A novel program of infiltrative control in astrocytomas: ADAM23 depletion promotes cell invasion by activating γ-secretase complex.

Background: Infiltration is a life-threatening growth pattern in malignant astrocytomas and a significant cause of therapy resistance. It results in the tumor cell spreading deeply into the surrounding brain tissue, fostering tumor recurrence and making complete surgical resection impossible. We need to thoroughly understand the mechanisms underlying diffuse infiltration to develop effective therapies. Methods: We integrated in vitro and in vivo functional assays, RNA sequencing, clinical, and expression information from public data sets to investigate the role of ADAM23 expression coupling astrocytoma's growth and motility. Results: ADAM23 downregulation resulted in increased infiltration, reduced tumor growth, and improved overall survival in astrocytomas. Additionally, we show that ADAM23 deficiency induces γ-secretase (GS) complex activity, contributing to the production and deposition of the Amyloid-ß and release of NICD. Finally, GS ablation in ADAM23-low astrocytomas induced a significant inhibitory effect on the invasive programs. Conclusions: Our findings reveal a role for ADAM23 in regulating the balance between cell proliferation and invasiveness in astrocytoma cells, proposing GS inhibition as a therapeutic option in ADAM23 low-expressing astrocytomas.

Exercise training attenuates skeletal muscle fat infiltration and improves insulin pathway of patients with immune-mediated necrotizing myopathies and dermatomyositis.

Objectives: This study aims to evaluate the effects of exercise training on intramuscular lipid content and genes related to insulin pathway in patients with systemic autoimmune myopathies (SAMs). Patients and methods: Between January 2016 and May 2019, a total of seven patients with dermatomyositis (DM; 3 males, 4 females; mean age: 49.8±2.3 years; range, 43 to 54 years), six with immune mediated necrotizing myopathy (IMNM; 3 males, 3 females; mean age: 58.5±10.6 years; range, 46 to 74 years), and 10 control individuals (CTRL group; 4 males, 6 females; mean age: 48.7±3.9 years; range, 41 to 56 years) were included. The muscle biopsy before and after the intervention was performed to evaluate the intramuscular lipid content. Patients underwent a combined exercise training program for 12 weeks. Skeletal muscle gene expression was analyzed and the DM versus CTRL group, DM pre- and post-, and IMNM pre- and post-intervention were compared. Results: The DM group had a higher intramuscular lipid content in type II muscle fibers compared to the CTRL group. After the intervention, there was a reduction of lipid content in type I and II fibers in DM and IMNM group. The CTRL group showed a significantly higher expression of genes related to insulin and lipid oxidation pathways (AMPKß2, AS160, INSR, PGC1-α, PI3K, and RAB14) compared to the DM group. After exercise training, there was an increase gene expression related to insulin pathway and lipid oxidation in DM group (AMPKß2, AS160, INSR, PGC1-α, PI3K, and RAB14) and in IMNM group (AKT2, AMPKß2, RAB10, RAB14, and PGC1-α). Conclusion: Exercise training attenuated the amount of fat in type I and II muscle fibers in patients with DM and IMNM and increased gene expression related to insulin pathways and lipid oxidation in DM and IMNM. These results suggest that exercise training can improve the quality and metabolic functions of skeletal muscle in these diseases.

GBM Cells Exhibit Susceptibility to Metformin Treatment According to TLR4 Pathway Activation and Metabolic and Antioxidant Status.

