NMDA glutamate receptor antagonist MK-801 induces proteome changes in adult human brain slices which are partially counteracted by haloperidol and clozapine.

Deciphering the molecular pathways associated with N-methyl-D-aspartate receptor (NMDAr) hypofunction and its interaction with antipsychotics is necessary to advance our understanding of the basis of schizophrenia, as well as our capacity to treat this disease. In this regard, the development of human brain-derived models that are amenable to studying the neurobiology of schizophrenia may contribute to filling the gaps left by the widely employed animal models. Here, we assessed the proteomic changes induced by the NMDA glutamate receptor antagonist MK-801 on human brain slice cultures obtained from adult donors submitted to respective neurosurgery. Initially, we demonstrated that MK-801 diminishes NMDA glutamate receptor signaling in human brain slices in culture. Next, using mass-spectrometry-based proteomics and systems biology in silico analyses, we found that MK-801 led to alterations in proteins related to several pathways previously associated with schizophrenia pathophysiology, including ephrin, opioid, melatonin, sirtuin signaling, interleukin 8, endocannabinoid, and synaptic vesicle cycle. We also evaluated the impact of both typical and atypical antipsychotics on MK-801-induced proteome changes. Interestingly, the atypical antipsychotic clozapine showed a more significant capacity to counteract the protein alterations induced by NMDAr hypofunction than haloperidol. Finally, using our dataset, we identified potential modulators of the MK-801-induced proteome changes, which may be considered promising targets to treat NMDAr hypofunction in schizophrenia. This dataset is publicly available and may be helpful in further studies aimed at evaluating the effects of MK-801 and antipsychotics in the human brain.

Somatic mutational profiling and clinical impact of driver genes in Latin-Iberian medulloblastomas: Towards precision medicine.

Medulloblastoma (MB) is the most prevalent malignant brain tumor in children, known for its heterogeneity and treatment-associated toxicity, and there is a critical need for new therapeutic targets. We analyzed the somatic mutation profile of 15 driver genes in 69 Latin-Iberian molecularly characterized medulloblastomas using the Illumina TruSight Tumor 15 panel. We classified the variants based on their clinical impact and oncogenicity. Among the patients, 66.7% were MBSHH, 13.0% MBWNT, 7.3% MBGrp3, and 13.0% MBGrp4. Among the 63 variants found, 54% were classified as Tier I/II and 31.7% as oncogenic/likely oncogenic. We observed 33.3% of cases harboring at least one mutation. TP53 (23.2%, 16/69) was the most mutated gene, followed by PIK3CA (5.8%, 4/69), KIT (4.3%, 3/69), PDGFRA (2.9%, 2/69), EGFR (1.4%, 1/69), ERBB2 (1.4%, 1/69), and NRAS (1.4%, 1/69). Approximately 41% of MBSHH tumors exhibited mutations, TP53 (32.6%) being the most frequently mutated gene. Tier I/II and oncogenic/likely oncogenic TP53 variants were associated with relapse, progression, and lower survival rates. Potentially actionable variants in the PIK3CA and KIT genes were identified. Latin-Iberian medulloblastomas, particularly the MBSHH, exhibit higher mutation frequencies than other populations. We corroborate the TP53 mutation status as an important prognostic factor, while PIK3CA and KIT are potential therapeutic targets.

Primary neural leprosy: clinical, neurophysiological and pathological presentation and progression.

Disability in leprosy is a direct consequence of damage to the peripheral nervous system which is usually worse in patients with no skin manifestations, an underdiagnosed subtype of leprosy known as primary neural leprosy. We evaluated clinical, neurophysiological and laboratory findings of 164 patients with definite and probable primary neural leprosy diagnoses. To better understand the disease progression and to improve primary neural leprosy clinical recognition we compared the characteristics of patients with short (≤12 months) and long (>12 months) disease duration. Positive and negative symptoms mediated by small-fibres were frequent at presentation (∼95%), and symptoms tend to manifest first in the upper limbs (∼68%). There is a consistent phenotypic variability between the aforementioned groups. Deep sensory modalities were spared in patients evaluated within the first 12 months of the disease, and were only affected in patients with longer disease duration (∼12%). Deep tendon reflexes abnormalities were most frequent in patients with longer disease duration (P < 0.001), as well as motor deficits (P = 0.002). Damage to large fibres (sensory and motor) is a latter event in primary neural leprosy. Grade-2 disability and nerve thickening was also more frequent in cases with long disease duration (P < 0.001). Primary neural leprosy progresses over time and there is a marked difference in clinical phenotype between patients with short and long disease duration. Patients assessed within the first 12 months of symptom onset had a non-length-dependent predominant small-fibre sensory neuropathy, whilst patients with chronic disease presented an asymmetrical all diameter sensory-motor neuropathy and patchily decreased/absent deep tendon reflexes.

