The American Heart Association previously recommended implementation of cardiac resuscitation systems of care that consist of interconnected community, emergency medical services, and hospital ...efforts to measure and improve the process of care and outcome for patients with cardiac arrest. In addition, the American Heart Association proposed a national process to develop and implement evidence-based guidelines for cardiac resuscitation systems of care. Significant experience has been gained with implementing these systems, and new evidence has accumulated. This update describes recent advances in the science of cardiac resuscitation systems and evidence of their effectiveness, as well as recent progress in dissemination and implementation throughout the United States. Emphasis is placed on evidence published since the original recommendations (ie, including and since 2010).
Abstract
Registration of data to a common frame of reference is an essential step in the analysis and integration of diverse neuroscientific data. To this end, volumetric brain atlases enable ...histological datasets to be spatially registered and analyzed, yet accurate registration remains expertise-dependent and slow. In order to address this limitation, we have trained a neural network, DeepSlice, to register mouse brain histological images to the Allen Brain Common Coordinate Framework, retaining registration accuracy while improving speed by >1000 fold.
Children and young adults with glioblastoma (GBM) have a median survival rate of only 12 to 15 months, and these GBMs are clinically and biologically distinct from histologically similar cancers in ...older adults. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A , occurring either at or close to key residues marked by methylation for regulation of transcription—K27 and G34. Here, we show that the cerebral hemisphere-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem-cell maintenance, cell-fate decisions, and self-renewal.Critically, H3F3A G34 mutations cause profound upregulation of MYCN , a potent oncogene that is causative of GBMs when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population through inhibiting kinases responsible for stabilization of the protein.
We provide the mechanistic explanation for how the fi rst histone gene mutation inhuman disease biology acts to deliver MYCN, a potent tumorigenic initiator, into a stem-cell compartment of the developing forebrain, selectively giving rise to incurable cerebral hemispheric GBM. Using synthetic lethal approaches to these mutant tumor cells provides a rational way to develop novel and highly selective treatment strategies
Novel therapies for paediatric-type diffuse high-grade glioma (PDHGG) are urgently required. Orthotopic models using patient-derived material are invaluable tools in preclinical drug development as ...they retain key genetic/epigenetic features, eg. histone H3G34 or H3K27 alterations. Their evaluation
in situ
is vital and requires sensitive imaging techniques such as MRI. 12 diffuse hemispheric glioma (DHG; 2 DHG-G34) and 21 diffuse midline glioma (DMG; 17 DMG-K27M) tumours have been characterised using MRI following site-specific orthotopic implantation of patient-derived cells directly from tumour material or after minimal expansion as stem cell cultures. Of the 62 models implanted; 3 DHG and 10 DMG samples were not tumourigenic and 13 DHG/3 DMG models are currently under MRI surveillance. Tumours identified on T
2
-weighted (T
2
w)-images varied from a diffuse hyperintense signal to well-defined high contrast masses. Tumour growth in 5 DMG models was too diffuse for longitudinal monitoring with T
2
w-MRI. Once established, diffusion-weighted, T
1
/T
2
mapping and contrast-enhanced MRI were used to further assess tumour phenotype. Quantitative data from 15 DMG models demonstrated higher water diffusivity and T
2
than 10 DHG tumours, which suggests less tightly packed tumour cells but may also reflect the closer proximity of tumours growing in the thalamus/pons/cerebellum to the ventricular system. Lack of contrast-agent enhancement in 11 DMG and 6/10 DHG models indicated an intact blood-brain barrier (BBB), with heterogeneous disruption observed in 4 DHGs; H3-G34 had no bearing on BBB integrity. Upon serial re-implantation survival was shortened in 3/4 DHG and 2/6 DMG models, while quantitative MRI parameters remained similar. Likewise, when 2 DHG and 2 DMG models grown in 2D/3D
in vitro
were implanted in parallel, poorer survival/improved penetrance was associated with 3D-cultured cells with no difference in imaging phenotype. The study highlights the potential of non-invasive MRI to accurately evaluate the efficacy of novel therapeutics in these PDHGG models.
