The aim of the study is to identify the epidemiology of CNS tumours within the West Yorkshire region in comparison to other regions. The study aims to identify if any links can be made between the type or site of CNS tumour and the initial clinical symptoms. It also aims to identify if there is any link between occupation and incidence of CNS tumour.
All CNS pathology reports created at Leeds Teaching Hospital between 01/01/2019 and 09/03/2020 were analysed. The histological diagnosis and biopsy site were recorded and the corresponding medical records were used to identify age, gender, occupation and presenting clinical symptoms. Duplicate biopsies and cases where the corresponding medical records could not be identified were excluded. A total of 448 reports were included in the study.
The results showed that 13.12% of the samples included in the study represent metastatic disease. The majority being a breast, lung or melanoma as a primary. The most prevalent histological subtypes were meningioma and glioblastoma. The most prevalent group of primary CNS tumours with a total of 26.79% of all the samples was the diffuse astrocytic and oligodendroglial tumours. In this group the top 3 occupations identified were in the healthcare sector, IT and administration, and the retail sector. The most prevalent symptoms in this group were headaches, dysarthria and weakness. Further comparisons and analysis have been drawn across all CNS tumours identified in the study.
Introduction:
IDH mutations are classically regarded as driver mutations in diffuse glioma but have recently been identified in medulloblastoma (MB). Interestingly, only specific IDH mutations (IDH1 R132C/S) have been found in MB, in particular in SHH- and WNT-activated groups. SHH MB may also often carry TERT promoter mutations.
Case:
The patient is a 35 year old female with a right cerebellar lesion radiologically suggestive of medulloblastoma, astrocytoma or lymphoma.
Histology showed a neuroepithelial, mitotically active neoplasm including both embryonal and glial differentiation, with immunoprofile reflecting the phenotype. In view of this, comprehensive molecular analysis for glioma and embryonal neoplasia was carried out, including special immunohistochemistry, cytogenetics, gene sequencing, MIMIC and EPIC methylation arrays.
While histologically, a diagnosis of medulloblastoma, classic type, was favoured, early sequencing revealed IDH1 R132C mutation, in combination with TERT promoter mutation (C228T), raising a differential diagnosis of diffuse glioma.
However, methylation array analysis (MIMIC and EPIC) confidently classified the neoplasm as MB (SHH group). The same MB group was also confirmed on immunoprofile.
Conclusion:
IDH mutation does not automatically indicate diffuse glioma, as IDH1 R132C/S mutations may (rarely) occur in medulloblastoma (1,2).
In view of the divergent therapeutic regimes, an accurate diagnosis is crucial to deliver optimal patient care.
The exact frequency and prognostic implication of IDH mutation in MB remain to be elucidated.
References:
Northcott PA et al, Nature (2017) 547:311-7
Snuderl M et al, J Clin Oncology (2015) 33: e27-31
The DNA mismatch repair (MMR) system is a critical mechanism in maintaining genomic stability by correcting errors generated during DNA replication. Although acquired MMR deficiency has emerged as a mechanism for temozolomide resistance, a standard treatment option in gliomas, the incidence of MMR deficiency in treatment naïve tumours is unknown and routine screening is not currently recommended. Here we present a series of 3 patients from a single centre, with treatment naïve gliomas and MMR deficiency.
As part of routine clinical care, targeted sequencing of 50 cancer associated genes was performed in 33 patients with high-grade gliomas. Nine tumours harboured frameshift mutations, characteristic of MMR deficiency, or multiple point mutations. MMR protein expression was then analysed by immunohistochemistry and DNA microsatellite instability (MSI) analysis performed using five mononucleotide-repeat microsatellite markers as defined by the Revised Bethesda panel.
Three of the nine patients analysed showed loss of expression of one or more MMR proteins by immunohistochemistry. MSI, a hallmark of MMR deficiency, was present in one of the three gliomas with loss of MMR proteins, demonstrating only subtle variance. Germline analysis revealed a pathogenic MSH6 mutation indicative of the hereditary cancer syndrome Lynch/Turcot in one individual.
Our findings suggest that MMR immunohistochemical analysis is easy to interpret on high-grade gliomas and more sensitive than MSI analysis using the Revised Bethesda panel. The identification of high-grade gliomas with MMR deficiency may have significant management implications for these patients (i.e. genetic counselling) and should be considered in tumours with a high mutational burden.
This 69 year old patient, with a known history of sarcoidosis, presented with a suspected seizure. T1 MRI imaging with contrast showed a small avidly enhancing right temporal lesion with surrounding oedema. The main differential diagnosis included metastasis and sarcoidosis. Over a 4 month period the lesion had increased in size and a decision was made to operate and resect
The lesion was resected en bloc. Macroscopically, a well circumscribed, ovoid tan lesion (~11 x 4 x 3 mm) with a homogenous tan cut surface was received. Histology showed a fibrotic and inflammatory nodule with surrounding reactive brain parenchyma. The lesion was characterised by plump histiocyte-like spindle cells forming vague fascicles and admixed with areas of fibrosis. There was no mitotic activity, no well-formed granulomas were seen and there was no necrosis. The Ziehl-Neelsen staining was very striking. The cytoplasm of the described spindle cells was filled with numerous acid fast bacilli. The diagnosis of mycobacterial spindle cell pseudotumour (MSP) was confirmed.
