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WHO 2016 classified glioblastomas into IDH-mutant and IDH-wildtype with the former having a better prognosis but there was no study on IDH-mutant primary glioblastomas only, as previous series included secondary glioblastomas. We recruited a series of 67 IDH-mutant primary glioblastomas/astrocytoma IV without a prior low-grade astrocytoma and examined them using DNA-methylation profiling, targeted sequencing, RNA sequencing and TERT promoter sequencing, and correlated the molecular findings with clinical parameters. The median OS of 39.4 months of 64 cases and PFS of 25.9 months of 57 cases were better than the survival data of IDH-wildtype glioblastomas and IDH-mutant secondary glioblastomas retrieved from datasets. The molecular features often seen in glioblastomas, such as EGFR amplification, combined +7/-10, and TERT promoter mutations were only observed in 6/53 (11.3%), 4/53 (7.5%), and 2/67 (3.0%) cases, respectively, and gene fusions were found only in two cases. The main mechanism for telomere maintenance appeared to be alternative lengthening of telomeres as ATRX mutation was found in 34/53 (64.2%) cases. In t-SNE analyses of DNA-methylation profiles, with an exceptional of one case, a majority of our cases clustered to IDH-mutant high-grade astrocytoma subclass (40/53; 75.5%) and the rest to IDH-mutant astrocytoma subclass (12/53; 22.6%). The latter was also enriched with G-CIMP high cases (12/12; 100%). G-CIMP-high status and MGMT promoter methylation were independent good prognosticators for OS (p = 0.022 and p = 0.002, respectively) and TP53 mutation was an independent poor prognosticator (p = 0.013) when correlated with other clinical parameters. Homozygous deletion of CDKN2A/B was not correlated with OS (p = 0.197) and PFS (p = 0.278). PDGFRA amplification or mutation was found in 16/59 (27.1%) of cases and was correlated with G-CIMP-low status (p = 0.010). Aside from the three well-known pathways of pathogenesis in glioblastomas, chromatin modifying and mismatch repair pathways were common aberrations (88.7% and 20.8%, respectively), the former due to high frequency of ATRX involvement. We conclude that IDH-mutant primary glioblastomas have better prognosis than secondary glioblastomas and have major molecular differences from other commoner glioblastomas. G-CIMP subgroups, MGMT promoter methylation, and TP53 mutation are useful prognostic adjuncts.

Adult medulloblastomas are clinically and molecularly understudied due to their rarity. We performed molecular grouping, targeted sequencing, and TERT promoter Sanger sequencing on a cohort of 99 adult medulloblastomas. SHH made up 50% of the cohort, whereas Group 3 (13%) was present in comparable proportion to WNT (19%) and Group 4 (18%). In contrast to paediatric medulloblastomas, molecular groups had no prognostic impact in our adult cohort (p = 0.877). Most frequently mutated genes were TERT (including promoter mutations, mutated in 36% cases), chromatin modifiers KMT2D (31%) and KMT2C (30%), TCF4 (31%), PTCH1 (27%) and DDX3X (24%). Adult WNT patients showed enrichment of TP53 mutations (6/15 WNT cases), and 3/6 TP53-mutant WNT tumours were of large cell/anaplastic histology. Adult SHH medulloblastomas had frequent upstream pathway alterations (PTCH1 and SMO mutations) and few downstream alterations (SUFU mutations, MYCN amplifications). TERT promoter mutations were found in 72% of adult SHH patients, and were restricted to this group. Adult Group 3 tumours lacked hallmark MYC amplifications, but had recurrent mutations in KBTBD4 and NOTCH1. Adult Group 4 tumours harboured recurrent mutations in TCF4 and chromatin modifier genes. Overall, amplifications of MYC and MYCN were rare (3%). Since molecular groups were not prognostic, alternative prognostic markers are needed for adult medulloblastoma. KMT2C mutations were frequently found across molecular groups and were associated with poor survival (p = 0.002). Multivariate analysis identified histological type (p = 0.026), metastasis (p = 0.031) and KMT2C mutational status (p = 0.046) as independent prognosticators in our cohort. In summary, we identified distinct clinical and mutational characteristics of adult medulloblastomas that will inform their risk stratification and treatment.

