Diagnostic Neuropathology Research Articles

DIPG-53. AN EXCEPTIONALLY RARE CASE OF A DIFFUSE MIDLINE GLIOMA WITH CONCOMITANT H3.1 K27M AND G34R MUTATIONS IN THEHIST1H3C (H3C3) GENE

Abstract BACKGROUND Histone mutations (H3 K27M, H3 G34R/V) define subtypes of paediatric-type diffuse high-grade gliomas (HGG) (diffuse midline glioma (DMG), H3 K27-altered, diffuse hemispheric glioma (DHG), H3 G34-mutant). The WHO classification recognises exceptional cases where these mutations co-occur. We report one such case of a 2-year-old female presenting with neurological symptoms and lethargy. METHODS MRI imaging identified a brainstem lesion; a stereotactic needle biopsy was undertaken, followed by standard diagnostic neuropathology workflows including histology review, panel immunohistochemistry, DNA methylation profiling (Illumina EPIC BeadArrays, brain tumour classifier (MNP v12.5 R package)) and WES/WGS. Patient-derived in vitro cell cultures were established allowing further characterisation using RNAseq and chromatin immunoprecipitation assays with sequencing (ChIP-seq). RESULTS Histology showed diffusely infiltrating pleomorphic astrocytes, multinucleated cells, and conspicuous mitotic activity; the diagnosis was DMG, H3 K27-altered (immunohistochemistry: H3K27me3 loss, H3K27M positivity). DNA methylation profiling classified the tumour as ‘DMG, H3 K27-altered’ (calibrated score=0.99). WES/WGS revealed concurrent H3.1 K27M and G34R mutations (clonal, in the same reads) of HIST1H3C (H3C3), somatic variants in FGF11 and PIK3CA, and a pathogenic germline NBN variant. The RNAseq profile of the primary tumour and cell cultures clustered with H3 K27M-mutant tumours. Unbiased in vitro drug screening showed no selective sensitivities. ChIP-seq (H3K27ac, H3K27me3, H3K36me3, RNApol2 marks) showed features in keeping with DMG H3 K27M-mutant tumours (H3K27ac loci including OLIG2, IRX1/2, PKDCC). The patient was treated with adjuvant radiotherapy and everolimus, but progressed and passed away 13 months post-diagnosis. CONCLUSION This case is an exceptionally rare, complex variant of histone-mutant paediatric HGG, and the first reported occurrence in HIST1H3C (H3C3). It illustrates that in cis, the H3.1 K27M mutation demonstrates a dominance over molecular and clinical profiles compared to G34R, and highlights the importance of broad molecular profiling to identify such examples for further study.

1209 Comparison of Competition in Recruitment into Surgical and Non-Surgical Specialties

Abstract Aim Despite notoriety for strenuous training with significant out of hours commitments, surgical specialties maintain a reputation for competitive entry with rigorous portfolio requirements. This study aimed to compare recruitment data between surgical and non-surgical specialties to examine whether surgical specialties are more competitive for entry. Method Recruitment data from 2022 published by Health Education England was collated. Specialties included were those recruiting for registrar level training post (ST3/ST4 depending on specialty). Fill rates and competition ratios for all surgical specialties (with an ST3 entry-point) were compared with all non-surgical specialties for which there was data. Statistical analysis was completed using the Mann-Whitney U test. Results Median fill rates were 94.99% (IQR 23.81%) for non-surgical specialties and 100% (IQR 0%) for surgical specialties (p < 0.05). Median competition ratios were 3.17 (IQR 2.86) for non-surgical specialties and 3.92 (IQR 1.58) for surgical specialties (p < 0.05). Highest competition ratio overall was in immunology (46), with cardiothoracics having the highest surgical competitions ratio (23.5). Only genitourinary medicine and diagnostic neuropathology had a competition ratio <1. Conclusions All specialties at registrar level training had high fill rates and competition ratios, however this varied greatly between specialties perhaps due to lifestyle factors and exposure as students. Surgical sub-specialties demonstrated significantly higher competition ratios and fill rates, suggesting that despite discussion around quality of life and burnout in recent years, surgery continues to be an attractive career. It would be interesting to compare the rates of those leaving training posts with recruitment data.