MSPs are very rare benign lesions, which usually occur on a background of immunocompromise. They are characterised by local proliferation of spindle-shaped histiocytes, caused by mycobacterial infection (typically but not exclusively Mycobacterium avium intracellulare). Cerebral MSPs are often misdiagnosed clinically and radiologically as brain tumours. This case highlights an important, potentially treatable, differential diagnosis of a spindle cell lesion in the brain in an immunosuppressed patient. To the best our knowledge this represents the third reported case of an MSP in a patient with sarcoidosis.
Atypical teratoid/rhabdoid tumours of the central nervous system are highly aggressive neoplasms that typically arise in young children, with cases in adults being exceptionally rare. A small number of cases, with an overwhelming female predominance, have been reported to develop in the sellar region around the fifth decade of life. We present the case of a 43-year-old female who presented with a short history of headaches and was found to have complete right-sided ptosis and ophthalmoplegia with cranial nerve palsies of CN III, IV and VI bilaterally. MR imaging demonstrated an aggressively enlarging pituitary lesion that extended into the right cavernous sinus with complete encasement of the right ICA. She underwent endoscopic transsphenoidal debulking of the tumour and post-operative radiotherapy. Histopathological examination demonstrated a highly cellular tumour consisting of markedly atypical cells with loss of normal expression of INI-1. Methylation array demonstrated that this tumour segregated into the AT/RT-MYC subclass of paediatric AT/RT, a finding consistent with previous literature. Gene sequencing identified two different pathogenic variants in the SMARCB1 gene. We will present the pathological features of this case and set it in the context of what is known about this curious cohort of AT/RTs of the sellar region.
Anaplastic large cell lymphoma is a type of T-cell lymphoma (ALCL). Involvement of the central nervous system is rare and it is exceptionally so in children. We present the case of a 12-year-old boy who presented with a very short history of headaches and vomiting and was found to have an isolated, partially necrotic large right frontal contrast-enhancing mass that was thought to be a malignant glioma, a view that the intraoperative smear appearances supported. However, paraffin-embedded sections showed florid astrocytic gliosis and macrophage infiltration with a relatively minor population of large atypical cells with pleomorphic nuclei, multiple nucleoli and variable amounts of cytoplasm that expressed CD30, CD43, LCA, vimentin and ALK-1. We will present the pathological features of this case and discuss the diagnostic challenges and differential diagnosis.
Introduction: MN1-altered brain tumours represent rare molecularly-defined CNS tumours with heterogeneous morphological presentation, often mimicking other entities and causing differential diagnostic problems. We present the histopathological and clinical features of three unusual paediatric tumours, all reclassified as high-grade CNS neuroepithelial tumour, MN1- altered.
Methods: A retrospective study was performed on 417 paediatric cases diagnosed between 2014 and 2020 in our department to reclassify all tumours according the current diagnostic guidelines. Cases with unusual histological features and immunoprofile were reviewed and investigated by high-throughput sequencing techniques.
Results: We identified three cases of high-grade CNS neuroepithelial tumour, MN1-altered by DNA methylation array. Histologically, they were all heterogeneous but focal sclerotic/hyalinised and astroblastomatous areas were identified retrospectively. In addition, predominant rhabdoid and epithelial differentiation (Case 1) and solid embryonal areas (Case 2 and 3) were also seen. Immunohistochemistry revealed an unusual profile with focal positivity for EMA, synaptophysin, GFAP, SMA and cytokeratin. An EWSR1-BEND2 fusion was detected by RNA Fusion Panel in Case 3. The patients presented with meningeal (Case 1, 16-year old female), frontal (Case 2, 11-year old female) and posterior fossa (Case 3, 18-year old male) tumours and had multiple recurrences with 41, 10 and 6 years of follow up, respectively.
Conclusion: Rapid improvement of molecular techniques allowed discovery of rare CNS tumours characterized by MN1 rearrangement. These cases may present with a recognisable astroblastoma pattern but with heterogeneous morphology and unusual immunoprofile requiring extensive molecular investigations for a firm diagnosis. Multiple recurrences can be expected with long survival.
Pituitary Neuro-Endocrine Tumours (PitNETs) are adenohypophyseal, neoplastic growths associated with increased morbidity and mortality due to their extensive clinical and pathological presentations. The current 2017 WHO Classification recognizes a Ki-67/MIB-1 index of ≥ 3%, positive p53 immuno-staining and a mitotic count of > 2 per 10 HPF as prognostic criteria indicative of PitNET invasiveness and recurrence, but studies have found this classification ineffective and too vague when considering the prognostic significance of Ki-67 and p53. Therefore, a universally accepted understanding of Ki-67 and p53’s role in recurrence is needed to accurately diagnose and treat atypical PitNETs.
We aimed to identify whether the Ki-67/MIB-1 index and p53 staining in LTHT patients with PitNETs was consistent with the 2017 WHO classification and whether Ki-67 and p53 were effective predictors of recurrence based on this classification.
Assessments of clinical and histological data collated over a 10-year span for all 119 cases of PitNETs in LTHT patients were conducted. Patients were divided into 2 groups according to the presence or absence of recurrence and Ki-67/MIB-1 values and the extent of p53 staining was compared between the groups.
