DNA methylation-based classification of malformations of cortical development in the human brain.

Malformations of cortical development (MCD) comprise a broad spectrum of structural brain lesions frequently associated with epilepsy. Disease definition and diagnosis remain challenging and are often prone to arbitrary judgment. Molecular classification of histopathological entities may help rationalize the diagnostic process. We present a retrospective, multi-center analysis of genome-wide DNA methylation from human brain specimens obtained from epilepsy surgery using EPIC 850 K BeadChip arrays. A total of 308 samples were included in the study. In the reference cohort, 239 formalin-fixed and paraffin-embedded (FFPE) tissue samples were histopathologically classified as MCD, including 12 major subtype pathologies. They were compared to 15 FFPE samples from surgical non-MCD cortices and 11 FFPE samples from post-mortem non-epilepsy controls. We applied three different statistical approaches to decipher the DNA methylation pattern of histopathological MCD entities, i.e., pairwise comparison, machine learning, and deep learning algorithms. Our deep learning model, which represented a shallow neuronal network, achieved the highest level of accuracy. A test cohort of 43 independent surgical samples from different epilepsy centers was used to test the precision of our DNA methylation-based MCD classifier. All samples from the test cohort were accurately assigned to their disease classes by the algorithm. These data demonstrate DNA methylation-based MCD classification suitability across major histopathological entities amenable to epilepsy surgery and age groups and will help establish an integrated diagnostic classification scheme for epilepsy-associated MCD.

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.

Papillary glioneuronal tumor (PGNT) exhibits a characteristic methylation profile and fusions involving PRKCA.

Papillary glioneuronal tumor (PGNT) is a WHO-defined brain tumor entity that poses a major diagnostic challenge. Recently, SLC44A1-PRKCA fusions have been described in PGNT. We subjected 28 brain tumors from different institutions histologically diagnosed as PGNT to molecular and morphological analysis. Array-based methylation analysis revealed that 17/28 tumors exhibited methylation profiles typical for other tumor entities, mostly dysembryoplastic neuroepithelial tumor and hemispheric pilocytic astrocytoma. Conversely, 11/28 tumors exhibited a unique profile, thus constituting a distinct methylation class PGNT. By screening the extended Heidelberg cohort containing over 25,000 CNS tumors, we identified three additional tumors belonging to this methylation cluster but originally histologically diagnosed otherwise. RNA sequencing for the detection of SLC44A1-PRKCA fusions could be performed on 19 of the tumors, 10 of them belonging to the methylation class PGNT. In two additional cases, SLC44A1-PRKCA fusions were confirmed by FISH. We detected fusions involving PRKCA in all cases of this methylation class with material available for analyses: the canonical SLC44A1-PRKCA fusion was observed in 11/12 tumors, while the remaining case exhibited a NOTCH1-PRKCA fusion. Neither of the fusions was found in the tumors belonging to other methylation classes. Our results point towards a high misclassification rate of the morphological diagnosis PGNT and clearly demonstrate the necessity of molecular analyses. PRKCA fusions are highly diagnostic for PGNT, and detection by RNA sequencing enables the identification of rare fusion partners. Methylation analysis recognizes a unique methylation class PGNT irrespective of the nature of the PRKCA fusion.

Primary intracranial spindle cell sarcoma with rhabdomyosarcoma-like features share a highly distinct methylation profile and DICER1 mutations.

Patients with DICER1 predisposition syndrome have an increased risk to develop pleuropulmonary blastoma, cystic nephroma, embryonal rhabdomyosarcoma, and several other rare tumor entities. In this study, we identified 22 primary intracranial sarcomas, including 18 in pediatric patients, with a distinct methylation signature detected by array-based DNA-methylation profiling. In addition, two uterine rhabdomyosarcomas sharing identical features were identified. Gene panel sequencing of the 22 intracranial sarcomas revealed the almost unifying feature of DICER1 hotspot mutations (21/22; 95%) and a high frequency of co-occurring TP53 mutations (12/22; 55%). In addition, 17/22 (77%) sarcomas exhibited alterations in the mitogen-activated protein kinase pathway, most frequently affecting the mutational hotspots of KRAS (8/22; 36%) and mutations or deletions of NF1 (7/22; 32%), followed by mutations of FGFR4 (2/22; 9%), NRAS (2/22; 9%), and amplification of EGFR (1/22; 5%). A germline DICER1 mutation was detected in two of five cases with constitutional DNA available. Notably, none of the patients showed evidence of a cancer-related syndrome at the time of diagnosis. In contrast to the genetic findings, the morphological features of these tumors were less distinctive, although rhabdomyoblasts or rhabdomyoblast-like cells could retrospectively be detected in all cases. The identified combination of genetic events indicates a relationship between the intracranial tumors analyzed and DICER1 predisposition syndrome-associated sarcomas such as embryonal rhabdomyosarcoma or the recently described group of anaplastic sarcomas of the kidney. However, the intracranial tumors in our series were initially interpreted to represent various tumor types, but rhabdomyosarcoma was not among the typical differential diagnoses considered. Given the rarity of intracranial sarcomas, this molecularly clearly defined group comprises a considerable fraction thereof. We therefore propose the designation "spindle cell sarcoma with rhabdomyosarcoma-like features, DICER1 mutant" for this intriguing group.

