Evaluation of a novel antibody to define histone 3.3 G34R mutant brain tumours

Missense somatic mutations affecting histone H3.1 and H3.3 proteins are now accepted as the hallmark of paediatric diffuse intrinsic pontine gliomas (DIPG), non-brain stem paediatric high grade gliomas (pHGG) as well as a subset of adult glioblastoma multiforme (GBM). Different mutations give rise to one of three amino acid substitutions at two critical positions within the histone tails, K27M, G34R/V. Several studies have highlighted gene expression and epigenetic changes associated with histone H3 mutations; however their precise roles in tumourigenesis remain incompletely understood. Determining how such amino acid substitutions in a protein affect its properties can be challenging because of difficulties in detecting and tracking mutant proteins within cells and tissues. Here we describe a strategy for the generation of antibodies to discriminate G34R and G34V mutant histone H3 proteins from their wild-type counterparts. Antibodies were validated by western blotting and immunocytochemistry, using recombinant H3.3 proteins and paediatric GBM cell lines. The H3-G34R antibody demonstrated a high degree of selectivity towards its target sequence. Accordingly, immunostaining on a cohort of 22 formalin-fixed paraffin embedded tumours with a previously known H3.3 G34R mutation status, detected successfully the corresponding mutant protein in 11/11 G34R cases. Since there was a high concordance between genotype and immunohistochemical analysis of G34R mutant tumour samples, we analysed a series of tissue microarrays (TMAs) to assess the specificity of the antibody in a range of paediatric brain tumours, and noted immunoreactivity in 2/634 cases. Importantly, we describe the generation and validation of highly specific antibodies for G34 mutations. Overall our work adds to an extremely valuable portfolio of antibodies, not only for histopathologic detection of tumour-associated mutant histone sequences, but also facilitating the study of spatial/anatomical aspects of tumour formation and the identification of downstream targets and pathways in malignant glioma progression

A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas

Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00401-014-1338-3) contains supplementary material, which is available to authorized users

Current paradigm of the 18-kDa translocator protein (TSPO) as a molecular target for PET imaging in neuroinflammation and neurodegenerative diseases

Neuroinflammation is a process characterised by drastic changes in microglial morphology and by marked upregulation of the 18-kDa translocator protein (TSPO) on the mitochondria. The continual increase in incidence of neuroinflammation and neurodegenerative diseases poses a major health issue in many countries, requiring more innovative diagnostic and monitoring tools. TSPO expression may constitute a biomarker for brain inflammation that could be monitored by using TSPO tracers as neuroimaging agents. From medical imaging perspectives, this review focuses on the current concepts related to the TSPO, and discusses briefly on the status of its PET imaging related to neuroinflammation and neurodegenerative diseases in humans

Clinical Efficacy of ONC201 in H3K27M-Mutant Diffuse Midline Gliomas Is Driven by Disruption of Integrated Metabolic and Epigenetic Pathways

UNLABELLED Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction. SIGNIFICANCE The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction. This article is featured in Selected Articles from This Issue, p. 2293

Protein Structure along the Order–Disorder Continuum

Thermal fluctuations cause proteins to adopt an ensemble of conformations wherein the relative stability of the different ensemble members is determined by the topography of the underlying energy landscape. “Folded” proteins have relatively homogeneous ensembles, while “unfolded” proteins have heterogeneous ensembles. Hence, the labels “folded” and “unfolded” represent attempts to provide a qualitative characterization of the extent of structural heterogeneity within the underlying ensemble. In this work, we introduce an information-theoretic order parameter to quantify this conformational heterogeneity. We demonstrate that this order parameter can be estimated in a straightforward manner from an ensemble and is applicable to both unfolded and folded proteins. In addition, a simple formula for approximating the order parameter directly from crystallographic B factors is presented. By applying these metrics to a large sample of proteins, we show that proteins span the full range of the order–disorder axis.National Institutes of Health (U.S.) (NIH Grant 5R21NS063185-02

In vivo PET imaging of the neuroinflammatory response in rat spinal cord injury using the TSPO tracer [F-18]GE-180 and effect of docosahexaenoic acid

Centre for Trauma Sciences, funded by the Barts & The London Charity, GE Healthcare Ltd, the Experimental Medicine Awards from the Blizard Institute and the Imaging Centre at the Barts Cancer Institute

Mouse models of neurodegenerative disease: preclinical imaging and neurovascular component.

Neurodegenerative diseases represent great challenges for basic science and clinical medicine because of their prevalence, pathologies, lack of mechanism-based treatments, and impacts on individuals. Translational research might contribute to the study of neurodegenerative diseases. The mouse has become a key model for studying disease mechanisms that might recapitulate in part some aspects of the corresponding human diseases. Neurode- generative disorders are very complicated and multifacto- rial. This has to be taken in account when testing drugs. Most of the drugs screening in mice are very di cult to be interpretated and often useless. Mouse models could be condiderated a ‘pathway models’, rather than as models for the whole complicated construct that makes a human disease. Non-invasive in vivo imaging in mice has gained increasing interest in preclinical research in the last years thanks to the availability of high-resolution single-photon emission computed tomography (SPECT), positron emission tomography (PET), high eld Magnetic resonance, Optical Imaging scanners and of highly speci c contrast agents. Behavioral test are useful tool to characterize di erent ani- mal models of neurodegenerative pathology. Furthermore, many authors have observed vascular pathological features associated to the di erent neurodegenerative disorders. Aim of this review is to focus on the di erent existing animal models of neurodegenerative disorders, describe behavioral tests and preclinical imaging techniques used for diagnose and describe the vascular pathological features associated to these diseases

Microglial activation in Parkinson’s disease using [18F]-FEPPA

BACKGROUND: Neuroinflammatory processes including activated microglia have been reported to play an important role in Parkinson’s disease (PD). Increased expression of translocator protein (TSPO) has been observed after brain injury and inflammation in neurodegenerative diseases. Positron emission tomography (PET) radioligand targeting TSPO allows for the quantification of neuroinflammation in vivo. METHODS: Based on the genotype of the rs6791 polymorphism in the TSPO gene, we included 25 mixed-affinity binders (MABs) (14 PD patients and 11 age-matched healthy controls (HC)) and 27 high-affinity binders (HABs) (16 PD patients and 11 age-matched HC) to assess regional differences in the second-generation radioligand [(18)F]-FEPPA between PD patients and HC. FEPPA total distribution volume (V (T)) values in cortical as well as subcortical brain regions were derived from a two-tissue compartment model with arterial plasma as an input function. RESULTS: Our results revealed a significant main effect of genotype on [(18)F]-FEPPA V (T) in every brain region, but no main effect of disease or disease × genotype interaction in any brain region. The overall percentage difference of the mean FEPPA V (T) between HC-MABs and HC-HABs was 32.6% (SD = 2.09) and for PD-MABs and PD-HABs was 43.1% (SD = 1.21). CONCLUSIONS: Future investigations are needed to determine the significance of [(18)F]-FEPPA as a biomarker of neuroinflammation as well as the importance of the rs6971 polymorphism and its clinical consequence in PD