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Purpose: RNA sequencing (RNA-seq) has recently proved to be effective for revealing novel virus–tumor associations. To get a thorough investigation of virus–glioma associations, we screened viruses in gliomas with RNA-seq data from the Chinese Glioma Genome Atlas project. Experimental Design: In total, 325 samples were enrolled into this study. Reads that failed to map to the human genome were aligned to viral genomes and screened for potential virus-derived transcripts. For quantification, VPKM was calculated according to mapped reads weighted by genome sizes and sequencing depth. Results: We observed that viruses tended to concertedly express in a certain subgroup of patients. Survival analysis revealed that individuals who were infected with Simian virus 40 (SV40) or woolly monkey sarcoma virus (WMSV) had a significantly shorter overall survival than those uninfected. A multivariate Cox proportional hazards model, taking clinical and molecular factors into account, was applied to assess the prognostic value of SV40 and WMSV. Both SV40 and WMSV were independent prognostic factors for predicting patient's survival in lower-grade gliomas. Subsequent gene analysis demonstrated that SV40 was correlated with regulation of transcription, whereas WMSV was correlated with cell-cycle phase, which indicated frequent proliferation of tumor cells. Conclusions: RNA-seq was sufficient to identify virus infection in glioma samples. SV40 and WMSV were identified to be prognostic markers for patients with lower-grade gliomas and showed potential values for targeting therapy. Clin Cancer Res; 23(9); 2177–85. ©2016 AACR.

// Fan Yang 1, * , Pei Yang 1, * , Chuanbao Zhang 1, * , Yongzhi Wang 2 , Wei Zhang 2 , Huimin Hu 1 , Zhiliang Wang 1 , Xiaoguang Qiu 3 and Tao Jiang 1, 2, 4, 5 1 Department of Molecular Pathology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China 2 Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China 3 Department of Radiation Therapy, Beijing Tiantan Hospital, Capital Medical University, Beijing, China 4 China National Clinical Research Center for Neurological Diseases, Beijing, China 5 Center of Brain Tumor, Beijing Institute for Brain Disorders, Beijing, China * These authors have contributed equally to this work Correspondence to: Tao Jiang, email: taojiang1964@163.com Keywords: glioblastoma, prognosis, recursive partitioning analysis, molecular marker, MGMT Received: July 18, 2016 Accepted: December 16, 2016 Published: April 21, 2017 ABSTRACT Glioblastoma accounts for more than half of diffuse gliomas. The prognosis of patients with glioblastoma remains poor despite comprehensive and intensive treatments. Furthermore, the clinical significance of molecular parameters and routinely available clinical variables for the prognosis prediction of glioblastomas remains limited. The authors describe a novel model may help in prognosis prediction and clinical management of glioblastoma patients. We performed a recursive partitioning analysis to generate three independent prognostic classes of 103 glioblastomas patients from TCGA dataset. Class I (MGMT promoter methylated, age 59, KPS ≥70), class III (MGMT promoter unmethylation, age 54-58, KPS ≥70; MGMT promoter unmethylation, KPS

Accurate and precise cortisol measurements are requisite for ensuring appropriate diagnosis and management of diseases related with adrenal or pituitary gland disorders. Prompted by the needs in characterization of certified reference materials and quality assurance for serum cortisol measurements, we developed and evaluated a highly reliable measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) combined with dextran sulfate-Mg

The commutability of pooled patient sera (PPS) and control materials (CM) should be evaluated to investigate their suitability for use in an external quality assessment (EQA) program. Individual human samples, PPS and CM were analyzed by four routine methods and inductively coupled plasma mass spectrometry reference methods for magnesium and calcium measurements. The commutability was analyzed according to EP14-A3 protocol and the difference in bias approach, respectively. For magnesium measurements, all PPS were commutable and 3/5 CM were commutable for all measurement systems according to the EP14-A3 protocol. For calcium measurements, most PPS were commutable for all measurement systems, but the CM were only commutable with the Cobas c702 system. The IFCC approach produced similar commutability profiles, except that a large number of inconclusive results appeared. The routine methods exhibited excellent linearity and precision. The majority of relative biases between the routine and reference methods were beyond the bias limits. The commutability of the CM and PPS vary depending on which evaluation approach and criterion is applied. Superiority in the commutability of PPS over CM was observed whichever evaluation approach is applied.

Gliomas are the most common type of primary brain tumor in adults with a high mortality rate. Low-grade gliomas progress to glioblastoma multiforme (GBM) in the majority of cases, forming secondary GBM (sGBM), followed by rapid fatal clinical outcomes. Protein tyrosine phosphatase receptor type Z1 (PTPRZ1)-MET proto-oncogene receptor tyrosine kinase (MET) (ZM) fusion has been identified as a biomarker for sGBM that is involved in glioma progression, but the mechanism of gliomagenesis and pathology of ZM-negative sGBM has remained to be fully elucidated. A whole-transcriptome signature is thus required to improve the outcome prediction for patients with sGBM without ZM fusion. In the present study, whole-transcriptome sequencing on 42 sGBM samples with or without ZM fusion from the Chinese Glioma Genome Atlas database identified mRNAs with differential expression between patients with and without ZM fusion and the most significant survival-associated genes were identified. A 6-gene signature was identified as a novel prognostic model reflecting survival probability in patients with ZM-negative sGBM. Clinical characteristics in patients with a high or low risk score value were analyzed with the Kaplan-Meier method and a two-sided log-rank test. In addition, ZM-negative sGBM patients with a high risk score exhibited an increase in immune cells, NF-κB-induced pathway activation and a decrease in endothelial cells compared with those with a low risk score. The present study demonstrated the potential use of a next-generation sequencing-based cancer gene signature in patients with ZM-negative sGBM, indicating possible clinical therapeutic strategies for further treatment of such patients.