BRAF, TERT and HLA-G Status in the Papillary Thyroid Carcinoma: A Clinicopathological Association Study.

As BRAF, TERT, HLA-G, and microRNAs have been individually associated with papillary thyroid carcinoma (PTC), we aimed to evaluate the individual and collaborative role of these markers in PTC in the same patient cohort. HLA-G and BRAF tumor expression was evaluated by immunohistochemistry. Using molecular methods, BRAFV600E and TERT promoter mutations were evaluated in thyroid fine needle aspirates. MicroRNA tumor profiling was investigated using massively parallel sequencing. We observed strong HLA-G (67.96%) while BRAF (62.43%) staining was observed in PTC specimens. BRAF overexpression was associated with poor response to therapy. The BRAFV600E (52.9%) and TERTC228T (13%) mutations were associated with extrathyroidal extension, advanced-age, and advanced-stage cancer. The TERT rs2853669 CC+TC genotypes (38%) were overrepresented in metastatic tumors. Nine modulated microRNAs targeting the BRAF, TERT, and/or HLA-G genes were observed in PTC and involved with cancer-related signaling pathways. The markers were individually associated with PTC features, emphasizing the synergistic effect of BRAFV600E and TERTC228T; however, their collaborative role on PTC outcome was not fully demonstrated. The differentially expressed miRNAs targeting the BRAF and/or HLA-G genes may explain their increased expression in the tumor milieu.

Single nCounter assay for prediction of MYCN amplification and molecular classification of medulloblastomas: a multicentric study.

PURPOSE: Medulloblastoma is the most frequent pediatric malignant brain tumor, and is divided into four main subgroups: WNT, SHH, group 3, and group 4. MYCN amplification is an important medulloblastoma prognostic biomarker. We aimed to molecular classify and predict MYCN amplification in a single assay. METHODS: It was included 209 medulloblastomas from 205 patients (Brazil, Argentina, and Portugal), divided into training (n = 50) and validation (n = 159) sets. A nCounter assay was carried out using a custom panel for molecular classification, with additional genes, including MYCN. nSolver 4.0 software and the R environment were used for profiling and MYCN mRNA analysis. MYCN amplification by FISH was performed in 64 cases. RESULTS: The 205 medulloblastomas were classified in SHH (44.9%), WNT (15.6%), group 3 (18.1%) and group 4 (21.4%). In the training set, MYCN amplification was detected in three SHH medulloblastomas by FISH, which showed significantly higher MYCN mRNA counts than non-FISH amplified cases, and a cutoff for MYCN amplification was established ([Formula: see text] + 4σ = 11,124.3). Applying this threshold value in the validation set, we identified MYCN mRNA counts above the cutoff in three cases, which were FISH validated. CONCLUSION: We successfully stratified medulloblastoma molecular subgroups and predicted MYCN amplification using a single nCounter assay without the requirement of additional biological tissue, costs, or bench time.

MicroRNA expression profile predicts prognosis of pediatric adrenocortical tumors.