The proposed Ki-67/MIB-1 index of ≥ 3% and extensive p53 staining was not associated with recurrent PitNETs. Non-recurrent PitNETs were associated with significantly higher Ki-67/MIB-1 indexes and more abundant p53 staining.
There is a need to redefine characteristics indicating recurrence in PitNETs. These findings contradict the 2017 WHO Classification guidelines, suggesting that Ki-67 and p53 are ineffectual markers of recurrence.
Background: CD8-encephalitis in treated HIV patients is recognised as a steroid responsive bilateral diffuse leukoencephalitis with polyclonal CD8 T cell parenchymal infiltrates. We present two cases, both receiving antiretroviral therapy (ARV), characterised by progressive neurological deterioration and unilateral cortical atrophy.
Methods: Case one (50 year old male), had focal onset of seizures, resistant to drug treatment culminating in episodes of focal and generalised status. Case two (51 year old female) with poor ARV compliance, presented with episodes of fluctuating consciousness, viral escape and sepsis with partial steroid response but no recorded seizures. Serial MRI in both showed right cortical and subcortical atrophy progressing over 2 (case 2) to 5 years (case 1).
Results: Post-mortem examination showed focal to confluent regions of cortical laminar atrophy, spongiosis and gliosis with subcortical myelin and axonal loss. Neuronal preservation was evident even in the more damaged cortical regions but no neuronophagia, giant cells, vascular pathology HIV-encephalitis or opportunistic infection was seen. Similar features but more patchy regions of cortical atrophy and focal CD8 infiltrates were evident in Case 2, including in the hippocampus. The left hemisphere in both cases showed minimal pathological change.
Conclusions: These cases broaden the spectrum of pathological phenotypes encountered in CD8-encephalitis and lend support to the concept of an autoimmune pathogenesis, with similarities to Rasmussen’s encephalitis.
A 56 year old female presented to her local hospital in Ghana with right sided sensory seizures. Local imaging demonstrated a ring enhancing lesion with extensive oedema involving the left post-central gyrus. On imaging review in Oxford, the features were in keeping with a high grade glioma. Following a course of methylprednisolone, repeat imaging showed reduction in the size of the lesion and surrounding oedema. The differential diagnosis included high grade glioma, inflammatory or infective lesions. Post multidisciplinary team meeting, she proceeded to excisional biopsy.
The intraoperative smear demonstrated reactive brain parenchyma and a solid component with round multilamellar objects, favouring parasitic or fungal infection, with no evidence of glioma. Histology confirmed the presence of a parasitic infection, with numerous scolices and the remnants of a cyst wall. A typical scolex showed a rostellum with hooklets with one to three suckers. Following review with genetic sequencing elsewhere, Coenurosis due to Taenia serialis was initially confirmed, but repeat genetic testing locally has raised the possibility of Taenai multiceps, instead (review ongoing).
Coenurosis is a parasitic infection resulting from ingesting the eggs of tapeworm species T. multiceps, T. serialis, T. brauni, or T. glomerata. Humans ingesting eggs from these species become intermediate hosts. Eggs can mature into larvae but not into adult worms. The larvae group within cysts, ‘coenuri’ can be seen in muscles, subcutaneous tissue and the central nervous system (CNS). CNS involvement is usually due to T. multiceps infestation and we present a rare example of CNS coenurosis histology.
We present the case of a 68-year-old retired engineer who presented with a short history of asymmetrical ascending sensorimotor symptoms in the distal lower limbs, having suffered a cutaneous insect bite one month prior. On investigation, he was found to have a painful asymmetrical length-dependent axonal polyneuropathy that was limited to the lower limbs. He was investigated extensively and as a definite cause was not found a sural nerve biopsy was performed, which showed a florid non-caseating granulomatous perineuritis and neuritis. There was a highly variable pattern of fibre involvement and axonal loss, with some fascicles preserved alongside some strikingly devoid of fibres, resulting in a severe patchy axonal neuropathy. After discussion with the clinicians, a diagnosis of isolated sarcoid neuropathy was made and treatment was commenced, with good effect. We present the pathological features of this case and discuss the differential diagnosis as well as the possible aetiological role of the insect bite.
Introduction: Alpers-Huttenlocher syndrome (AHS) is a fatal paediatric mitochondrial disease resulting from depletion of mitochondrial DNA and characterised by intractable epilepsy and severe neurodegeneration. A severe, consistent loss of parvalbumin (PV)-positive interneurons is suggested to underlie seizure-associated cortical activity in the occipital cortex (BA17) of patients with AHS. This is accompanied by a relative preservation of calretinin (CR)-positive interneurons. We hypothesised changes in mitochondrial respiratory chain expression may underlie the selective vulnerability of PV interneurons in AHS.
Methods: Post-mortem brain tissues were obtained for BA17 from 6 patients with AHS and 5 age-matched controls. Quadruple immunofluorescence permitted visualisation and quantification of expression of mitochondrial respiratory complexes I and IV subunits (NDUFB8 and COXI, respectively) within mitochondria (porin) in PV and CR interneurons. Control respiratory chain data were used to derive z-scores in order to make inferences about interneuron dysfunction in AHS. Classifications were based on standard deviation limits: z>2SD = overexpression, z<-4SD = severe deficiency.
