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

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

Altered Extracellular Matrix as an Alternative Risk Factor for Epileptogenicity in Brain Tumors

Seizures are one of the most common symptoms of brain tumors. The incidence of seizures differs among brain tumor type, grade, location and size, but paediatric-type diffuse low-grade gliomas/glioneuronal tumors are often highly epileptogenic. The extracellular matrix (ECM) is known to play a role in epileptogenesis and tumorigenesis because it is involved in the (re)modelling of neuronal connections and cell-cell signaling. In this review, we discuss the epileptogenicity of brain tumors with a focus on tumor type, location, genetics and the role of the extracellular matrix. In addition to functional problems, epileptogenic tumors can lead to increased morbidity and mortality, stigmatization and life-long care. The health advantages can be major if the epileptogenic properties of brain tumors are better understood. Surgical resection is the most common treatment of epilepsy-associated tumors, but post-surgery seizure-freedom is not always achieved. Therefore, we also discuss potential novel therapies aiming to restore ECM function

High frequency oscillations associate with neuroinflammation in low-grade epilepsy associated tumors

Objective: High frequency oscillations (HFOs) in intraoperative electrocorticography (ioECoG) are thought to be generated by hyperexcitable neurons. Inflammation may promote neuronal hyperexcitability. We investigated the relation between HFOs and inflammation in tumor-related epilepsy. Methods: We identified HFOs (ripples 80–250 Hz, fast ripples 250–500 Hz) in the preresection ioECoG of 32 patients with low-grade tumors. Localization of recorded HFOs was classified based on magnetic resonance imaging reconstructions: in tumor, in resected non-tumorous area and outside the resected area. We tested if the following inflammatory markers in the tumor or peritumoral tissue were related to HFOs: activated microglia, cluster of differentiation 3 (CD3)-positive T-cells, interleukin 1-beta (IL1β), toll-like receptor 4 (TLR4) and high mobility group box 1 protein (HMGB1). Results: Tumors that generated ripples were infiltrated by more CD3-positive cells than tumors without ripples. Ripple rate outside the resected area was positively correlated with IL1β/TLR4/HMGB1 pathway activity in peritumoral area. These two areas did not directly overlap. Conclusions: Ripple rates may be associated with inflammatory processes. Significance: Our findings support that ripple generation and spread might be associated with synchronized fast firing of hyperexcitable neurons due to certain inflammatory processes. This pilot study provides arguments for further investigations in HFOs and inflammation

MULTINODULAR AND VACUOLATING NEURONAL TUMOURS IN EPILEPSY: DYSPLASIA OR NEOPLASIA?

Multinodular and vacuolating neuronal tumour (MVNT) is a new pattern of neuronal tumour included in the recently revised WHO 2016 classification of tumours of the CNS. There are fifteen reports in the literature to date. They are typically associated with late onset epilepsy and a neoplastic versus malformative biology has been questioned. We present a series of ten cases and compare their pathological and genetic features to better characterised epilepsy associated malformations including focal cortical dysplasia type II (FCDII) and low-grade epilepsy associated tumours (LEAT). Clinical and neuroradiology data were reviewed and a broad immunohistochemistry panel was applied to explore neuronal and glial differentiation, interneuronal populations, mTOR pathway activation and neurodegenerative changes. Next generation sequencing was performed for targeted multi-gene analysis to identify mutations common to epilepsy lesions including FCDII and LEAT. All of the surgical cases in this series presented with seizures, and were located in the temporal lobe. There was a lack of any progressive changes on serial pre-operative MRI and a mean age at surgery of 45 years. The vacuolated cells of the lesion expressed mature neuronal markers (neurofilament/SMI32, MAP2, synaptophysin). Prominent labelling of the lesional cells for developmentally regulated proteins (OTX1, TBR1, SOX2, MAP1b, CD34, GFAPδ) and oligodendroglial lineage markers (OLIG2, SMI94) was observed. No mutations were detected in the mTOR pathway genes, BRAF, FGFR1 or MYB. Clinical, pathological and genetic data could indicate that MVNT aligns more with a malformative lesion than a true neoplasm with origin from a progenitor neuro-glial cell type showing aberrant maturation. This article is protected by copyright. All rights reserve

MULTINODULAR AND VACUOLATING NEURONAL TUMOURS IN EPILEPSY: DYSPLASIA OR NEOPLASIA?