Pediatric adrenocortical tumors (ACT) are rare aggressive neoplasms with heterogeneous prognosis. Despite extensive efforts, identifying reliable prognostic factors for pediatric patients with ACT remains a challenge. MicroRNA (miRNA) signatures have been associated with cancer diagnosis, treatment response, and prognosis of several types of cancer. However, the role of miRNAs has been poorly explored in pediatric ACT. In this study, we performed miRNA microarray profiling on a cohort of 37 pediatric ACT and nine nonneoplastic adrenal (NNA) samples and evaluated the prognostic significance of abnormally expressed miRNAs using Kaplan-Meier plots, log-rank test, and Cox regression analysis. We identified a total of 98 abnormally expressed miRNAs; their expression profile discriminated ACT from NNAs. Among the 98 deregulated miRNAs, 17 presented significant associations with patients' survival. In addition, higher expression levels of hsa-miR-630, -139-3p, -125a-3p, -574-5p, -596, -564, -1321, and -423-5p and lower expression levels of hsa-miR-377-3p, -126-3p, -410, -136-3p, -29b-3p, -29a-3p, -337-5p, -143-3p, and 140-5p were significantly associated with poor prognosis, tumor relapse, and/or death. Importantly, the expression profile of these 17 miRNAs stratified patients into two groups of ACTs with different clinical outcomes. Although some individual miRNAs exhibit potential prognostic values in ACTs, only the 17 miRNA-based expression clustering was considered an independent prognostic factor for 5-year event-free survival (EFS) compared to other clinicopathological features. In conclusion, our study reports for the first time associations between miRNA profiles and childhood ACT prognosis, providing evidence that miRNAs could be useful biomarkers to discriminate patients with favorable and unfavorable clinical outcomes.

NTRK2 gene fusions are uncommon in pilocytic astrocytoma.

BACKGROUND: Pilocytic astrocytoma is the most frequent pediatric glioma. Despite its overall good prognosis, complete surgical resection is sometimes unfeasible, especially for patients with deep-seated tumors. For these patients, the identification of targetable genetic alterations such as NTRK fusions, raised as a new hope for therapy. The presence of gene fusions involving NTRK2 has been rarely reported in pilocytic astrocytoma. The aim of the present study was to investigate the frequency of NTRK2 alterations in a series of Brazilian pilocytic astrocytomas. METHODS: Sixty-nine pilocytic astrocytomas, previously characterized for BRAF and FGFR1 alterations were evaluated. The analysis of NTRK2 alterations was performed using a dual color break apart fluorescence in situ hybridization (FISH) assay. RESULTS: NTRK2 fusions were successfully evaluated by FISH in 62 of the 69 cases. Neither evidence of NTRK2 gene rearrangements nor NTRK2 copy number alterations were found. CONCLUSIONS: NTRK2 alterations are uncommon genetic events in pilocytic astrocytomas, regardless of patients' clinicopathological and molecular features.

The T-box transcription factor brachyury behaves as a tumor suppressor in gliomas.

The oncogene brachyury (TBXT) is a T-box transcription factor that is overexpressed in multiple solid tumors and is associated with tumor aggressiveness and poor patient prognosis. Gliomas comprise the most common and aggressive group of brain tumors, and at the present time the functional and clinical impact of brachyury expression has not been investigated previously in these neoplasms. Brachyury expression (mRNA and protein) was assessed in normal brain (n = 67), glioma tissues (n = 716) and cell lines (n = 42), and further in silico studies were undertaken using genomic databases totaling 3115 samples. Our glioma samples were analyzed for copy number (n = 372), promoter methylation status (n = 170), and mutation status (n = 1569 tissues and n = 52 cell lines) of the brachyury gene. The prognostic impact of brachyury expression was studied in 1524 glioma patient tumors. The functional impact of brachyury on glioma proliferation, viability, and cell death was evaluated both in vitro and in vivo. Brachyury was expressed in the normal brain, and significantly downregulated in glioma tissues. Loss of brachyury was associated with tumor aggressiveness and poor survival in glioma patients. Downregulation of brachyury was not associated with gene deletion, promoter methylation, or inactivating point mutations. Brachyury re-expression in glioma cells was found to decrease glioma tumorigenesis by induction of autophagy. These data strongly suggest that brachyury behaves as a tumor suppressor gene in gliomas by modulating autophagy. It is important to note that brachyury constitutes an independent positive biomarker of patient prognosis. Our findings indicate that the role of brachyury in tumorigenesis may be tissue-dependent and demands additional investigation to guide rational interventions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Evaluation of the prognostic potential of EGFL7 in pilocytic astrocytomas.