Results: Immunofluorescence analysis revealed a severe deficiency of NDUFB8 and COXI in 87% and 96% of surviving PV interneurons (z<-4), respectively, in AHS. This was accompanied by a significant increase in mitochondrial mass in 79% of PV interneurons (z>2). Whereas only 31% and 11% of CR interneurons showed severe deficiencies in NDUFB8 and COXI, respectively.
Conclusion: Severe respiratory chain impairment within PV interneurons in BA17 may underlie the severe, selective vulnerability of PV interneurons in AHS. This could result in loss of inhibitory neurotransmission leading to seizures in patients with AHS.
Background
Alexander’s Disease (AxD) is rare neurodegenerative disorder caused by mutations in the gene encoding a major intermediate filament protein in astrocytes, GFAP. It is characterized by white matter degeneration related to the loss of myelin, and abnormal protein aggregates in astrocytes, called Rosenthal Fibres. In animal models of AxD, upregulation of proinflammatory chemokines and microgliosis have been shown, particularly surrounding astrocytes. In humans, microglia are known to acquire a more reactive morphology with thickened processes and enlarged cell bodies, and evidence suggests they may phagocytose astrocytes. However the phenotype of microglia in human AxD brains has not been extensively characterised.
Methods
To characterise the neuroinflammatory environment in AxD, we acquired FFPE brain tissue from 4 AxD patients and 6 gender and age matched controls, via BRAIN UK. Using immunohistochemistry, tissue was stained with 5 microglial markers (Iba1, HLA-DR, CD68, CD16 and CD64) and 3 astrocytic markers (GFAP, Glutamine Synthetase and EAAT2). We assessed the protein load of these markers in regions of white and grey matter with evidence of pathological Rosenthal fibres.
Results
CD16 protein load was significantly increased in the grey (p=0.0087) and white matter (p=0.001) in AxD; while the other microglial markers were unaffected. GFAP was significantly increased in the grey matter (p=0.0021) and Glutamine Synthetase in the white matter (p=0.0007). EAAT2 showed no difference between the groups.
Conclusion
Our findings imply a role for antibody-dependent cellular toxicity and impaired glutamate synthesis in AxD, potentially as the result of a dysregulation in the microglia-astrocyte communication.
Astrocytes provide support for neurons and change early in Alzheimer’s disease. Astrocytes surrounding fibrillar Aβ in plaques may be protective, but astrocytes also mediate Aβ toxicity. We hypothesise that different forms of Aβ can drive astrocytes to neurodegenerative or neuroprotective phenotypes during disease.
We generated stable monomeric Aβ1-42 preparations, from which we derived oligomeric and fibrillar Aβ1-42. The morphology of Aβ1-42 was confirmed using transmission electron microscopy, asymmetric flow field flow fractionation, immunoblotting, and size exclusion chromatography. To compare with an acute stress response, the astrocyte DNA damage response (DDR) was characterised. Human primary astrocytes (Sciencell®) were treated with 100μM H2O2 and analysed for γH2AX, pChk2, p21 and cdc2 expression at multiple time points.
Oligomer preparations contained >50% oligomers of varying sizes, with no observable Aβ fibrils. Fibril preparations comprised large numbers of uniform fibrils. Treatment of human astrocytes with H2O2 resulted in a rapid DDR detected 1 hour post-treatment, which is associated with double-strand DNA breaks, formation of γH2AX-positive DNA foci, and cell cycle arrest. Current work is focused on characterising the responses of human primary astrocytes to oligomeric and fibrillar Aβ1-42, including measuring cell death and the formation of γH2AX-positive DNA foci. This is carried out using immunocytochemistry and toxicity assays.
We have generated well-validated preparations of oligomeric and fibrillar Aβ to determine their impact on astrocyte responses; and characterised the DDR in human astrocytes. Understanding how astrocytes respond to Aβ is important in understanding the aetiology of Alzheimer’s disease, and in designing new therapeutic targets.
Background: Systemic inflammation in patients with Alzheimer’s disease (AD) has been associated with an exacerbation in cognitive decline, but the underlying mechanisms remain largely unknown. In AD, intraneuronal hyperphosphorylated tau spreads through the brain via trans-synaptic prion-like propagation. Evidence suggests that propagation of tau pathology is linked to neuroinflammation. Here we aim to investigate if a low-grade systemic bacterial infection drives neuroinflammation and exacerbates the spread of tau using an experimental model.
Methods: C57BL/6 mice underwent intracerebral injection of vehicle or AT8-positive tau lysate, isolated from post-mortem AD tissue. Two months post-injection, all mice were given a systemic injection of saline or Salmonella Typhimurium. Body and spleen weight was measured to determine susceptibility to infection and brain tissue was collected 4 weeks post-infection to assess tau pathology and markers of neuroinflammation.
Results: We observed tau pathology in all mice exposed to human tau lysate. Pathology in the dorsal fornix and the supramammillary nuclei was observed only in mice exposed to Salmonella Typhimurium. We also examined the inflammatory markers MHCII and CD64 and detected increased expression in mice exposed to a bacterial infection. The inflammatory response was localised to regions where pathology was observed, such as the CA3-dentate gyrus axis.