Abstract
Novel therapies for paediatric-type diffuse high-grade glioma (PDHGG) are urgently required. Orthotopic models using patient-derived material are invaluable tools in preclinical drug ...development as they retain key genetic/epigenetic features, eg. histone H3G34 or H3K27 alterations. Their evaluation in situ is vital and requires sensitive imaging techniques such as MRI. 12 diffuse hemispheric glioma (DHG; 2 DHG-G34) and 21 diffuse midline glioma (DMG; 17 DMG-K27M) tumours have been characterised using MRI following site-specific orthotopic implantation of patient-derived cells directly from tumour material or after minimal expansion as stem cell cultures. Of the 62 models implanted; 3 DHG and 10 DMG samples were not tumourigenic and 13 DHG/3 DMG models are currently under MRI surveillance. Tumours identified on T2-weighted (T2w)-images varied from a diffuse hyperintense signal to well-defined high contrast masses. Tumour growth in 5 DMG models was too diffuse for longitudinal monitoring with T2w-MRI. Once established, diffusion-weighted, T1/T2 mapping and contrast-enhanced MRI were used to further assess tumour phenotype. Quantitative data from 15 DMG models demonstrated higher water diffusivity and T2 than 10 DHG tumours, which suggests less tightly packed tumour cells but may also reflect the closer proximity of tumours growing in the thalamus/pons/cerebellum to the ventricular system. Lack of contrast-agent enhancement in 11 DMG and 6/10 DHG models indicated an intact blood-brain barrier (BBB), with heterogeneous disruption observed in 4 DHGs; H3-G34 had no bearing on BBB integrity. Upon serial re-implantation survival was shortened in 3/4 DHG and 2/6 DMG models, while quantitative MRI parameters remained similar. Likewise, when 2 DHG and 2 DMG models grown in 2D/3D in vitro were implanted in parallel, poorer survival/improved penetrance was associated with 3D-cultured cells with no difference in imaging phenotype. The study highlights the potential of non-invasive MRI to accurately evaluate the efficacy of novel therapeutics in these PDHGG models.
Abstract
Diffuse intrinsic pontine glioma (DIPG) are incurable childhood brain tumours marked by alterations in histone 3 in the form of somatic K27M mutations and loss of H3K27 trimethylation ...(H3K27me3). These highly aggressive tumours display extensive intratumoral heterogeneity, despite their relatively modest mutational burden. We recently proposed a model whereby co-operative subclonal interactions may exist to promote tumorigenesis in DIPG, and identified rare cancer stem cell populations to harbour mutations in the histone H4 lysine methyltransferase KMT5B. Although not previously described in glioma, loss of H4K20me2/3 caused by abrogated H4 methyltransferase function has been linked to defects in the DNA damage response and enhanced invasion in breast and pancreatic cancer. In order to explore whether dysregulation of H4 methylation may be directly responsible for tumorigenic phenotypes in DIPG, we targeted the methyltransferases by genetically engineering numerous isogenic cells from patient-derived in vitro models using CRISPR/Cas9 to knock out KMT5B and/or KMT5C, alongside pharmacological treatment with the small molecule KMT5B/C inhibitor A-196. We observed the expected reduction in H4K20me2 and H4K20me3 in response to inhibition of KMT5B/C, with an attendant increase in H3K27me3. Knock-out of KMT5B caused a significant increase in tumour cell migration and invasion in vitro, whilst KMT5C-deficient cells had reduced motility. Double knock-outs presented an intermediate phenotype. With H4K20me2/3 playing an important role in DNA repair via 53BP1 recruitment, we further observed a significantly enhanced efficacy of the PARP inhibitors olaparib and talazoparib in knock-out DIPG cells in vitro. Ongoing analysis of differential binding of post-translational modifications by ChIP-seq will provide important insights into the underlying mechanisms of epigenetic dysregulation, particularly in respect of the cross-talk between H3 and H4 marks. Although often present only in small tumour cell populations, H4K20me2/3 and KMT5B/C may represent an important novel aspect of DIPG tumorigenesis.