FGFR1:TACC1 fusion is a frequent event in molecularly defined extraventricular neurocytoma.

Extraventricular neurocytoma (EVN) is a rare primary brain tumor occurring in brain parenchyma outside the ventricular system. Histopathological characteristics resemble those of central neurocytoma but exhibit a wider morphologic spectrum. Accurate diagnosis of these histologically heterogeneous tumors is often challenging because of the overlapping morphological features and the lack of defining molecular markers. Here, we explored the molecular landscape of 40 tumors diagnosed histologically as EVN by investigating copy number profiles and DNA methylation array data. DNA methylation profiles were compared with those of relevant differential diagnoses of EVN and with a broader spectrum of diverse brain tumor entities. Based on this, our tumor cohort segregated into different groups. While a large fraction (n = 22) formed a separate epigenetic group clearly distinct from established DNA methylation profiles of other entities, a subset (n = 14) of histologically diagnosed EVN grouped with clusters of other defined entities. Three cases formed a small group close to but separated from the epigenetically distinct EVN cases, and one sample clustered with non-neoplastic brain tissue. Four additional samples originally diagnosed otherwise were found to molecularly resemble EVN. Thus, our results highlight a distinct DNA methylation pattern for the majority of tumors diagnosed as EVN, but also indicate that approximately one third of morphological diagnoses of EVN epigenetically correspond to other brain tumor entities. Copy number analysis and confirmation through RNA sequencing revealed FGFR1-TACC1 fusion as a distinctive, recurrent feature within the EVN methylation group (60%), in addition to a small number of other FGFR rearrangements (13%). In conclusion, our data demonstrate a specific epigenetic signature of EVN suitable for characterization of these tumors as a molecularly distinct entity, and reveal a high frequency of potentially druggable FGFR pathway activation in this tumor group.

Characteristics of glioblastomas and immune microenvironment in a Chinese family with Lynch syndrome and concurrent porokeratosis.

Background: Lynch syndrome (LS)-associated glioblastoma (GBM) is rare in clinical practice, and simultaneous occurrence with cutaneous porokeratosis is even rarer. In this study, we analyzed the clinicopathological and genetic characteristics of LS-associated GBMs and concurrent porokeratosis, as well as evaluated the tumor immune microenvironment (TIME) of LS-associated GBMs. Methods: Immunohistochemical staining was used to confirm the histopathological diagnosis, assess MMR and PD-1/PD-L1 status, and identify immune cell subsets. FISH was used to detect amplification of EGFR and PDGFRA, and deletion of 1p/19q and CDKN2A. Targeted NGS assay analyzed somatic variants, MSI, and TMB status, while whole-exome sequencing and Sanger sequencing were carried out to analyze the germline mutations. Results: In the LS family, three members (I:1, II:1 and II:4) were affected by GBM. GBMs with loss of MSH2 and MSH6 expression displayed giant and multinucleated bizarre cells, along with mutations in ARID1A, TP53, ATM, and NF1 genes. All GBMs had TMB-H but not MSI-H. CD8+ T cells and CD163+ macrophages were abundant in each GBM tissue. The primary and recurrent GBMs of II:1 showed mesenchymal characteristics with high PD-L1 expression. The family members harbored a novel heterozygous germline mutation in MSH2 and FDPS genes, confirming the diagnosis of LS and disseminated superficial actinic porokeratosis. Conclusion: LS-associated GBM exhibits heterogeneity in clinicopathologic and molecular genetic features, as well as a suppressive TIME. The presence of MMR deficiency and TMB-H may serve as predictive factors for the response to immune checkpoint inhibitor therapy in GBMs. The identification of LS-associated GBM can provide significant benefits to both patients and their family members, including accurate diagnosis, genetic counseling, and appropriate screening or surveillance protocols. Our study serves as a reminder to clinicians and pathologists to consider the possibility of concurrent genetic syndromes in individuals or families.

DNA Methylation Profiling Discriminates between Malignant Pleural Mesothelioma and Neoplastic or Reactive Histologic Mimics.