Glioblastoma (GBM) is an aggressive brain cancer associated with poor overall survival. The metabolic status and tumor microenvironment of GBM cells have been targeted to improve therapeutic strategies. TLR4 is an important innate immune receptor capable of recognizing pathogens and danger-associated molecules. We have previously demonstrated the presence of TLR4 in GBM tumors and the decreased viability of the GBM tumor cell line after lipopolysaccharide (LPS) (TLR4 agonist) stimulation. In the present study, metformin (MET) treatment, used in combination with temozolomide (TMZ) in two GBM cell lines (U87MG and A172) and stimulated with LPS was analyzed. MET is a drug widely used for the treatment of diabetes and has been repurposed for cancer treatment owing to its anti-proliferative and anti-inflammatory actions. The aim of the study was to investigate MET and LPS treatment in two GBM cell lines with different metabolic statuses. MET treatment led to mitochondrial respiration blunting and oxidative stress with superoxide production in both cell lines, more markedly in U87MG cells. Decreased cell viability after MET + TMZ and MET + LPS + TMZ treatment was observed in both cell lines. U87MG cells exhibited apoptosis after MET + LPS + TMZ treatment, promoting increased ER stress, unfolded protein response, and BLC2 downregulation. LPS stimulation of U87MG cells led to upregulation of SOD2 and genes related to the TLR4 signaling pathway, including IL1B and CXCL8. A172 cells attained upregulated antioxidant gene expression, particularly SOD1, TXN and PRDX1-5, while MET treatment led to cell-cycle arrest. In silico analysis of the TCGA-GBM-RNASeq dataset indicated that the glycolytic plurimetabolic (GPM)-GBM subtype had a transcriptomic profile which overlapped with U87MG cells, suggesting GBM cases exhibiting this metabolic background with an activated inflammatory TLR4 pathway may respond to MET treatment. For cases with upregulated CXCL8, coding for IL8 (a pro-angiogenic factor), combination treatment with an IL8 inhibitor may improve tumor growth control. The A172 cell line corresponded to the mitochondrial (MTC)-GBM subtype, where MET plus an antioxidant inhibitor, such as anti-SOD1, may be indicated as a combinatory therapy.

Impact of a cell cycle and an extracellular matrix remodeling transcriptional signature on tumor progression and correlation with EZH2 expression in meningioma.

OBJECTIVE: The authors searched for genetic and transcriptional signatures associated with tumor progression and recurrence in their cohort of patients with meningiomas, combining the analysis of targeted exome, NF2-LOH, transcriptome, and protein expressions. METHODS: The authors included 91 patients who underwent resection of intracranial meningioma at their institution between June 2000 and November 2007. The search of somatic mutations was performed by Next Generation Sequencing through a customized panel and multiplex ligation-dependent probe amplification for NF2 loss of heterozygosity. The transcriptomic profile was analyzed by QuantSeq 3' mRNA-Seq. The differentially expressed genes of interest were validated at the protein level analysis by immunohistochemistry. RESULTS: The transcriptomic analysis identified an upregulated set of genes related to metabolism and cell cycle and downregulated genes related to immune response and extracellular matrix remodeling in grade 2 (atypical) meningiomas, with a significant difference in recurrent compared with nonrecurrent cases. EZH2 nuclear positivity associated with grade 2, particularly with recurrent tumors and EZH2 gene expression level, correlated positively with the expression of genes related to cell cycle and negatively to genes related to immune response and regulation of cell motility. CONCLUSIONS: The authors identified modules of dysregulated genes in grade 2 meningiomas related to the activation of oxidative metabolism, cell division, cell motility due to extracellular remodeling, and immune evasion that were predictive of survival and exhibited significant correlations with EZH2 expression.

Activation of Hedgehog signaling by the oncogenic RELA fusion reveals a primary cilia-dependent vulnerability in supratentorial ependymoma.

BACKGROUND: Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. METHODS: Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. RESULTS: Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. CONCLUSION: Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

Aquaporin-4 Expression in Meningioma Malignancy Progression / Expressão da aquaporina-4 na progressão da malignidade dos meningiomas

Objectives The aim of the present study is to analyze if aquaporin-4 (AQP4) may also be a tumor progression marker for meningiomas. Methods This is an immunohistochemistry study realized at the Universidade de São Paulo, São Paulo, state of São Paulo, Brazil: frozen meningioma samples from 81 patients (57 females and 24 males, age range from 22 to 81 years old, average 56.5 14.1 years old), including 57 meningiomas World Health Organization (WHO) grade I (GI); 19 grade II (GII), and 5 grade III (GIII) were analyzed. The relative expression level of AQP4 was analyzed by quantitative polymerase chain reaction (qPCR), using the SYBR Green approach and for staining detection. Tissue sections were routinely processed and subjected to antigen retrieval. Results The expression of AQP4 in meningioma samples ranged from 0 to 10.26, with a median of 0.001 in GI cases, of 0.008 in GII cases, and of 0.006 in GIII cases. Although not statistically significant (p » 0.942), GI meningiomas have a lower median AQP4 expression level than higher malignant grade cases. Conclusion The AQP4 gene and protein expressions presented no association with meningioma malignant progression.