Pilocytic astrocytoma (PA) is the most frequent solid neoplasm in childhood. It has a good 5-year overall survival (90% in childhood and 52% in adults). However, up to 20% of patients experience residual tumor growth, recurrence, and death. Although the main genetic alteration of PAs, including KIAA1549:BRAF fusion, involves chromosome 7q34, we previously found frequent loss in chr9q34.3 locus in a small subset of these tumors. Among the genes present in this locus, EGFL7 is related to poor prognosis in several tumor types. In this study, we aimed to assess EGFL7 expression through immunohistochemistry, and to evaluate its prognostic value in a series of 64 clinically and molecularly well-characterized pilocytic astrocytomas. We found high expression of EGFL7 in 71.9% of patients. Low EGFL7 expression was associated with older patients, the mean age mainly older than 11 years (P = 0.027). EGFL7 expression was not associated with presence of KIAA1549:BRAF fusion, BRAF mutation, FGFR1 mutation, nor FGFR1 duplication. Moreover, high EGFL7 expression was associated with high FGFR1 (P = 0.037) and 5'-deoxy-5'-methyltioadenosine phosphorylase (MTAP) (P = 0.005) expression, and with unfavorable outcome of patients (P = 0.047). Multivariate analysis revealed low EGFL7 expression related to older patients and high EGFL7 expression related to retained expression of MTAP. In addition, we found a borderline significance of unfavorable outcome and high EGFL7 expression. Finally, EGFL7 expression was not associated with overall or event-free survival of PA patients. Our findings point to EGFL7 expression as a novel candidate prognostic marker in PA, which should be further investigated.

Adult giant cerebellar cavernous malformations: case report and review of the literature.

Cavernous malformations are vascular malformations that can occur anywhere in the central nervous system (CNS). Giant cavernous malformations (GCM) are extremely rare in adults, especially in the posterior fossa. Herein, we described a 48-year-old male who presented with vertigo and postural instability for three months. Neuroimaging revealed a 131.15 cm3 heterogeneous midline upper cerebellar lesion. After a suboccipital craniotomy, a gross total resection (GTR) was accomplished. Histopathologic examination revealed a huge cavernous malformation. Only 27 GCM adult cases were reported in the English-based literature. Only two patients had cerebellar lesions and, to the best of our knowledge, this is the first case of cerebellar vermis GCM. We concluded that cerebellar GCM (CGCM) in adults are exceedingly rare and indolent lesions. These lesions can radiologically and clinically mimic neoplastic lesions that have to be considered in the differential diagnosis. GTR is the mainstay of treatment and, whenever possible, should be attempted.

Somatic Copy Number Alterations and Associated Genes in Clear-Cell Renal-Cell Carcinoma in Brazilian Patients.

Somatic copy number aberrations (CNAs) have been associated with clear-cell renal carcinoma (ccRCC) pathogenesis and are a potential source of new diagnostic, prognostic and therapeutic biomarkers. Recurrent CNAs include loss of chromosome arms 3p, 14q, 9p, and gains of 5q and 8q. Some of these regional CNAs are suspected of altering gene expression and could influence clinical outcomes. Despite many studies of CNAs in RCC, there are currently no descriptions of genomic copy number alterations in a Brazilian ccRCC cohort. This study was designed to evaluate the chromosomal profile of CNAs in Brazilian ccRCC tumors and explore clinical associations. A total of 92 ccRCC Brazilian patients that underwent nephrectomy at Barretos Cancer Hospital were analyzed for CNAs by array comparative genomic hybridization. Most patients in the cohort had early-stage localized disease. The most significant alterations were loss of 3p (87.3%), 14q (35.8%), 6q (29.3%), 9p (28.6%) and 10q (25.0%), and gains of 5q (59.7%), 7p (29.3%) and 16q (20.6%). Bioinformatics analysis revealed 19 genes mapping to CNA significant regions, including SETD2, BAP1, FLT4, PTEN, FGFR4 and NSD1. Moreover, gain of 5q34-q35.3 (FLT4 and NSD1) and loss of 6q23.2-q23.3 (MYB) and 9p21.3 (MLLT3) had gene expression levels that correlated with TCGA data and was also associated with advanced disease features, such as larger tumors, Fuhrman 3, metastasis at diagnosis and death. The loss of region 14q22.1 which encompasses the NIN gene was associated with poor overall survival. Overall, this study provides the first CNA landscape of Brazilian patients and pinpoints genomic regions and specific genes worthy of more detailed investigations. Our results highlight important genes that are associated with copy number changes involving large chromosomal regions that are potentially related to ccRCC tumorigenesis and disease biology for future clinical investigations.

Phosphorylation of S6 Protein as a Potential Biomarker in Surgically Treated Refractory Epilepsy.

