Inhibition of BMP signaling pathway induced senescence and calcification in anaplastic meningioma.

PURPOSE: Meningiomas are the most common type of brain tumors and are generally benign, but malignant atypical meningiomas and anaplastic meningiomas frequently recur with poor prognosis. The metabolism of meningiomas is little known, so few effective treatment options other than surgery and radiation are available, and the targets for treatment of recurrence are not well defined. The Aim of this paper is to find the therapeutic target. METHODS: The effects of bone morphogenetic protein (BMP) signal inhibitor (K02288) and upstream regulator Gremlin2 (GREM2) on meningioma's growth and senescence were examined. In brief, we examined as follows: 1) Proliferation assay by inhibiting BMP signaling. 2) Comprehensive analysis of forced expression GREM2.3) Correlation between GREM2 mRNA expression and proliferation marker in 87 of our clinical samples. 4) Enrichment analysis between GREM2 high/low expressed groups using RNA-seq data (42 cases) from the public database GREIN. 5) Changes in metabolites and senescence markers associated with BMP signal suppression. RESULTS: Inhibitors of BMP receptor (BMPR1A) and forced expression of GREM2 shifted tryptophan metabolism from kynurenine/quinolinic acid production to serotonin production in malignant meningiomas, reduced NAD + /NADH production, decreased gene cluster expression involved in oxidative phosphorylation, and caused decrease in ATP. Finally, malignant meningiomas underwent cellular senescence, decreased proliferation, and eventually formed psammoma bodies. Reanalyzed RNA-seq data of clinical samples obtained from GREIN showed that increased expression of GREM2 decreased the expression of genes involved in oxidative phosphorylation, similar to our experimental results. CONCLUSIONS: The GREM2-BMPR1A-tryptophan metabolic pathway in meningiomas is a potential new therapeutic target.

Comprehensive genetic screening for vascular Ehlers-Danlos syndrome through an amplification-based next-generation sequencing system.

Vascular Ehlers-Danlos syndrome (vEDS) is a hereditary connective tissue disorder (HCTD) characterized by arterial dissection/aneurysm/rupture, sigmoid colon rupture, or uterine rupture. Diagnosis is confirmed by detecting heterozygous variants in COL3A1. This is the largest Asian case series and the first to apply an amplification-based next-generation sequencing through custom panels of causative genes for HCTDs, including a specific method of evaluating copy number variations. Among 429 patients with suspected HCTDs analyzed, 101 were suspected to have vEDS, and 33 of them (32.4%) were found to have COL3A1 variants. Two patients with a clinical diagnosis of Loeys-Dietz syndrome and/or familial thoracic aortic aneurysm and dissection were also found to have COL3A1 variants. Twenty cases (57.1%) had missense variants leading to glycine (Gly) substitutions in the triple helical domain, one (2.9%) had a missense variant leading to non-Gly substitution in this domain, eight (22.9%) had splice site alterations, three (8.6%) had nonsense variants, two (5.7%) had in-frame deletions, and one (2.9%) had a multi-exon deletion, including two deceased patients analyzed with formalin-fixed and paraffin-embedded samples. This is a clinically useful system to detect a wide spectrum of variants from various types of samples.

Methionine regulates self-renewal, pluripotency, and cell death of GIC through cholesterol-rRNA axis.

BACKGROUND: Glioma-initiating cells (GICs) are the source of glioma cells that can self-renew, have pluripotency, and are treatment-resistant, so are the starting point for relapse and eventual death despite multimodality therapy. L-[methyl-11C] methionine PET has observed high accumulation at the time of recurrence, it is important to understand the mechanism of tumor cell activation caused by the reorganization of methionine metabolism.  METHODS: We cultured cells in methionine-deprived culture medium for comprehensive analysis. Based on the obtained results, the possible target molecules were chemically inhibited and the respective markers were analyzed. RESULTS: Methionine depletion markedly decreased proliferation and increased cell death of GICs. Decreased S-adenosyl-methionine, which is synthesized intracellularly by catalyzed by methionine adenosyltransferase using methionine, triggered the following: (i) global DNA demethylation, (ii) hyper-methylation of signaling pathways regulating pluripotency of stem cells, (iii) decreased expression of the core-genes and pluripotent markers of stem cells including FOXM1, SOX2, SOX4, PROM1, and OLIG2, (iv) decreased cholesterol synthesis and increased excretion mainly through decreased SREBF2, and (v) down-regulation of the large subunit of ribosomal protein configured 28S and ACA43, small nucleolar RNA guiding the pseudouridylation of 28S rRNA, which is essential for translation. In addition, inhibition of cholesterol synthesis with statin resulted in a phenotype similar to that of methionine depletion and decreases in stem cell markers and small nucleolar RNA ACA43. Moreover, suppression of FOXM1 decreased stem cell markers such as SOX4 and PROM1. The gene expression profile for cholesterol production was obtained from the Ivy Glioblastoma Atlas Project database and compared between tumor cells with relatively low methionine levels in areas of pseudopalisading arrangement around necrosis and tumor cells in the infiltrating region, showing that cells in the infiltrating region have higher capacity to produce cholesterol. CONCLUSIONS: Methionine metabolism is closely related with self-renewal, pluripotency, and cell death in GICs through modification of cholesterol biosynthesis, especially in the SREBF2-FOXM1 and ACA43 axis with modification of rRNA.