Objetivo O objetivo do presente estudo é analisar se a aquaporina-4 (AQP4) também pode ser um marcador de progressão tumoral para meningiomas. Métodos Trata-se de um estudo imunohistoquímico realizado na Universidade de São Paulo, SP, Brasil. Amostras congeladas de meningioma de 81 pacientes (57 mulheres e 24 homens, faixa etária de 22 a 81 anos, média de 56,5 14,1 anos), incluindo 57 meningiomas grau I (GI) da Organização Mundial da Saúde (OMS); 19 grau II (GII) e 5 grau III (GIII) foram analisados. O nível de expressão relativa de AQP4 foi analisado por reação em cadeia de polimerase quantitativa (qPCR, sigla em inglês), usando a abordagem SYBR Green e para detecção de manchas. As seções de tecido foram rotineiramente processadas e sujeitas a recuperação de antígeno. Resultados A expressão de AQP4 em amostras de meningioma variou de 0 a 10,26, com mediana de 0,001 nos casos GI; 0,008 nos casos GII; e 0,006 nos casos GIII. Embora não sejam estatisticamente significantes (p » 0,942), os meningiomas GI apresentam mediana mais baixa do nível de expressão de AQP4 do que os casos de grau maligno mais alto. Conclusão Expressões de genes e proteínas AQP4 apresentadas na associação com progressão maligna do meningioma.

Correlation of Matrisome-Associatted Gene Expressions with LOX Family Members in Astrocytomas Stratified by IDH Mutation Status.

Tumor cell infiltrative ability into surrounding brain tissue is a characteristic of diffusely infiltrative astrocytoma and is strongly associated with extracellular matrix (ECM) stiffness. Collagens are the most abundant ECM scaffolding proteins and contribute to matrix organization and stiffness. LOX family members, copper-dependent amine oxidases, participate in the collagen and elastin crosslinking that determine ECM tensile strength. Common IDH mutations in lower-grade gliomas (LGG) impact prognosis and have been associated with ECM stiffness. We analyzed the expression levels of LOX family members and matrisome-associated genes in astrocytoma stratified by malignancy grade and IDH mutation status. A progressive increase in expression of all five LOX family members according to malignancy grade was found. LOX, LOXL1, and LOXL3 expression correlated with matrisome gene expressions. LOXL1 correlations were detected in LGG with IDH mutation (IDHmut), LOXL3 correlations in LGG with IDH wild type (IDHwt) and strong LOX correlations in glioblastoma (GBM) were found. These increasing correlations may explain the increment of ECM stiffness and tumor aggressiveness from LGG-IDHmut and LGG-IDHwt through to GBM. The expression of the mechanosensitive transcription factor, ß-catenin, also increased with malignancy grade and was correlated with LOXL1 and LOXL3 expression, suggesting involvement of this factor in the outside-in signaling pathway.

Alpha2beta1 Integrin Polymorphism in Diffuse Astrocytoma Patients.

Integrins are heterodimeric transmembrane glycoproteins resulting from the non-covalent association of an α and ß chain. The major integrin receptor for collagen/laminin, α2ß1 is expressed on a wide variety of cell types and plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Integrin-triggered signaling pathways promote the invasion and survival of glioma cells by modifying the brain microenvironment. In this study, we investigated the association of a specific genetic polymorphism of integrin α2ß1 with the incidence of diffusely infiltrating astrocytoma and the progression of these tumors. Single-nucleotide polymorphism in intron 7 of the integrin ITGA2 gene was examined in 158 patients and 162 controls using polymerase chain reaction and restriction enzyme analysis. The ITGA2 genotype +/+ (with a BglII restriction site in both alleles) exhibited higher frequency in grade II astrocytoma compared to control (P = 0.02) whereas the genotype -/- (lacking the BglII site) correlated with the poorest survival rate (P = 0.04). In addition, in silico analyses of ITGA2 expression from low-grade gliomas (LGG, n = 515) and glioblastomas (GBM, n = 159) indicated that the higher expression of ITGA2 in LGG was associated with poor overall survival (P < 0.0001). However, the distribution of integrin ITGA2 BglII genotypes (+/+, +/-, -/-) was not significantly different between astrocytoma subgroups III and IV (P = 0.65, 0.24 and 0.33; 0.29, 0.48, 0.25, respectively) compared to control. These results suggest a narrow association between the presence of this SNP and indicate that further studies with larger samples are warranted to analyze the relation between tumor grade and overall survival, highlighting the importance of determining these polymorphisms for prognosis of astrocytomas.