Conclusion: Propagation of tau was induced both dorsal and ventral to the injection site. The rate of propagation was exaggerated by the presence of low-grade systemic inflammation. This suggests that systemic inflammatory episodes in AD patients may induce propagation of tau pathology.
Background
Glucocerebrosidase (GBA) mutations are a recognized risk factor for developing not only Parkinson’s disease (PD) but also Lewy body dementia (LBD), including dementia with Lewy body (DLB), and PD dementia (PDD). Currently, there is no clear understanding of the mechanistic relationships between GBA mutations and α-synuclein accumulation, although, GBA mutations are thought to accelerate α-synuclein accumulation. Therefore, we aimed to investigate whether post-translational modifications of α-synuclein will give rise to species that differ between PD cases with N370S GBA mutation and WT-PD.
Methods
14 PD/LBD cases with and without GBA N370S were obtained from QSBB and 2 from SWBB. Brain regions of anterior cingulate cortex (ACC), substantia nigra (SN), and ventral tegmental area (VTA) were examined using immunohistochemistry with 5 different α-synuclein antibodies: 4 novel epitopes at the n-and c-terminal of a-synuclein and 1 well-known phosphorylation site and analysed semiquantitatively using Mckeith criteria.
Results
In cingulate cortex, although, Lewy body (LB) inclusions were higher in N370S PD/LBD compared with WT PD/LBD, a significant difference was found only with the n-terminal antibody. In SN, LB-pathology was increased in the GBA group compared with WT-PD. In VTA, several different structures including LBs, pale bodies, and cytoplasmic staining were significantly higher in the GBA group compared with WT-PD using different antibodies employed in this study.
Conclusion
Immunohistochemical results suggest that N370S GBA mutation exacerbate α-syn pathology. These findings may explain why clinically, GBA mutation carriers manifest early disease onset, increased cognitive decline, and rapid disease progression compared to non-carriers.
Introduction: Progressive supranuclear palsy (PSP) is a fatal, multifactorial, neurodegenerative disease. DNA methylation is an epigenetic modification that leads to changes in expression of target genes and is implicated in a large range of diseases, including neurodegenerative diseases. Weber et al. (2018) investigated the DNA methylation profile of PSP patients and found several DNA methylation alterations. We investigated this publicly available methylation dataset using a different DNA methylation analysis pipeline, and also performed weighted correlation network analysis to identify modules of co-methylated genes associated with PSP.
Methods: Raw methylation data, derived from post-mortem frontal cortex tissue, was downloaded from the NCBI-Gene Expression Omnibus depository (GEO accession GSE75704). After quality control assessment and normalization of the data, we included 164 samples in downstream analysis (93 PSP patients and 71 controls). Differential methylation analysis was performed, and we then carried out weighted gene correlation network analysis (WGCNA) using those probes showing the highest variance of methylation levels, after adjustment for possible confounding factors (e.g. age and sex).
Results: In the differential methylation analysis, similar to the results from Weber et al. (2018), we found DNA methylation changes associated with PSP, including several CpGs mapping to DLX1 that are significantly hypermethylated in PSP cases compared to controls. In the network analysis, we found clusters of highly correlated CpGs (co-methylation modules) and we are evaluating their correlation with the disease status.
Conclusion: Our results support a role for DNA methylation alterations in PSP.
Background
Mutations in glucocerebrosidase gene (GBA) are a common risk factor for Parkinson’s disease (PD) and Lewy body dementia (LBD). <70% of PD patients also develop dementia. To date, the molecular mechanisms underlying these diseases are unknown. Synaptic protein changes have been shown to correlate with cognitive decline in Alzheimer’s disease (AD). Here we examined a range of synaptic proteins in alpha-synucleinopathies with & without GBA mutations.
Methods
Brain tissue was obtained from QSBB and SWDBB. Synaptic marker proteins synaptophysin, SNAP25, PSD95, neurogranin, Rab3a, VAMP2 and VAMP3 were examined in 7 GBA N370S PD/LBD, 21 WT PD/LBD, and 10 controls. Four cortical regions were assayed using standard immunoblot procedures.
Results
Post-synaptic protein, neurogranin was significantly decreased in the frontal and parietal cortices of GBA PD/LBD. Whilst the cingulate cortex showed a significant increase in PD/LBD compared to controls. Presynaptic protein synaptophysin was significantly increased in WT PD/LBD compared with control in cingulate cortex. SNAP25 was significantly increased in GBA PD/LBD compared to both control and WT PD/LBD in frontal cortex. VAMP2 was significantly reduced in both diseased groups compared to controls in frontal cortex. Additionally, the astrocytic protein VAMP3 was significantly reduced in PD/LBD compared to control and GBA groups.
Conclusion
Regional changes in synaptic proteins were observed in disease versus control groups. Our GBA cases showed differential expression pattern of certain synaptic proteins compared with WT, suggesting that synaptic changes may underlie cognitive impairment in PD with N370S GBA mutation.
Purpose: Recent Parkinson’s disease (PD) genetic studies have implied vesicular dysfunction as one of the pathogenic causes of PD. It is likely that these dysfunctions occur in very early stages of disease. How these changes relate to PD pathology have not been explored. We have examined three recent genome wide association study nominated genes for PD namely Rab29, syt11 and ATP13A2 and how this correlated with PD progression.