The study area is located in the Langgu sag of Northern Jizhong depression, Bohai Bay Basin, East China. In order to achieve exploration breakthrough in deep buried hill, key engineering technologies are developed and used to accurately demonstrate important target identification by recognizing new hydrocarbon accumulation patterns resulting from the analysis of multi-stage structure-controlled trap mechanism and the detailed study of controlling factors over high-quality Ordovician reservoirs based on new high-accuracy 3D seismic data. This study reveals a new evolution mechanism of buried hill controlled by structural superposition, experiencing "the uplift from thrusting in Indo-Chinese to early Yanshan epoch, uplifted block faulting into horsts in middle Yanshan epoch, horsts tilting into belt in Eocene, and belt reversion into trap", and thus puts forward a new mechanism for reservoir forming controlled by a superposition of "dolomite, karsting, and faulting". Three types of reservoir development are identified, including "regional layered pore, local block micropore-fracture, and fracture hole pore layer-block composite", and an accumulation pattern in deep buried hill is constructed, characterized by "efficient hydrocarbon supply from gas-type source rock, predominant migration through fractured surface-nonconformity surface, and stratum- and mass-controlled accumulation", which has guided the 40 years' exploration of Ordovician Yangshuiwu buried hill zone and made a great breakthroughs. Novel relevant exploration technologies have been developed, involving high-accuracy imaging, high-precision well logging identification of hydrocarbon reservoir, ultra-high temperature deep drilling and completion, ultra-high temperature carbonate reservoir stimulation, etc, which solve a worldwide problem that has restricted the exploration of the ultra-high temperature buried hill for many years. These technologies make possible the highest daily production of over 100 m3 oil and 0.5 million m3 gas respectively and sustain a high and stable production for a long term, which guarantee the clean energy supply for Beijing-Tianjin-Hebei region.

Purpose: We aimed to develop a diagnostic platform to capture the transcriptomic resemblance of individual adult diffuse gliomas of WHO grades II to IV to neural development and the genomic signature associated with glioma progression. Experimental Design: Based on the EM/PM classification scheme, we designed a RT-PCR–based TaqMan low-density array (TLDA) containing 44 classifier and 4 reference genes. Samples of a training dataset (GSE48865), characterized by RNA-sequencing, were utilized to optimize the TLDA design and to develop a support vector machine (SVM)-based prediction model. Complemented with Sanger sequencing for IDH1/2, and low coverage whole genome sequencing (WGS), the TLDA and SVM prediction model were tested in a validation (31 gliomas) and a test (121 gliomas) dataset. Results: Independent of morphologically defined subtypes and grades, gliomas can be individually assigned into the EM and PM glioma subtypes with the respective areas under ROC curves at 0.86 and 0.85 in the validation dataset. The EM gliomas showed a medium overall survival (OS) of 15.6 months, whereas the medium OS for PM gliomas was not reached (HR = 3.55; 95% confidence interval, 1.96–6.45). The EM and PM gliomas showed distinct patterns of genomic alterations, with IDH mutation and 1p19q codeletion in the PM gliomas and gain of chromosome 7/loss of chromosome 10 in the EM gliomas. Extensive chromosomal abnormalities marked the progression of PM gliomas. Conclusions: The integration of EM/PM subtyping, IDH sequencing, and low coverage WGS may improve the risk stratification and selection of treatment regimens for patients with glioma.

CTLA-4, identified as an immune checkpoint, has been widely reported to contribute to downregulation of immune responses to anti-tumor activity. Anti-CTLA-4 provides a novel strategy to improve the outcome of cancer immunotherapy, but the role of CTLA-4 needs to be addressed in glioma. To investigate the role of CTLA-4 in diffuse gliomas, we analyzed the transcriptome level data of CTLA-4 and evaluated its clinical prognostic value. 1022 glioma samples with transcriptome data were enrolled into this study, including 325 RNA-seq data from CGGA and 697 RNA-seq data from TCGA. Clinical and molecular data were also obtained. R project was the main tool for statistical analysis and graphical work. High-grade gliomas exhibited high expression of CTLA-4 while IDH wildtype gliomas showed CTLA-4 enrichment. According to TCGA transcriptional classification in gliomas, mesenchymal subtype displayed significantly higher CTLA-4 expression than other molecular subtypes. Gene ontology analysis demonstrated that CTLA-4 and correlated genes were involved in immune responses and inflammatory activities in diffuse gliomas. Through the analysis of transcriptomic data, CTLA-4 expression strongly correlated with immune cell populations and inflammatory activity. Survival analysis suggested that high expression of CTLA-4 was associated with poor prognosis in gliomas and may serve as an indicator of outcome in gliomas. High CTLA-4 expression is closely related to malignancy in diffuse gliomas and predicts poor outcome. Meanwhile, CTLA-4 plays an important role in regulating T cell activation and antitumor responses in gliomas. Therefore, blocking CTLA-4 is a potential target for developing the immunotherapy of gliomas. Funding: This work was supported by the capital health research and development of special (2014-2-1072); Beijing Municipal Natural Science Foundation (7142054) and National Natural Science Foundation of China (81471229). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by Capital Medical University Institutional Review Board (IRB).

Background: Discovering the biological functions of Aldehyde Dehydrogenases (ALDH) is the hot topic in current cancer researches. Despite the large amount of relative research results, the clinical translational research of these results was difficult to achieve. One of the most important reasons was lack of systematic understanding of ALDHs. Methods: Based on transcriptome data from 999 glioma patients, functional clustering analysis was performed to reveal the functional phenotypes of ALDH isoforms. Subsequently, ALDH subgroups closely related to gliomas were identified by expression and COX survival analysis. Finally, Gene Set Variation Analysis and pearson correlation analysis were used to exploring the biological functions of ALDHs. Findings: The present study found that ALDHs could be classified into five subgroups based on their biological functions in glioma. Three groups were closely related to malignant progression and the prognosis of gliomas and ALDHs in these groups were closely related to gene mutations, which most likely caused by changes in DNA repair functions. Further studies revealed that ALDHs play their roles by affecting tumor immune functions. The effectiveness of immune checkpoints inhibitors in treating glioma might be improved by altering the expression of ALDHs in certain subgroups. Interpretation: Our data not only comprehensively revealed the biological functions of ALDHs in glioma, but also provided a way for the potential clinical application of ALDHs targeted therapy. Funding: The National Natural Science Foundation of China; The National Key Research and Development Plan; Capital Medical Development Research Fund; Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special. Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: This study was approved by the Beijing Tiantan Hospital institutional review board (IRB).

