Summary Medulloblastoma is an aggressively-growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and displays tremendous biological and clinical heterogeneity1. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently discriminated2,3. WNT tumours, displaying activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens4. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis2. Group 3 & 4 tumours are molecularly less well-characterised, and also present the greatest clinical challenges2,3,5. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs. Tetraploidy was identified as a frequent early event in Group 3 & 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA-sequencing confirmed these alterations, and revealed the expression of the first medulloblastoma fusion genes. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 & 4 patients.

Gene signatures can improve current stratification of patients with RMS but will require complex assays to be developed and extensive validation before clinical application. A significant majority of their prognostic value was encapsulated by the fusion gene status. A continuous risk score derived from the combination of clinical parameters with the presence or absence of PAX3/FOXO1 represents a robust approach to improving current risk-adapted therapy for RMS.

SUMMARY Oncogenic transcription factors such as RUNX1/ ETO, which is generated by the chromosomal translocation t(8;21), subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq) to identify the core RUNX1/ ETO-responsive transcriptional network of t(8;21) cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

Medulloblastoma is curable in approximately 70 % of patients. Over the past decade, progress in improving survival using conventional therapies has stalled, resulting in reduced quality of life due to treatment-related side effects, which are a major concern in survivors. The vast amount of genomic and molecular data generated over the last 5–10 years encourages optimism that improved risk stratification and new molecular targets will improve outcomes. It is now clear that medulloblastoma is not a single-disease entity, but instead consists of at least four distinct molecular subgroups: WNT/Wingless, Sonic Hedgehog, Group 3, and Group 4. The Medulloblastoma Down Under 2013 meeting, which convened at Bunker Bay, Australia, brought together 50 leading clinicians and scientists. The 2-day agenda included focused sessions on pathology and molecular stratification, genomics and mouse models, high-throughput drug screening, and clinical trial design. The meeting established a global action plan to translate novel biologic insights and drug targeting into treatment regimens to improve outcomes. A consensus was reached in several key areas, with the most important being that a novel classification scheme for medulloblastoma based on the four molecular subgroups, as well as histopathologic features, should be presented for consideration in the upcoming fifth edition of the World Health Organization’s classification of tumours of the central nervous system. Three other notable areas of agreement were as follows: (1) to establish a central repository of annotated mouse models that are readily accessible and freely available to the international research community; (2) to institute common eligibility criteria between the Children’s Oncology Group and the International Society of Paediatric Oncology Europe and initiate joint or parallel clinical trials; (3) to share preliminary high-throughput screening data across discovery labs to hasten the development of novel therapeutics. Medulloblastoma Down Under 2013 was an effective forum for meaningful discussion, which resulted in enhancing international collaborative clinical and translational research of this rare disease. This template could be applied to other fields to devise global action plans addressing all aspects of a disease, from improved disease classification, treatment stratification, and drug targeting to superior treatment regimens to be assessed in cooperative international clinical trials.

Focal high-level amplifications of MYC (or MYCC) define a subset of high-risk medulloblastoma patients. However, the prognostic role of MYCN oncogene amplification remains unresolved. We aimed to evaluate the prognostic value of this alteration alone and in combination with biological modifiers in 67 pediatric medulloblastomas with MYCN amplification (MYCN-MB). Twenty-one MYCN-MB were examined using gene expression profiling and array-CGH, whereas for 46 tumors immunohistochemical analysis and FISH were performed. All 67 tumors were further subjected to mutational analyses. We compared molecular, clinical, and prognostic characteristics both within biological MYCN-MB groups and with non-amplified tumors. Transcriptomic analysis revealed SHH-driven tumorigenesis in a subset of MYCN-MBs indicating a biological dichotomy of MYCN-MB. Activation of SHH was accompanied by variant-specific cytogenetic aberrations including deletion of 9q in SHH tumors. Non-SHH MB were associated with gain of 7q and isochromosome 17q/17q gain. Among clinically relevant variables, SHH subtype and 10q loss for non-SHH tumors comprised the most powerful markers of favorable prognosis in MYCN-MB. In conclusion, we demonstrate considerable heterogeneity within MYCN-MB in terms of genetics, tumor biology, and clinical outcome. Thus, assessment of disease group and 10q copy-number status may improve risk stratification of this group and may delineate MYCN-MB with the same dismal prognosis as MYC amplified tumors. Furthermore, based on the enrichment of MYCN and GLI2 amplifications in SHH-driven medulloblastoma, amplification of these downstream signaling intermediates should be taken into account before a patient is enrolled into a clinical trial using a smoothened inhibitor.

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jV-Nitrosoatrazine (NNAT) was reported to be produced by the nitrosation of atrazine but was not fully characterized. We report here an improved synthesis of NNAT by nitrosation of atrazine in glacial acetic acid followed by high-performance liquid chromatography. The mass and infrared spectra confirmed that NNAT is a mononitroso derivative. The and 13C nuclear magnetic resonance spectra of atrazine and NNAT were measured, including the use of selective decoupling, two-dimensional and homonuclear and heteronuclear correlation spectra, and variable-temperature experiments. These indicated that four syn-anti conformers of atrazine are present, attributed to restricted rotation of the two exocylic bonds (interconversion energy, 16.5 keal/mol) and that NNAT contains the nitroso group attached to the N-ethyl nitrogen and exists as two conformers, attributed to restricted rotation about the exocylic bond to the N-isopropyl nitrogen (interconversion energy, 18.3 keal/mol). NNAT was relatively stable in alkali and was photolabile. Nitrosation of atrazine in 1:1 ethanol-water followed third-order kinetics (first order for atrazine, HNO2, and H+), with a relatively low rate constant of 4.6x "3 "2 s"1 at 25 °C.

We agree with Missiaglia et al 1 that the presence or absence of PAX3/FOXO1 may serve as a prognostic molecular marker in RMS that is treated according to the Malignant Mesenchymal Tumor-SIOP or COG protocols. We also agree that heterogeneity in clinical practices between SIOP and COG may cause a bias in the combined evaluation of the two gene-expression data sets. However, our results, which were obtained with a different therapeutic strategy, point out that the potential prognostic relevance of PAX3/FOXO1 cannot be seen outside of the treatment context. Caution should be exercised before assuming that PAX3/FOXO1 fusion gene status is the universal key prognostic molecular marker in RMS.