The tuberous sclerosis complex (TSC), focal cortical dysplasia IIB (FCD IIB), and hemimegalencephaly (HME) exhibit similar molecular features that are dependent on the hyperactivation of the mTOR pathway. They are all associated with refractory epilepsy and the need for surgical resection with varying outcomes. The phosphorylated protein S6 (pS6) is a downstream target of mTOR, whose increased expression might indicate mTOR hyperactivation, but which is also present when there is no alteration in the pathway (such as in FCD type I). We have performed immunohistochemical marking and quantification of pS6 in resected brain specimens of 26 patients clinically and histologically diagnosed with TSC, FCD IIB, or HME and compared this data to a control group of 25 patients, to measure the extent of pS6 positivity and its correlation with clinical aspects. Our results suggest that pS6 may serve as a reliable biomarker in epilepsy and that a greater percentage of pS6 marking can relate to more severe forms of mTOR-dependent brain anomalies.

Drebrin expression patterns in patients with refractory temporal lobe epilepsy and hippocampal sclerosis.

OBJECTIVE: Drebrins are crucial for synaptic function and dendritic spine development, remodeling, and maintenance. In temporal lobe epilepsy (TLE) patients, a significant hippocampal synaptic reorganization occurs, and synaptic reorganization has been associated with hippocampal hyperexcitability. This study aimed to evaluate, in TLE patients, the hippocampal expression of drebrin using immunohistochemistry with DAS2 or M2F6 antibodies that recognize adult (drebrin A) or adult and embryonic (pan-drebrin) isoforms, respectively. METHODS: Hippocampal sections from drug-resistant TLE patients with hippocampal sclerosis (HS; TLE, n = 33), of whom 31 presented with type 1 HS and two with type 2 HS, and autopsy control cases (n = 20) were assayed by immunohistochemistry and evaluated for neuron density, and drebrin A and pan-drebrin expression. Double-labeling immunofluorescences were performed to localize drebrin A-positive spines in dendrites (MAP2), and to evaluate whether drebrin colocalizes with inhibitory (GAD65) and excitatory (VGlut1) presynaptic markers. RESULTS: Compared to controls, TLE patients had increased pan-drebrin in all hippocampal subfields and increased drebrin A-immunopositive area in all hippocampal subfields but CA1. Drebrin-positive spine density followed the same pattern as total drebrin quantification. Confocal microscopy indicated juxtaposition of drebrin-positive spines with VGlut1-positive puncta, but not with GAD65-positive puncta. Drebrin expression in the dentate gyrus of TLE cases was associated negatively with seizure frequency and positively with verbal memory. TLE patients with lower drebrin-immunopositive area in inner molecular layer (IML) than in outer molecular layer (OML) had a lower seizure frequency than those with higher or comparable drebrin-immunopositive area in IML compared with OML. SIGNIFICANCE: Our results suggest that changes in drebrin-positive spines and drebrin expression in the dentate gyrus of TLE patients are associated with lower seizure frequency, more preserved verbal memory, and a better postsurgical outcome.

High-throughput microRNA profile in adult and pediatric primary glioblastomas: the role of miR-10b-5p and miR-630 in the tumor aggressiveness.

Glioblastoma (GBM) is the most common primary malignant neoplasm of the central nervous system and, despite the standard therapy; the patients' prognoses remain dismal. The miRNA expression profiles have been associated with patient prognosis, suggesting that they may be helpful for tumor diagnosis and classification as well as predictive of tumor response to treatment. We described the microRNA expression profile of 29 primary GBM samples (9 pediatric GBMs) and 11 non-neoplastic white matter samples as controls (WM) by microarray analysis and we performed functional in vitro assays on these 2 most differentially expressed miRNAs. Hierarchical clustering analysis showed 3 distinct miRNA profiles, two of them in the GBM samples and a group consisting only of cerebral white matter. When adult and pediatric GBMs were compared to WM, 37 human miRNAs were found to be differentially expressed, with miR-10b-5p being the most overexpressed and miR-630 the most underexpressed. The overexpression of miR-630 was associated with reduced cell proliferation and invasion in the U87 GBM cell line, whereas the inhibition of miR-10b-5p reduced cell proliferation and colony formation in the U251 GBM cell line, suggesting that these miRNAs may act as tumor-suppressive and oncogenic miRNAs, respectively. The present study highlights the distinct epigenetic profiling of adult and pediatric GBMs and underscores the biological importance of mir-10b-5p and miR-630 for the pathobiology of these lethal tumors.