Background: Immunotherapy is a promising therapeutic approach for cancers. However, the immune background and the interactions between pituitary adenomas (PAs) and the host immune system remain unclear. Methods: Poly-A enriched RNA sequencing was performed on 115 snap frozen PA samples. Estimating the Proportion of Immune and Cancer cells (EPIC) was used to detect all the main cell types (B cells, CD8+ T cells, CD4+ T cells, macrophages and NK cells) in a tumor sample from the expression of genes. Immunohistochemical (IHC) staining was used to validate the main findings. Findings: In RNA sequencing data, PA samples showed immune infiltration, including B cells, CD8+ T cells, CD4+ T cells, macrophages and NK cells. The results were further validated by IHC. Meanwhile, pathway analysis showed that TNFR superfamily (TNFRSF), IL-10 family and TGF beta (TGFβ) family were the potential mechanisms of immune infiltration in PAs. In order to search how tumor making progression with high infiltration of immune cells, gene-expression data revealed that tumor progression was related to increased expression of PD-1/PD-L1 and was associated with higher infiltration of CD8+ T cells. Besides, pathway enrichment analysis showed that regulation of death/apoptosis processes and the JAK-STAT pathway were found to be significantly enriched in samples with higher immune infiltration. At last, analysis of cancer-testis antigens (CTAs) expression in CD8+ T cells suggested that CTAG2 and TSPYL6 were potential immunotherapeutic targets in GH and non-functioning PAs, respectively. Interpretation: Our work provides new angles for understanding tumor-infiltrating immune cells. The results may inform effective checkpoint blockade and cancer vaccine therapies and make it possible to take immunotherapy into invasive functioning PAs. Funding Statement: This work was supported by the National Natural Science Foundation of China under Grant number 81471229; Capital Medical Development Research Foundation under Grant number 201811071. Declaration of Interests: No potential conflicts of interest are declared. Ethics Approval Statement: The PA tissue collection was approved by the Institutional Review Board of Beijing Tiantan Hospital, Capital Medical University. All procedures involving human participants conformed to the ethical standards of the national research committee and the 2013 Helsinki Declaration, which were approved by the Institutional Review Board of Beijing Tiantan Hospital, Capital Medical University. Informed consent was obtained from all patients prior to their enrollments in this study.

Growth-hormone-secreting pituitary adenoma (GHSPA) is a benign tumour with a high incidence and large economic burden, which greatly affects quality of life. The aetiological factors are yet to be clarified for GHSPA. Conventional two-dimensional (2D) monolayer culture of tumour cells cannot ideally reflect the growth status of tumours in the physiological environment, and insufficiencies of in vitro models have severely restricted the progress of cancer research. Three-dimensional (3D) bioprinting technology is being increasingly used in various fields of biology and medicine, which allows recapitulation of the in vivo growth environment of tumour cells. In this study, a GHSPA microtissue model was established using 3D bioprinting. Tumour cells in the 3D environment exhibited more active cell cycle progression, secretion, proliferation, invasion, and tumourigenesis compared with those in the 2D environment. Furthermore, the molecular mechanisms of the 3D-printed microtissue model were explored. We demonstrated that the 3D-printed microtissue provides an excellent in vitro model at the tissue level for oncological research and may facilitate in-depth studies on the aetiology, treatment, drug resistance, and long-term prognosis of GHSPA .

As aldehyde dehydrogenase (ALDH) is a novel stem cell marker, increasing studies have confirmed that high ALDH activity promotes tumorigenesis and progression in cancers. Some preliminary studies have found that ALDH1A3 may play an important role in glioma malignant progression, but so far there was no conclusive conclusion. The purpose of our study was to elucidate the mechanisms by which ALDH1A3 regulated in glioma and to provide practical tools for clinical application. Aldefluor, flow cytometry sorting and qRT-PCR were performed to verify the role of ALDH1A3 in ALDH activity maintenance. Transwell, immunofluorescence, glycolytic assays, and orthotopic xenograft models were used to explore ALDH1A3 bio-functions in GBM. LASSO-COX, COX survival analysis and Kaplan–Meier analysis were used to establish the prognostic evaluation system and predict postoperative chemotherapy sensitivity of GBMs. Our integrated study found that (1) ALDH1A3 associates with mesenchymal differentiation of GBM in Eastern and Western world patients. (2) ALDH1A3 plays a critical role in ALDH activity maintenance. (3) ALDH1A3 is an activator of mesenchymal transformation in GBM. (4) ALDH1A3-derived PMT markers’ molecular signature can predict 1-, 2-, and 3-year survival rates of GBMs precisely. In conclusion, ALDH1A3 was a major contributor to ALDH activity and a key driver in triggering mesenchymal transformation in GBM. ALDH1A3-based molecular classification scheme can help to improve guidance for prognosis forecasting and individualized treatment decision making for GBM patients.

// Zheng Wang 1, 2, 6 , Chuanbao Zhang 1, 2, 6 , Lihua Sun 1, 6 , Jingshan Liang 1, 6 , Xing Liu 1, 2, 6 , Guanzhang Li 1, 2, 6 , Kun Yao 3, 6 , Wei Zhang 2, 4, 5, 6 , Tao Jiang 1, 2, 4, 5, 6 1 Beijing Neurosurgical Institute, Capital Medical University, Beijing, China 2 Beijing Tiantan Hospital, Capital Medical University, Beijing, China 3 Sanbo Brain Hospital, Capital Medical University, Beijing, China 4 Center of Brain Tumor, Beijing Institute for Brain Disorders, Beijing, China 5 China National Clinical Research Center for Neurological Diseases, Beijing, China 6 Chinese Glioma Genome Atlas network (CGGA), Beijing, China Correspondence to: Tao Jiang, email: taojiang1964@163.com Keywords: FGFR3, receptor tyrosine kinase, FGFR-TACC fusion genes, glioma Received: March 21, 2016 Accepted: October 26, 2016 Published: November 05, 2016 ABSTRACT Background: Activation of receptor tyrosine kinases is common in Malignancies. FGFR3 fusion with TACC3 has been reported to have transforming effects in primary glioblastoma and display oncogenic activity in vitro and in vivo . We set out to investigate the role of FGFR3 in glioma through transcriptomic analysis. Results: FGFR3 increased in Classical subtype and Neural subtype consistently in CGGA and TCGA cohort. Similar patterns of FGFR3 distribution through subtypes were observed in CGGA and TCGA samples. Gene ontology analysis was performed with genes that were significantly correlated with FGFR3 expression. We found that positively associated biological processes of FGFR3 were focused on differentiated cellular functions and neuronal activities, while negatively correlated biological processes focused on mitosis and cell cycle phase. Clinical investigation showed that higher FGFR3 expression predicted improved survival for glioma patients, especially in Proneural subtype. Moreover, FGFR3 showed very limited relevance with other receptor tyrosine kinases in glioma at transcriptome level. Materials and Methods: FGFR3 expression data of glioma was obtained from Chinese Glioma Genome Atlas (CGGA) and TCGA (The Cancer Genome Atlas). In total, RNA sequencing data of 325 glioma samples and mRNA microarray data of 301 samples from CGGA dataset were enrolled into this study. To consolidate the findings that we have revealed in CGGA dataset, RNA-seq data of 672 glioma samples from TCGA dataset were used as a validation cohort. R language was used as the main tool to perform statistical analysis and graphical work. Conclusions: FGFR3 expression increased in classical and neural subtypes and was associated with differentiated cellular functions. FGFR3 showed very limited correlation with other common receptor tyrosine kinases, and predicted improved survival for glioma patients.