Aberrant Protein Glycosylation in Brain Cancers, with Emphasis on Glioblastoma.

Aberrant glycosylation has been associated with several processes of tumorigenesis from cell signaling, migration and invasion, to immune regulation and metastasis formation. The biosynthesis of glycoconjugates is regulated through concerted and finely tuned enzymatic reactions. This includes the levels and activity of glycosyltransferases and glycosidases, nucleotide sugar metabolism, substrate availability, epigenetic condition, and cellular functional state. Glioblastoma (GBM) is the most aggressive brain tumor, frequently occurring in adults with overall survival not surpassing 17 months after diagnosis. GBM has been classified by the World Health Organization (WHO) as a grade 4 astrocytoma and stratified into G-CIMP, proneural, classical, and mesenchymal subtypes. Several biomolecular features associated with GBM aggressiveness have been elucidated; however, more studies are needed to elucidate the role of glycosylation in GBM pathology, looking at their potential as cancer targets. Here, we focus on the alteration of genes involved in protein N- and O-linked glycosylation in GBM. Specifically, the mRNA levels of glycogenes were analyzed using astrocytoma-TCGA-RNAseq datasets from public repositories. A total of 68 genes were differentially regulated in the most aggressive, mesenchymal subtype of GBM compared to the proneural and classical subtypes, and the expression of these genes was compared to normal brain tissues. Among them, we focused on 38 genes coding for proteins that belong to: 1) asparagine glycosylation (ALG); 2) glycosyltransferases (B3T, B4T); 3) fucosyltransferase (FUT); 4) acetylgalactosaminyltransferases (GALNT); 5) hexosaminidase (HEX); 6) mannosidase (MAN); 7) acetylglucosaminyltransferase (MGAT); 8) sialidase or neuraminidase (NEU); 9) solute carrier 35 family (SLC); and 10) sialyltransferase (ST). The differential expression of some genes was already reported in several solid tumors; however, several of them were found to be dysregulated in GBM for the first time. These data represent an important starting point to perform further orthogonal and functional validations to pinpoint the role of these glycogenes in GBM as diagnostic and therapeutic targets.

Cellular Model of Malignant Transformation of Primary Human Astrocytes Induced by Deadhesion/Readhesion Cycles.

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG-a human glioblastoma-derived cell lineage-reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/ß-catenin signaling pathways, in addition to upregulation of genes related to epithelial-mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

The chromatin remodeler complex ATRX-DAXX-H3.3 and telomere length in meningiomas.

ATRX-DAXX-H3.3 chromatin remodeler complex is a well known epigenetic factor responsible for the heterochromatin maintenance and control. ATRX is an important nucleosome controller, especially in tandem repeat regions, and DAXX is a multi-function protein with particular role in histone H3.3 deposition due to its chaperone characteristic. Abnormalities in this complex have been associated with telomere dysfunction and consequently with activation of alternative lengthening of telomeres mechanism, genomic instability, and tumor progression in different types of cancer. However, the characterization of this complex is still incomplete in meningioma. We analyzed ATRX, DAXX and H3.3 expressions and the telomere length in a cohort of meningioma of different malignant grades. We observed ATRX upregulation at gene and protein levels in grade II/III meningiomas. A low variability of telomere length was observed in meningiomas across different ages and malignant grades, in contrast to the shortening of telomere length with aging in normal controls.

Extracellular Matrix Proteome Remodeling in Human Glioblastoma and Medulloblastoma.