MicroRNA profile of pediatric pilocytic astrocytomas identifies two tumor-specific signatures when compared to non-neoplastic white matter.

PURPOSES: Pilocytic astrocytoma (PA) is a low-grade neoplasm frequently found in childhood. PA is characterized by slow growth and a relatively good prognosis. Genetic mechanisms such as activation of MAPK, BRAF gene deregulation and neurofibromatosis type 1 (NF1) syndrome have been associated with PA development. Epigenetic signature and miRNA expression profile are providing new insights about different types of tumor, including PAs. METHODS: In the present study we evaluated global miRNA expression in 16 microdissected pediatric PA specimens, three NF1-associated PAs and 11 cerebral white matter (WM) samples by the microarray method. An additional cohort of 20 PAs was used to validate by qRT-PCR the expression of six miRNAs differentially expressed in the microarray data. RESULTS: Unsupervised hierarchical clustering analysis distinguished one cluster with nine PAs, including all NF1 cases and a second group consisting of the WM samples and seven PAs. Among 88 differentially expressed miRNAs between PAs and WM samples, the most underexpressed ones regulate classical pathways of tumorigenesis, while the most overexpressed miRNAs are related to pathways such as focal adhesion, P53 signaling pathway and gliomagenesis. The PAs/NF1 presented a subset of underexpressed miRNAs, which was also associated with known deregulated pathways in cancer such as cell cycle and hippo pathway. CONCLUSIONS: In summary, our data demonstrate that PA harbors at least two distinct miRNA signatures, including a subgroup of patients with NF1/PA lesions.

Impact of the Canonical Wnt Pathway Activation on the Pathogenesis and Prognosis of Adamantinomatous Craniopharyngiomas.

CTNNB1 mutations and abnormal ß-catenin distribution are associated with the pathogenesis of adamantinomatous craniopharyngioma (aCP). We evaluated the expression of the canonical Wnt pathway components in aCPs and its association with CTNNB1 mutations and tumor progression. Tumor samples from 14 aCP patients and normal anterior pituitary samples from eight individuals without pituitary disease were studied. Gene expression of Wnt pathway activator (WNT4), inhibitors (SFRP1, DKK3, AXIN1, and APC), transcriptional activator (TCF7), target genes (MYC, WISP2, and, CDH1), and Wnt modulator (TP53) was evaluated by qPCR. ß-Catenin, MYC, and WISP2 expression was determined by immunohistochemistry (IHC). The transcription levels of all genes studied, except APC, were higher in aCPs as compared to controls and TCF7 mRNA levels correlated with CTNNB1 mutation. CDH1 mRNA was overexpressed in tumor samples of patients with disease progression in comparison to those with stable disease. ß-Catenin was positive and aberrantly distributed in 11 out of 14 tumor samples. Stronger ß-catenin immunostaining associated positively with tumor progression. MYC positive staining was found in 10 out of 14 cases, whereas all aCPs were negative for WISP2. Wnt pathway genes were overexpressed in aCPs harboring CTNNB1 mutations and in patients with progressive disease. Recurrence was associated with stronger staining for ß-catenin. These data suggest that Wnt pathway activation contributes to the pathogenesis and prognosis of aCPs.

Reproducibility of the NanoString 22-gene molecular subgroup assay for improved prognostic prediction of medulloblastoma.