Tumors can use metabolic reprogramming to survive nutrient stress. Epigenetic regulators play a critical role in metabolic adaptation. Here we screened a sgRNA library to identify epigenetic regulators responsible for the vulnerability of colorectal cancer (CRC) cells to glucose deprivation and found that more EZH2-knockout cells survived glucose deprivation. Then, we showed that EZH2 expression was significantly downregulated in response to glucose deprivation in a glucose-sensitive CRC cell line, and EZH2-knockdown cells were more resistant to glucose deprivation. Mechanistically, EZH2 deficiency upregulated the expression of glutaminase (GLS) and promoted the production of glutamate, which in turn led to increased synthesis of intracellular glutathione (GSH) and eventually attenuated the reactive oxygen species (ROS)-mediated cell death induced by glucose deprivation. Although EZH2 functioned as an oncogene in cancer progression and EZH2 knockout abolished colorectal cancer development in a mouse model, here we revealed a mechanistic link between EZH2 and metabolic reprogramming via the direct regulation of GLS expression and observed a negative correlation between EZH2 and GLS expression in colorectal cancer tissues. These findings further confirmed the importance of heterogeneity, provided an explanation for the clinical tolerance of cancer cells to EZH2 inhibitors from the perspective of metabolism, and proposed the possibility of combining EZH2 inhibitors and glutamine metabolism inhibitors for the treatment of cancer.

Background: To investigate: (1) what are the prognosis in patients treated with this chemotherapy scheme and limb salvage; (2) what are the clinical outcomes of expandable endoprosthesis reconstruction in child?Methods: From January 2009 to December 2014, we retropected 29 skeletally immature kids ( mean age, 10.5 years; range, 6-15 years) with osteosarcoma around knee. Of the 29 patients who underwent neoadjuvant chemotherapy and limb salvage surgery, this expandable prosthesis was implanted to reconstruct. These 29 patients were included in our retrospection. No patients were missed during follow-up. The evaluation index involved follow-up time, complication, functional results, lengthening procedures. The survivorship and recurrence were assessed by SPSS software. The function was assessed by Musculoskeletal Tumor Society (MSTS) scoring system.Results: A mean of follow-up time was 8.9 years (range, 6-12years). The overall 5-year survival was 89.1% based on Kaplan-Meier. Relapse suffered in three patients and one underwent amputation. Lung metastasis developed in one patient. At six months after operation, patients had a mean MSTS score of 27 points (range, 24-29). Two patients underwent revision surgery--one for implant infection and one for aseptic loosening.Conclusions: Chemotherapy scheme and limb salvage could achieved high survival rate. Use of this expandable prosthesis was associated with good function and low complication. The character of expandability could be a method to overcome discrepancy in the growth period.Level of Evidence Level IV, therapeutic study.

There have only been rare studies of IDH-mutant primary glioblastomas (IDH-mutant astrocytoma IV); there were one or two studies on secondary glioblastomas. In a cohort of 70 cases, we conducted clinical analysis, methylation profiling, RNA sequencing, targeted sequencing, and TERTp seqeuncing on available FFPE tissues. Median follow-up was 58.2 months (n= 60). IDH-mutant primary glioblastomas had longer median OS (30.4 months) and median PFS (25.9 months) than IDH-mutant secondary glioblastomas as in the literature or established databases. MGMTp methylated cases had better OS (p= 0.001) and it was an independent prognosticator. We previously showed G-CIMP to be an independent prognostic marker for IDH-mutant glioblastomas (NOA 2019). Although CDKN2A deletion was an independent prognostic marker for poorer OS (p= 0.036) and PFS (p= 0.005), MGMTp methylation had a trend of superseding CDKN2A deletion (p= 0.055) for prognostication and G-CIMP subgroups could similarly partially supersede CDKN2A deletion (p= 0.582). Hence, CDKN2A deletion should be supplemented with these two biomarkers for finer prognostication. Targeted sequencing (n= 55) showed that there were more ATRX (35/55, 64%), TP53 (31/55, 56%), KMT2D (18/55, 33%), POLE (11/55, 20%) and MSH6 (7/55, 13%) mutations, but fewer TERTp (3/69, 4%) and PTEN (1/55, 2%) mutations than IDH-wildtype glioblastomas as from literature and databases. CNVs revealed by methylomes (n= 53) and mutations (n= 55) showed that there were more PDGFRA (amplification: 9/53, 17%, mutation: 10/55, 18%) alterations, but fewer MET (amplification: 3/53, 6%, mutation: 4/55, 7%) alterations and hypermutated (6/55, 11%) cases than IDH-mutant secondary glioblastomas from literature. GISTIC analysis revealed amplifications of CCND2, CDK4, MYC, and PDGFRA, deletions of CDKN2A, RB1, and chromosome 10q to be significant CNVs (q< 0.05). There were few EGFR amplifications (2/53, 4%), which was different from regular glioblastomas. RNA sequencing (n= 42) showed few fusions (4/42, 10%), which was different from IDH-mutant secondary glioblastomas.