Introduction

The 2021 WHO Classification of Central Nervous System tumors is a major advance in the field of neuro-oncology and neuropathology and represents a good example of translation of current molecular findings into a new classification system and more precise diagnostic criteria. Compared to the previous edition, in this fifth edition the integration of phenotypic and molecular genetic information in diagnostic categories has significatively increased. Moreover, the integration of epigenetic data through methylation profiling is the distinctive feature of this edition. In this issue of Pathologica, six review articles provide updates in the classification of major tumor categories relevant to diagnostic neuropathology according to the new WHO Classification. Antonelli and Poliani 1 discuss the current classification of diffuse gliomas in adults, including the role of ancillary molecular testing in identifying distinct tumor types (e.g., EGFR amplification, TERT promoter mutation, and +7/10 in glioblastoma, IDH wildtype) and grading (homozygous CDKN2A/B homozygous deletion in IDH mutant astrocytomas)

A Targeted Next-Generation Sequencing Panel to Genotype Gliomas.

Gliomas account for the majority of primary brain tumors. Glioblastoma is the most common and malignant type. Based on their extreme molecular heterogeneity, molecular markers can be used to classify gliomas and stratify patients into diagnostic, prognostic, and therapeutic clusters. In this work, we developed and validated a targeted next-generation sequencing (NGS) approach to analyze variants or chromosomal aberrations correlated with tumorigenesis and response to treatment in gliomas. Our targeted NGS analysis covered 13 glioma-related genes (ACVR1, ATRX, BRAF, CDKN2A, EGFR, H3F3A, HIST1H3B, HIST1H3C, IDH1, IDH2, P53, PDGFRA, PTEN), a 125 bp region of the TERT promoter, and 54 single nucleotide polymorphisms (SNPs) along chromosomes 1 and 19 for reliable assessment of their copy number alterations (CNAs). Our targeted NGS approach provided a portrait of gliomas’ molecular heterogeneity with high accuracy, specificity, and sensitivity in a single workflow, enabling the detection of variants associated with unfavorable outcomes, disease progression, and drug resistance. These preliminary results support its use in routine diagnostic neuropathology.

Neurodegeneration: 2022 update.

Here, we review a collection of recent manuscripts and research trends on the neuropathology of neurodegeneration that are considered by the author to be among the potentially most impactful. To the greatest extent possible, we chose to focus on histopathological studies that are most relevant to experimental and diagnostic neuropathology. While there has been an abundance of important recent discoveries and developments in neurodegenerative disease research, there was a deliberate effort here to provide balance to prevent disease categories and experimental approaches from overshadowing the others. The result is a diverse series of outstanding studies, together showing the landscape of progress across neurodegenerative disorders. One is a stereological study examining dystrophic microglia in aging. We highlight the first large genetic study of primary age-related tauopathy, showing convergence and divergence from classical Alzheimer's disease. There were further advances in the neuropathological criteria and staging of chronic traumatic encephalopathy. Links suggesting a causal role for TMEM106B in TDP-43 proteinopathy emerged. Attempts to subtype Alzheimer's disease on the molecular level were made. Evidence for a role for the VEGF family in cognitive impairment was advanced. Comparison of gene expression profiles from myeloid cells in peripheral blood and brain tissues from Parkinson's disease patients revealed pathways that may lead to new mechanistic insights and biomarkers. A large autopsy series identified an increased frequency of central nervous system developmental malformations in Huntington's disease. A robust and reliable system for assessing Lewy body pathology was proposed. Finally, we continue to be plagued by the COVID-19 pandemic, with lingering concerns of a long-term link with neurodegeneration.

Dementia in the Forensic Setting: Diagnoses Obtained Using a Condensed Protocol at the Office of Chief Medical Examiner, New York City.

