Somatic variants as a cause of drug-resistant epilepsy including mesial temporal lobe epilepsy with hippocampal sclerosis.

OBJECTIVE: The contribution of somatic variants to epilepsy has recently been demonstrated, particularly in the etiology of malformations of cortical development. The aim of this study was to determine the diagnostic yield of somatic variants in genes that have been previously associated with a somatic or germline epilepsy model, ascertained from resected brain tissue from patients with multidrug-resistant focal epilepsy. METHODS: Forty-two patients were recruited across three categories: (1) malformations of cortical development, (2) mesial temporal lobe epilepsy with hippocampal sclerosis, and (3) nonlesional focal epilepsy. Participants were subdivided based on histopathology of the resected brain. Paired blood- and brain-derived DNA samples were sequenced using high-coverage targeted next generation sequencing to high depth (585× and 1360×, respectively). Variants were identified using Genome Analysis ToolKit (GATK4) MuTect-2 and confirmed using high-coverage Amplicon-EZ sequencing. RESULTS: Sequence data on 41 patients passed quality control. Four somatic variants were validated following amplicon sequencing: within CBL, ALG13, MTOR, and FLNA. The diagnostic yield across 41 patients was 10%, 9% in mesial temporal lobe epilepsy with hippocampal sclerosis and 20% in malformations of cortical development. SIGNIFICANCE: This study provides novel insights into the etiology of mesial temporal lobe epilepsy with hippocampal sclerosis, highlighting a potential pathogenic role of somatic variants in CBL and ALG13. We also report candidate diagnostic somatic variants in FLNA in focal cortical dysplasia, while providing further insight into the importance of MTOR and related genes in focal cortical dysplasia. This work demonstrates the potential molecular diagnostic value of variants in both germline and somatic epilepsy genes.

Intratumoral histological and molecular heterogeneity in an adult diffuse glioma.

Adult-type diffuse gliomas are the most prevalent type of malignant adult brain tumors. Intratumoral heterogeneity can hinder accurate diagnosis and subsequent treatment. This case report documents a tumor with intratumoral heterogeneity, both histologically and by methylation analysis, located within the left cerebral hemisphere of a 29-year-old female. She presented after a witnessed generalized tonic clonic seizure at home. Two years prior she had a witnessed seizure; however, no brain imaging was done at the time. Magnetic resonance imaging (MRI), on this admission, showed a mass lesion in the left frontal operculum with poorly identified margins and right-sided midline shift. Sampling from the left temporal lobe showed an IDH-mutant, ATRX-mutant astrocytoma, which appeared grade 4 in the enhancing anterior portion and grade 2 in the left temporal lobe. Methylation analysis confirmed this heterogeneity. In summary, this is an excellent example of tumor heterogeneity both histologically and by molecular analysis. It is probable, given the clinical history of presentation 2 years prior, that this tumor originated as a low-grade glioma and subsequently evolved.

CPEB4-CLOCK crosstalk during temporal lobe epilepsy.

OBJECTIVE: Posttranscriptional mechanisms are increasingly recognized as important contributors to the formation of hyperexcitable networks in epilepsy. Messenger RNA (mRNA) polyadenylation is a key regulatory mechanism governing protein expression by enhancing mRNA stability and translation. Previous studies have shown large-scale changes in mRNA polyadenylation in the hippocampus of mice during epilepsy development. The cytoplasmic polyadenylation element-binding protein CPEB4 was found to drive epilepsy-induced poly(A) tail changes, and mice lacking CPEB4 develop a more severe seizure and epilepsy phenotype. The mechanisms controlling CPEB4 function and the downstream pathways that influence the recurrence of spontaneous seizures in epilepsy remain poorly understood. METHODS: Status epilepticus was induced in wild-type and CPEB4-deficient male mice via an intra-amygdala microinjection of kainic acid. CLOCK binding to the CPEB4 promoter was analyzed via chromatin immunoprecipitation assay and melatonin levels via high-performance liquid chromatography in plasma. RESULTS: Here, we show increased binding of CLOCK to recognition sites in the CPEB4 promoter region during status epilepticus in mice and increased Cpeb4 mRNA levels in N2A cells overexpressing CLOCK. Bioinformatic analysis of CPEB4-dependent genes undergoing changes in their poly(A) tail during epilepsy found that genes involved in the regulation of circadian rhythms are particularly enriched. Clock transcripts displayed a longer poly(A) tail length in the hippocampus of mice post-status epilepticus and during epilepsy. Moreover, CLOCK expression was increased in the hippocampus in mice post-status epilepticus and during epilepsy, and in resected hippocampus and cortex of patients with drug-resistant temporal lobe epilepsy. Furthermore, CPEB4 is required for CLOCK expression after status epilepticus, with lower levels in CPEB4-deficient compared to wild-type mice. Last, CPEB4-deficient mice showed altered circadian function, including altered melatonin blood levels and altered clustering of spontaneous seizures during the day. SIGNIFICANCE: Our results reveal a new positive transcriptional-translational feedback loop involving CPEB4 and CLOCK, which may contribute to the regulation of the sleep-wake cycle during epilepsy.