INTRODUCTION Adult medulloblastomas are clinically and molecularly understudied due to their rarity. METHODS We studied a cohort of 101 adult medulloblastomas grouped by Nanostring assay. We performed targeted sequencing on exonic regions of 69 genes implicated in medulloblastoma, and Sanger sequencing for TERT promoter hotspot mutations. RESULTS Median age was 27 (range 19-63), and 9.5% were metastatic at diagnosis. SHH accounted for 50% of cases. Unlike previous studies, Group 3 comprised a significant proportion (14%) of adult medulloblastomas, comparable to WNT (19%) and Group 4 (18%). Median follow-up was 51.1 months. Median OS and PFS were 102 and 99 months respectively. In contrast to paediatric medulloblastomas, molecular groups had no prognostic impact in adults, for both OS (p=0.956) and PFS (p=0.162). Most frequently mutated genes were TERT (including promoter, mutated in 35% cases), chromatin modifiers KMT2C (32%) and KMT2D (31%), TCF4 (31%), PTCH1 (26%) and DDX3X (24%). Adult WNT patients showed enrichment of TP53 mutations (6/15 WNT cases), and 3/6 TP53-mutant WNT tumours were of large cell/anaplastic histology. 5 WNT cases harboured concurrent CTNNB1 and PTCH1 mutations. Adult SHH medulloblastomas had frequent upstream pathway alterations (PTCH1 and SMO mutations) and seldom downstream alterations (SUFU mutations, MYCN amplifications). TERT promoter mutations were found in 71% of adult SHH patients, and were restricted to this group. Adult Group 3 patients lacked hallmark MYC amplifications, but had recurrent mutations in KBTBD4 and NOTCH1. Adult Group 4 patients had recurrent mutations in TCF4 and chromatin modifier genes. Overall, amplifications of MYC and MYCN were rare (3%) in adult medulloblastomas. CONCLUSIONS We identified distinct clinical and mutational characteristics of adult medulloblastomas that will inform their treatment and risk stratification. Importantly, molecular groups were not prognostic in adult medulloblastoma, TP53 mutations were enriched in adult WNT, and MYC amplifications were absent in adult Group 3.