Individuals with dementia may come to forensic autopsy, partly because of non-natural deaths (e.g. fall-related), and/or concerns of abuse/neglect. At the New York City Office of Chief Medical Examiner (NYC OCME), brains from such cases are submitted for neurodegenerative disease (ND) work-up. Seventy-eight sequential cases were evaluated using a recently published condensed protocol for the NIA-AA guidelines for the neuropathologic assessment of Alzheimer disease (AD), a cost-cutting innovation in diagnostic neuropathology. ND was identified in 74 (94.9%) brains; the most common were AD (n = 41 [52.5%]), primary age-related tauopathy (n = 26 [33.3%]), and Lewy body disease ([LBD], n = 25 [32.1%]). Others included age-related tau astrogliopathy, hippocampal sclerosis of aging, progressive supranuclear palsy, multiple system atrophy, amyotrophic lateral sclerosis, argyrophilic grain disease, and Creutzfeldt-Jakob disease. 26.8% of AD cases involved a non-natural, dementia-related death, versus 40.0% for LBD. Finally, 70 (89.7%) cases had chronic cerebrovascular disease, 53 (67.9%) being moderate-to-severe. We present a diverse distribution of NDs notable for a high rate of diagnoses associated with falls (e.g. LBD), a potential difference from the hospital neuropathology experience. We also report a high burden of cerebrovascular disease in demented individuals seen at the NYC OCME. Finally, we demonstrate that the aforementioned condensed protocol is applicable for a variety of ND diagnoses.

Molecular Diagnostics of Adult Gliomas in Neuropathological Practice.

This review focuses on adult gliomas, highlighting the most relevant biomarkers in the diagnosis of these tumours and the use of DNA methylation arrays to complement conventional molecular diagnostic techniques. The discovery and characterisation of diagnostic and prognostic biomarkers in brain tumours has significantly changed the neuropathological landscape over the last decade. These include mutations in the IDH1 and IDH2 genes in astrocytomas and oligodendrogliomas, histone H3 K27M mutations in midline gliomas, or BRAF mutations in a range of low-grade and high-grade glial and glioneuronal tumours. Other biomarkers of relevance are mutations in the TERT promoter, the ATRX gene, and genomic alterations such as 1p/19q codeletion, EGFR amplification, and chromosome 7 gain and 10 loss. The development of DNA methylation profiling and algorithmic classification of brain tumours has further enhanced the diagnostic abilities of neuropathologists. Methylation profiling is particularly useful for the diagnostic workup of biopsies with an inconclusive molecular test results, small samples, or samples with indistinctive low-grade or high-grade histology. This technology has become indispensable for the risk stratification of ependymal tumours, medulloblastomas and meningiomas. CONCLUSION: This review highlights the importance of an integrated approach to brain tumour diagnostics and gives a balanced view of the relevance and choice of conventional and molecular techniques in the workup of adult gliomas in diagnostic neuropathology practice.

Neurodegeneration: 2021 update.

This article reviews a collection of manuscripts in the field of neurodegenerative disease chosen from what are considered by the author to be among the 10 most important and potentially impactful topics or research trends of 2020 relevant to the field of experimental and diagnostic neuropathology. A deliberate effort was made to provide balance among disease categories covered. The result is a varied selection that includes not just individual papers but also research topics and trends. The association of COVID-19 with longer-term neurological symptoms has launched a research trend fueled by speculation that the SARS-CoV-2 might trigger neurodegenerative changes. The onslaught of transcriptomic studies has begun to give way to proteomics, with three transformative studies published examining glial contributions to Alzheimer disease, cerebral atherosclerosis in cognitive decline, and the complex sequence of post-translational modifications of the tau protein. Plasma biomarkers for Alzheimer disease have continued to make rapid advances, especially around highly sensitive assays capable of detecting different forms of abnormal hyperphosphorylated tau in peripheral blood. Two studies using cryo-electron microscopy showed the power of the approach by continuing to elucidate the diversity of filamentous tau inclusions, and a third study gave the first glimpse of α-synuclein aggregates at near atomic resolution. Another study continued to delineate how different α-synuclein conformers ("strains") target specific brain regions and lead to neurodegeneration. In Huntington's disease, we saw compelling molecular data showing how cells adapt to endoplasmic reticulum stress through the unfolded protein response. Finally, the role of astrocytes in chronic traumatic encephalopathy has emerged as a critical area of interest.