The diagnosis of malignant pleural mesothelioma (MPM) is challenging because of its potential overlap with other neoplasms or even with reactive conditions. DNA methylation analysis is effective in diagnosing tumors. In the present study, this approach was tested for use in MPM diagnosis. The DNA methylation patterns of a discovery cohort and an independent-validation cohort of MPMs were compared to those of 202 cases representing malignant and benign diagnostic mimics (angiosarcoma, desmoid-type fibromatosis, epithelioid sarcoma, leiomyosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, nodular fasciitis, reactive mesothelial hyperplasia, sclerosing fibrous pleuritis, solitary fibrous tumor, and synovial sarcoma). By both unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analysis, MPM samples in the discovery cohort exhibited a DNA methylation profile different from those of other neoplastic and reactive mimics. These results were confirmed in the independent validation cohort and by in silico analysis of the MPM-The Cancer Genome Atlas data set. Copy number variation profiles were also inferred to identify molecular hallmarks of MPM, including CDKN2A and NF2 deletions. Methylation profiling was effective in the diagnosis of MPM, although caution is advised in samples with low tumor cell content.

Nrf2 inhibits NLRP3 inflammasome activation through regulating Trx1/TXNIP complex in cerebral ischemia reperfusion injury.

The nod-like receptor protein 3 (NLRP3) inflammasome has a critical role in inflammation damage in ischemic injury, and the activation of the inflammasome is closely related to the interaction with thioredoxin interacting protein (TXNIP), which dissociates from the thioredoxin1 (Trx1)/TXNIP complex under oxidative stress. However, the negative regulator of NLRP3 inflammasome activation has not been fully investigated. Nuclear factor erythroid 2-related factor 2 (Nrf2) takes on a critical part in the antioxidant stress system, that controls the driven genes of antioxidant response element (ARE). Activate Nrf2 could inhibit the activation of NLRP3 inflammasome in acute liver injury and severe lupus nephritis. We aimed to explore the protective effect of Nrf2 in inhibiting the NLPR3 inflammasome formulation through the Trx1/TXNIP complex in cerebral ischemia reperfusion (cerebral I/R) injury. Middle cerebral artery occlusion/reperfusion (MCAO/R) model was used to imitate ischemic insult. Nrf2 was activated by tert-butylhydroquinone (tBHQ) intraperitoneally (i.p.) injection (16.7mg/kg), Nrf2,Trx1 and NLRP3 siRNAs were infused into the left paracele (12µl per rat), protein and mRNA levels were assessed by Western blot, qRT-PCR. ELISA was used for IL-1ß and IL-18 activity measurements. After upregulating Nrf2, the expression of TXNIP in cytoplasm, NLRP3 inflammasome, and downstream factors caspase-1, IL-18, and IL-1ß were significantly reduced, and Nrf2 knockdown yielded the opposite results. Trx1 knockdown produced the same effect of Nrf2 inhibition and the protective effect of Nrf2 was mostly abolished. Our results suggested that Nrf2 acted as a protective regulator against NLRP3 inflammasome activation by regulating the Trx1/TXNIP complex, which could possibly represent an innovative insight into the treatment of ischemia and reperfusion injury.

Resveratrol alleviates cerebral ischemia/reperfusion injury in rats by inhibiting NLRP3 inflammasome activation through Sirt1-dependent autophagy induction.

Resveratrol has been reported to protect against cerebral ischemia/reperfusion (I/R) injury in rats, but the underlying mechanism is unclear. In the current study, we examined whether resveratrol ameliorates cerebral I/R injury by inhibiting NLRP3 inflammasome-derived inflammation and whether autophagy is involved in this process. In addition, we explored the role of Sirt1 in resveratrol-mediated protective effects. To answer these questions, healthy male Sprague-Dawley rats were exposed to middle cerebral artery occlusion for 1h followed by 24h reperfusion. We found that cerebral I/R increased levels of activated NLRP3 inflammasome, caspase-1, IL-1ß, and IL-18 and enhanced autophagy activity (ratio of LC3B-II/LC3B-I and p62/SQSTM1). Treatment with resveratrol, a specific Sirt1 agonist, attenuated I/R-induced NLRP3 inflammasome-derived inflammation but upregulated autophagy. Furthermore, resveratrol treatment clearly reduced cerebral infarct volume, decreased brain water content, and improved neurological scores. In addition, inhibition of autophagy using 3-MA intracerebroventricular injection blocked the inhibitory effect of resveratrol on NLRP3 inflammasome activation. Finally, Sirt1 knockdown with siRNA significantly blocked resveratrol-induced enhancement of autophagy activity and suppression of NLRP3 inflammasome activation. In conclusion, our results demonstrate that resveratrol protects against cerebral I/R injury by inhibiting NLRP3 inflammasome activation through Sirt1-dependent autophagy activity.