Patients with hepatocellular carcinoma (HCC) have different prognoses depending on whether or not they also have fibrosis. Since long non‑coding RNAs (lncRNAs) affect tumor formation and progression, the present study aimed to investigate whether their expression might help predict the survival of patients with HCC. Expression profiles downloaded from The Cancer Genome Atlas database were examined to identify lncRNAs differentially expressed (DElncRNAs) between HCC patients with or without fibrosis. These DElncRNAs were then used to develop a risk scoring system to predict overall survival (OS) or recurrence‑free survival (RFS). A total of 142 significant DElncRNAs were identified using data from 135 patients with fibrosis and 72 without fibrosis. For HCC patients with fibrosis, a risk scoring system to predict OS was constructed based on five lncRNAs (AL359853.1, Z93930.3, HOXA‑AS3, AL772337.1 and AC012640.3), while the risk scoring system to predict RFS was based on 12 lncRNAs (PLCE1‑AS1, Z93930.3, LINC02273, TRBV11‑2, HHIP‑AS1, AC004687.1, LINC01857, AC004585.1, AP000808.1, CU638689.4, AC090152.1 and AL357060.1). For HCC patients without fibrosis, the risk scoring system to predict OS was established based on seven lncRNAs (LINC00239, AC104971.4, AP006285.2, HOXA‑AS3, AC079834.2, NRIR and LINC01929), and the system to predict RFS was based on five lncRNAs (AC021744.1, NRIR, LINC00487, AC005858.1 and AC107398.3). Areas under the receiver operating characteristic curves for all risk scoring systems exceeded 0.7. Uni‑ and multivariate Cox analyses showed that the risk scoring systems were significant independent predictors of OS for HCC patients with fibrosis, or of OS and RFS for HCC patients without fibrosis, after adjusting for clinical factors. Functional enrichment analysis suggested that, depending on the risk scoring system, highly associated genes were involved in pathways mainly associated with the cell cycle, chemokines, Th17 cell differentiation or thermogenesis. The findings of the present study indicate that risk scoring systems based on lncRNA expression can effectively predict the OS of HCC patients with fibrosis as well as the OS or RFS of HCC patients without fibrosis.

Background Epstein-Barr virus (EBV) actively contributes to the pathological process of nasopharyngeal carcinoma (NPC) by enabling NPC cells to acquire various capacities required for their malignant biological actions. Our earlier works demonstrated that EBV-encoded latent membrane protein 1 (LMP1) enhanced vascular endothelial growth factor (VEGF)-mediated angiogenesis by boosting store-operated Ca2+ entry (SOCE) upon extracellular epidermal growth factor (EGF) stimulation. However, the antagonistic effects of SOCE blockage on EBV-promoted angiogenesis must be appropriately evaluated in vivo, and the global effect of EBV infection on the EGF-elicited cytosolic Ca2+ signaling, which regulates VEGF-mediated angiogenesis remains to be further clarified. Materials and methods Two EBV-infected NPC cell lines, CNE2-EBV and HK1-EBV, along with their parental cell lines were employed in the present study. Dynamic cytosolic Ca2+ changes were measured in individual fluorescent Ca2+ indicator-loaded cells. Amounts of VEGF production were determined by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs)-formed tube networks were quantitatively evaluated as an in vitro angiogenesis assay. A mouse model concurrently bearing EBV-positive/negative xenografts was utilized to evaluate the tumor growth and angiogenesis in vivo. Results EBV infection reliably promoted transplanted tumor growth while enhancing angiogenesis. Introduction of EBV into EBV-negative NPC cells increased the EGF-stimulated VEGF production while amplifying the EGF-evoked Ca2+ responses. Inhibition of the EBV-boosted Ca2+ signaling using 2-aminoethyl diphenylborinate (2-APB), a specific SOCE inhibitor, effectively antagonized the EBV-promoted VEGF production and endothelial tube formation in vitro. Pharmacological blockage of SOCE exhibited anti-angiogenic effect in the EBV-positive xenografts. Conclusion SOCE can serve as a candidate pharmacological target for treating NPC, as blockage of the Ca2+ signaling via SOCE is a feasible strategy to suppress the EBV-driven malignant profiles in NPC cells.

