diagnostic classification of common paediatric brain tumours. A key contribution of this thesis was to provide evidence that diagnostic classification could be optimised by extending the analysis to include three-dimensional features obtained from multiple MR imaging slices. In addition to this, TA was shown to have a good cross-centre transferability, which is essential for long-term clinical adoption of the technique. Finally, fifteen textural features extracted from T2-weighted MRI were identified to be of significant prognostic value for paediatric medulloblastoma. Conclusion: It was shown that MRI TA provides valuable quantifiable information that can supplement qualitative assessments conducted by radiologists, for the characterisation of paediatric brain tumours. TA can potentially have a large clinical impact, since MR imaging is routinely used in the brain cancer clinical work-flow worldwide, providing an opportunity to improve personalised healthcare and decision-support at low cost

Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas

The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. FGFR1 with the duplication induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs and LGGNTs.Jinghui Zhang, Gang Wu, Claudia P Miller, Ruth G Tatevossian, James D Dalton, Bo Tang, Wilda Orisme, Chandanamali Punchihewa, Matthew Parker, Ibrahim Qaddoumi, Fredrick A Boop, Charles Lu, Cyriac Kandoth, Li Ding, Ryan Lee, Robert Huether, Xiang Chen, Erin Hedlund, Panduka Nagahawatte, Michael Rusch, Kristy Boggs, Jinjun Cheng, Jared Becksfort, Jing Ma, Guangchun Song, Yongjin Li, Lei Wei, Jianmin Wang, Sheila Shurtleff, John Easton, David Zhao, Robert S Fulton, Lucinda L Fulton, David J Dooling, Bhavin Vadodaria, Heather L Mulder, Chunlao Tang, Kerri Ochoa, Charles G Mullighan, Amar Gajjar, Richard Kriwacki, Denise Sheer, Richard J Gilbertson, Elaine R Mardis, Richard K Wilson, James R Downing, Suzanne J Baker and David W Elliso

TRANSCRIPTIONAL AND POST-TRANSLATIONAL MECHANISMS CONTRIBUTE TO MAINTENANCE OF REST IN NEURAL TUMORS

The RE-1 silencing transcription factor (REST) is an important regulator of normal nervous system development. It negatively regulates neuronal lineage specification in neural progenitors by binding to its consensus RE-1 element(s) located in the regulatory region of its target neuronal differentiation genes. The developmentally coordinated down-regulation of REST mRNA and protein in neural progenitors triggers terminal neurogenesis. REST is overexpressed in pediatric neural tumors such as medulloblastoma and neuroblastoma and is associated with poor neuronal differentiation. High REST protein correlate with poor prognosis for patients with medulloblastoma, however similar studies have not been done with neuroblastoma patients. Mechanism(s) underlying elevated REST levels medulloblastoma and neuroblastoma are unclear, and is the focus of this thesis project. We discovered that transcriptional and post-translational mechanisms govern REST mis-regulation in medulloblastoma and neuroblastoma. In medulloblastoma, REST transcript is aberrantly elevated in a subset of patient samples. Using loss of function and gain of function experiments, we provide evidence that the Hairy Enhancer of Split (HES1) protein represses REST transcription in medulloblastoma cell lines, modulates the expression of neuronal differentiation genes, and alters the survival potential of these cells in vitro. We also show that REST directly represses its own expression in an auto-regulatory feedback loop. Interestingly, our studies identified a novel interaction between REST and HES1. We also observed their co-occupancy at the RE-1 sites, thereby suggesting potential for co-regulation of REST expression. Our pharmacological studies in neuroblastoma using retinoic acid revealed that REST levels are controlled by transcriptional and post-transcriptional mechanisms. Post-transcriptional mechanisms are mediated by modulation of E3 ligase or REST, SCFβ-TRCP, and contribute to resistance of some cells to retinoic acid treatment

Molecular Mechanisms of Oncogenesis & Precision Medicine Approaches for Pediatric Low-Grade Gliomas

