Search

BACKGROUND Medulloblastoma is the most common yet challenging paediatric brain tumour. No longer considered a single disease entity, medulloblastoma is comprised of at least four distinct genetically and molecularly defined subgroups. OBJECTIVES Retrospectively assess the performance and outcomes of all patients diagnosed with medulloblastoma between 1990-2017 according to molecular subgroups. METHODS After obtaining ethical approval, confirming sample adequacy, 76 samples (FFPE and Fresh Frozen) were retrieved from the two Victorian children’s oncology providers, Monash Children’s Hospital and The Royal Children’s Hospital. Blinded review of histology and immunohistochemistry using subgroups specific antibodies (INI1, CTNNB1, GAB, YAP, DKK1, SFRP1), is performed by a neuropathologist. For molecular determination of the subgroups, DNA extraction and restoration (FFPE samples only), and bisulphite modification is performed prior to analysis on the Infinium MethylationEPIC BeadChip, containing >850k methylation sites, performed at the Australian Genome Research Facility. Whilst several methods exist, Methylation Array is the current gold standard for subgroup analysis. Data analysis is performed using the Heidelberg groups (MolecularNeuropathology) algorithm and locally using R-studio. Molecular information is paired with clinical variables and international data (where available). RESULTS 24 samples analysed to date show no discrepancy with regards to incidence amongst subgroups, gender distribution, outcomes and international data. Further analysis will examine trends in mutational burden within subgroups, change in population trends with respect to incidence, relapse and survival. Acknowledgements: CCF, Australian Lions, Baileys Day.

Malignant Rhabdoid Tumor (MRT) is a rare pediatric cancer predominantly occurring in the kidney and CNS that is highly resistant to current treatment protocols. MRT is almost exclusively characterized by homozygous inactivation of SMARCB1, a critical subunit of the SWI/SNF chromatin-remodeling complex, implicating epigenetic deregulation in the pathogenesis of the disease. Recently, we showed that sustained treatment of human MRT cell lines with the Histone deacetylase inhibitor, Panobinostat, at low-dose, inhibited tumor growth by driving multi-lineage differentiation in vitro and in vivo. Furthermore, re-expression of SMARCB1 in MRT cells phenocopied low-dose Panobinostat treatment and led to growth inhibition, senescence and terminal differentiation in vitro and in vivo, suggesting similar mechanistic functionality. Enhancer of Zeste homolog 2 (EZH2), a core subunit of the Polycomb Repressive Complex 2, confers transcriptional silencing via the addition of methyl groups to Lysine 27 of Histone 3 (H3K27me3), and is a transcriptional target of SMARCB1. EZH2 expression and H3K27me3 were drastically reduced following sustained low-dose Panobinostat treatment and re-expression of SMARCB1 in MRT cells. Sustained siRNA knockdown of EZH2 in G401 cells resulted in reduced cell growth and changes in mRNA expression, mimicking low-dose Panobinostat treatment and SMARCB1 re-expression. Treatment of MRT cells with the EZH2-catalytic domain inhibitors, GSK126, GSK343 and UNC1999, had no effect on EZH2 expression and only partially reduced cell growth despite dose-dependent reductions in H3K27me3 implying important non-catalytic EZH2 function. Remarkably, co-treatment of MRT with low-dose Panobinostat and GSK126 resulted in reduced EZH2 and H3K27me3 expression, significantly reduced cell growth and increased differentiation in vitro and in vivo compared to single agent controls, demonstrating a synergistic relationship. This data suggests EZH2 is an important mediator of MRT proliferation and differentiation and provides evidence for improved efficacy of dual therapeutic targeting of EZH2 with low-dose Panobinostat.