Growing evidence has revealed that long noncoding RNAs (lncRNAs) have an important impact on tumorigenesis and tumor progression via a mechanism involving competing endogenous RNAs (ceRNAs). However, their use in predicting the survival of a patient with hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to develop a novel lncRNA expression-based risk score system to accurately predict the survival of patients with HCC. In our study, using expression profiles downloaded from The Cancer Genome Atlas database, the differentially expressed messenger RNAs (mRNAs), lncRNAs, and microRNAs (miRNAs) were explored in patients with HCC and normal liver tissues, and then a ceRNA network constructed. A risk score system was established between lncRNA expression of the ceRNA network and overall survival (OS) or recurrence-free survival (RFS); it was further analyzed for associations with the clinical features of patients with HCC. In HCC, 473 differentially expressed lncRNAs, 63 differentially expressed miRNAs, and 1417 differentially expressed mRNAs were detected. The ceRNA network comprised 41 lncRNA nodes, 12 miRNA nodes, 24 mRNA nodes, and 172 edges. The lncRNA expression-based risk score system for OS was constructed based on six lncRNAs (MYLK-AS1, AL359878.1, PART1, TSPEAR-AS1, C10orf91, and LINC00501), while the risk score system for RFS was based on four lncRNAs (WARS2-IT1, AL359878.1, AL357060.1, and PART1). Univariate and multivariate Cox analyses showed the risk score systems for OS or RFS were significant independent factors adjusted for clinical factors. Receiver operating characteristic curve analysis showed the area under the curve for the risk score system was 0.704 for OS, and 0.71 for RFS. Our result revealed a lncRNA expression-based risk score system for OS or RFS can effectively predict the survival of patients with HCC and aid in good clinical decision-making.

It has been widely reported that the CT60 single-nucleotide polymorphism (SNP), which is in the 3′-untranslated region of the cytotoxic T lymphocyte associated 4 (CTLA4) gene, is strongly correlated with certain immune-mediated diseases. The present case-control study aimed to investigate the genetic association between the CT60 SNP within the CTLA4 gene and Graves' disease (GD). A total of 288 patients with GD and 290 control subjects were recruited for the study. The CT60 SNP of the CTLA4 gene was detected by direct DNA sequencing. The results indicated that the frequencies of the GG genotype and G allele in the case group were evidently higher than that in the control group (P=4×10−6 and P=2.9×10−5, respectively). Furthermore, the G/G genotype of the CT60 SNP was associated with an increased risk for GD (odds ratio=2.223). In conclusion, these results suggested that the CT60 SNP is associated with susceptibility to GD. The frequency of the disease-susceptible G allele of CT60 was significantly associated with an increased risk of GD development.

Background Osteosarcoma is the most common bone malignancy in children and adolescents, and 20%–30% of the patients suffer from poor prognosis because of individual chemoresistance. The Hippo/yes-associated protein (YAP) signaling pathway has been shown to play a role in tumor chemoresistance, but no previous report has focused on its involvement in osteosarcoma chemoresistance. This study aimed to investigate the role of the Hippo/YAP signaling pathway in osteosarcoma chemoresistance and to determine potential treatment targets. Methods Using the Cell Titer-Glo Luminescent cell viability assay and flow cytometry analysis, we determined the proliferation and chemosensitivity of YAP-overexpressing and YAP-knockdown osteosarcoma cells. In addition, using western blotting and the real-time polymerase chain reaction technique, we investigated the alteration of the Hippo/YAP signaling pathway in osteosarcoma cells treated with chemotherapeutic agents. Results Mammalian sterile 20-like kinase 1 (MST1) degradation was increased, and large tumor suppressor kinase 1/2 (LATS1/2) total protein levels were decreased by methotrexate and doxorubicin, which increased activation and nuclear translocation of YAP. Moreover, YAP increased the proliferation and chemoresistance of MG63 cells. Conclusions The Hippo/YAP signaling pathway plays a role in osteosarcoma chemoresistance, and YAP is a potential target for reducing chemoresistance.

Dengue virus (DENV) causes self‑limiting dengue fever (DF), severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). It is generally considered that cytokine storm leads to the increased plasma leakage characteristic of DHF/DSS. In the present study, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of healthy volunteers and infected with DENV serotype 2 (DENV2). Culture supernatants of DENV2‑infected and -uninfected PBMCs were analyzed using a human cytokine array. Between a 6‑12 h post‑infection, levels of CCL5, IL‑6 and IL‑8 were markedly elevated, while those of TNF‑α decreased. Total RNA isolated from these PBMCs was analyzed by human miRNA microarray to identify differentially expressed microRNAs (miRNAs). Quantitative reverse transcription polymerase chain reaction was used to validate 11 upregulated and 4 downregulated miRNAs. Sanger mibase, miRanda and TargetScan were used to identify 261 common predicted genes. Databases were used to identify homologous sequences on mRNAs of putative target genes that may be directly bound by the miRNAs identified. We found that cytokines and epigenetic regulators may be putative target genes of these miRNAs. Using ingenuity pathway analysis, we noted that canonical pathways, including biological regulation, may be modulated by these miRNAs.