Comparing Differential Gene Expression in Chronic Traumatic Encephalopathy, Parkinson's Disease, and Bipolar Disorder

Introduction. Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder that isdefined, neuropathologically, by the presence of aggregated hyperphosphorylated tau in the neurons andastrocytes of the perivascular area that is located deep in the cerebral sulci. The lesion is associated withrepetitive brain trauma, from the spectrum of asymptomatic subconcussive head injury to grossly identifiablefeatures of concussion. Although the diagnostic neuropathology of CTE is well-characterized, the precisemechanism that causes this to occur in CTE is not yet clearly elucidated. The features of hyperphosphorylatedtau in CTE is quite similar with Alzheimer’s Disease (AD), as is the reduced expression of certain genes thatare required to dephosphorylate tau, which is the putative culprit in the generation of amyloid aggregatesand hyperphosphorylated tau.1 In comparison, Parkinson’s Disease (PD) is a neurodegenerative disease thatis caused by accumulation of misfolded alpha-synuclein (α-syn) that causes the formation of intraneuronalLewy Body aggregates. The pattern of accumulation for α-syn involves the olfactory bulb and the gut withprogressive involvement of the posterior part of the brain.2 Despite establishing the presence of two differentintraneuronal inclusions for CTE and PD, contact sports associated with the clinical spectrum of CTE has beenshown to present with Parkinsonian features along with dementia. Mood disorders has been reported to occurin patients with these neurologic conditions. Several studies have documented that patients had a previousexperience of traumatic brain injury prior to the diagnosis of Bipolar Disorder (BD). A review of electronicliterature suggested that having an earlier diagnosis of BD increased the likelihood of having a diagnosis ofPD in the future.3,4 Objectives. This research aimed to compare the over- and underexpressed genes in cases with Parkinson'sDisease (PD), cases with Bipolar Disorder (BD), and cases with Chronic Traumatic Encephalopathy (CTE) versusnormal controls. This was done to determine if parallel overexpression in certain genes may indicate the possible association at the level of gene expression. Identifying similar RNA sequence establishing gene expression may provide an insight to the relationship of the diseases in terms of pathobiological behavior. Determining the similar over- or underexpression pattern may provide an insight on the common pathobiologic mechanisms that may be the reason for the three disorders being associated by way of pre-morbid or co-morbid condition. Methodology. Transcripts from the public domain archive of the NCBI SRA were identified for the RNA sequence (RNAseq) of interest using the search string “Chronic Traumatic Encephalopathy,” “Bipolar Disorder,” and “Parkinson.” Only public domain transcriptome files of post-mortem brain samples labeled as RNAseq dataextracted thru the Illumina platform that have a paired normal control were selected. A total of ten (10)cases for each disorder and thirty (30) normal subjects for control in the NCBI SRA RNAseq database witha whole exome sequence file that was available for public domain use was utilized for differential geneexpression analysis.6,7,8 Results and Discussion. Among 21,122 identified genes from the RNAseq, the analysis was able to identify26 genes exhibiting increased expression of up to >15 log2 fold change among cases with CTE, PD, andBD compared with normal controls. In contradistinction, only 6 well-described genes exhibited a decreasedexpression among cases with CTE and BD compared to normal controls. However, there were no identified genes that exhibited underexpression in cases with PD compared with normal controls. The identification of parallel gene overexpression among the CTE, BD, and PD groups with respect to structural integrity, cellular metabolism, homeostasis, and apoptosis may indicate a common pathway that have been initiated as part of the response to maintain tissue function or as a consequence of the underlying pathobiologic mechanism that caused the primary lesion.

Rapid Validation of Telepathology by an Academic Neuropathology Practice During the COVID-19 Pandemic.