Methods: Brain tissue samples were obtained from Queen Square Brain Bank following ethical approval from local research ethics committee. Flash frozen and formalin fixed brain tissues were obtained from 4 neurologically normal controls, 4 incidental LB cases, 4 limbic and 4 neocortical PD cases for immunohistochemistry and immunoblotting for RAB29, syt11, and ATP13A2 using standard procedures. Medulla, substantia nigra, basal ganglia and cingulate brain regions were studied.
Results: Rab29 levels were low while syt11 and ATP13A2 were relatively higher in all 4 regions examined. Immunohistochemistry demonstrated that ATP13A2 was expressed in neuronal cell bodies while syt11 was expressed in some cell bodies but more in neuronal processes. Syt11 appeared to show some Lewy body and Lewy neurite staining in the substantia nigra. Rab29 was also expressed in astrocytes in the substantia nigra of early disease cases.
Conclusion: Differential vesicular protein expression may contribute to synaptic dysfunction in PD. Expression of Rab29 in nigral astrocytes indicate that cell type specific expression may contribute to PD pathogenesis. Possible Lewy body and Lewy neurite staining may indicate a role for syt11 in PD pathogenesis.
Neuroinflammation is a prominent feature of the Apolipoprotein E knockout (ApoE-/-) mouse model of atherosclerosis. The current study tested the hypothesis that microvascular damage related to systemic atherosclerosis induces cell specific changes which lead to neurovascular unit (NVU) dysfunction, contributing to neuroinflammation and neurodegeneration.
Detailed immunohistological assessment of astrocytes (GFAP), microglia (IBA-1) and endothelial cells (ICAM-1) was performed on ApoE-/-mice fed on a high fat diet (HFD) (n = 6-12) and on a low fat diet (n = 7-13). The hippocampus was isolated using laser capture microdissection and the transcriptomic profile characterised using microarray analysis.
GFAP+ astrocytes (p = 0.03) and ICAM1+ endothelial cells (p= 0.01) were significantly higher in the corpus collosum of animals fed on a HFD. Significantly higher levels of Iba-1+ microglia were detected in the hippocampus (p = 0.002), corpus callosum (p = 0.004), and cerebral cortex (p = 0.004).
Transcriptomic analysis of the hippocampus indicated significant downregulation of the endoplasmic reticulum associated degradation pathway (p = 0.003) and calcium signalling (p = 0.002); and a significant upregulation of metabolic (p < 0.001) and inflammatory pathways (p = 0.006) in animals fed on a HFD. These findings indicate that systemic atherosclerosis is associated with changes in the cerebral microvasculature, affecting astrocyte, microglial and endothelial cell responses. The gene expression changes associated with HFD and inflammation suggest that systemic atherosclerosis may be associated with cellular pathway alterations in the NVU. Future work is required to establish the importance of this in producing alterations in the brain.
HnRNP K is a member of the functionally diverse family of RNA-binding proteins called heterogeneous nuclear ribonucleoproteins (hnRNPs). HnRNPs, including TDP-43 and FUS have been inextricably linked to the pathomechanistic underpinnings of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). HnRNP K, by contrast, remains underexplored in neurodegeneration. The hnRNP K localisation profile in neurons of the frontal cortex was examined in a large, comprehensive pathological analyses including FTLD-TDP type A (n=28), type C (n=12), FTLD-Tau (n=5), ALS (n=7) and control (n=35) brain tissue. Immunohistochemically stained hnRNP K sections were digitally scanned. Neurons with normally localised (predominantly nuclear) hnRNP K were classified by an automated analytical pipeline utilising deep learning. Abnormally localised neurons (nuclear depletion and cytoplasmic accumulation) were counted manually. Cytoplasmic mislocalisation of hnRNP K was observed in many FTLD cases within pyramidal neurons independent of TDP-43 or tau pathology. FTLD-TDP A and FTLD-Tau frontal regions exhibited significantly less normally localised hnRNP K (p=0.01) and significantly more abnormally localised hnRNP K (p<0.01) than controls. Mislocalisation frequency correlated with age at death across the cohort (r=0.44, p<0.0001). However, the median value of total mislocalisation in the FTLD-TDP A group was observed 23 years earlier than controls (63 vs 86 years of age) hinting at an advanced ageing-like phenotype. Neuronal mislocalisation of hnRNP K is a potentially novel pathological event to be described in the context of neurodegeneration. The potential transcriptomic changes that may accompany such a dramatic subcellular redistribution in FTLD patient brain warrants further study.