Comparative analysis of deeply phenotyped GBM cohorts of 'short-term' and 'long-term' survivors.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). METHODS: Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed. RESULTS: Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. CONCLUSION: Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.

Brain biopsy in neurological disease of unknown etiology: A single-center 12-year retrospective analysis.

There are no international guidelines for brain biopsy in neurological disease of unknown etiology, yet most practicing neurologists will encounter difficult cases in which biopsy is considered. This patient cohort is heterogenous, and it is unclear in which circumstances biopsy is most useful. We performed an audit of brain biopsies reviewed in our neuropathology department from 2010 to 2021. Of 9,488 biopsies, 331 biopsies undertaken for an undiagnosed neurological disease were identified. Where documented, the commonest symptoms were hemorrhage, encephalopathy, and dementia. 29% of biopsies were non-diagnostic. The most common clinically relevant findings on biopsy were infection, cerebral amyloid angiopathy with or without angiitis, and demyelination. Rarer conditions included CNS vasculitis, non-infectious encephalitis, and Creutzfeldt Jakob Disease. We highlight the value of brain biopsy in the workup of cryptogenic neurological disease despite recent advances in less invasive diagnostics.

MicroRNA inhibition using antimiRs in acute human brain tissue sections.

Antisense inhibition of microRNAs is an emerging preclinical approach to pharmacoresistant epilepsy. A leading candidate is an "antimiR" targeting microRNA-134 (ant-134), but testing to date has used rodent models. Here, we develop an antimiR testing platform in human brain tissue sections. Brain specimens were obtained from patients undergoing resective surgery to treat pharmacoresistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF) and dissected for clinical neuropathological examination, and unused material was transferred for sectioning. Individual sections were incubated in oxygenated ACSF, containing either ant-134 or a nontargeting control antimiR, for 24 h at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels were measured using quantitative reverse transcriptase polymerase chain reaction. Specimens transported in ACSF could be used for neuropathological diagnosis and had good RNA integrity. Ant-134 mediated a dose-dependent knockdown of miR-134, with approximately 75% reduction of miR-134 at 1 µmol L-1 and 90% reduction at 3 µmol L-1 . These doses did not have off-target effects on expression of a selection of three other miRNAs. This is the first demonstration of ant-134 effects in live human brain tissues. The findings lend further support to the preclinical development of a therapy that targets miR-134 and offer a flexible platform for the preclinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain.

Melanoma brain metastases in Ireland: surgical and systemic considerations.

BACKGROUND: Cerebral metastases is a common complication in patients with melanoma. There is a paucity of information in the Republic of Ireland regarding the factors associated with melanoma brain metastases (MBM). METHODS: Patients diagnosed with melanoma brain metastases in Ireland were retrospectively identified in Beaumont Hospital between 1999 and 2018. Patient demographics; age at diagnosis of primary melanoma, age at detection of MBM, year of detection of MBM, anatomical location of primary melanoma, BRAF mutation analysis and the number of metastases were investigated. Follow-up data were also derived, including overall survival. RESULTS: There has being a 158% increase in the incidence of primary melanoma from 1999 compared to 2016. Over the same time period 128 patients with melanoma brain metastases were diagnosed. There was a significant male predominance (n = 77/128; 60%; p < 0.0001). BRAF mutation and leptomeningeal disease were independent prognostic factors in our cohort with a median survival 8 months and 0.5 months, respectively. CONCLUSIONS: Male predominance, leptomeningeal disease and BRAF mutation represent important considerations in this population group. The results of this study add to our knowledge concerning outcomes in melanoma brain metastases and may be useful in clinical planning and future treatments.