// Chuanbao Zhang 1, * , Jiye Li 1, 2, 3, * , Haoyuan Wang 4 , Sonya Wei Song 1, 2, 3 1 Beijing Neurosurgical Institute, Capital Medical University, TiantanXili, Dongcheng District, Beijing 100050, China 2 Beijing Institute for Brain Disorders, Youanmen, Beijing, 100069, China 3 Center for Brain Disorders Research, Capital Medical University, Youanmen, Beijing, 100069, China 4 Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China * These authors contributed equally to this work Correspondence to: Sonya Wei Song, email: sonyasongbj2016@163.com , sonyasong@ccmu.edu.cn Keywords: immune system, tumor-infiltrating B cells, prognosis, biomarker, glioma Received: April 11, 2016 Accepted: September 27, 2016 Published: October 12, 2016 ABSTRACT High grade gliomas contribute to most brain tumor mortality. A few studies reported that the immune system affected glioma development, and immune biomarkers helped understand the disease and formulate effective immunotherapy for patients. Currently, no B lymphocyte-based prognostic signature was reported in gliomas. By applying 78 B cell lineage-specific genes, we conducted a whole-genome gene expression analysis in 782 high grade gliomas derived from three independent datasets by Cox regression analysis and risk score method for signature identification, and then used Gene Ontology, Gene Set Enrichment Analysis, and other statistical methods for functional annotations of the signature-defined differences. We developed a five B cell-associated gene signature for prognosis of high grade glioma patients, which is independent of clinicopathological and genetic features. The signature identified high risk patients suitable for chemoradiotherapy, whereas low risk patients should rule out chemotherapy with radiotherapy only. We found that tumors of TCGA Mesenchymal subtype and wild type IDH1 were preferentially stratified to the high risk group, which bore strong immunosuppressive microenvironment, while tumors of TCGA Proneural subtype and mutated IDH1 were significantly accumulated to the low risk group, which exhibited less immunosuppressive state. The five B cell-associated gene signature predicts poor survival of high risk patients bearing strong immunosuppression and helps select optimal therapeutic regimens for glioma patients.

Background Calcium is an important serum ion which is frequently assayed in clinical laboratories. Since quality assurance requires reference methods, the establishment of a candidate reference method for serum calcium measurement is important. Method An inductively coupled plasma mass spectrometry (ICP-MS) method was developed. Serum samples were spiked gravimetrically with aluminum internal standard, digested with 69% ultrapure nitric acid and diluted to test concentration. Then the 44Ca/27Al ratios were measured by ICP-MS in hydrogen mode. The method was calibrated using 5% nitric acid matrix calibrators and the calibration function was established with bracketing method. Results The correlation coefficients between the measured 44Ca/27Al ratios and the analyte concentrations ratios were all > 0.9999. The coefficients of variation of the measurements were 0.27% and 0.16% for two spiked serum. The analytical recovery was 100.24%. The accuracy of the measurement was also verified through measurement of certified reference materials. Comparison with recognized reference method and international inter-laboratory comparisons gave satisfied results. Conclusion New ICP-MS method is specific, precise, simple, and low in cost, and may be used as a candidate reference method in the standardization of serum calcium measurement.

Isocitrate dehydrogenase (IDH)1 mutation is one of the most important genetic aberrations in glioma. Even several genetic events have refined its prognostic value, the genome-wide expression alteration has not been systematically profiled. In this work, RNA-seq expression data from 310 patients in the Chinese Glioma Genome Atlas database were included as training set, while another 297 patients with microarray data were used as internal validation set. An independent cohort of GSE16011 (n = 205) constituted an external validation set. Approximately one fifth of the genes were differentially expressed in LGG according to IDH1 mutation status, yielding distinct gene expression profiles. A six-gene risk signature was established for IDH1-mutant LGG to distinguish low- from high-risk cases, which had distinct prognoses. The six-gene signature was an independent prognostic factor for IDH1-mutant LGG and had superior predictive value as compared to traditional clinicopathologic factors. Moreover, we depicted the differential expression pattern in GBM attributing to various IDH1 status, which was similar to that of LGG. It suggested that the effect of IDH1 mutation is conserved across histological classifications. The six-gene signature had equal prognostic value for IDH1-mutant GBM. By combining glioma grade, IDH1 status, and the six-gene signature, all glioma patients could be classified into six subgroups. These six subgroups could be further summarized into three sets with distinct prognosis. Taken together, a gene expression profile associated with IDH1 status was identified in LGG and GBM; a risk signature based on six genes was developed with equal prognostic value for IDH1-mutant LGG and GBM. When combined with clinicopathologic factors, the six-gene signature is a tool that enables precise risk stratification and can improve clinical management.

Recurrence and progression to higher grade lesions are key biological events and characteristic behaviors in the evolution process of glioma. Malignant astrocytic tumors such as glioblastoma (GBM) are the most lethal intracranial tumors. However, the clinical practicability and significance of molecular parameters for the diagnostic and prognostic prediction of astrocytic tumors is still limited. In this study, we detected ATRX, IDH1-R132H and Ki-67 by immunohistochemistry and observed the association of IDH1-R132H with ATRX and Ki-67 expression. There was a strong association between ATRX loss and IDH1-R132H (p

Accurate and precise glucose measurements are requisite for ensuring appropriate diagnosis and management of diseases related to hyperglycemia or hypoglycemia. It is necessary to have a higher order method to provide an accuracy base to which routine methods can be compared. We developed and evaluated a highly reliable measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) with a simple one-step derivatization. An appropriate amount of serum was accurately weighed and spiked with an isotope-labeled internal standard. After protein precipitation, the supernatant was reacted with 1-phenyl-3-methyl-5-pyrazolone for chemical structural transformation. The glucose derivatives were analyzed with LC-MS/MS in positive electrospray ionization mode. The within-run and total CVs ranged from 0.28 to 0.42 % and from 0.42 to 0.76 %, respectively, for a concentration range of 1.691 to 15.676 mmol/L. A regression comparison of the presented method to an existing RMP based on ID GC-MS showed agreement with no statistical difference (Y = 0.9985X-0.008; 95 % CI for the slope, 0.9966 to 1.001; 95 % CI for the intercept, -0.012 to 0.019). The structural analogs of glucose with a molecular mass of 180 were tested, and no significant interference effect was found. The limit of quantification was estimated to 0.8 ng glucose in absolute amount. This method is accurate, simple, and can serve as a candidate reference measurement procedure (RMP) in the establishment of a serum glucose reference system.

