Medulloblastoma is the most common malignant brain tumour of childhood. Transcriptomic classification of the disease has indicated the existence of discrete molecular subgroups of medulloblastoma, although the precise number, nature and clinical significance of these subgroups remains unclear. Two groups, characterised by activation of the WNT and SHH signalling pathways, are common to all published studies. An assay for the rapid diagnosis of medulloblastoma subgroups was therefore designed, using transcriptomic gene signatures of pathway activation for the WNT and SHH signalling pathways. The successful validation of these gene signatures in vitro and in silico enabled a meta-analysis of 173 new and published cases to be performed, which defined the molecular and clinico-pathological correlates of the disease subgroups more precisely. WNT subgroup cases were associated with CTNNB1 mutation, chromosome 6 loss and classic histology and were diagnosed > 5 years of age. SHH cases predominated in infants and showed an age-dependent relationship to desmoplastic / nodular histology. WNT / SHH independent tumours showed all histologies, peaked at 3 to 6 years and were associated with chromosome 17p loss. A novel DNA methylation array-based approach was next applied to disease subclassification. Using consensus clustering, based on non-negative matrix factorisation, four methylomic subgroups were identified in a training cohort (n = 100), which were robustly validated in a test cohort (n = 130). The subgroups were characterised by significant relationships to specific clinico-pathological and molecular markers. Two subgroups were characterised by activation of the WNT and SHH signalling pathways and showed equivalent clinico-pathological and molecular characteristics to the previously defined transcriptomic subgroups. For the WNT / SHH independent subgroups, group I was associated with a loss of chromosome 17p, whereas group II was enriched for large cell / anaplastic (LCA) histology. The WNT subgroup was associated with a favourable prognosis, while no survival differences were apparent between the remaining subgroups (SHH, group I, group II). Specific methylation biomarkers were identified for the discrimination of all subgroups. Assays of DNA methylation status were robust in derivatives of FFPE tissues, enabling testing in routinely-collected clinical material. Finally, the prognostic potential of methylomic biomarkers was investigated in a large clinical trials-based cohort (n = 191), with particular focus on the non-WNT subgroups (n = 163), where subgroup membership was not prognostic. Using the Cox Boost algorithm, which adds high dimensional data to mandatory clinical covariates to form cross-validated prognostic Cox survival models, the methylation status of MXI1 and IL8 were each identified as independent prognostic markers. These were incorporated into a novel risk stratification scheme, based on the cumulative assessment of disease risk using clinical (metastatic disease; poor prognosis), pathological (LCA pathology, poor prognosis) and methylomic variables (WNT subgroup, favourable prognosis; MXI1 and IL8 status). Importantly, this scheme assigns 46% of cases to a low risk group of patients (>90% survival) who could potentially be treated less intensively, with the aim of reducing therapy-associated late effects. This model out-performed the current clinical and other state-of-the-art medulloblastoma risk classification schemes. These data provide clear precedent for the utility of DNA methylation biomarkers for disease subclassification and prognostication in medulloblastoma, and their clinical application in diagnostic tumour biopsies.

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Objectives: To investigate cognitive and motor functioning in children treated for brain tumours. To also assess processing speed as a possible mediator in the outcomes of children treated for medulloblastoma in the posterior fossa. Design: A cross-sectional design was used. There were two groups. Children treated for medulloblastomas, and an age and gender matched non-CNS control group of children treated for Wilm's tumours. There were 14 participants in each group. Participants were aged between nine and 16 years old. Methods: Participants were assessed using measures for processing speed, attention, fine motor and visual motor co-ordination. Long-term outcomes were assessed using measures of intelligence (IQ), academic achievement, adaptive functioning, social functioning, and resilience. Results: There were significant differences between the two groups. Participants treated for medulloblastoma were significantly impaired on measures of processing speed, attention, fine motor and visual motor co-ordination. The medulloblastoma group performance was also significantly worse on measures of IQ, academic, adaptive, and social functioning. Interestingly, processing speed was found to be a significant mediator in the outcomes measured. When processing speed was co- varied, the group differences were no longer significant. Conclusion: The results of this study offer information on the impact of medulloblastoma on cognitive and motor abilities. It also offers novel information concerning the processing speed as a possible mediator in long-term outcomes of 3 children treated for medulloblastoma. This information will help with the design of interventions to target specific deficits. This will also provide additional information on the links between processing speed and outcomes. 4

Medulloblastoma is the most commonly occurring malignant brain tumour of childhood. The RASSF1A tumour suppressor is potentially the most important candidate gene identified in medulloblastoma to date, being epigenetically silenced in over 79% of primary tumours. In other tumour types, RASSF1A has been shown to play key roles in both cell cycle regulation and apoptotic signalling pathways. However, its functional role has not been previously addressed in medulloblastoma. UW228-3 medulloblastoma cells, stably expressing RASSF1A displayed increased sensitivity to an anti-CD95 antibody and to the chemotherapeutic agents, etoposide, cisplatin or vincristine, compared to vector control cells. In RASSF1A-expressing UW228-3 cells, increased cleavage of key caspases and of the DNA repair protein, PARP was observed following treatment with anti-CD95 or etoposide in comparison to vector control cells. In addition, inhibition of caspases in RASSF1A-expressing UW228-3 cells by Z-VAD-FMK, prevented apoptotic DNA fragmentation following death receptor activation (by anti-CD95) or DNA damage (induced by etoposide). Increased phosphorylation of H2AX was also observed in RASSF1A-expressing UW228-3 cells during apoptosis, suggesting a possible novel function for this tumour suppressor in the regUlation of histone modification during apoptotic cell death. Increased activation of the pro-apoptotic BCL-2 family member, BAX was also observed in UW228-3 cells stably expressing RASSF1A following activation of both the extrinsic and intrinsic apoptotic pathways. Further investigation of BAX activation in UW228-3 cells revealed that apoptotic signalling can also be promoted in the absence of RASSF1A using the BH3-only mimetic, ABT-737 (BAD-mimetic) in combination with chemotherapeutic agents (etoposide, cisplatin and vincristine). ABT- 737 increased susceptibility to apoptosis induced by DNA damage regardless of RASSF1A expression status through increased activation of BAX. In conclusion, data from this study shows that RASSF1A has a proapoptotic function in vitro and may potentially modulate therapeutic response via the BCL-2 family (and BAX in particular). This family of proteins therefore represent tractable therapeutic targets in medulloblastoma.

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