Medulloblastomas (MBs) and glioblastomas (GBMs) are high-incidence central nervous system tumors. Different origin sites and changes in the tissue microenvironment have been associated with the onset and progression. Here, we describe differences between the extracellular matrix (ECM) signatures of these tumors. We compared the proteomic profiles of MB and GBM decellularized tumor samples between each other and their normal decellularized brain site counterparts. Our analysis revealed that 19, 28, and 11 ECM proteins were differentially expressed in MBs, GBMs, and in both MBs and GBMs, respectively. Next, we validated key findings by using a protein tissue array with 53 MB and 55 GBM cases and evaluated the clinical relevance of the identified differentially expressed proteins through their analysis on publicly available datasets, 763 MB samples from the GSE50161 and GSE85217 studies, and 115 GBM samples from RNAseq-TCGA. We report a shift toward a denser fibrillary ECM as well as a clear alteration in the glycoprotein signature, which influences the tumor pathophysiology. MS data have been submitted to the PRIDE repository, project accession: PXD023350.

LOXL3 Silencing Affected Cell Adhesion and Invasion in U87MG Glioma Cells.

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family, is responsible for the crosslinking in collagen or elastin. The cellular localization of LOXL3 is in the extracellular space by reason of its canonical function. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among tumors with the highest LOXL3 expression levels. LOXL3 silencing of U87MG cells by siRNA led to the spreading of the tumor cell surface, and the transcriptome analysis of these cells revealed an upregulation of genes coding for extracellular matrix, cell adhesion, and cytoskeleton components, convergent to an increase in cell adhesion and a decrease in cell invasion observed in functional assays. Significant correlations of LOXL3 expression with genes coding for tubulins were observed in the mesenchymal subtype in the TCGA RNA-seq dataset of glioblastoma (GBM). Conversely, genes involved in endocytosis and lysosome formation, along with MAPK-binding proteins related to focal adhesion turnover, were downregulated, which may corroborate the observed decrease in cell viability and increase in the rate of cell death. Invasiveness is a major determinant of the recurrence and poor outcome of GBM patients, and downregulation of LOXL3 may contribute to halting the tumor cell invasion.

Glutaminolysis dynamics during astrocytoma progression correlates with tumor aggressiveness.

BACKGROUND: Glioblastoma is the most frequent and high-grade adult malignant central nervous system tumor. The prognosis is still poor despite the use of combined therapy involving maximal surgical resection, radiotherapy, and chemotherapy. Metabolic reprogramming currently is recognized as one of the hallmarks of cancer. Glutamine metabolism through glutaminolysis has been associated with tumor cell maintenance and survival, and with antioxidative stress through glutathione (GSH) synthesis. METHODS: In the present study, we analyzed the glutaminolysis-related gene expression levels in our cohort of 153 astrocytomas of different malignant grades and 22 non-neoplastic brain samples through qRT-PCR. Additionally, we investigated the protein expression profile of the key regulator of glutaminolysis (GLS), glutamate dehydrogenase (GLUD1), and glutamate pyruvate transaminase (GPT2) in these samples. We also investigated the glutathione synthase (GS) protein profile and the GSH levels in different grades of astrocytomas. The differential gene expressions were validated in silico on the TCGA database. RESULTS: We found an increase of glutaminase isoform 2 gene (GLSiso2) expression in all grades of astrocytoma compared to non-neoplastic brain tissue, with a gradual expression increment in parallel to malignancy. Genes coding for GLUD1 and GPT2 expression levels varied according to the grade of malignancy, being downregulated in glioblastoma, and upregulated in lower grades of astrocytoma (AGII-AGIII). Significant low GLUD1 and GPT2 protein levels were observed in the mesenchymal subtype of GBM. CONCLUSIONS: In glioblastoma, particularly in the mesenchymal subtype, the downregulation of both genes and proteins (GLUD1 and GPT2) increases the source of glutamate for GSH synthesis and enhances tumor cell fitness due to increased antioxidative capacity. In contrast, in lower-grade astrocytoma, mainly in those harboring the IDH1 mutation, the gene expression profile indicates that tumor cells might be sensitized to oxidative stress due to reduced GSH synthesis. The measurement of GLUD1 and GPT2 metabolic substrates, ammonia, and alanine, by noninvasive MR spectroscopy, may potentially allow the identification of IDH1mut AGII and AGIII progression towards secondary GBM.