Pediatric low-grade gliomas (PLGGs) are a heterogeneous group of tumors that collectively represent the most common childhood brain cancer. Despite favorable outcomes with surgical and adjuvant therapies, majority of patients suffer from long-term treatment-related morbidities and recurrent/inoperable disease. This necessitates a deeper understanding of PLGG biology to aid development of molecular diagnostics and low-toxicity targeted therapeutics. Hitherto, PLGGs have been defined by activating mutations that dysregulate the MAPK signaling pathway, leading to clinical testing of RAF/MAPK inhibitors for PLGGs. Interestingly, recent large-scale sequencing efforts discovered novel gene fusions in PLGGs and we identified the unique recurrent association of tumor suppressor Quaking (QKI) with distinct proto-oncogenes, MYB and RAF1, in different PLGG sub-types. We hypothesized that MYB-QKI and QKI-RAF1 function via novel oncogenic mechanisms invoking a two-hit mechanism of gain-of-function in the MYB/RAF1 oncoproteins collaborating with QKI loss of putative tumor suppressor function, describing for the first time a unique gene fusion setting involving both fusion partners with implications for therapeutic targeting. Utilizing heterologous cell model systems and in vivo mouse models, we found MYB-QKI and QKI-RAF1 are capable of driving oncogenesis. Furthermore, MYB-QKI is a specific driver mutation defining angiocentric gliomas and mediates tumorigenesis via a tri-partite mechanism: (1)MYB oncogenic activation via truncation, (2)rearrangement led enhancer translocation that drives MYB-QKI expression and (3)LOH of QKI tumor suppressor. In contrast, QKI-RAF1 drives some pilocytic astrocytomas via aberrant activation of the MAPK pathway in a QKI-dimerization dependent manner. We also found differential response to RAF targeted therapy in QKI-RAF1, compared to BRAF fusions in PLGGs, due to QKI-mediated dimerization. Hence, our study highlights distinct roles for the same gene, QKI in supporting the oncogenic functions of MYB and RAF1 in different PLGG-gene fusions. Overall, our study has uncovered distinct molecular mechanisms associated with different QKI gene fusions in PLGGs. We show that MYB-QKI is specific to angiocentric gliomas and mediates a unique oncogenic program, and with QKI-RAF1 we demonstrate how mutational context guides differential response to targeted therapy. Therefore, our study has important clinical implications on molecular diagnostics and targeted therapy for these rather understudied class of childhood brain tumors

Diffusion and Perfusion MRI in Paediatric Posterior Fossa Tumours

Brain tumours in children frequently occur in the posterior fossa. Most undergo surgical resection, after which up to 25% develop cerebellar mutism syndrome (CMS), characterised by mutism, emotional lability and cerebellar motor signs; these typically improve over several months. This thesis examines the application of diffusion (dMRI) and arterial spin labelling (ASL) perfusion MRI in children with posterior fossa tumours. dMRI enables non-invasive in vivo investigation of brain microstructure and connectivity by a computational process known as tractography. The results of a unique survey of British neurosurgeons’ attitudes towards tractography are presented, demonstrating its widespread adoption and numerous limitations. State-of-the-art modelling of dMRI data combined with tractography is used to probe the anatomy of cerebellofrontal tracts in healthy children, revealing the first evidence of a topographic organization of projections to the frontal cortex at the superior cerebellar peduncle. Retrospective review of a large institutional series shows that CMS remains the most common complication of posterior fossa tumour resection, and that surgical approach does not influence surgical morbidity in this cohort. A prospective case-control study of children with posterior fossa tumours treated at Great Ormond Street Hospital is reported, in which children underwent longitudinal MR imaging at three timepoints. A region-of-interest based approach did not reveal any differences in dMRI metrics with respect to CMS status. However, the candidate also conducted an analysis of a separate retrospective cohort of medulloblastoma patients at Stanford University using an automated tractography pipeline. This demonstrated, in unprecedented spatiotemporal detail, a fine-grained evolution of changes in cerebellar white matter tracts in children with CMS. ASL studies in the prospective cohort showed that following tumour resection, increases in cortical cerebral blood flow were seen alongside reductions in blood arrival time, and these effects were modulated by clinical features of hydrocephalus and CMS. The results contained in this thesis are discussed in the context of the current understanding of CMS, and the novel anatomical insights presented provide a foundation for future research into the condition

Transforming cancer molecular diagnostics: Molecular subgrouping of medulloblastoma via lowdepth whole genome bisulfite sequencing