BackgroundBrain stem lesions are common in patients with acute disseminated encephalomyelitis (ADEM), neuromyelitis optica (NMO), and multiple sclerosis (MS).ObjectivesTo investigate comparative brain stem lesions on magnetic resonance imaging (MRI) among adult patients with ADEM, NMO, and MS.MethodsSixty-five adult patients with ADEM (n = 17), NMO (n = 23), and MS (n = 25) who had brain stem lesions on MRI were enrolled. Morphological features of brain stem lesions among these diseases were assessed.ResultsPatients with ADEM had a higher frequency of midbrain lesions than did patients with NMO (94.1% vs. 17.4%, PConclusionsBrain stem lesions showed various morphological features among adult patients with ADEM, NMO, and MS. The different lesion locations may be helpful in distinguishing these diseases.

The hallmark of the dengue hemorrhagic fever/dengue shock syndrome is hematologic abnormality. The pathogenesis of dengue hemorrhagic fever/dengue shock syndrome remains unknown. Our work showed that the dengue virus serotype-2 induced apoptosis in human umbilical vein endothelial cells. Fas (CD95), Tumor Necrosis Factor receptors, and Tumor Necrosis Factor-related apoptosis-inducing ligand receptors are the most common death receptors, which can induce apoptosis. Compared with the untreated human umbilical vein endothelial cells, Fas expression was increased both in the mRNA level and on the surface of infected human umbilical vein endothelial cells. FasL was expressed at similar levels on human umbilical vein endothelial cells over a course of dengue virus serotype-2 infection, but the expression in mRNA level was increased in infected human umbilical vein endothelial cells. It is possible that there is soluble FasL secreted from human umbilical vein endothelial cells in the supernatant. Tumor Necrosis Factor-related apoptosis-inducing ligand receptor 1 and Tumor Necrosis Factor receptors 1–2 were constantly very low, whereas Tumor Necrosis Factor-related apoptosis-inducing ligand receptors 2–4 decreased after dengue virus serotype-2 infection. This result suggested that dengue virus serotype-2 may inhibit Tumor Necrosis Factor-related apoptosis-inducing ligand receptors-induced apoptosis. The apoptotic rates in human umbilical vein endothelial cells were decreased upon the addition of caspase family inhibitors. In addition, activated caspase 8 and caspase 3 were also observed by Western blot following dengue virus serotype-2 infection. Thus, it is shown that the Fas/FasL pathway may participate in dengue virus-induced apoptosis of vascular endothelial cells in vitro. J. Med. Virol. 82:1392–1399, 2010. © 2010 Wiley-Liss, Inc.

OBJECTIVES Our previous report showed that beta-cell antigen-specific CD56+ T-cells and cytokine TRAIL mediate destruction of human pancreatic [beta] cells in vitro. To determine whether CD56 and TRAIL are present during islet cell destruction at the onset of clinical symptoms of type 1 diabetes mellitus (T1D), we studied cell marker and cytokine expression in the pancreatic islets of 2 children who died at presentation of acute-onset T1D and in T-cell lines derived from a group of children with new-onset T1D. METHODS TRAIL, CD56, and other T-cell markers and cytokine expression were studied using immunohistochemistry on pancreatic sections from 2 children with acute-onset T1D. TRAIL and CD56 expression was analyzed by flow cytometry in the antigen-activated T-cell lines derived from 29 children with new-onset T1D. RESULTS TRAIL+, CD56+, CD45RO+, and CD3+ cells were present in the islets of acute-onset T1D patients, while none were present in the normal islets. T-cell lines from new-onset T1D expressed TRAIL and CD56 in response to stimulation with beta-cell antigens GAD, IA-2 and insulin beta chain. CONCLUSION The presence of TRAIL and CD56 markers is part of the T-cell response repertoire in beta-cell destruction.