The coronavirus disease 19 (COVID-19) pandemic is placing unparalleled burdens on regional and institutional resources in medical facilities across the globe. This disruption is causing unprecedented downstream effects to traditionally established channels of patient care delivery, including those of essential anatomic pathology services. With Washington state being the initial North American COVID-19 epicenter, the University of Washington in Seattle has been at the forefront of conceptualizing and implementing innovative solutions in order to provide uninterrupted quality patient care amidst this growing crisis. To conduct a rapid validation study assessing our ability to reliably provide diagnostic neuropathology services via a whole slide imaging (WSI) platform as part of our departmental COVID-19 planning response. This retrospective study assessed diagnostic concordance of neuropathologic diagnoses rendered via WSI as compared to those originally established via traditional histopathology in a cohort of 30 cases encompassing a broad range of neurosurgical and neuromuscular entities. This study included the digitalization of 93 slide preparations, which were independently examined by groups of board-certified neuropathologists and neuropathology fellows. There were no major or minor diagnostic discrepancies identified in either the attending neuropathologist or neuropathology trainee groups for either the neurosurgical or neuromuscular case cohorts. Our study demonstrates that accuracy of neuropathologic diagnoses and interpretation of ancillary preparations via WSI are not inferior to those generated via traditional microscopy. This study provides a framework for rapid subspecialty validation and deployment of WSI for diagnostic purposes during a pandemic event.

Schizophrenia in a genomic era: a review from the pathogenesis, genetic and environmental etiology to diagnosis and treatment insights.

Schizophrenia is a common multigenic and debilitating neurological disorder characterized by chronic psychotic symptoms and psychosocial impairment. Complex interactions of genetics and environmental factors have been implicated in etiology of schizophrenia. There is no central pathophysiology mechanism, diagnostic neuropathology, or biological markers have been defined for schizophrenia. However, a number of different hypotheses including neurodevelopmental and neurochemical hypotheses have been proposed to explain the neuropathology of schizophrenia. This review provides an overview of pathogenesis, genetic and environmental etiologies to diagnosis and treatment insights in clinical management of schizophrenia in light of the recent discoveries of genetic loci associated with susceptibility to schizophrenia.

Carbonic anhydrase IX as a marker of hypoxia in gliomas: A narrative review

Hypoxia is a powerful driver of the malignant phenotype in solid tumors including gliomas. A major, though not sole, driver of this effect is the hypoxia-inducible factors (HIF) which promote the expression of hundreds of downstream genes through binding with hypoxia-responsive elements in the promoter regions of targeted genes. HIF-2α drives the cancer stem cell phenotype that has been shown to promote chemo- and radioresistance. HIF-1α drives the transcription of a number of genes, the most prolific and important of which appears to be that of CAIX, but also drives the transcription of VEGF and a number of glycolytic enzymes, thus participating in driving the Warburg effect. This brief review introduces how the localization of CAIX by immunohistochemistry has, though still in its early phases, allowed the identification of gliomas with worse prognosis, an application of significant importance in diagnostic neuropathology. The future of hypoxia research will manipulate these downstream pathways to provide further biomarkers through which the presence of hypoxia and its effects can be established, analyzed, and exploited.

Top ten discoveries of the year: Neuroinflammation.

Ten neuropathological studies, published in 2019, are discussed, which address important aspects of neuroimmunology and inflammatory brain disease. They include topics related to new mechanisms of inflammation and immune mediated neurodegeneration, which are relevant for multiple sclerosis (publications 1 to 4) and discuss the role of specific autoantibodies against myelin oligodendrocyte glycoprotein or aquaporin 4 in neuromyelitis optica spectrum disorders (publications 5 and 6). Other studies highlight the discovery of new virus induced diseases of the nervous system and their relevance for clinical neurology and diagnostic neuropathology (publications 7 and 8). Finally, very interesting studies are discussed dealing with microglia and immune mechanisms in neurodegeneration (publication 9) and the neuropathological long-term outcome of Aß vaccination in Alzheimer's disease (publication 10). All these studies highlight the central role of neuropathology in neurological disease research.

