Summary Accurate pathological diagnosis is crucial for optimal management of cancer patients. For the ~100 known central nervous system (CNS) tumour entities, standardization of the diagnostic process has been shown to be particularly challenging - with substantial inter-observer variability in the histopathological diagnosis of many tumour types. We herein present the development of a comprehensive approach for DNA methylation-based CNS tumour classification across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that availability of this method may have substantial impact on diagnostic precision compared with standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility we have designed a free online classifier tool (www.molecularneuropathology.org) requiring no additional onsite data processing. Our results provide a blueprint for the generation of machine learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Glioma growth and progression depend on a specialized subpopulation of tumour cells, termed tumour stem cells. Thus, tumour stem cells represent a critical therapeutic target, but the molecular mechanisms that regulate them are poorly understood. Hypoxia plays a key role in tumour progression and in this study we provide evidence that the hypoxic tumour microenvironment also controls tumour stem cells. We define a detailed molecular signature of tumour stem cell genes, which are overexpressed by tumour cells in vascular and perinecrotic/hypoxic niches. Mechanistically, we show that hypoxia plays a key role in the regulation of the tumour stem cell phenotype through hypoxia-inducible factor 2alpha and subsequent induction of specific tumour stem cell signature genes, including mastermind-like protein 3 (Notch pathway), nuclear factor of activated T cells 2 (calcineurin pathway) and aspartate beta-hydroxylase domain-containing protein 2. Notably, a number of these genes belong to pathways regulating the stem cell phenotype. Consistently, tumour stem cell signature genes are overexpressed in newly formed gliomas and are associated with worse clinical prognosis. We propose that tumour stem cells are maintained within a hypoxic niche, providing a functional link between the well-established role of hypoxia in stem cell and tumour biology. The identification of molecular regulators of tumour stem cells in the hypoxic niche points to specific signalling mechanisms that may be used to target the glioblastoma stem cell population.

The establishment of polarity is an essential process in early neuronal development. Although a number of molecules controlling neuronal polarity have been identified, genetic evidence about their physiological roles in this process is mostly lacking. We analyzed the consequences of loss of Cdc42, a central regulator of polarity in multiple systems, on the polarization of mammalian neurons. Genetic ablation of Cdc42 in the brain led to multiple abnormalities, including striking defects in the formation of axonal tracts. Neurons from the Cdc42 null animals sprouted neurites but had a strongly suppressed ability to form axons both in vivo and in culture. This was accompanied by disrupted cytoskeletal organization, enlargement of the growth cones, and inhibition of filopodial dynamics. Axon formation in the knock-out neurons was rescued by manipulation of the actin cytoskeleton, indicating that the effects of Cdc42 ablation are exerted through modulation of actin dynamics. In addition, the knock-outs showed a specific increase in the phosphorylation (inactivation) of the Cdc42 effector cofilin. Furthermore, the active, nonphosphorylated form of cofilin was enriched in the axonal growth cones of wild-type, but not of mutant, neurons. Importantly, cofilin knockdown resulted in polarity defects quantitatively analogous to the ones seen after Cdc42 ablation. We conclude that Cdc42 is a key regulator of axon specification, and that cofilin is a physiological downstream effector of Cdc42 in this process.

Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons.