Distinct DNA Methylation Patterns of Subependymal Giant Cell Astrocytomas in Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a monogenic disorder caused by mutations in either the TSC1 or TSC2 gene, two key regulators of the mechanistic target of the rapamycin complex pathway. Phenotypically, this leads to growth and formation of hamartomas in several organs, including the brain. Subependymal giant cell astrocytomas (SEGAs) are low-grade brain tumors commonly associated with TSC. Recently, gene expression studies provided evidence that the immune system, the MAPK pathway and extracellular matrix organization play an important role in SEGA development. However, the precise mechanisms behind the gene expression changes in SEGA are still largely unknown, providing a potential role for DNA methylation. We investigated the methylation profile of SEGAs using the Illumina Infinium HumanMethylation450 BeadChip (SEGAs n = 42, periventricular control n = 8). The SEGA methylation profile was enriched for the adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. More interestingly, we identified two subgroups in the SEGA methylation data and show that the differentially expressed genes between the two subgroups are related to the MAPK cascade and adaptive immune response. Overall, this study shows that the immune system, the MAPK pathway and extracellular matrix organization are also affected on DNA methylation level, suggesting that therapeutic intervention on DNA level could be useful for these specific pathways in SEGA. Moreover, we identified two subgroups in SEGA that seem to be driven by changes in the adaptive immune response and MAPK pathway and could potentially hold predictive information on target treatment response.

MicroRNA-34a activation in tuberous sclerosis complex during early brain development may lead to impaired corticogenesis.

AIMS: Tuberous sclerosis complex (TSC) is a genetic disorder associated with dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. Neurodevelopmental disorders, frequently present in TSC, are linked to cortical tubers in the brain. We previously reported microRNA-34a (miR-34a) among the most upregulated miRs in tubers. Here, we characterised miR-34a expression in tubers with the focus on the early brain development and assessed the regulation of mTORC1 pathway and corticogenesis by miR-34a. METHODS: We analysed the expression of miR-34a in resected cortical tubers (n = 37) compared with autopsy-derived control tissue (n = 27). The effect of miR-34a overexpression on corticogenesis was assessed in mice at E18. The regulation of the mTORC1 pathway and the expression of the bioinformatically predicted target genes were assessed in primary astrocyte cultures from three patients with TSC and in SH-SY5Y cells following miR-34a transfection. RESULTS: The peak of miR-34a overexpression in tubers was observed during infancy, concomitant with the presence of pathological markers, particularly in giant cells and dysmorphic neurons. miR-34a was also strongly expressed in foetal TSC cortex. Overexpression of miR-34a in mouse embryos decreased the percentage of cells migrated to the cortical plate. The transfection of miR-34a mimic in TSC astrocytes negatively regulated mTORC1 and decreased the expression of the target genes RAS related (RRAS) and NOTCH1. CONCLUSIONS: MicroRNA-34a is most highly overexpressed in tubers during foetal and early postnatal brain development. miR-34a can negatively regulate mTORC1; however, it may also contribute to abnormal corticogenesis in TSC.

The coding and non-coding transcriptional landscape of subependymal giant cell astrocytomas.

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited neurocutaneous disorder caused by inactivating mutations in TSC1 or TSC2, key regulators of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. In the CNS, TSC is characterized by cortical tubers, subependymal nodules and subependymal giant cell astrocytomas (SEGAs). SEGAs may lead to impaired circulation of CSF resulting in hydrocephalus and raised intracranial pressure in patients with TSC. Currently, surgical resection and mTORC1 inhibitors are the recommended treatment options for patients with SEGA. In the present study, high-throughput RNA-sequencing (SEGAs n = 19, periventricular control n = 8) was used in combination with computational approaches to unravel the complexity of SEGA development. We identified 9400 mRNAs and 94 microRNAs differentially expressed in SEGAs compared to control tissue. The SEGA transcriptome profile was enriched for the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation and survival. Analysis at the protein level confirmed that extracellular signal-regulated kinase (ERK) is activated in SEGAs. Subsequently, the inhibition of ERK independently of mTORC1 blockade decreased efficiently the proliferation of primary patient-derived SEGA cultures. Furthermore, we found that LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4 and LAMTOR5 were overexpressed at both gene and protein levels in SEGA compared to control tissue. Taken together LAMTOR1-5 can form a complex, known as the 'Ragulator' complex, which is known to activate both mTORC1 and MAPK/ERK pathways. Overall, this study shows that the MAPK/ERK pathway could be used as a target for treatment independent of, or in combination with mTORC1 inhibitors for TSC patients. Moreover, our study provides initial evidence of a possible link between the constitutive activated mTORC1 pathway and a secondary driver pathway of tumour growth.

Mosaic trisomy of chromosome 1q in human brain tissue associates with unilateral polymicrogyria, very early-onset focal epilepsy, and severe developmental delay.

Polymicrogyria (PMG) is a developmental cortical malformation characterized by an excess of small and frustrane gyration and abnormal cortical lamination. PMG frequently associates with seizures. The molecular pathomechanisms underlying PMG development are not yet understood. About 40 genes have been associated with PMG, and small copy number variations have also been described in selected patients. We recently provided evidence that epilepsy-associated structural brain lesions can be classified based on genomic DNA methylation patterns. Here, we analyzed 26 PMG patients employing array-based DNA methylation profiling on formalin-fixed paraffin-embedded material. A series of 62 well-characterized non-PMG cortical malformations (focal cortical dysplasia type 2a/b and hemimegalencephaly), temporal lobe epilepsy, and non-epilepsy autopsy controls was used as reference cohort. Unsupervised dimensionality reduction and hierarchical cluster analysis of DNA methylation profiles showed that PMG formed a distinct DNA methylation class. Copy number profiling from DNA methylation data identified a uniform duplication spanning the entire long arm of chromosome 1 in 7 out of 26 PMG patients, which was verified by additional fluorescence in situ hybridization analysis. In respective cases, about 50% of nuclei in the center of the PMG lesion were 1q triploid. No chromosomal imbalance was seen in adjacent, architecturally normal-appearing tissue indicating mosaicism. Clinically, PMG 1q patients presented with a unilateral frontal or hemispheric PMG without hemimegalencephaly, a severe form of intractable epilepsy with seizure onset in the first months of life, and severe developmental delay. Our results show that PMG can be classified among other structural brain lesions according to their DNA methylation profile. One subset of PMG with distinct clinical features exhibits a duplication of chromosomal arm 1q.

Precise detection of low-level somatic mutation in resected epilepsy brain tissue.

Low-level somatic mutations have been shown to be the major genetic etiology of intractable epilepsy. The extents thereof, however, have yet to be systematically and accurately explored in a large cohort of resected epilepsy brain tissues. Moreover, clinically useful and precise analysis tools for detecting low-level somatic mutations from unmatched formalin-fixed paraffin-embedded (FFPE) brain samples, the most clinically relevant samples, are still lacking. In total, 446 tissues samples from 232 intractable epilepsy patients with various brain pathologies were analyzed using deep sequencing (average read depth, 1112x) of known epilepsy-related genes (up to 28 genes) followed by confirmatory site-specific amplicon sequencing. Pathogenic mutations were discovered in 31.9% (74 of 232) of the resected epilepsy brain tissues and were recurrently found in only eight major focal epilepsy genes, including AKT3, DEPDC5, MTOR, PIK3CA, TSC1, TSC2, SCL35A2, and BRAF. Somatic mutations, two-hit mutations, and germline mutations accounted for 22.0% (51), 0.9% (2), and 9.1% (21) of the patients with intractable epilepsy, respectively. The majority of pathogenic somatic mutations (62.3%, 33 of 53) had a low variant allelic frequency of less than 5%. The use of deep sequencing replicates in the eight major focal epilepsy genes robustly increased PPVs to 50-100% and sensitivities to 71-100%. In an independent FCDII cohort of only unmatched FFPE brain tissues, deep sequencing replicates in the eight major focal epilepsy genes identified pathogenic somatic mutations in 33.3% (5 of 15) of FCDII individuals (similar to the genetic detecting rate in the entire FCDII cohort) without any false-positive calls. Deep sequencing replicates of major focal epilepsy genes in unmatched FFPE brain tissues can be used to accurately and efficiently detect low-level somatic mutations, thereby improving overall patient care by enriching genetic counseling and informing treatment decisions.