Medulloblastoma is the most frequent malignant brain tumor in children. Four medulloblastoma molecular subgroups, MBSHH , MBWNT , MBGRP3 and MBGRP4 , have been identified by integrated high-throughput platforms. Recently, a 22-gene panel NanoString-based assay was developed for medulloblastoma molecular subgrouping, but the robustness of this assay has not been widely evaluated. Mutations in the gene for human telomerase reverse transcriptase (hTERT) have been found in medulloblastomas and are associated with distinct molecular subtypes. This study aimed to implement the 22-gene panel in a Brazilian context, and to associate the molecular profile with patients' clinical-pathological features. Formalin-fixed, paraffin-embedded (FFPE) medulloblastoma samples (n = 104) from three Brazilian centers were evaluated. Expression profiling of the 22-gene panel was performed by NanoString and a Canadian series (n = 240) was applied for training phase. hTERT mutations were analyzed by PCR followed by direct Sanger sequencing and the molecular profile was associated with patients' clinicopathological features. Overall, 65% of the patients were male, average age at diagnosis was 18 years and 7% of the patients presented metastasis at diagnosis. The molecular classification was attained in 100% of the cases, with the following frequencies: MBSHH (n = 51), MBWNT (n = 19), MBGRP4 (n = 19) and MBGRP3 (n = 15). The MBSHH and MBGRP3 subgroups were associated with older and younger patients, respectively. The MBGRP4 subgroup exhibited the lowest 5-year cancer-specific overall survival (OS), yet in the multivariate analysis, only metastasis at diagnosis and surgical resection were associated with OS. hTERT mutations were detected in 29% of the cases and were associated with older patients, increased hTERT expression and MBSHH subgroup. The 22-gene panel provides a reproducible assay for molecular subgrouping of medulloblastoma FFPE samples in a routine setting and is well-suited for future clinical trials.

Congenital Zika Virus Infection Induces Severe Spinal Cord Injury.

We report 2 fatal cases of congenital Zika virus (ZIKV) infection. Brain anomalies, including atrophy of the cerebral cortex and brainstem, and cerebellar aplasia were observed. The spinal cord showed architectural distortion, severe neuronal loss, and microcalcifications. The ZIKV proteins and flavivirus-like particles were detected in cytoplasm of spinal neurons, and spinal cord samples were positive for ZIKV RNA.

The DNA methyltransferase inhibitor zebularine exerts antitumor effects and reveals BATF2 as a poor prognostic marker for childhood medulloblastoma.

Medulloblastoma (MB) is the most common solid tumor among pediatric patients and corresponds to 20 % of all pediatric intracranial tumors in this age group. Its treatment currently involves significant side effects. Epigenetic changes such as DNA methylation may contribute to its development and progression. DNA methyltransferase (DNMT) inhibitors have shown promising anticancer effects. The agent Zebularine acts as an inhibitor of DNA methylation and shows low toxicity and high efficacy, being a promising adjuvant agent for anti-cancer chemotherapy. Several studies have reported its effects on different types of tumors; however, there are no studies reporting its effects on MB. We analyzed its potential anticancer effects in four pediatric MB cell lines. The treatment inhibited proliferation and clonogenicity, increased the apoptosis rate and the number of cells in the S phase (p < 0.05), as well as the expression of p53, p21, and Bax, and decreased cyclin A, Survivin and Bcl-2 proteins. In addition, the combination of zebularine with the chemotherapeutic agents vincristine and cisplatin resulted in synergism and antagonism, respectively. Zebularine also modulated the activation of the SHH pathway, reducing SMO and GLI1 levels and one of its targets, PTCH1, without changing SUFU levels. A microarray analysis revealed different pathways modulated by the drug, including the Toll-Like Receptor pathway and high levels of the BATF2 gene. The low expression of this gene was associated with a worse prognosis in MB. Taken together, these data suggest that Zebularine may be a potential drug for further in vivo studies of MB treatment.

Expression signatures of DNA repair genes correlate with survival prognosis of astrocytoma patients.

Astrocytomas are the most common primary brain tumors. They are very resistant to therapies and usually progress rapidly to high-grade lesions. Here, we investigated the potential role of DNA repair genes in astrocytoma progression and resistance. To this aim, we performed a polymerase chain reaction array-based analysis focused on DNA repair genes and searched for correlations between expression patters and survival prognoses. We found 19 genes significantly altered. Combining these genes in all possible arrangements, we found 421 expression signatures strongly associated with poor survival. Importantly, five genes (DDB2, EXO1, NEIL3, BRCA2, and BRIP1) were independently correlated with worse prognoses, revealing single-gene signatures. Moreover, silencing of EXO1, which is remarkably overexpressed, promoted faster restoration of double-strand breaks, while NEIL3 knockdown, also highly overexpressed, caused an increment in DNA damage and cell death after irradiation of glioblastoma cells. These results disclose the importance of DNA repair pathways for the maintenance of genomic stability of high-grade astrocytomas and suggest that EXO1 and NEIL3 overexpression confers more efficiency for double-strand break repair and resistance to reactive oxygen species, respectively. Thereby, we highlight these two genes as potentially related with tumor aggressiveness and promising candidates as novel therapeutic targets.