A coordinated approach for the assessment of molecular subgroups in pediatric ependymomas using low-cost methods.

Although ependymoma (EPN) molecular subgroups have been well established by integrated high-throughput platforms, low- and middle-income countries still need low-cost techniques to promptly classify these molecular subtypes. Here, we applied low-cost methods to classify EPNs from a Brazilian cohort with 60 pediatric EPN patients. Fusion transcripts (C11orf95-RELA, YAP1-MAMLD1, and YAP1-FAM118B) were investigated in supratentorial EPN (ST-EPNs) samples through RT-PCR/Sanger sequencing and immunohistochemistry (IHC) for p65/L1CAM. qRT-PCR and IHC were used to evaluate expression profiling of CXorf67, LAMA2, NELL2, and H3K27me3 in posterior fossa EPN (PF-EPNs) samples. In silico analysis was performed using public microarray data to validate the molecular assignment PF-EPNs with LAMA2/NELL2 markers. RELA cases and YAP1-MAMLD1 fusions were identified in nine and four ST-EPNs, respectively. An additional RELA case was identified by IHC. Of note, LAMA2 and NELL2 gene expression and immunoprofiling were less accurate for classifying PF-EPNs, which were confirmed by in silico analysis. Yet, H3K27me3 staining was sufficient to classify PF-EPN subgroups. Our results emphasize the feasibility of a simplified strategy to molecularly classify EPNs in the vast majority of cases (49/60; 81.7%). A coordinated combination of simple methods can be effective to screen pediatric EPN with the available laboratory resources at most low-/mid-income countries, giving support for clinical practice in pediatric EPN. KEY MESSAGES: Low- and middle-income countries need effective low-cost approaches to promptly distinguish between EPN molecular subgroups. RT-PCR plus Sanger sequencing is able to recognize the most common types of RELA and YAP1 fusion transcripts in ST-EPNs. Genetic and protein expressions of LAMA2 and NELL2 are of limited value to accurately stratify PF-EPNs. Immunohistochemical staining for H3K27me3 may be used as a robust method to accurately diagnose PF-EPNs subgroups. A coordinated flow diagram based on these validated low-cost methods is proposed to help clinical-decision making and to reduce costs with NGS assessment outside research protocols.

Late p65 nuclear translocation in glioblastoma cells indicates non-canonical TLR4 signaling and activation of DNA repair genes.

Glioblastoma (GBM) is the most aggressive brain primary malignancy. Toll-like receptor 4 (TLR4) has a dual role in cell fate, promoting cell survival or death depending on the context. Here, we analyzed TLR4 expression in different grades of astrocytoma, and observed increased expression in tumors, mainly in GBM, compared to non-neoplastic brain tissue. TLR4 role was investigated in U87MG, a GBM mesenchymal subtype cell line, upon LPS stimulation. p65 nuclear translocation was observed in late phase, suggesting TLR4-non-canonical pathway activation. In fact, components of ripoptosome and inflammasome cascades were upregulated and they were significantly correlated in GBMs of the TCGA-RNASeq dataset. Moreover, an increased apoptotic rate was observed when the GBM-derived U87MG cells were co-treated with LPS and Temozolomide (TMZ) in comparison to TMZ alone. Increased TLR4 immunostaining was detected in nuclei of U87MG cells 12 h after LPS treatment, concomitant to activation of DNA repair genes. Time-dependent increased RAD51, FEN1 and UNG expression levels were confirmed after LPS stimulation, which may contribute to tumor cell fitness. Moreover, the combined treatment with the RAD51 inhibitor, Amuvatinib in combination with, TMZ after LPS stimulation reduced tumor cell viability more than with each treatment alone. In conclusion, our results suggest that stimulation of TLR4 combined with pharmacological inhibition of the DNA repair pathway may be an alternative treatment for GBM patients.

Cyclin E1 expression and malignancy in meningiomas.