// Pei Yang 1,2,* , Chuanbao Zhang 2,* , Jinquan Cai 9 , Gan You 1,2 , Yinyan Wang 1,2 , Xiaoguang Qiu 3 , Shouwei Li 5 , Chenxing Wu 5 , Kun Yao 6 , Wenbin Li 7 , Xiaoxia Peng 10 , Wei Zhang 1,2 and Tao Jiang 2,4,8 1 Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China 2 Beijing Neurosurgical Institute, Capital Medical University, Beijing, China 3 Department of Radiation Therapy, Beijing Tiantan Hospital, Capital Medical University, Beijing, China 4 China National Clinical Research Center for Neurological Diseases, Beijing, China 5 Department of Neurosurgery, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing, China 6 Department of Pathology, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing, China 7 Department of Oncology, Beijing Shijitan Hospital, Capital Medical University, Beijing, China 8 Center of Brain Tumor, Beijing Institute for Brain Disorders, Beijing, China 9 Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, China 10 Department of Epidemiology and Biostatistics, School of Public Health and Family Medicine, Capital Medical University, Beijing, China * The first two authors contributed equally to this work Correspondence to: Wei Zhang, email: // Tao Jiang, email: // Keywords : high-grade glioma, young adult, radiation, temozolomide, survival Received : November 16, 2015 Accepted : August 22, 2016 Published : August 31, 2016 Abstract Purpose: Age is a major prognostic factor for malignant gliomas. However, few studies have investigated the management of gliomas in young adults. We determined the role of survival and treatment in young adults with advanced gliomas in a large population from the Chinese Glioma Genome Atlas (CGGA). Methods: This study included 726 adults (age ≥ 18) with histologically proven anaplastic glioma or glioblastoma multiforme (GBM). The overall and progression-free survival was determined in young (age < 50) and older groups (age ≥ 50). Results: The study included an older group (OP) of 264 patients and a younger group (YP) of 462patients. In the OP group with GBM and anaplastic glioma, patients treated with RT combined with temozolomide (TMZ) manifested significantly longer OS and PFS compared with patients assigned to RT alone (P < 0.05). In contrast, the YP group diagnosed with anaplastic glioma failed to show any survival advantage with RT plus TMZ compared with RT alone. Conclusions: We observed no survival benefit in young adults (age < 50) with anaplastic glioma when treated with TMZ combined with RT. Our findings warrant further investigation of younger patients diagnosed with anaplastic glioma treated with radiotherapy plus TMZ chemotherapy.

The Chinese Glioma Cooperative Group (CGCG) Guideline Panel for adult diffuse gliomas provided recommendations for diagnostic and therapeutic procedures. The Panel covered all fields of expertise in neuro-oncology, i.e. neurosurgeons, neurologists, neuropathologists, neuroradiologists, radiation and medical oncologists and clinical trial experts. The task made clearer and more transparent choices about outcomes considered most relevant through searching the references considered most relevant and evaluating their value. The scientific evidence of papers collected from the literature was evaluated and graded based on the Oxford Centre for Evidence-based Medicine Levels of Evidence and recommendations were given accordingly. The recommendations will provide a framework and assurance for the strategy of diagnostic and therapeutic measures to reduce complications from unnecessary treatment and cost. The guideline should serve as an application for all professionals involved in the management of patients with adult diffuse glioma and also as a source of knowledge for insurance companies and other institutions involved in the cost regulation of cancer care in China.

Human telomerase reverse transcriptase (TERT) promoter mutations are important genetic alterations in many kinds of human malignancies, including glioma. The current study aimed to investigate the anatomical specificity of TERT promoter mutations in glioblastomas (GBMs). Clinical information and preoperative magnetic resonance images of 203 patients with GBMs were reviewed. TERT promoter mutation status was assessed by Sanger sequencing in all cases. Tumor lesions were manually segmented and then registered to a standard brain atlas. Then the specific brain regions associated with TERT promoter mutation status were subsequently identified by voxel-based regression analysis. TERT promoter mutations were detected in 94 (46.3 %) of the 203 patients. Voxel-based statistical analysis demonstrated that GBMs with TERT promoter mutations were much more likely to locate in the right temporal lobe, while those with wild-type TERT promoters were more likely to occur in the anterior region of the right lateral ventricle. No significant difference was found in the lesion volumes of the T2-identified tumor or in the contrast enhancement areas between the two groups. The current study demonstrated the anatomic specificity of TERT promoter mutation status in GBM. These findings may provide new insight into the molecular classification of GBM and further our understanding of the associations between tumor-specific molecular alterations and tumor location.

Kinesin family member 23 (KIF23), a nuclear protein and a key regulator of cellular cytokinesis, has been found to be overexpressed as an oncogene in glioma. However, the prognostic and clinicopathological features of glioma with KIF23 expression was not clear yet. Here, we analyzed KIF23 expression pattern by using whole genome mRNA expression microarray data from Chinese Glioma Genome Atlas (CGGA) database (http://www.cgga.org.cn), and found that KIF23 overexpression was significantly associated with high grade glioma as well as the higher mortality in survival analysis (log-rank test, p

Recurrence and progression to higher grade lesions are key biological events and characteristic behaviors in the evolution process of glioma. A small residual population of cells always escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence or progression. IDH mutation (isocitrate dehydrogenase) and ATRX (alpha-thalassemia/mental retardation, X-linked) loss/mutation occur in association and may represent early genetic alterations in the development of gliomas. However, their prognostic value in the evolution of gliomas still needs further investigation. Two hundreds and eleven serial sampling of gliomas were included in our study. We used immunohistochemistry (IHC) to detect IDH1-R132H mutation and ATRX status and showed that the IDH1-R132H and (or) ATRX status could be necessary to provide the basic molecular information for the “integrated diagnosis” of gliomas. We illustrated an evaluation formula for the evolution of gliomas by IDH1-R132H combined with ATRX immunohistochemistry and identified the association of IDH1-R132H/ATRX loss accompanied by longer progression time interval of patients with gliomas. Furthermore, we observed that most recurrences had a consistent IDH1 and ATRX status with their matched primary tumors and demonstrated the progressive pattern of grade II astrocytoma/oligodendroglial tumors and anaplastic oligoastrocytoma with or without IDH1-R132H. Identification of IDH1-R132H and ATRX loss status in the primary-recurrent gliomas may aid in treatment strategy selection, therapeutic trial design, and clinical prognosis evaluation.