Background
IBM is a complex muscle disease with unknown cause. However, it is believed that genetic factors may influence disease susceptibility. We hypothesize that whole exome sequencing (WES) is effective to study rare variants and this is important particularly for rare disease like IBM, but this approach will miss genome wide variants. GWAS can contribute to understand the genetic events underling IBM. The major limitations, particularly in rare disease like IBM, is the need for large numbers of well-defined cases
Aims
Methods
WES was performed on the initial collection of IBM and controls to study rare and common coding variants. Sample and variant quality control were performed to ensure high quality data for association analysis. Candidate gene approach was performed to investigate rare variants in VCP and SQSTM1. Single variant association analysis was performed based on χ2 test. Logistic regression was performed with including sex as a covariate. Multiple testing correction was applied to validate the associations found
Results
Our data analysis has identified rare variants in VCP and SQSTM1, which emphasizes the hypothesis of a pathogenic mechanism involving impaired autophagy-lysosome pathways. Single variant association analysis has demonstrated statistical significance of several common variants on chromosome 6p21, where inflammatory genes are located
Conclusion
We present results from exome Burden analysis. These results identified candidate loci and will be complemented by an ongoing GWAS on larger cohort
Introduction: Mitochondrial diseases comprise the largest group of inherited metabolic disorders. Neurological symptoms include epilepsy, stroke-like episodes, ataxia and cognitive impairment. This study aims to model early-onset neurological symptoms in mitochondrial disease using a novel murine model and to test the hypothesis that underlying hyperexcitability may arise due to neuronal network disinhibition, following mitochondrial dysfunction in parvalbumin-expressing (PV+) inhibitory interneurons.
Methods: A mouse model of mitochondrial DNA (mtDNA) depletion specifically within the PV+ cells was generated by a knockout of mitochondrial transcription factor A (Tfam) and characterised at behavioural, electrophysiological, neuropathological and molecular levels. Neuronal network activity was probed within the hippocampal CA3 area in vitro by inducing gamma oscillations using carbachol (cholinergic agonist).
Results and conclusions: A downregulation of complexes I, III and IV of the mitochondrial respiratory chain was confirmed within the PV+ cells of the knockout brain tissues. The most affected neurons were Purkinje neurons of the cerebellum, that showed a reduction in mtDNA copy number and an upregulation of anaplerosis enzyme pyruvate carboxylase. Mutant mice exhibited progressive phenotype, initiating at 8 weeks of age with tremor, cognitive impairment (assessed by novel object recognition test) and anxiety behaviour (in the elevated plus maze test). Hyper-locomotion (in the open field test) and stargazing (absence-like seizures) were detected at 10 weeks, with severe ataxia observed by 12-13 weeks. Electrophysiological analysis of mutant mice revealed hippocampal network perturbation. The novel mouse model recapitulates the early-onset phenotype of mitochondrial disease and highlights the role of mitochondrial function within the PV+ interneurons.
Case:
A 21 year old female presented with right frontoparietal lesion radiologically suggestive of meningioma or metastasis. Coincidentally, a rectal lesion was identified on CT scan. No family history or past medical history was recorded.
Histologically, the cerebral lesion showed a densely cellular high grade glioma with ATRX and p53 mutation, and a rare IDH1 R132S mutation but no CDKN2A/B deletion. MGMT promoter was highly methylated (53.75%)
In view of the rectal lesion, immunohistochemistry for MMR was performed on the brain tumour specimen. This revealed total loss of PMS2 expression in the tumour but retained PMS2 expression in non-neoplastic cells, consistent with Lynch syndrome. The patient has been referred to a geneticist.
Conclusions:
We present a case of high grade glioma (HGG) with a rare IDH mutation and mismatch repair deficiency (MMRD) in a young adult, reminiscent of a tumour type recently described as ‘primary mismatch repair deficient IDH-mutant astrocytoma (PMMRDIA)’.
Despite IDH mutation, this glial tumour is predicted to be highly aggressive and does not imply a better prognosis within the grade IV category.
HGG with MMRD show a hypermutational status reducing efficacy of Temozolomide therapy, even with highly methylated MGMT status.
There is an interest in using checkpoint inhibitors for these patients in view of high mutational load.
References:
Suwala et al. 2021 Acta Neuropathologica 141:85–100
Bouffet et al. 2016 Journal of Clinical Oncology 34:2206-2211
The heterogeneity of tumor infiltrating lymphocytes is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune cell subsets in brain metastasis tissues to test which immunosuppressive routes are involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on twenty formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and Iba1, and analyzed an average of 15000 cells per sample. We classified tumours as either high (>30%) or low (<30%) tumour infiltrating lymphocytes (TILs) and found that increased TILs density correlated with survival. We next sought out to phenotype these TILs using mIF. The tumours with low TILs (n=9) had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p<0.01) as well as in their microenvironment (p<0.001). Contrastingly, the brain metastatic tumours with high TILs (n=8) displayed higher levels of activated microglia, as measured by Iba1 expression. Low TILs-tumours displayed CD8+ T-cells that co-express VISTA (p<0.01) significantly more compared to high TILs group, where CD8+ T-cells significantly co-express Iba1 (p<0.05). Interestingly, no definite phenotypes of CD4+ subsets were observed. These results were supported by RNA analysis of a publicly available, independent cohort. In conclusion, our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.
Background: Overactivation of the classical renin-angiotensin system (cRAS) correlates with cognitive decline and disease pathology in AD. ACE1, a gene recently identified by GWAS as a genetic risk factor for AD, encodes ACE-1, a rate-limiting enzyme in the cRAS pathway. The role of ACE-1 in AD remains unclear as it is known to degrade Aβ and facilitate its clearance whilst producing angiotensin II - a potential mediator of AD pathology. We tested the association between the ACE1 variant (rs4343, indel proxy polymorphism) and disease pathology and ACE-1 protein level in a large post-mortem study.