OBJECTIVE: The aim of the present study was to analyze if the pathway Skp2-p27-cyclin E1 could also be a tumor progression marker for meningiomas. PATIENTS AND METHODS: We used quantitative real-time PCR to assess the relative expression levels of the genes coding for cyclin E1 (CCNE1), Skp2 (SKP2), and p27 (P27). The expression levels were compared in grades I to III meningiomas and among different histological subtypes of grade I meningiomas. RESULTS: Anaplastic meningiomas accounted for 4.9%, atypical meningiomas for 23.5% and grade I meningiomas for 71.6%.CCNE1 expression level was significantly higher in grade II compared to grade I meningiomas (p = 0.0027), and its expression level reliably predicts grade II meningiomas (ROC AUC = 0.731, p = 0.003). CCNE1 expression also correlated with SKP2 and P27 expression levels in grade I meningiomas (r = 0.539, p < 0.0001 and r = 0.687, p = <0.0001, respectively for CCNE1/SKP2 and CCNE1/P27, Spearman's test). Fibrous subtype among grade I meningiomas presented the highest expression levels of CCNE1, SKP2 and P27. Higher expression of cyclin E1 protein was detected in the nuclei of atypical meningiomas compared to grade I meningiomas. CONCLUSIONS: CCNE1 expression level predicts meningioma malignancy, and the fibrous subtype presents the highest gene expression levels among grade I meningiomas.

The TP53 p.R337H mutation is uncommon in a Brazilian cohort of pediatric patients diagnosed with ependymoma.

BACKGROUND: Ependymoma (EPN) is the third most common childhood cancer of the central nervous system. RELA fusion-positive EPN accounts for approximately 70% of all childhood supratentorial tumors and shows the worst prognosis among the supratentorial EPNs. TP53 mutation is infrequent in RELA fusions EPNs. In the population from the Southern region of Brazil, there is a high incidence of the germline TP53 p.R337H mutation that predisposes carriers to develop early-onset tumors. However, despite this high incidence, the frequency of this mutation among EPN patients remains to be determined. Here, we investigated the presence of the TP53 p.R337H mutation in a larger cohort of pediatric EPNs of three institutions located in the state of São Paulo, Brazil. METHODS: The TP53 p.R337H mutation was screened by conventional RT-PCR and Sanger sequencing in 49 pediatric EPNs diagnosed during the period from 1995 to 2016. RESULTS: We described for the first time a case of a 5-year-old girl with RELA fusion EPN with a heterozygous TP53 p.R337H mutation. CONCLUSIONS: The present finding indicates that the TP53 p.R337H germline mutation is uncommon in patients with EPN in Brazil and screening of pediatric patients RELA fusion EPN may be informative to better understand the role of TP53 germline mutations in the development and prognosis of these tumors.

SELAdb: A database of exonic variants in a Brazilian population referred to a quaternary medical center in São Paulo

OBJECTIVES: High-throughput sequencing of genomes, exomes, and disease-focused gene panels is becoming increasingly common for molecular diagnostics. However, identifying a single clinically relevant pathogenic variant among thousands of genetic polymorphisms is a challenging task. Publicly available genomic databases are useful resources to filter out common genetic variants present in the population and enable the identification of each disease-causing variant. Based on our experience applying these technologies at Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), São Paulo, Brazil, we recognized that the Brazilian population is not adequately represented in widely available genomic databases. METHODS: Here, we took advantage of our 5-year experience as a high-throughput sequencing core facility focused on individuals with putative genetic disorders to build a genomic database that may serve as a more accurate reference for our patient population: SELAdb. RESULTS/CONCLUSIONS: Currently, our database comprises a final cohort of 523 unrelated individuals, including patients or family members managed by different clinics of HCFMUSP. We compared SELAdb with other publicly available genomic databases and demonstrated that this population is very heterogeneous, largely resembling Latin American individuals of mixed origin, rather than individuals of pure European ancestry. Interestingly, exclusively through SELAdb, we identified a spectrum of known and potentially novel pathogenic variants in genes associated with highly penetrant Mendelian disorders, illustrating that pathogenic variants circulating in the Brazilian population that is treated in our clinics are underrepresented in other population databases. SELAdb is freely available for public consultation at: http://intranet.fm.usp.br/sela