Protective effects of PGC-1α via the mitochondrial pathway in rat brains after intracerebral hemorrhage.

UNLABELLED: Peroxisome-proliferator-activated receptor co-activator-1α (PGC-1α) is a transcriptional co-activator that coordinately regulates genes required for mitochondrial biogenesis, which stimulates mitochondrial activity. It is also a major factor in the up-regulation of antioxidant activities that are a response to oxidative stress. However, the role of PGC-1α after intracerebral hemorrhage (ICH) has not been studied. The purpose of the present work was to investigate the effects and mechanism of PGC-1α after ICH in the brain. Brain injury was induced by injecting autologous arterial blood (50µL) into the rat brain. PGC-1α siRNAs were injected into rat brains 24h prior to ICH. Then, 72h after ICH, brains were collected for investigation. Post-assessment included western blot analysis, RT-PCR assay, neurobehavioral function testing, measurement of brain water content, high-performance liquid chromatography (HPLC), and projection electron microscopy on ICH rat models. The concentration of PGC-1α was higher in the ipsilateral striatum after ICH, peaking around 72h after ICH. The expression of NRF-1, TFAM, SOD2, UCP2, mitochondrial DNA, ATP concentration, mitochondrial quantity, and brain water content were increased 72h after ICH. However, the neurological score was decreased 72h after ICH. Treatment with PGC-1α siRNAs significantly decreased the neurological score, ATP concentration, number of mitochondria, expression of NRF-1, TFAM, SOD2, UCP2, and mitochondrial DNA, and increased brain water content and formation of mitochondrial myelin layer structures. In conclusion, our data suggest that PGC-1α protects rat brains via a mitochondrial pathway following ICH. KEY WORDS: PGC-1α intracerebral hemorrhage(ICH); mitochondrial; neuroprotection.

Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1α Pathway in Rats.

Sestrin2 (Sesn2) exerts neuroprotective properties in some neurodegenerative diseases. However, the role of Sesn2 in stroke is unclear. The AMP-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α (AMPK/PGC-1α) pathway plays an important role in regulating mitochondrial biogenesis, which helps prevent cerebral ischemia/reperfusion (I/R) injury. Here, we aimed to determine whether Sesn2 alleviated I/R damage by regulating mitochondrial biogenesis through the AMPK/PGC-1α signaling pathway. To be able to test this, Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h with Sesn2 silencing. At 24 h after reperfusion, we found that neurological deficits were exacerbated, infarct volume was enlarged, and oxidative stress and neuronal damage were greater in the Sesn2 siRNA group than in the MCAO group. To explore protective mechanisms, an AMPK activator was used. Expression levels of Sesn2, p-AMPK, PGC-1α, NRF-1, TFAM, SOD2, and UCP2 were significantly increased following cerebral I/R. However, upregulation of these proteins was prevented by Sesn2 small interfering RNA (siRNA). In contrast, activation of AMPK with 5'-aminoimidazole-4-carboxamide riboside weakened the effects of Sesn2 siRNA. These results suggest that Sesn2 silencing may suppress mitochondrial biogenesis, reduce mitochondrial biological activity, and finally aggravate cerebral I/R injury through inhibiting the AMPK/PGC-1α pathway.

Foxo1-mediated inflammatory response after cerebral hemorrhage in rats.

The forkhead box O (Foxo) family of transcription factors plays a crucial role in cell apoptosis, immune regulation, and tissue development. Foxo1, as the foremost member of the Foxo family, regulates a wide range of molecular signals in many tissues, including tumor, liver, and brain. This study investigated Foxo1 expression at different time points and in different brain areas, and the role of Foxo1 in vivo in regulating inflammatory injury in a rat model of autologous blood-injected cerebral hemorrhage injury. We found that Foxo1 expression peaked at 12h post-intracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Foxo1 knockdown by Foxo1 siRNA decreased ICH injury, improved neurological function, and decreased the expression of inflammatory factors downstream of the Foxo1 pathway, including TLR4, NF-κB, TNF-α, IL-1ß, and IL-18. Foxo1 knockdown also decreased the expression and activity of myeloperoxidase, IL-1ß, and IL-18. In conclusion, our findings demonstrate that Foxo1 is a key regulator of inflammatory injury in rats after ICH. By identifying the molecular mechanisms of Foxo1/TLR4/NF-κB signaling, we provide a novel rationale for therapeutic approaches to managing inflammatory injury after ICH.