ADAM22/LGI1 complex as a new actionable target for breast cancer brain metastasis.

BACKGROUND: Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood-brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets. METHODS: Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target's natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis. RESULTS: Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis. CONCLUSION: ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease.

Desmoplastic myxoid tumor, SMARCB1-mutant: clinical, histopathological and molecular characterization of a pineal region tumor encountered in adolescents and adults.

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly occurring in infants. Mutations of the SMARCB1 gene are the characteristic genetic lesion. SMARCB1-mutant tumors in adolescents and adults are rare and may show uncommon histopathological and clinical features. Here we report seven SMARCB1-deficient intracranial tumors sharing distinct clinical, histopathological and molecular features. Median age of the four females and three males was 40 years (range 15-61 years). All tumors were located in the pineal region. Histopathologically, these tumors displayed spindled and epithelioid cells embedded in a desmoplastic stroma alternating with a variable extent of a loose myxoid matrix. All cases showed loss of nuclear SMARCB1/INI1 protein expression, expression of EMA and CD34 was frequent and the Ki67/MIB1 proliferation index was low in the majority of cases (median 3%). Three cases displayed heterozygous SMARCB1 deletions and two cases a homozygous SMARCB1 deletion. On sequencing, one tumor showed a 2 bp deletion in exon 4 (c.369_370del) and one a short duplication in exon 3 (c.237_276dup) both resulting in frameshift mutations. Most DNA methylation profiles were not classifiable using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-SNE analysis and hierarchical clustering analysis, however, all tumors grouped closely together and showed similarities with ATRT-MYC. After a median observation period of 48 months, three patients were alive with stable disease, whereas one patient experienced tumor progression and three patients had succumbed to disease. In conclusion, our series represents an entity with distinct clinical, histopathological and molecular features showing epigenetic similarities with ATRT-MYC. We propose the designation desmoplastic myxoid tumor (DMT), SMARCB1-mutant, for these tumors.

CNS lymphoma, the Irish experience: A retrospective review of neuropathologically confirmed cases over 10 years.

BACKGROUND: Central nervous system (CNS) lymphoma is rare, representing 2% of all brain tumors. The commonest subtype is diffuse large B-cell lymphoma (DLBCL), with primary T-cell lymphomas (PCNSTL) accounting for ~ 2%. OBJECTIVE: To determine the frequency and describe the key features of CNS lymphoma over a 10-year period in an Irish population. MATERIALS AND METHODS: This retrospective review was carried out using the neuropathology database in Beaumont hospital, the largest of two national neurosurgical centers, to identify all cases of CNS lymphoma from 2007 to 2017. Clinical, radiological, morphological, immunophenotype, and molecular information was recorded where available. RESULTS: We identified 149 cases of CNS lymphoma from 2007 to 2017, which equated to a cumulative incidence rate of 0.4/100,000 persons. Median age at diagnosis was 66 years, and 46% were male. 86% were classified as DLBCL (n = 128), 10% immunodeficiency-associated CNS lymphoma (n = 15), 3% PCNSTL (n = 4), and 1% (n = 2) cases of intravascular large B-cell lymphoma. Location in declining frequency was as follows: supratentorial (n = 125), infratentorial (n = 22), spinal (n = 1), and orbital (n = 1). CONCLUSION: This is the first study in an Irish population to determine a cumulative incidence rate of CNS lymphoma, which is in line with larger international population-based registries. No significant trends in incidence rate have been observed from 2007 to 2017. DLBCL is the commonest subtype encountered. Rare variants including PCNSTL can pose a significant diagnostic challenge, and in this setting, molecular studies can be useful to confirm diagnoses.

IgG4 hypophysitis - a rare and underdiagnosed cause of pituitary gland and stalk mass-like thickening.

Our aim is to present a typical case of IgG4-related hypophysitis, which will offer insight into the aetiology and pathogenesis of this relatively newly described disease. IgG4 Related Disease is a protean systemic condition that mimics inflammatory, infectious, and malignant processes. Biopsy of affected organs will show a typical histopathological pattern.

Machine learning: a useful radiological adjunct in determination of a newly diagnosed glioma's grade and IDH status.