T2-fluid-attenuated inversion recovery mismatch sign in lower grade gliomas: correlation with pathological and molecular findings.

After the new molecular-based classification was reported to be useful for predicting prognosis, the T2-fluid-attenuated inversion recovery (FLAIR) mismatch sign has gained interest as one of the promising methods for detecting lower grade gliomas (LGGs) with isocitrate dehydrogenase (IDH) mutations and chromosome 1p/19q non-codeletion (IDH mut-Noncodel) with high specificity. Although all institutions could use T2-FLAIR mismatch sign without any obstacles, this sign was not completely helpful because of its low sensitivity. In this study, we attempted to uncover the mechanism of T2-FLAIR mismatch sign for clarifying the cause of this sign's low sensitivity. Among 99 patients with LGGs, 22 were T2-FLAIR mismatch sign-positive (22%), and this sign as a marker of IDH mut-Noncodel showed a sensitivity of 55.6% and specificity of 96.8%. Via pathological analyses, we could provide evidence that not only microcystic changes but the enlarged intercellular space was associated with T2-FLAIR mismatch sign (p = 0.017). As per the molecular analyses, overexpression of mTOR-related genes (m-TOR, RICTOR) were detected as the molecular events correlated with T2-FLAIR mismatch sign (p = 0.020, 0.030. respectively). Taken together, we suggested that T2-FLAIR mismatch sign could pick up the IDH mut-Noncodel LGGs with enlarged intercellular space or that with overexpression of mTOR-related genes.

High-resolution melting effectively pre-screens for TP53 mutations before direct sequencing in patients with diffuse glioma.

TP53 mutations are important molecular markers in diffuse astrocytic tumors and medulloblastomas. We examined the efficacy of a pre-screening method for high-resolution melting (HRM) analysis of TP53 mutation before direct sequencing using samples from patients with diffuse glioma. Surgical samples from 64 diffuse gliomas were classified based on the 2016 World Health Organization (WHO) histopathological grading system and the cIMPACT-NOW (consortium to inform molecular and practical approaches to CNS tumor taxonomy-not official WHO) update. TP53 mutations from exon 5 to exon 8 were assessed by direct sequencing. The results of HRM and p53 immunohistochemistry (IHC) analysis were compared by recording the sensitivity, specificity, and false negative and false positive rates. Direct sequencing detected TP53 mutations in 18 of 64 samples (28.1%): diffuse astrocytoma, IDH-mutant (n = 3); diffuse astrocytoma, IDH-wild type (n = 1); anaplastic astrocytoma, IDH-mutant (n = 3); anaplastic astrocytoma, IDH-wild type (n = 4); and glioblastoma, IDH-wild type (n = 7). A total of 22 mutations was detected in the 18 samples; 4 samples exhibited duplicate missense mutations. Sensitivity and specificity were 0.96 and 0.96, respectively, for HRM analysis; they were 0.89 and 0.52, respectively, for p53 IHC. Overall accuracy was 0.98 for HRM and 0.63 for IHC. HRM analysis is a good pre-screening method for the detection of TP53 mutation before direct sequencing.

Ecotropic viral integration site 1 regulates EGFR transcription in glioblastoma cells.

PURPOSE: Ecotropic viral integration site-1 (EVI1) is a transcription factor that contributes to the unfavorable prognosis of leukemia, some epithelial cancers, and glial tumors. However, the biological function of EVI1 in glioblastoma multiforme (GBM) remains unclear. Based on microarray experiments, EVI1 has been reported to regulate epidermal growth factor receptor (EGFR) transcription. Signal transduction via EGFR plays an essential role in glioblastoma. Therefore, we performed this study to clarify the importance of EVI1 in GBM by focusing on the regulatory mechanism between EVI1 and EGFR transcription. METHODS: We performed immunohistochemical staining and analyzed the EVI1-expression in glioma tissue. To determine the relationship between EVI1 and EGFR, we induced siRNA-mediated knockdown of EVI1 in GBM cell lines. To investigate the region that was essential for the EVI1 regulation of EGFR expression, we conducted promoter reporter assays. We performed WST-8 assay to investigate whether EVI1 affected on the proliferation of GBM cells or not. RESULTS: It was observed that 22% of GBM tissues had over 33% of tumor cells expressing EVI1, whereas no lower-grade glioma tissue had over 33% by immunohistochemistry. In A172 and YKG1 cells, the expression levels of EGFR and EVI1 correlated. Analysis of the EGFR promoter region revealed that the EGFR promoter (from - 377 to - 266 bp) was essential for the EVI regulation of EGFR expression. We showed that EVI1 influenced the proliferation of A172 and YKG1 cells. CONCLUSION: This is the first study reporting the regulation of EGFR transcription by EVI1 in GBM cells.