INTRODUCTION: International consensus recognises four molecular subgroups of medulloblastoma, each with distinct molecular features and clinical outcomes. Assigning molecular subgroup is typically achieved via the Illumina DNA methylation microarray. Given the rapidly-expanding WGS capacity in healthcare institutions, there is an unmet need to develop platform-independent, sequence-based subgrouping assays. Whole genome bisulfite sequencing (WGBS) enables the assessment of genome-wide methylation status at single-base resolution. To date, its routine application for subgroup assignment has been limited, due to high economic cost and sample input requirements and currently no optimised pipeline exists that is tailored for handling samples sequenced at low-pass (i.e., 1-10x depth). METHODOLOGY: Two datasets were utilised; 36 newly-sequenced low-depth (10x) and 42 publicly available high-depth (30x) WGBS medulloblastoma and cerebellar samples, all with matched DNA methylation microarray data. We applied imputation to low-pass WGBS data, assessed inter-platform correlation and identified molecular subgroups by directly integrating WGBS sample data with preexisting array-trained models. We developed machine learning WGBS-based classifiers and compared performance against microarray. We optimised reference-free aneuploidy detection with low-pass WGBS and assessed concordance with microarray-derived aneuploidy calls. RESULTS: We optimised a pipeline for processing and analysis of low-pass WGBS data, suitable for routine molecular subgrouping and aneuploidy assessment. Using down-sampling, we showed that subgroup assignment remains robust at low depths and identified additional regions of differential methylation that are not assessed by methylation microarray. WGBS data can be integrated into existing array-trained models with high assignment probabilities, and WGBS-derived classifier performance measures exceeded microarray-derived classifiers. CONCLUSION: We describe a platform-independent WGBS assay for molecular subgrouping of medulloblastoma. It performs equivalently to array-based methods at increasingly comparable cost (currently ~$396 vs ~$584) and provides proof-of-concept for routine clinical adoption using standard WGS technology. Finally, the full methylome enabled elucidation of additional biological heterogeneity that has hitherto been inaccessible

Multi-aspect testing and ranking inference to quantify dimorphism in the cytoarchitecture of cerebellum of male, female and intersex individuals: a model applied to bovine brains

The dimorphism among male, female and freemartin intersex bovines, focusing on the vermal lobules VIII and IX, was analyzed using a novel data analytics approach to quantify morphometric differences in the cytoarchitecture of digitalized sections of the cerebellum. This methodology consists of multivariate and multi-aspect testing for cytoarchitecture-ranking, based on neuronal cell complexity among populations defined by factors, such as sex, age or pathology. In this context, we computed a set of shape descriptors of the neural cell morphology, categorized them into three domains named size, regularity and density, respectively. The output and results of our methodology are multivariate in nature, allowing an in-depth analysis of the cytoarchitectonic organization and morphology of cells. Interestingly, the Purkinje neurons and the underlying granule cells revealed the same morphological pattern: female possessed larger, denser and more irregular neurons than males. In the Freemartin, Purkinje neurons showed an intermediate setting between males and females, while the granule cells were the largest, most regular and dense. This methodology could be a powerful instrument to carry out morphometric analysis providing robust bases for objective tissue screening, especially in the field of neurodegenerative pathologies

Multi-aspect testing and ranking inference to quantify dimorphism in the cytoarchitecture of cerebellum of male, female and intersex individuals: a model applied to bovine brains.

The dimorphism among male, female and freemartin intersex bovines, focusing on the vermal lobules VIII and IX, was analyzed using a novel data analytics approach to quantify morphometric differences in the cytoarchitecture of digitalized sections of the cerebellum. This methodology consists of multivariate and multi-aspect testing for cytoarchitecture-ranking, based on neuronal cell complexity among populations defined by factors, such as sex, age or pathology. In this context, we computed a set of shape descriptors of the neural cell morphology, categorized them into three domains named size, regularity and density, respectively. The output and results of our methodology are multivariate in nature, allowing an in-depth analysis of the cytoarchitectonic organization and morphology of cells. Interestingly, the Purkinje neurons and the underlying granule cells revealed the same morphological pattern: female possessed larger, denser and more irregular neurons than males. In the Freemartin, Purkinje neurons showed an intermediate setting between males and females, while the granule cells were the largest, most regular and dense. This methodology could be a powerful instrument to carry out morphometric analysis providing robust bases for objective tissue screening, especially in the field of neurodegenerative pathologies