Recent observations from metabonomic studies have consistently found that branched-chain amino acids (BCAAs), aromatic amino acids (AAAs), glutamine (Gln), glutamic acid (Glu), Gln/Glu ratio, carnitine, and several species of acylcarnitines and lysophosphatidylcholines (LPCs) are possible risk factors for metabolic diseases such as diabetes mellitus (DM) and cardiovascular diseases (CVD). We described here a simple and reliable method for simultaneous quantification of these metabolic risk factors by liquid chromatography tandem mass spectrometry (LC-MS/MS). Serum samples were extracted with isopropanol, and the extracted metabolites were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospary ionization (ESI) inpositive ion mode with multiple reaction monitor (MRM) mode. All the metabolites were effectively separated within 5.5min. Analytical recoveries were in the range of 92.8-106.9%, with an average of 100.6%. The intra- run and total imprecisions for the measurement of these metabolites were 1.2-3.8% and 1.5-7.4%, respectively. Serum concentrations of the metabolites were analyzed in 123 apparently healthy volunteers. Significant associations between the metabolites and traditional CVD risk factors were observed. The newly developed LC-MS/MS method was simple, precise, and accurate and can be used as an efficient tool in CVD research and studies.

Recent evidence has confirmed that a mutation of the isocitrate dehydrogenase (IDH) gene occurs early in gliomagenesis and contributes to suppressed immunity. The present study aimed to determine the candidate genes associated with IDH mutation status that could serve as biomarkers of immune suppression for improved prognosis prediction. Clinical information and RNA-seq gene expression data were collected for 932 glioma samples from the CGGA and TCGA databases, and differentially expressed genes in both lower-grade glioma (LGG) and glioblastoma (GBM) samples were identified according to IDH mutation status. Only one gene, interferon-stimulated exonuclease gene 20 (ISG20), with reduced expression in IDH mutant tumors, demonstrated significant prognostic value. ISG20 expression level significantly increased with increasing tumor grade, and its high expression was associated with a poor clinical outcome. Moreover, increased ISG20 expression was associated with increased infiltration of monocyte-derived macrophages and neutrophils, and suppressed adaptive immune response. ISG20 expression was also positively correlated with PD-1, PD-L1, and CTLA4 expression, along with the levels of several chemokines. We conclude that ISG20 is a useful biomarker to identify IDH-mediated immune processes in glioma and may serve as a potential therapeutic target.

Shale gas usually distributes in dense porous media reservoirs, which is not easy to develop without external force. Hydraulic fracturing has been an effective technology utilized to improve the yield of shale gas. However, the performance of this method depends on large displacement and liquid volume, resulting in the blockage of the flowing channels in the reservoir and huge waste of water resources. Thus, it is particularly important to increase the rate of fracturing fluid return. The contact angle test, owens two-liquids method and spontaneous imbibition were conducted to investigate the effect of gas-wetting alteration on the shale wettability and liquid flow. Moreover, the energy dispersive spectroscopy was used to analyze elemental changes on the shale surface. The results showed that the contact angles of water and n-decane on the shale surface increased from 36o and 0o to 128o and 115o, respectively. The surface free energy rapidly reduced from primeval 71 mN/m to 5.6 mN/m after treated by 0.4 wt.% gas-wetting reversal agent. The results were further verified by spontaneous imbibition. The analysis of energy dispersive spectroscopy (EDS) indicated that the gas-wetting reversal agent could product adsorption layer on shale surface, which played an important role in super gas-wetting. The wettability of shale surface could be reversed from liquid-wetting to super gas-wetting by the novel gas-wetting reversal agent with double-chain hydrophobic and olephobic structure, which could significantly enhance the flowback rate of the fracturing fluid due to super gas-wetting alteration.

Background This study observed and analyzed the internal quality control (IQC) of cystatin C (CysC) so that we can have overall knowledge about imprecision levels in Chinese medical laboratories. Methods Using the software developed by the National Center for Clinical Laboratories (NCCL), we can get the IQC information of CysC from 2014 to 2017. Then the proportion of laboratories meeting five quality specifications (pass rates) were calculated and the current CVs (coefficient of variation) were also compared among subgroups and years. Results We find that the current CVs between 2014 and 2017 show significant differences (p = 0.016) and the proportion of laboratories meeting the 1/3 TEa specification distributes randomly from 2014 to 2017 but all of them exceed 80 percent. When the optimum specification is applied, the pass rates all become very low and the distributions are wide spread (from 3.63% to 6.74%). Beckman, Roche, and Hitachi are mainstream analyzers, making up as much as 78% to 85% of all. We can see a significant difference of the pass rate between Beckman and Hitachi in 2014 (p = 0.005). The primary means of detecting CysC is particle-enhanced turbidimetric immunoassay (PETIA) which makes up 30.57% - 32.64% of detection methods. The good news is that the IQC practice has improved greatly from 2014 to 2017 in laboratories in China. Conclusions Medical laboratories have made some progress in IQC from 2014 to 2017, but it is still not satisfactory. As a result, there is a long way to go to improve detection quality of laboratories in China.

Recent studies have found that carnitine and acylcarnitines (AcyCNs) are potential biomarkers for many metabolic diseases, including inherited disorders, diabetes and coronary atherosclerotic artery disease (CAD). Here we presented and validated a method for measurement of free carnitine and twelve AcyCNs in human serum by using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Serum samples were mixed with isotopic labeled internal standards and extracted with isopropanol. Carnitine and AcyCNs were separated within 5min by isocratic elution on a hydrophilic interaction liquid chromatographic column (HILIC) and detected with electrospray ionization (ESI) in positive ion mode with multiple reaction monitor (MRM) mode. This method was linear in response with correlation coefficients higher than 0.999. Analytical recoveries were 89.4%–105.4% with an average of 98.7%. The intra-run and total imprecisions were 0.9–4.7% and 1.5–14.1%, respectively. Stability tests showed that all the compounds were stable in serum for at least 6 month at −80 °C, but unstable when stored at room temperature (RM) and 4 °C.Serum carnitine and AcyCNs concentrations of 472 apparently healthy subjects were analyzed. Significant associations between the metabolites and traditional CAD risk factors were observed. The established HILIC–ESI-MS/MS method was simple, precise, and sensitive and may be used as an efficient tool in CAD risk assessment and research.