Cognitive functioning after epilepsy surgery in children with mild malformation of cortical development and focal cortical dysplasia.

Mild malformation of cortical development (mMCD) and focal cortical dysplasia (FCD) subtypes combined are by far the most common histological diagnoses in children who undergo surgery as treatment for refractory epilepsy. In patients with refractory epilepsy, a substantial burden of disease is due to cognitive impairment. We studied intelligence quotient (IQ) or developmental quotient (DQ) values and their change after epilepsy surgery in a consecutive series of 42 children (median age at surgery: 4.5, range: 0-17.0 years) with refractory epilepsy due to mMCD/FCD. Cognitive impairment, defined as IQ/DQ below 70, was present in 51% prior to surgery. Cognitive impairment was associated with earlier onset of epilepsy, longer epilepsy duration, and FCD type I histology. Clinically relevant improvement of ≥10 IQ/DQ points was found in 24% of children and was related to the presence of presurgical epileptic encephalopathy (EE). At time of postsurgical cognitive testing, 59% of children were completely seizure-free (Engel 1A). We found no association between cognitive outcome and seizure or medication status at two years of follow-up. Epilepsy surgery in children with mMCD or FCD not only is likely to result in complete and continuous seizure freedom, but also improves cognitive function in many.

miR147b: A novel key regulator of interleukin 1 beta-mediated inflammation in human astrocytes.

Astrocytes are important mediators of inflammatory processes in the brain and seem to play an important role in several neurological disorders, including epilepsy. Recent studies show that astrocytes produce several microRNAs, which may function as crucial regulators of inflammatory pathways and could be used as therapeutic target. We aim to study which miRNAs are produced by astrocytes during IL-1ß mediated inflammatory conditions in vitro, as well as their functional role and to validate these findings in human epileptogenic brain tissue. Sequencing was used to assess miRNA and mRNA expression in IL-1ß-stimulated human fetal astrocyte cultures. miRNAs were overexpressed in cell cultures using miRNA mimics. Expression of miRNAs in resected brain tissue from patients with tuberous sclerosis complex or temporal lobe epilepsy with hippocampal sclerosis was examined using in situ hybridization. Two differentially expressed miRNAs were found: miR146a and miR147b, which were associated with increased expression of genes related to the immune/inflammatory response. As previously reported for miR146a, overexpression of miR147b reduced the expression of the pro-inflammatory mediators IL-6 and COX-2 after IL-1ß stimulation in both astrocyte and tuberous sclerosis complex cell cultures. miR146a and miR147b overexpression decreased proliferation of astrocytes and promoted neuronal differentiation of human neural stem cells. Similarly to previous evidence for miR146a, miR147b was increased expressed in astrocytes in epileptogenic brain. Due to their anti-inflammatory effects, ability to restore aberrant astrocytic proliferation and promote neuronal differentiation, miR146a and miR147b deserve further investigation as potential therapeutic targets in neurological disorders associated with inflammation, such as epilepsy.

Predominance of Nonatherosclerotic Internal Elastic Lamina Calcification in the Intracranial Internal Carotid Artery.

BACKGROUND AND PURPOSE: Calcification of the intracranial internal carotid artery (iICA) is an independent risk factor for stroke. These calcifications are generally seen as manifestation of atherosclerosis, but histological investigations are limited. The aim of this study is to determine whether calcifications in the iICA are present in atherosclerotic plaques, or in other parts of the arterial wall. METHODS: Thirty-nine iICAs were histologically assessed, using digital microscopy to quantify the amount of calcification in the different layers of the arterial wall. RESULTS: Calcifications were found in the intima, around the internal elastic lamina and in the medial layer of the arterial wall. In 71% of the arteries, internal elastic lamina calcification contributed most to the total calcified cross-sectional surface area. Internal elastic lamina calcification was unrelated to the occurrence of atherosclerotic intimal lesions. Intimal calcifications were most often associated with atherosclerotic lesions, but also many noncalcified atherosclerotic lesions were found. CONCLUSIONS: In the iICA, calcifications are predominantly present around the internal elastic lamina, suggesting that this nonatherosclerotic type of calcification contributes to the previously observed increased risk of stroke in patients with iICA calcifications.

Lines of Baillarger in vivo and ex vivo: Myelin contrast across lamina at 7T MRI and histology.

The human cerebral cortex is characterized by a number of features that are not uniformly distributed, such as the presence of multiple cytoarchitectonic elements and of myelinated layers running tangentially to the cortex surface. The presence and absence of these features are the basis of the parcellation of the cerebral cortex in several areas. A number of areas show myelin increases localized within the cortex, e.g., the stria of Gennari located in layer IV of the primary visual cortex. Sub-millimeter MRI can resolve myelin variations across the human cortex and may allow in vivo parcellation of these brain areas. Here, we image within-area myelination. We modified a T1-weighted (T1-w) MPRAGE sequence to enhance myelin visualization within the cortex. First, we acquired images from an ex vivo sample, and compared MRI laminar profiles from calcarine (corresponding to primary visual cortex) and extra-calcarine areas with histology sections from the same locations. Laminar profiles between myelin stained sections and the T1-w images were similar both in calcarine as well as extra-calcarine cortex. In calcarine cortex, the profile reveals the stria of Gennari. In extra-calcarine cortex, a similar profile exists which we suggest corresponds to the lines of Baillarger. Next, we adapted the same sequence to image within-area myelination in vivo. Also in in vivo data, we discriminated similar laminar profiles in calcarine and extra-calcarine cortex, extending into parietal and frontal lobes. We argue that this myelin pattern outside the calcarine cortex represents the lines of Baillarger.

Functional aspects of early brain development are preserved in tuberous sclerosis complex (TSC) epileptogenic lesions.

Tuberous sclerosis complex (TSC) is a rare multi-system genetic disease characterized by several neurological disorders, the most common of which is the refractory epilepsy caused by highly epileptogenic cortical lesions. Previous studies suggest an alteration of GABAergic and glutamatergic transmission in TSC brain indicating an unbalance of excitation/inhibition that can explain, at least in part, the high incidence of epilepsy in these patients. Here we investigate whether TSC cortical tissues could retain GABAA and AMPA receptors at early stages of human brain development thus contributing to the generation and recurrence of seizures. Given the limited availability of pediatric human brain specimens, we used the microtransplantation method of injecting Xenopus oocytes with membranes from TSC cortical tubers and control brain tissues. Moreover, qPCR was performed to investigate the expression of GABAA and AMPA receptor subunits (GABAA α1-5, ß3, γ2, δ; GluA1, GluA2) and cation chloride co-transporters NKCC1 and KCC2. The evaluation of nine human cortical brain samples, from 15 gestation weeks to 15years old, showed a progressive shift towards more hyperpolarized GABAA reversal potential (EGABA). This shift was associated with a differential expression of the chloride cotransporters NKCC1 and KCC2. Furthermore, the GluA1/GluA2 mRNA ratio of expression paralleled the development process. On the contrary, in oocytes micro-transplanted with epileptic TSC tuber tissue from seven patients, neither the GABAA reversal potential nor the GluA1/GluA2 expression showed similar developmental changes. Our data indicate for the first time, that in the same cohort of TSC patients, the pattern of both GABAAR and GluA1/GluA2 functions retains features that are typical of an immature brain. These observations support the potential contribution of altered receptor function to the epileptic disorder of TSC and may suggest novel therapeutic approaches. Furthermore, our findings strengthen the novel hypothesis that other developmental brain diseases can share the same hallmarks of immaturity leading to intractable seizures.

FLAIR images at 7 Tesla MRI highlight the ependyma and the outer layers of the cerebral cortex.

OBJECTIVES: Fluid-attenuated inversion recovery (FLAIR) imaging is an important clinical 'work horse' for brain MRI and has proven to facilitate imaging of both intracortical lesions as well as cortical layers at 7T MRI. A prominent observation on 7T FLAIR images is a hyperintense rim at the cortical surface and around the ventricles. We aimed to clarify the anatomical correlates and underlying contrast mechanisms of this hyperintense rim. MATERIALS AND METHODS: Two experiments with post-mortem human brain tissue were performed. FLAIR and T2-weighted images were obtained at typical in vivo (0.8mm isotropic) and high resolution (0.25mm isotropic). At one location the cortical surface was partly removed, and scanned again. Imaging was followed by histological and immunohistochemical analysis. Additionally, several simulations were performed to evaluate the potential contribution from an artifact due to water diffusion. RESULTS: The hyperintense rim corresponded to the outer - glia rich - layer of the cortex and disappeared upon removal of that layer. At the ventricles, the rim corresponded to the ependymal layer, and was not present at white matter/fluid borders at an artificial cut. The simulations supported the hypothesis that the hyperintense rim reflects the tissue properties in the outer cortical layers (or ependymal layer for the ventricles), and is not merely an artifact, although not all observations were explained by the simulated model of the contrast mechanism. CONCLUSIONS: 7T FLAIR seems to amplify the signal from layers I-III of the cortex and the ependyma around the ventricles. Although diffusion of water from layer I into CSF does contribute to this effect, a long T2 relaxation time constant in layer I, and probably also layer II-III, is most likely the major contributor, since the rim disappears upon removal of that layer. This knowledge can help the interpretation of imaging results in cortical development and in patients with cortical pathology.

ALS-associated mutations in FUS disrupt the axonal distribution and function of SMN.

Mutations in the RNA binding protein fused in sarcoma/translated in liposarcoma (FUS/TLS) cause amyotrophic lateral sclerosis (ALS). Although ALS-linked mutations in FUS often lead to a cytosolic mislocalization of the protein, the pathogenic mechanisms underlying these mutations remain poorly understood. To gain insight into these mechanisms, we examined the biochemical, cell biological and functional properties of mutant FUS in neurons. Expression of different FUS mutants (R521C, R521H, P525L) in neurons caused axonal defects. A protein interaction screen performed to explain these phenotypes identified numerous FUS interactors including the spinal muscular atrophy (SMA) causing protein survival motor neuron (SMN). Biochemical experiments showed that FUS and SMN interact directly and endogenously, and that this interaction can be regulated by FUS mutations. Immunostaining revealed co-localization of mutant FUS aggregates and SMN in primary neurons. This redistribution of SMN to cytosolic FUS accumulations led to a decrease in axonal SMN. Finally, cell biological experiments showed that overexpression of SMN rescued the axonal defects induced by mutant FUS, suggesting that FUS mutations cause axonal defects through SMN. This study shows that neuronal aggregates formed by mutant FUS protein may aberrantly sequester SMN and concomitantly cause a reduction of SMN levels in the axon, leading to axonal defects. These data provide a functional link between ALS-linked FUS mutations, SMN and neuronal connectivity and support the idea that different motor neuron disorders such as SMA and ALS may be caused, in part, by defects in shared molecular pathways.

Landscape of chromosomal copy number aberrations in gangliogliomas and dysembryoplastic neuroepithelial tumours.

AIM: Gangliogliomas (GGs) and dysembryoplastic neuroepithelial tumours (DNTs) represent the most common histological entities within the spectrum of glioneuronal tumours (GNTs). The wide variability of morphological features complicates histological classification, including discrimination from prognostically distinct diffuse low-grade astrocytomas (AIIs). This study was performed to increase our understanding of these tumours. METHODS: We studied chromosomal copy number aberrations (CNAs) by genome-wide sequencing in a large cohort of GNTs and linked these to comprehensive histological analysis and clinical characteristics. One hundred fourteen GNTs were studied: 50 GGs and 64 DNTs. Also, a data set of CNAs from 38 diffuse AIIs was included. RESULTS: The most frequent CNAs in both GGs and DNTs were gains at chromosomes 5 and 7, often concurrent, and gain at chromosome 6. None of the CNAs was linked to histological subtype, immunohistochemical features or to clinical characteristics. Comparison of AIIs and diffuse GNTs revealed that gain at whole chromosome 5 is only observed in GNTs. CNA patterns indicative of chromothripsis were detected in three GNTs. CONCLUSION: We conclude that GNTs with diverse morphologies share molecular features, and our findings support the need to improve classification and differential diagnosis of tumour entities within the spectrum of GNTs, as well as their distinction from other gliomas.

Ischaemic cavities in the cerebellum: an ex vivo 7-tesla MRI study with pathological correlation.

BACKGROUND: Small cerebellar cavities (≤15 mm) are often observed coincidentally in ageing subjects and have also been associated with migraine. Although generally assumed to be of ischaemic origin, descriptive imaging studies are sparse and imaging findings have not been correlated with histopathology. We aimed to investigate whether small ischaemic cavities in the cerebellum show characteristic infarct patterns that might be helpful for diagnostic imaging. METHODS: We examined 40 whole postmortem cerebella with 7-tesla MRI ex vivo for the presence of small ischaemic cavities. The scan protocol included a T2-, T2*- and fluid-attenuated inversion recovery-weighted sequence for all specimens. We investigated to which degree small ischaemic cavities affect the cortical, juxtacortical and/or deep subcortical regions of the cerebellum. In a subset of the cavities identified, we correlated the imaging data with histopathological findings. This was performed by cutting the particular cerebellar specimen into 5-mm-thick slices. Serial sections were performed if cavities remained unidentified macroscopically. RESULTS: Twenty-two cavities were seen on ex vivo MRI in 8 out of 40 examined cerebella. Twenty out of 22 cerebellar cavities were located in the cortex, and only 2 in the deep white matter, with no cavities located in the juxtacortical white matter. None of the 20 cerebellar cortical cavities showed extension into the juxtacortical white matter on MRI, although in 1 cortical cavity some surrounding gliosis was seen to extend into the juxtacortical white matter. Nine out of 22 cavities were sampled for pathological correlation, including 7 cerebellar cortical cavities and both cavities or lacunes in the deep white matter. Three out of 7 cortical and both the deep cavities were histopathologically verified as cavities of ischaemic origin, while the remaining cortical cavities could not be retrieved upon histopathologic examination. Some microscopic gliosis was seen to extend into the juxtacortical white matter of all confirmed cortical cavities. CONCLUSION: Knowledge of typical infarct patterns may facilitate the detection and characterisation of cerebellar ischaemic cavities in vivo. Cerebellar cortical cavities appeared to be much more common than deep cavities and presented on imaging as a full-thickness defect in the cerebellar cortex without extension in the adjacent white matter.

Frameless image-guided stereotactic brain biopsies: emphasis on diagnostic yield.

BACKGROUND: Studies regarding frameless stereotactic brain biopsy mainly report high diagnostic yield (DY) as opposed to relatively low diagnostic accuracy. This discrepancy raises the question of the certainty and precision of obtained diagnoses. This article proposes a DY definition encompassing diagnostic certainty and precision according to the World Health Organization (WHO) central nervous system (CNS) tumour classification system. Furthermore, our eight-year experience with this procedure is reviewed and evaluated. METHODS: A consecutive series of 235 frameless biopsy procedures was reviewed. Criteria were set up for categorising obtained diagnoses. All cases were included in a predictive factor analysis of inconclusive biopsy and postoperative complications. RESULTS: According to our predefined DY criteria, the DY was 72.8 %. The inconclusive biopsy outcome measured 21.7 %; the non-diagnostic biopsy outcome was 5.5 %. The only predictive factor found for inconclusive biopsy procedures was age under 30. Predictive factors for postoperative complications, which were found statistically significant after multivariable analysis, were glucose level and intra-operative haemorrhage. The total morbidity rate was 8.5 %, including a mortality rate of 0.9 %. CONCLUSIONS: Although frameless stereotactic brain biopsy procedures are considered to be relatively safe, the true DY is significantly less than previously reported, most probably due to the lack of standardised DY criteria. Based on our DY definition and subsequent DY findings, standardisation of DY criteria and definition is paramount for biopsy diagnosis interpretation.

Differential expression of major histocompatibility complex class I in developmental glioneuronal lesions.

PURPOSE: The expression of the major histocompatibility complex class I (MHC-I) in the brain has received considerable interest not only because of its fundamental role in the immune system, but also for its non-immune functions in the context of activity-dependent brain development and plasticity. METHODS: In the present study we evaluated the expression and cellular pattern of MHC-I in focal glioneuronal lesions associated with intractable epilepsy. MHC-I expression was studied in epilepsy surgery cases with focal cortical dysplasia (FCD I, n = 6; FCD IIa, n = 6 and FCD IIb, n = 15), tuberous sclerosis complex (TSC, cortical tubers; n = 6) or ganglioglioma (GG; n = 15) using immunocytochemistry. Evaluation of T lymphocytes with granzyme-B+ granules and albumin immunoreactivity was also performed. RESULTS: All lesions were characterized by MHC-I expression in blood vessels. Expression in both endothelial and microglial cells as well as in neurons (dysmorphic/dysplastic neurons) was observed in FCD II, TSC and GG cases. We observed perivascular and parenchymal T lymphocytes (CD8+, T-cytotoxic) with granzyme-B+ granules in FCD IIb and TSC specimens. Albumin extravasation, with uptake in astrocytes, was observed in FCD IIb and GG cases. CONCLUSIONS: Our findings indicate a prominent upregulation of MHC-I as part of the immune response occurring in epileptogenic glioneuronal lesions. In particular, the induction of MHC-I in neuronal cells appears to be a feature of type II FCD, TSC and GG and may represent an important accompanying event of the immune response, associated with blood-brain barrier dysfunction, in these developmental lesions.

Activation of Toll-like receptor, RAGE and HMGB1 signalling in malformations of cortical development.

Recent evidence in experimental models of seizures and in temporal lobe epilepsy support an important role of high-mobility group box 1 and toll-like receptor 4 signalling in the mechanisms of hyperexcitability leading to the development and perpetuation of seizures. In this study, we investigated the expression and cellular distribution of toll-like receptors 2 and 4, and of the receptor for advanced glycation end products, and their endogenous ligand high-mobility group box 1, in epilepsy associated with focal malformations of cortical development. Immunohistochemistry showed increased expression of toll-like receptors 2 and 4 and receptor for advanced glycation end products in reactive glial cells in focal cortical dysplasia, cortical tubers from patients with the tuberous sclerosis complex and in gangliogliomas. Toll-like receptor 2 was predominantly detected in cells of the microglia/macrophage lineage and in balloon cells in focal cortical dysplasia, and giant cells in tuberous sclerosis complex. The toll-like receptor 4 and receptor for advanced glycation end products were expressed in astrocytes, as well as in dysplastic neurons. Real-time quantitative polymerase chain reaction confirmed the increased receptors messenger RNA level in all pathological series. These receptors were not detected in control cortex specimens. In control cortex, high-mobility group box 1 was ubiquitously detected in nuclei of glial and neuronal cells. In pathological specimens, protein staining was instead detected in the cytoplasm of reactive astrocytes or in tumour astrocytes, as well as in activated microglia, predictive of its release from glial cells. In vitro experiments in human astrocyte cultures showed that nuclear to cytoplasmic translocation of high-mobility group box 1 was induced by interleukin-1ß. Our findings provide novel evidence of intrinsic activation of these pro-inflammatory signalling pathways in focal malformations of cortical development, which could contribute to the high epileptogenicity of these developmental lesions.

STAT5b is a marker of poor prognosis, rather than a therapeutic target in glioblastomas.

The copy number and mRNA expression of STAT5b were assessed in samples from the TCGA repository of glioblastomas (GBM). The activation of this transcription factor was analyzed on tissue microarrays comprising 392 WHO 2016 GBM samples from our clinical practice. These data were correlated with patient survival using multivariable Cox analysis and, for a subset of 167 tumors, with signs of tumor invasiveness on the MRI. The effects of STAT5b knockdown by siRNA were assessed on the growth, therapeutic resistance, invasion and migration of GBM cell lines U87, U87­EGFRVIII and LN18 and primary cultures GM2 and GM3. The activation, but not the copy number or the mRNA expression of nuclear transcription factor STAT5b expression correlated inversely with patient survival independently of IDH1R132H status, age, Karnofsky Performance Score, treatment and tumor volume. STAT5b inhibition neither altered the cell proliferation nor reduced the clonogenic proliferative potency of GBM cells, and did not sensitize them to the cytotoxic effect of ionizing radiation and temozolomide in vitro. STAT5b inhibition significantly increased GBM cell migration, but decreased the invasion of some GBM cells in vitro. There was no correlation between the activation of STAT5b in clinical tumors and the extent of invasion on MRI OF patients. In conclusion, STAT5b is frequently activated in GBM and correlates inversely with patient survival. It does not contribute to the growth and resistance of these tumors, and is thus rather a potential prognostic marker than a therapeutic target in these tumors.