Multinodular and vacuolating neuronal tumour (MVNT) is a new pattern of neuronal tumour included in the recently revised WHO 2016 classification of tumours of the CNS. There are fifteen reports in the literature to date. They are typically associated with late onset epilepsy and a neoplastic versus malformative biology has been questioned. We present a series of ten cases and compare their pathological and genetic features to better characterised epilepsy associated malformations including focal cortical dysplasia type II (FCDII) and low-grade epilepsy associated tumours (LEAT). Clinical and neuroradiology data were reviewed and a broad immunohistochemistry panel was applied to explore neuronal and glial differentiation, interneuronal populations, mTOR pathway activation and neurodegenerative changes. Next generation sequencing was performed for targeted multi-gene analysis to identify mutations common to epilepsy lesions including FCDII and LEAT. All of the surgical cases in this series presented with seizures, and were located in the temporal lobe. There was a lack of any progressive changes on serial pre-operative MRI and a mean age at surgery of 45 years. The vacuolated cells of the lesion expressed mature neuronal markers (neurofilament/SMI32, MAP2, synaptophysin). Prominent labelling of the lesional cells for developmentally regulated proteins (OTX1, TBR1, SOX2, MAP1b, CD34, GFAPδ) and oligodendroglial lineage markers (OLIG2, SMI94) was observed. No mutations were detected in the mTOR pathway genes, BRAF, FGFR1 or MYB. Clinical, pathological and genetic data could indicate that MVNT aligns more with a malformative lesion than a true neoplasm with origin from a progenitor neuro-glial cell type showing aberrant maturation. This article is protected by copyright. All rights reserve

Impaired myelin production due to an intrinsic failure of oligodendrocytes in mTORpathies

AIMS: We aim to evaluate if the myelin pathology observed in epilepsy-associated focal cortical dysplasia type 2B (FCD2B) and-histologically indistinguishable-cortical tubers of tuberous sclerosis complex (TSC) is primarily related to the underlying malformation or constitutes a secondary phenomenon due to the toxic microenvironment created by epileptic seizures. We also aim to investigate the possible beneficial effect of the mTOR pathway regulator everolimus on white matter pathology. METHODS: Primary mixed glial cell cultures derived from epilepsy surgery specimens of one TSC and seven FCD2B patients were grown on polycaprolactone fibre matrices and analysed using immunofluorescence and electron microscopy. Unaffected white matter from three age-matched epilepsy patients with mild malformations of cortical development (mMCD) and one with FCD3D served as controls. Additionally, TSC2 knock-out was performed using an oligodendroglial cell line. Myelination capacities of nanofibre grown cells in an inflammatory environment after mTOR-inhibitor treatment with everolimus were further investigated. RESULTS: Reduced oligodendroglial turnover, directly related to a lower myelin content was found in the patients' primary cells. In our culture model of myelination dynamics, primary cells grown under 'inflammatory condition' showed decreased myelination, that was repaired by treatment with everolimus. CONCLUSIONS: Results obtained in patient-derived primary oligodendroglial and TSC2 knock-out cells suggest that maturation of oligodendroglia and production of a proper myelin sheath seem to be impaired as a result of mTOR pathway disturbance. Hence, oligodendroglial pathology may reflect a more direct effect of the abnormal genetic programme rather than to be an inactive bystander of chronic epilepsy

Myelin Pathology Beyond White Matter in Tuberous Sclerosis Complex (TSC) Cortical Tubers

Tuberous sclerosis complex (TSC) is a monogenetic disease that arises due to mutations in either the TSC1 or TSC2 gene and affects multiple organ systems. One of the hallmark manifestations of TSC are cortical malformations referred to as cortical tubers. These tubers are frequently associated with treatment-resistant epilepsy. Some of these patients are candidates for epilepsy surgery. White matter abnormalities, such as loss of myelin and oligodendroglia, have been described in a small subset of resected tubers but mechanisms underlying this phenomenon are unclear. Herein, we analyzed a variety of neuropathologic and immunohistochemical features in gray and white matter areas of resected cortical tubers from 46 TSC patients using semi-automated quantitative image analysis. We observed divergent amounts of myelin basic protein as well as numbers of oligodendroglia in both gray and white matter when compared with matched controls. Analyses of clinical data indicated that reduced numbers of oligodendroglia were associated with lower numbers on the intelligence quotient scale and that lower amounts of myelin-associated oligodendrocyte basic protein were associated with the presence of autism-spectrum disorder. In conclusion, myelin pathology in cortical tubers extends beyond the white matter and may be linked to cognitive dysfunction in TSC patients

Subependymal giant cell astrocytomas are characterized by mTORC1 hyperactivation, a very low somatic mutation rate, and a unique gene expression profile

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5–10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2–46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0–7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation

Author Correction: Coding and small non-coding transcriptional landscape of tuberous sclerosis complex cortical tubers: implications for pathophysiology and treatment (Scientific Reports, (2017), 7, 1, (8089), 10.1038/s41598-017-06145-8)

This Article contains an error in the Acknowledgements section. “This work was supported by European Union’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement no. 602391 (project acronym EPISTOP; A.I., J.v.S., A.M., E.A., F.I., K.K., S.K., A.J., L.L., P.C., D.K.); Epilepsiefonds (WAR 15-05; RJP); European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642881 (ECMED; A.E., J.G.). None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.” should read: “This work was supported by European Union’s Seventh Framework Programme (FP7/2007-2013) under the Grant Agreement no. 602391 (project acronym EPISTOP; A.I., J.v.S., A.M., E.A., F.I., K.K., S.J., A.J., L.L., P.C., D.K.) and partially supported by the Polish Ministerial funds for science (years 2013–2019) for the implementation of international co-financed project (K.K, S.J.); Epilepsiefonds (WAR 15-05; RJP); European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642881 (ECMED; A.E., J.G.). None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.”