CMTM6, a previously uncharacterized protein, was identified as a critical regulator of PD-L1, which is reported as an immune checkpoint inhibitor, to modulate the T cell activities both in vitro and in vivo of other tumors. However, the role of CMTM6 has so far remained unclear in glioma. To investigate the role of CMTM6 in gliomas, we analyzed the transcriptome level, genomic profiles and its relationship with clinical practice. 1862 glioma samples with transcriptome data were enrolled in this study, including CGGA RNA-seq, TCGA RNA-seq, CGGA-microarray, GSE16011 and IVY GBM databases. Clinical information and genomic profiles containing somatic mutations and DNA copy numbers were also obtained. We found that CMTM6 expression was highly correlated with major clinical and molecular characteristics. Cases with high CMTM6 expression were more likely to be predicted as malignant entities and frequent with genomic aberrations of driver oncogenes. Moreover, gene ontology analysis based on significantly correlated genes of CMTM6 expression exhibited that CMTM6 was associated with immune responses and inflammatory activities. CMTM6 was synthetic with other immune checkpoint inhibitors. Additionally, CMTM6 was involved in immune functions via modulating T-lymphocyte-mediated anti-tumor immunity. Finally, high CMTM6 expression was associated with reduced survival time and may serve as a strong indicator of poor prognosis in gliomas. In brief, High level of CMTM6 expression is closely related to high malignant gliomas. Meanwhile, CMTM6 plays an important role in regulating T cell activation and antitumor responses. Therefore, CMTM6 is a promising target for developing immunotherapy of gliomas.

Background Immune checkpoint inhibitors have been shown to promote antitumor immunity and achieve durable tumor remissions. However, certain tumors are refractory to current immunotherapy. These negative results encouraged us to uncover other therapeutic targets and strategies. PTPN2 (protein tyrosine phosphatase, non-receptor type 2) has been newly identified as an immunotherapy target. Loss of PTPN2 sensitizes the tumor to immunotherapy via IFNγ signaling. Methods Here, we investigated the relationship between PTPN2 mRNA levels and clinical characteristics in gliomas. RNA-seq data of a cohort of 325 patients with glioma were available from the Chinese Glioma Genome Atlas and 671 from The Cancer Genome Atlas. R language, GraphPad Prism 5, and SPSS 22.0 were used to analyze data and draw figures. Results PTPN2 transcript levels increased significantly with higher grades of glioma and in isocitrate dehydrogenase (IDH) wild-type and mesenchymal subtype gliomas. A comprehensive biological analysis was conducted, which indicated a crucial role of PTPN2 in the immune and inflammation responses in gliomas. Specifically, PTPN2 was positively associated with HCK, LCK, MHC II, and STAT1 but negatively related to IgG and interferon. Moreover, canonical correlation analysis showed a positive correlation of PTPN2 with infiltrating immune cells, such as macrophages, neutrophils, and CD8+ T cells. Clinically, higher levels of PTPN2 were associated with a worse overall survival both in patients with gliomas and glioblastomas. Conclusion PTPN2 expression level was increased in glioblastomas and associated with gliomas of the IDH wild-type and mesenchymal subtype. There was a close correlation between PTPN2 and the immune response and inflammatory activity in gliomas. Our results show that PTPN2 is a promising immunotherapy target and may provide additional treatment strategies. Electronic supplementary material The online version of this article (10.1186/s12974-018-1187-4) contains supplementary material, which is available to authorized users.

Background: Gliomas are aggressive tumors with various molecular and clinical characteristics and exhibit strongly resistance to radio-chemotherapy. Programmed cell death 1 ligand 2 (PD-L2) is a cell surface protein, which was reported in many cancers, modulating cancer-associated immune responses, while the role of PD-L2 in gliomas remained unclear. Herein, we aimed to investigate the biological behaviors and clinical prognostic values of PD-L2 in gliomas. Methods: Totally, we enrolled RNA sequencing data of 325 glioma samples from Chinese Glioma Genome Atlas (CGGA) as training cohort and RNA expression data of 1032 samples from The Cancer Genome Atlas (TCGA) dataset as validation cohort in this research. Then, the clinical and molecular characteristics, and the prognostic value of PD-L2 were analyzed. Results: We found that PD-L2 expression level was significantly upregulated in higher grade glioma and IDH wild-type glioma. Receiver Operating Characteristic (ROC) analysis revealed that PD-L2 was a potential indicator of mesenchymal subtype. PD-L2 exhibited tight relationship with immune response and immune-modulating process in glioma. Moreover, PD-L2 expression level could predict unfavorable prognoses of patients independent of age, grade, IDH status and 1p/19q status. Conclusions: Our study revealed that PD-L2 was closely related with inflammation and immune response. Patients with lower PD-L2 expression level tended to experience improved survival. Targeting PD-L2 may become a valuable approach for the treatment of gliomas in clinical practice.

Background This study aimed to investigate and analyze the status of internal quality control (IQC) for BNP and NT-proBNP from 2014 to 2017, so as to have an integral understanding of quality performance of measurement in clinical laboratories in China. Methods The 4-year IQC information for BNP and NT-proBNP of participant laboratories were collected through EQA reporting system. Percentages of laboratories meeting different quality requirements (pass rates) for current coefficient of variations (CVs) were calculated afterwards. Further analysis for current CVs and pass rates among different years and measurement systems were conducted. Finally, we analyzed and summarized IQC practice and its changes in 4 years. Results The current CVs for BNP and NT-proBNP have decreased significantly from 2014 to 2017, and pass rates both presented significant increasing trends. NT-proBNP had higher pass rates than BNP regardless of 1/3TEa or 1/4TEa specification was used. The main measurement systems for two analytes were different. For NT-proBNP, current CVs of Roche has decreased significantly among 4 years and were significantly lower than Radiometer and BioMerieux in 2015. Current CVs of Abbott also had decreasing tendency for BNP. Analysis of IQC practice indicated that control rules and IQC frequency had made great progress in 4 years. Conclusion The imprecision performance of measurement of BNP and NT-proBNP has improved with decreasing of current CVs and increasing of pass rates in 4 years. However, it still needs continual improvement. Clinical laboratories in China should take active actions to promote performance of BNP and NT-proBNP measurement.

Background Serum uric acid is a critical clinical indicator, and results without equivalence among laboratories cause troubles for disease diagnosis and patient management. External quality assessment (EQA) is a common tool for enhancing harmonization/standardization, therefore, the National Center for Clinical Laboratories in China has initiated a category 1 EQA for serum uric acid measurement since 2010 for evaluating its process of standardization. Methods Commutable EQA samples with target values assigned by reference measurement procedures were sent to participant laboratories. Both concentrations were measured 15 times in 3 days then means and intra-laboratory coefficient of variations (CVs) were reported. Biological variation criteria were used for analysis with CLIA88 criteria as a comparison. Results A total of 1250 laboratories participated in EQA programs from 2010 to 2017, pass rates calculated according to desirable specifications in biological variation database were on a rise overall and inter-laboratory mean bias and CVs were on a decrease. Homogeneous systems showed better inter-laboratory CVs and pass rates than heterogeneous systems. For the mostly used measurement systems; Abbott, Beckman, Roche Modular, Siemens and Hitachi showed desirable performances other than Roche Cobas, according to biological variation criteria. Conclusions Our study provides reliable information on the standardization of measurement procedures for serum uric acid for manufacturers and laboratories. Further improvements for standardization are still needed to make laboratories more patient-centered.