Tumor-related molecular determinants of neurocognitive deficits in patients with diffuse glioma.

BACKGROUND: Cognitive impairment is a common and debilitating symptom in patients with diffuse glioma, and is the result of multiple factors. We hypothesized that molecular tumor characteristics influence neurocognitive functioning (NCF), and aimed to identify tumor-related markers of NCF in diffuse glioma patients. METHODS: We examined the relation between cognitive performance (executive function, memory, and psychomotor speed) and intratumoral expression levels of molecular markers in treatment-naive patients with diffuse glioma. We performed a single-center study in a consecutive cohort, through a two-step design: (1) hypothesis-free differential expression and gene set enrichment analysis to identify candidate oncogenetic markers for cognitive impairment. Nineteen molecular markers of interest were derived from this set of genes, as well as from prior knowledge; (2) correlation of cognitive performance to intratumoral expression levels of these nineteen molecular markers, measured with immunohistochemistry. RESULTS: From 708 included patients with immunohistochemical data, we performed an in-depth analysis of neuropsychological data in 197, and differential expression analysis in 65 patients. After correcting for tumor volume and location, we found significant associations between expression levels of CD3 and IDH-1 and psychomotor speed; between IDH-1, ATRX, NLGN3, BDNF, CK2Beta, EAAT1, GAT-3, SRF, and memory performance; and between IDH-1, P-STAT5b, NLGN3, CK2Beta, and executive functioning. P-STAT5b, CD163, CD3, and Semaphorin-3A were independently associated after further correction for histopathological grade. CONCLUSION: Molecular characteristics of glioma can be independent determinants of patients' cognitive functioning. This suggests that besides tumor volume, location, and histological grade, variations in glioma biology influence cognitive performance through mechanisms that include perturbation of neuronal communication. These results pave the way towards targeted cognition improving therapies in neuro-oncology.

Early Surgery Prolongs Professional Activity in IDH Mutant Low-Grade Glioma Patients: A Policy Change Analysis.

Background: Until 2015, Dutch guidelines recommended follow-up and biopsy rather than surgery as initial care for suspected low-grade gliomas (LGG). Given evidence that surgery could extend patient survival, our center stopped following this guideline on January 1, 2010 and opted for early maximal safe resection of LGG. The effects of early surgery on the ability of patients to work remains little documented. Methods: A total of 104 patients operated on at our center between January 2000 and April 2013 and diagnosed with the WHO 2016 grade 2 astrocytoma, IDH mutant or oligodendroglioma, IDH mutant and deleted 1p19q were included. The clinical characteristics, survival, and work history of patients operated on before or after January 2010 were obtained from the patients' records and compared. The minimal follow-up was 8 years. Results: As per policy change, the interval between radiological diagnosis and first surgery decreased significantly after 2010. Likewise, before 2010, 25.8% of tumors were initially biopsied, 51.6% were resected under anesthesia, and 22.5% under awake conditions versus 14.3%, 23.8%, and 61.9% after this date (p < 0.001). The severity of permanent postoperative neurological deficits decreased after 2010. In total, 82.5% of the patients returned to work postoperatively before 2010 versus 100% after 2010. The postoperative control of epilepsy increased significantly after 2010 (74.4% vs. 47.9%). The median time from diagnosis to a definitive incapacity to work increased by more than 2 years after 2010 (88.7 vs. 62.2 months). Conclusion: A policy shift towards early aggressive surgical treatment of IDH mutant LGG is safe and prolongs the patients' ability to work.