INTRODUCTION: Machine learning methods have been introduced as a computer aided diagnostic tool, with applications to glioma characterisation on MRI. Such an algorithmic approach may provide a useful adjunct for a rapid and accurate diagnosis of a glioma. The aim of this study is to devise a machine learning algorithm that may be used by radiologists in routine practice to aid diagnosis of both: WHO grade and IDH mutation status in de novo gliomas. METHODS: To evaluate the status quo, we interrogated the accuracy of neuroradiology reports in relation to WHO grade: grade II 96.49% (95% confidence intervals [CI] 0.88, 0.99); III 36.51% (95% CI 0.24, 0.50); IV 72.9% (95% CI 0.67, 0.78). We derived five MRI parameters from the same diagnostic brain scans, in under two minutes per case, and then supplied these data to a random forest algorithm. RESULTS: Machine learning resulted in a high level of accuracy in prediction of tumour grade: grade II/III; area under the receiver operating characteristic curve (AUC) = 98%, sensitivity = 0.82, specificity = 0.94; grade II/IV; AUC = 100%, sensitivity = 1.0, specificity = 1.0; grade III/IV; AUC = 97%, sensitivity = 0.83, specificity = 0.97. Furthermore, machine learning also facilitated the discrimination of IDH status: AUC of 88%, sensitivity = 0.81, specificity = 0.77. CONCLUSIONS: These data demonstrate the ability of machine learning to accurately classify diffuse gliomas by both WHO grade and IDH status from routine MRI alone-without significant image processing, which may facilitate usage as a diagnostic adjunct in clinical practice.

Reactive gliosis mimicking tumor recurrence - a case series documenting MRI abnormalities and neuropathological correlates.

The aim of this study is to identify, in our center, all cases of foreign-body reactions to hemostatic agents or other prostheses resulting in a radiological suspicion of tumor recurrence. We interrogated our internal database to identify all such cases and systematically evaluated the MRI brain scans of patients: (i) at the time of initial tumor diagnosis, (ii) postoperatively, (iii) and at the time of suspected tumor recurrence. In addition, we reviewed each patient's operative notes and reviewed the histology of all cases following a second surgical intervention. In total, we identified 8 patients, 7 of whom had a WHO grade II glioma at initial surgery. We did not identify any distinguishing radiological abnormalities from the initial diagnostic brain scan to the suspected recurrence, and histologically all cases were characterized by extensive gliosis; with both macrophages and reactive astrocytes present throughout. The cause of gliosis was identified as being relating to hemostatic agents in 4 cases; in the other 4 cases, the foreign-body reaction was presumed to be caused be materials used in a craniotomy or cranioplasty. This study highlights the difficulty in radiologically diagnosing a foreign-body reaction and also identifies that such a gliotic reaction may occur as a consequence of exogenous materials used in a craniotomy or cranioplasty.
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The DNA copy number landscape of a collision tumor.

Intracranial collision tumors are composed of two histologically distinct but merging components, and are rare. Their genetic profile has rarely been described. Comparative genome hybridization of a combined meningioma and oligodendroglioma demonstrated deletion of chromosome 22q and of 19q in both tumors. Somatic deletion of chromosome 22q and 19q is associated with development of an intracranial collision tumor.
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Absence of 1p/19q codeletion in oligodendroglioma-like areas of pilocytic astrocytomas.

The presence of oligodendroglioma-like areas in pilocytic astrocytoma may give rise to pathologic diagnostic uncertainty. This study aims to determine if the oligodendroglioma-like areas present in some pilocytic astrocytomas (PA) possess the signature 1p/19q codeletion that is characteristic of classical oligodendroglioma. Array comparative genomic hybridization was carried out on 12 PA samples, from which oligodendroglioma-like areas were microdissected and used as the template DNA source. 1p/19q codeletions were not found in any of the oligodendroglioma areas in PAs. We conclude that PAs with oligodendroglioma-like areas do not share the same molecular genetics as classic oligodendroglioma.
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Diverse phenotype of hypokalaemic periodic paralysis within a family.

Hypokalaemic periodic paralysis typically presents with intermittent mild-to-moderate weakness lasting hours to days. We report a case with an uncommon phenotype of late-onset myopathy without episodic paralytic attacks. Initial work-up including muscle biopsy was inconclusive. A subsequent review of the right deltoid biopsy, long exercise testing and repeated family history was helpful, followed by appropriate genetic testing. We identified a heterozygous pathogenic mutation in calcium ion channel (CACNA1S:c.1583G>A p.Arg528His) causing hypokalaemic periodic paralysis. Myopathy can present without episodic paralysis and the frequency of paralytic episodes does not correlate well with the development and progression of a fixed myopathy. Our report also highlights the intrafamilial phenotypic variation of hypokalaemic periodic paralysis secondary to a CACNA1S gene mutation.