A half century of change in diagnostic neuropathology: from the giants of yore to current brain tumor classification

The first issue of Human Pathology contains a laudatory review of one of the most treasured books in the history of neuropathology: Neurological Clinicopathological Conferences of the Massachusetts General Hospital, a collection of neurological cases that appeared first in the New England Journal of Medicine in the 1940s, 1950s, and 1960s. Each patient history is discussed by well-known neurologists, neurosurgeons, and neuropathologists. Review of these cases provides a framework to explore diagnostic shifts that have occurred over the past half century. Importantly, while the discussants of these cases were great diagnosticians, they were somewhat limited by the methods available to them at the time; subsequent novel technologies provided opportunities for new insights that were made by the next generation of experts. Today's pathologists (whether neuropathologists or any other pathology subspecialists) are similarly skilled at diagnosis, although their diagnoses are now more often made on biopsies (rather than autopsies) and informed by pre-operative imaging studies as well as post-operative molecular analyses. In turn, one would conclude that, even in the face of future technological changes brought about by disruptive innovations like artificial intelligence and deep molecular analyses, a need will continue for the expertise of pathologists and other clinical diagnosticians.

Parkinsonism in essential tremor cases: A clinicopathological study.

BackgroundEssential tremor and Parkinson's syndrome are two common movement disorders that may co‐occur in some individuals. There is no diagnostic neuropathology for essential tremor, but in PD and other Parkinson's syndrome variants, the neuropathology is well known. The spectrum of Parkinson's syndrome variants associated with essential tremor, their clinical features, and course have not been determined in autopsy‐confirmed cases.ObjectivesTo identify: diagnostic features of essential tremor/Parkinson's syndrome, different Parkinson's syndrome variants, and long‐term clinical profile in such cases.MethodsPatients that had an essential tremor diagnosis and a subsequent clinical or pathological diagnosis of Parkinson's syndrome seen in our clinic during 50 years were included. The diagnosis of parkinsonism was made when bradykinesia, rigidity, and resting tremor were all clinically evident.ResultsTwenty‐one cases were included. All the common variants of parkinsonism co‐occurred with essential tremor. The most common was PD (67%) followed by PSP. The pathological findings were not predicted clinically in 2 cases that had essential tremor/PD and in all 5 essential tremor/PSP cases.ConclusionIn most essential tremor/Parkinson's syndrome patients, the main motor features of parkinsonism—bradykinesia, rigidity, and resting tremor—were identifiable. All known degenerative Parkinson's syndrome variants co‐occurred in essential tremor patients. © 2019 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

The Science and Art of Nerve Fiber Teasing for Myelinated Nerves: Methodology and Interpretation.

Nerve fiber teasing is a sensitive technique utilized in diagnostic neuropathology practice, laboratory research, and animal toxicity studies for characterizing changes in single myelinated nerve fibers over extended distances. In animal toxicity studies, a nerve portion (approximately 10 mm in length) is stained with Sudan black for 24 to 48 hours and then transferred into a drop of viscous medium (eg, glycerin) mounted on an adhesive-coated glass slide, positioning it such that the proximodistal orientation is known. Individual fibers are removed using fine forceps while the sample is viewed under a stereomicroscope. In general, lesions can be identified during teasing, but more detailed characterization and photodocumentation is undertaken once nerve fibers have been dried and coverslipped. Nerve fiber teasing is particularly useful for distinguishing early stages of axonal degeneration (which presents as ovoid fiber fragments in the midinternodal region) from segmental demyelination (which presents as loss of original myelin segments and their replacement by thinner, shorter segments in the absence of axonal damage). The slow, laborious nature of nerve fiber teasing dictates that the technique will be employed on a few samples as an auxiliary method to better define the pathogenesis of nerve lesions first identified by conventional histopathologic assessment.

Diagnostic histochemistry in neuropathology.

Neuropathologists have a long and storied history of utilizing histochemistry in the characterization and diagnosis of neurological diseases. This review considers stains that are used for the identification of neurons, neuritic processes and axons, myelin sheaths, neuroglial cells, and connective tissue in the nervous system. Rapid histochemical-smear methods for intraoperative diagnosis are also discussed, along with possible roles for lectin-histochemistry in diagnostic neuropathology.

Diffuse Gliomas for Nonneuropathologists: The New Integrated Molecular Diagnostics.