Methods: We studied 318 dementia cases (AD, Mixed and vascular dementia) and 116 post-mortem controls from the South West Dementia Brain Bank, University of Bristol. ACE-1 level was measured by sandwich ELISA and parenchymal Aβ plaque and tau tangle load was quantified by computer-assisted field fraction analysis in frontal and parietal lobes. ACE1 (rs4343) polymorphism was genotyped by PCR.
Results: ACE-1 protein level was increased in AD and mixed dementia cases compared to controls in the parietal cortex (p<0.0001 and p=0.0244) but not the frontal cortex. Individuals who were homozygous I/I had lower ACE-1 protein level compared to homozygous (D/D) in the frontal cortex (p=0.0051) and the parietal cortex (p=0.0083).
Conclusion: We report that the disease-associated ACE1 I-allele is associated with reduced level of ACE-1, consistent with ACE-1 having a potentially protective effect in AD beyond its central deleterious role in cRAS activation.
Early and accurate diagnosis in Parkinsonian disorders may be difficult in the earliest disease stages. Moreover, disease progression varies from patient to patient. Genetics have provided tremendous insights into neurological diseases, largely based on studies in clinically diagnosed patients. The gold standard for diagnosis is neuropathology, and this defines the primary protein pathology which is the major target for therapy.
We aim to integrate clinical and genetic data with neuropathology on a large scale to better understand the genetic drivers of the disease process and improve the diagnostic accuracy of movement disorders. We hope to generate a resource to enable secondary analyses which use clinical, pathological and genetic information.
We will use the Illumina Neurobooster array to genotype 2800 neuropathologically confirmed cases with Parkinsonian disorders and 1000 controls in a collaborative project in the UK Brain Bank Network. We will conduct pathologically defined case-control, case-case and progression GWAS, and use the data to develop polygenic risk tools to enhance diagnostic accuracy. In a subset of 750 LBD cases, we will use digital pathology approaches to quantify the amyloid, tau and alpha-synuclein burden. We will carry out gene and transcript analyses to study the genetic drivers of pathology and integrate this with analyses of clinical heterogeneity to decode the different patterns of neurological disease, which may ultimately respond to different therapies.
We believe this study will improve our understanding of the neurobiology of movement disorders and contribute to the development of disease-modifying treatments.
Following the WHO declaration of the COVID-19 outbreak as a global pandemic, the UK governments declared 'lockdown' in March 2020. In line with the GMC guidance to the healthcare workers, we swiftly adopted new working practices to meet the need for flexible and remote working including virtual MDT clinical meetings.
The 'fresh' muscle biopsy sample was handled with necessary precautions as per the PHE safe handling guidance. The whole slide digital images were acquired using an advanced digital slide scanner. The images were analysed on a virtual microscopy platform with high resolution PC monitor. The relative small size of the muscle biopsy sample and high-throughput automatic scanning facility enabled quick turn around times.
The digital pathology reporting was introduced as per the UK RCPath guidelines, with initial self-validation, comparing digital images with the respective glass slides. In order to maintain clinical standards and patient safety, whenever in doubt, we cross-checked digital images for concordance with the glass slides.
The MDT clinical discussions were held on a virtual platform, taking appropriate measures to protect patient confidentiality. The virtual platform enabled expansion of clinical audience and facilitated better training opportunities to the junior doctors. Despite intermittent challenges such as scan failure, poor quality audio/video, the overall end-user feedback has been very positive.
Nearly a year after the digitalisation and adaptations to the workflow due to COVID-19 pandemic, we share our newly acquired set of skills and knowledge, which we hope will help to improve patient care in the neuromuscular services.
Introduction
Gliomas typically present as solitary lesions, and therefore, the synchronous occurrence of multiple primary brain tumours in the same patient is extremely rare; the majority of cases being of synchronous glioma and meningioma. We report the first known case of synchronous glioblastoma multiforme (GBM) and anaplastic oligodendroglioma (AO). Our case presents a 55-year-old woman referred to our service following a 3-week history of headaches, photophobia, confusion and dysphasia.
Results
CT and MRI revealed two space-occupying lesions; a cystic left temporal lobe lesion and a heterogenous right frontal lobe lesion. Immunohistochemical profiling confirmed that the two synchronous tumours originated from two distinct cell lines: GBM (IDH-1 wildtype, ATRX retained, loss of heterozygosity (LOH) at 19q locus) and AO (IDH-1 mutant, ATRX wildtype, and LOH at 1p/19q). Following resection of both tumours, the patient was commenced on a standard regimen of radiotherapy and Temozolomide (TMZ).
Discussion
Immunohistochemical analysis, particularly, the lack of IDH mutation in the Grade IV tumour supports its classification as a primary GBM, with both tumours originating from two different cell types with different mutational spectra. Prognostically, in AO, the LOH of 1p/19q is associated with longer overall survival, due to increased tumour sensitivity to radiotherapy and alkylating chemotherapy agents, therefore our patient was commenced on the combination of adjuvant TMZ and radiotherapy.
Conclusion
Despite the rare occurrence of synchronous primary brain tumours, this case demonstrates the importance of histological diagnosis and subsequent immunohistochemical profiling of each lesion to aid diagnosis, management and prognosis evaluation.