Abstract BACKGROUND Paediatric-type diffuse high-grade glioma (PDHGG) is recognised in the WHO2021 CNS Tumour Classification as biologically distinct from ‘adult-types’, and comprises four major ...categories based on distinct biology and clinical presentation, though sub-types within these are less well-defined. METHODS Published and unpublished genome/exome/targeted sequencing from n=1593 cases were integrated with n=2027 cases with methylation array profiling, with an overlap of n=761. These were supplemented with varying availability of bulk/single-cell RNAseq, ChIP-seq and ATAC-seq, and analysed alongside data from ~100 patient-derived models representing 16 PDHGG sub-types. RESULTS Around half of cases were diffuse midline glioma with H3K27 alterations (DMG-H3K27), which can be split into sub-types based upon either the specific histone alteration (DMG-H3.3K27M/-H3.1K27M/-EZHIP) or co-segregating pathway activating mutations (DMG-EGFR/-MAPK/-ACVR1). These showed distinct copy number, transcriptomic and epigenetic signatures depending on the anatomical location, replicated in ~50 models established as 2D/3D cultures and CDX/PDX. By contrast, diffuse hemispheric glioma with H3G34 mutations (DHG-H3G34) presented a homogenous genomic landscape (TP53/ATRX/PDGFRA) but with a higher degree of transcriptional diversity in both tumours and models. In high-grade glioma, H3/IDH-wild-type (HGG-WT) samples, receptor tyrosine kinase (RTK) subtypes are split by the presence or absence of co-segregating mutations (BCOR/SETD2/NF1) and copy number changes. Across methylation-based subtypes, genetic drivers are associated with distinct phenotypes or drug responses independent of location. EGFR mutations are largely confined to the highly infiltrative pedHGG_RTK2 hemispheric (enriched in gliomatosis cerebri) and DMG-EGFR (bithalamic glioma) subtypes, whilst PDGFRAmutations span DMG-H3.3K27M, DHG-H3G34, and pedHGG-RTK1, and exhibit a differential drug response to targeted therapies depending on the nature of the alteration. Hypermutant tumours are split between pedHGG-RTK1A and the emerging ‘HGG-E’ subtype. CONCLUSIONS These data aim to catalogue the diversity of PDHGG subtypes in order to identify biologically rational targets, and credential patient-derived models which can be exploited for mechanistic/preclinical studies.
Abstract
Immune cells have the potential to selectively eradicate high-risk brain tumours such as paediatric-type diffuse high-grade glioma (PDHGG). We aim to characterize the tumour immune ...microenvironment (TIME) of intra-cranial syngeneic mouse models of diffuse hemispheric glioma, H3G34 (DHG-H3G34) and diffuse midline glioma, H3K27 (DMG-H3K27). We also demonstrate how an oncolytic reovirus (Reolysin) can “heat-up” the TIME of our syngeneic models. Orthotopic immunocompetent mouse models of DHG-H3G34 (C57BL/6, NRASG12V + shp53 + shATRX +/- H3.3G34R) and DMG-H3K27 (Nestin-Tv-a/p53fl/fl, RCAS-ACVR1R206H + RCAS-H3.1K27M) were profiled using single-cell RNA-sequencing (scRNA-seq) (10x genomics), a 22-colour custom flow cytometry immune panel and spatial transcriptomics. Differential marker expression was validated with immunohistochemistry and immunofluorescence in tissue sections. Syngeneic mouse tumours treated systemically with Reolysin were also profiled to evaluate the effects of the oncolytic virus on the TIME. Cell type predictions in scRNA-seq using singleR, ssGSEA and expression of individual marker genes suggested that the predominant immune cell types within hemispheric tumours were monocytes (11-21%) and macrophages (10-19%) with much smaller proportions of CD4+ and CD8+ T-cells (4-10%). By contrast, much smaller proportions of monocytes (2%) and macrophages (3%) were observed in the H3.1K27M pontine model. Flow cytometry, immunohistochemistry and immunofluorescence validated scRNA-seq immune profiles and characterised signalling of the PD-1/PD-L1 checkpoint pathway. Spatial transcriptomics allowed immune cell populations to be positioned within tumour sections and showed significant co-localization of CD4+ and CD8+ lymphocytes at tumour margins. Treatment of syngeneic mouse tumours with Reolysin resulted in reduced tumour volumes and altered the TIME, in particular increasing cytotoxic T-cell tumour infiltration. Our results highlight immunological heterogeneity within molecular subgroups of PDHGG and demonstrate ability of a systemically delivered oncolytic virus, Reolysin, to “heat-up” the TIME, contributing to a more immune actionable profile. Future work will help to identify optimal combinations for the next generation of immunotherapies in PDHGG.
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