Diffuse gliomas comprise the bulk of "brain cancer" in adults. The recent update to the 4th edition of the World Health Organization's classification of tumors of the central nervous system reflects an unprecedented change in the landscape of the diagnosis and management of diffuse gliomas that will affect all those involved in the management and care of patients. Of the recently discovered gene alterations, mutations in the Krebs cycle enzymes isocitrate dehydrogenases (IDHs) 1 and 2 have fundamentally changed the way the gliomas are understood and classified. Incorporating information on a few genetic parameters (IDH, ATRX and/or p53, and chromosome 1p19q codeletion), a relatively straightforward diagnostic algorithm has been generated with robust and reproducible results that correlate with patients' survival far better than relying on conventional histology alone. Evidence also supports the conclusion that the vast majority of diffuse gliomas without IDH mutations (IDH-wild-type astrocytomas) behave like IDH-wild-type glioblastomas ("molecular GBM"). Together, these changes reflect a big shift in the practice of diagnostic neuropathology in which tumor risk stratification aligns better with molecular information than histology/grading. The purpose of this review is to provide the readers with a brief synopsis of the changes in the 2016 World Health Organization update with an emphasis on diffuse gliomas and to summarize key gene abnormalities on which these classifications are based. Practical points involved in day-to-day diagnostic workup are also discussed, along with a comparison of the various diagnostic tests, including immunohistochemistry, with an emphasis on targeted next-generation sequencing panel technology as a future universal approach.

A novel method to visualise the three-dimensional organisation of the human cerebral cortical vasculature.

Current tissue-clearing protocols for imaging in three dimensions (3D) are typically applied to optimally fixed, small-volume rodent brain tissue - which is not representative of the tissue found in diagnostic neuropathology laboratories. We present a method to visualise the cerebral cortical vasculature in 3D in human post-mortem brain tissue which had been preserved in formalin for many years. Tissue blocks of cerebral cortex from two control cases, two Alzheimer's brains and two cases from Alzheimer's patients immunised against Aβ42 were stained with fluorescent Lycopersicon esculentum agglutinin (Tomato lectin), dehydrated and cleared using an adapted three-dimensional imaging of solvent cleared organs (3DISCO) protocol to visualise the vascular endothelium. Tissue was imaged using light sheet and confocal microscopy and reconstructed in 3D using amira software. The method permits visualisation of the arrangement of the parallel penetrating cortical vasculature in the human brain. The presence of four vascular features including anastomosis, U-shaped vessels, spiralling and loops were revealed. In summary, we present a low cost and simple method to visualise the human cerebral vasculature in 3D compatible with prolonged fixation times (years), allowing study of vascular involvement in a range of normative and pathological states.

Brain biopsies requiring Creutzfeldt-Jakob disease precautions in the Republic of Ireland 2005-2016.

Creutzfeldt-Jakob disease (CJD) risk precautions are required when performing brain biopsies on patients with a dementing illness and in 'risk' groups. The impact on a diagnostic neuropathology service is considerable. We sought to determine if better case selection might reduce the necessity for application of CJD risk precautions. We reviewed the clinical information, contributory investigations and final neuropathologic diagnosis in a cohort of patients (n=21), referred to the National CJD Surveillance Centre between January 1, 2005, and December 31, 2016. Of this 21-patient cohort, five were positive for CJD, four belonged to the 'at risk of CJD' category requiring brain surgery, while the remaining 12 were referred to the National CJD Surveillance Unit with CJD as part of their differential diagnosis. CJD was confirmed in 5/21 (three sporadic [s]CJD, one variant [v]CJD and one iatrogenic [i] CJD). CJD was clinically probable in 4/5 proven CJD patients (80%). The patients (n=4) in the 'at risk of CJD' group were diagnosed with tumour (n=2), inflammation (n=1) and non-specific changes (n=1). Of the remaining 12 patients (in whom CJD was included in the differential diagnosis), the final neuropathologic diagnoses included tumour (n=2), neurodegenerative (n=2), inflammatory (n=1), metabolic (n=2), vascular (n=2) and non-specific gliosis (n=3). More often than not, the clinical suspicion of CJD was not borne out by the final neuropathological diagnosis. Failure by clinicians to adhere to the recommended CJD investigation algorithm impacts adversely on the neuropathology workload and causes unnecessary concern among operating theatre, laboratory and nursing personnel.