Detection of the KIAA1549-BRAF fusion gene in cells forming microvascular proliferations in pilocytic astrocytoma.

Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVPs in PA contained tumor-derived cells and/or phenotypically distinct tumor cells expressing vascular markers. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was identified by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP contained tumor derived cell and/or phenotypically distinct tumor cells expressing vascular markers.

Impact of PCR-based molecular analysis in daily diagnosis for the patient with gliomas.

The WHO2016 CNS update requires a combined histological and molecular assessment. To assess the major aberrations such as co-deletion of complete chromosome arms 1p and 19q (Co-del), isocitrate dehydrogenase and histone H3 mutations, direct sequencing, multiplex ligation-dependent probe amplification and/or FISH are methods considered to be "golden standard" in the community. However, these methods are expensive and complicated. The aim of this study is verification of the sensitivity of the simple PCR-based techniques for assessment of molecular information in daily diagnosis. We analyzed a total number of 80 patients with gliomas. FISH and PCR-based microsatellite analysis were compared for Co-del assessment. Direct sequencing and qPCR using hig-resolution melting (HRM) were compared for IDH and histone H3 mutations. The sensitivity and specificity of FISH were 0.71 and 0.79, respectively. FISH using a commercially available Vysis probe had a risk of high false-positive rate (0.25). For assessment of IDH1 mutations, the sensitivity and specificity of HRM were 1.0 and 0.96, respectively. For assessment of IDH2 and H3 mutations by HRM, both sensitivity and specificity were 1.0. We consider PCR-based molecular analysis to be a simple and accurate technique in daily diagnosis that is readily available for a small scientific facility.

MGMT promoter methylation in patients with glioblastoma: is methylation-sensitive high-resolution melting superior to methylation-sensitive polymerase chain reaction assay?

OBJECTIVE: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a prognostic factor in adults with glioblastoma (GBM); it also yields information that is useful for clinical decision-making in elderly GBM patients. While pyrosequencing is the gold standard for the evaluation of the methylation status of MGMT, methylation-sensitive polymerase chain reaction (MS-PCR) assay continues to be used widely. Although MS-PCR results exhibited a good correlation with the prognosis of patients with GBM treated under the Stupp protocol, interpretation of the bands is based on subjective judgment, and the assay cannot be used to analyze heterogeneously methylated samples. We assessed whether methylation-sensitive high-resolution melting (MS-HRM) is an alternative to MS-PCR. METHODS: The authors prepared 3 primer sets that covered CpG 72­89 for MS-HRM analysis to determine the methylation levels of 6 human glioma cell lines. The results were validated by bisulfite sequencing of cloned alleles. The authors also subjected surgical samples from 75 GBM patients treated with temozolomide (TMZ) to MS-HRM to assess the MGMT methylation status and compared the findings with MS-PCR results using receiver operating characteristic (ROC), univariate, and multivariate analyses. RESULTS: There was a strong correlation between the methylation levels of the 6 glioma cell lines evaluated by MSHRM and by bisulfite sequencing; with primers 1 and 2, the correlation was significant (r = 0.959 and r = 0.960, respectively, p < 0.01). Based on log-rank analysis, MS-HRM was significantly better than MS-PCR for predicting progressionfree survival (PFS) and overall survival (OS) based on the methylation status of the MGMT promoter (PFS predicted by MS-HRM and MS-PCR = 0.00023 and 0.0035, respectively; OS = 0.00019 and 0.00028, respectively). ROC analysis showed that the area under the curve was larger with MS-HRM than with MS-PCR (PFS: 0.723 vs 0.635; OS: 0.716 vs 0.695). Based on multivariate Cox regression analysis, MS-HRM was significantly better than MS-PCR for predicting the treatment outcome (MS-HRM vs MS-PCR: PFS, p = 0.001 vs 0.207; OS, p = 0.013 vs 0.135). CONCLUSIONS: The authors' findings show that MS-HRM is superior to MS-PCR for the detection of MGMT promoter methylation. They suggest MS-HRM as an alternative to MS-PCR for assessing the prognosis of patients with GBM.