CJD surveillance in the Republic of Ireland from 2005 to 2015: a suggested algorithm for referrals.

Definitive diagnosis of Creutzfeldt Jakob disease (CJD) remains tissue-based. Possible and probable CJD are useful clinical terms but may be used indiscriminately. The aim of this study was to assess the effectiveness of the Irish surveillance system and to ascertain how diagnostic accuracy in identifying clinically "definite" cases might be improved. We reviewed the clinical information, relevant investigations, and samples n = 100; (autopsy n = 87; biopsy n = 13) in 96 patients between January 1, 2005 and December 31, 2015. In 4 cases both a biopsy and autopsy were performed. CJD was confirmed in 50 patients (45 at autopsy and 5 at biopsy). Sporadic CJD (sCJD) accounted for 90% of cases (n = 45); variant CJD (vCJD) for 6% (n = 3) with 1 case each of familial CJD and iatrogenic CJD. CSF 14-3-3, EEG, and MRI investigations were helpful but not available on all patients. CJD mimics (n = 46) fell into the following categories: neurodegenerative (n = 22), immune mediated (n = 3), cerebrovascular disease (n = 5), tumor (n = 5), dual pathology (n = 3), and miscellaneous (n = 8). The Irish surveillance system fulfils its main objective as all clinically suspicious cases are being referred. CJD was confirmed in 52% (n = 50/96) of referrals. Based on this, we propose an algorithm for CJD referrals to reduce both infection control and diagnostic difficulties encountered in CJD surveillance.
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Gastrointestinal stromal tumor metastases to the pituitary: a rare entity.

Gastrointestinal stromal tumors (GISTs) are rare, but represent the most common mesenchymal tumor of the gastrointestinal tract. The paucity of intracranial metastasis makes treatment strategies difficult. This manuscript presents the first documented case report of a GIST that presented clinically with pituitary symptoms due to a pituitary metastasis.

Ten years on: genetic screening for mitochondrial disease in Ireland.

Mitochondrial DNA (mtDNA) analysis is centralized in the Department of Neuropathology, Beaumont Hospital. Services offered include analysis of common mtDNA point mutations, large scale mtDNA deletions/rearrangements, and sequencing of the nuclear gene POLG. The aims of this study were to audit the mtDNA diagnostic service over a 10-year period, to determine appropriate use of the service, and to improve efficient use of the service by devising a requisition form that includes diagnostic algorithms. Between July 2002 and October 2013, 716 samples were received for analysis. Overall, the number of confirmed mutations was low. Lack of diagnostic algorithms may result in expansive, unrefined requests, leading to costly investigations. We introduced a requisition form that extracts clinical, biochemical, and pathological data prior to analysis. With this information, diagnostic algorithms can be applied to select the most relevant mutations for initial analysis and also to increase the incidence of mutation detection.

Genetic features of oligodendrogliomas and presence of seizures. The relationship of seizures and genetics in LGOs.

Low grade oligodendrogliomas (LGO) are diffusely infiltrating World Health Organization (WHO) grade II gliomas, 20 - 30% of which show contrast enhancement. Seizures are a common presenting feature. It has been suggested that 1p19q co-deletion is associated with occurrence of seizures in adults, however, to date, the relationship of tumor genetics and seizure activity has not been extensively investigated. We sought to assess the influence of 1p19q co-deletion, IDH1-R132H positivity, and radiological variables on seizure activity in LGO patients. Specifically, we examined whether these characteristics were associated with seizure at initial presentation, or if they could predict outcome in terms of seizure free survival. In 62 LGOs, neither tumor location nor tumor enhancement were associated with seizures. 1p19q co-deletion status did not predict seizures when controlled for mutant IDH1-R132H expression, tumor location, or enhancement status (odds ratio (OR) 0.9, 95% confidence interval (CI) 0.1 - 4.3). This study, although of limited statistical power, did not demonstrate an association between 1p19q status and seizure occurrence in LGO's. Replication in a larger cohort would further support our hypothesis that 1p19q status alone cannot be used as a reliable predictor of seizure occurrence in LGO's.