BackgroundAlkaline phosphatase (ALP) is critical for various diseases. The International Federation of Clinical Chemistry and Laboratory Medicine had recommended the new reference procedure in 2011, but many manufacturers did not trace results to the higher procedure. Since 2012, the National Center for Clinical Laboratories (NCCL) in China has organized the trueness verification program (TV) with commutable materials. The present study summarizes the 4-year TV program to give an overview of the measurement standardization for ALP results.MethodsCommutable serum-based materials with different concentrations were prepared and sent to participating laboratories. The target values were assigned by the reference lab network.ResultsThe analytical performance was evaluated according to three indexes: trueness (bias), imprecision (CV) and accuracy (total error [TE]). The number of participating laboratories increased from 115 in 2012 to 287 in 2016. The pass rates of precision for homogeneous and heterogeneous systems were all above 85% over the 4 years; however, the pass rates of bias were much lower (ConclusionsThe PT/EQA program with commutable materials can be used to assess the trueness against target values assigned by reference procedures. For ALP, homogeneous systems did not perform better than heterogeneous systems. The bias for ALP performance was notable and was the main obstacle to its standardization in China.

Gliomas are characterized by a malignant phenotype with proliferation, cell cycle arrest and invasion. To explore the biological consequences of epigenetically regulated miRNAs, we performed a microarray-based screening (whose expression was affected by 5-AZA treatment) followed by bisulfite sequencing validation. We found that miR-134 as an epigenetically regulated suppressor gene with prognostic value in gliomas. MicroRNA-134 was downregulated in high-grade gliomas, especially in GBM samples. Functional studies in vitro and in vivo in mouse models showed that overexpression of miR-134 was sufficient to reduce cell cycle arrest, cell proliferation and invasion. Target analysis and functional assays correlated the malignant phenotype with miR-134 target gene KRAS, an established upstream regulator of ERK and AKT pathways. Overall, our results highlighted a role for miR-134 in explaining the malignant phenotype of gliomas and suggested its relevance as a target to develop for early diagnostics and therapy.

The blood‑brain barrier (BBB) constitutes an efficient organization of tight junctions that limits the delivery of tumor to the brain. The principal tight junction protein in BBB is claudin‑5 (CLDN5), but its mechanism of action remains largely unknown. Long non‑coding RNAs (lncRNAs) are aberrantly expressed in many cancers, some lncRNAs play key roles in regulating BBB permeability and are involved in tumor brain metastasis. In particular, lncRNAs can function as competing endogenous RNAs (ceRNAs). Herein, we investigated whether ceRNA dysregulation is associated with alterations of the level of CLDN5 in human brain vascular endothelial hCMEC/D3 cells. The Affymetrix Human Transcriptome Array 2.0 and Affymetrix GeneChip miRNA 4.0 Array were used to detect the expression levels of 2,578 miRNAs, 22,829 lncRNAs, and 44,699 mRNAs in pLL3.7‑CLDN5‑transfected and pLL3.7 control hCMEC/D3 cells. The distinctly expressed miRNAs, lncRNAs, and mRNAs were subjected to construction of miRNA‑lncRNA‑mRNA interaction network. A total of 41 miRNAs, 954 lncRNAs, and 222 mRNAs were found to be differentially expressed between the CLDN5‑overexpressing and control group. 148 lncRNA acting as ceRNAs were identified based on the miRNA‑lncRNA‑mRNA interaction network. The function of differential mRNA in the network was determined by GO and pathway analysis. The potential roles of the 27 ceRNAs were revealed, the possible biology functions of these regulatory ceRNAs mainly included tight junction, focal adhesion, cell‑cell adhesion, cell growth and apoptosis. The identified sets of miRNAs, lncRNAs and mRNAs specific to CLDN5‑overexpressing hCMEC/D3 cells were verified by quantitative real‑time RT‑PCR experiment. Our study predicts the biological functions of a multitude of ceRNAs associated with the alteration of CLDN5 in brain vascular endothelial cells. Our data suggest that these dysregulated ceRNAs, in conjunction with the high CLDN5 levels, could serve as useful targets of prevention of brain metastasis formation. Further studies are warranted to determine the role of these ceRNAs in facilitating the function of CLDN5 in brain‑tumor barrier.

Background This study aimed to evaluate the performance of HbA1c measurement systems quantitatively with sigma metric and promote the quality improvement. Methods 1066 laboratories enrolled external quality assessment (EQA) and reported their internal quality control (IQC) data for HbA1c in 2017 were included in this study. The bias and coefficient of variation were collected from EQA and IQC program designed by the National Center for Clinical Laboratory. The σ values was used to assess the performance of HbA1c, and combine Westgard Sigma Rules™ to select proper quality control rules in every laboratory. Results Totally, There were 388,190,185,100,59,55,89 participants used Bia-Rad HPLC, TOSOH G7/G8, ARKRAY HA8160/8180, HUIZHONG MQ-2000/2000PT, Roche, Primus HPLC and “Others” system, respectively. “HUIZHONG MQ-2000/2000PT” group had the smallest variation of bias. 56.19% (599/1066) of laboratories met bias criterion and 55.63% (593/1066) satisfied imprecision criterion. However, sigma metrics indicated that 80.21% (855/1066) of 1066 laboratories needed to improve their performance of HbA1c. Moreover, “TOSOH G7/G8” group had the highest constituent ration of σ ≥ 3. Conclusions The analytical quality of HbA1c in most laboratories need improving. Laboratories should pay more attention on the performance of HbA1c, and EQA organizers in China should improve evaluation criteria and push standardization work for HbA1c.

Background: Alternative splicing (AS) is assumed to play important roles in the progression and prognosis of cancer. Currently, the comprehensive analysis and clinical relevance of AS in lower-grade diffuse gliomas has not been systematically addressed. Methods: AS data were obtained from the SpliceSeq database, and the clinical information of glioma patients was downloaded from The Cancer Genome Atlas (TCGA). Significance analysis of microarray (SAM) was performed to identify differential AS events among subgroups. Unsupervised consensus clustering was used for class discovery based on the comparison of AS events. Cox proportional hazards model was applied to develop an AS-related signature for prognostic prediction. Results: Totally, 48050 AS events of 10787 genes in lower-grade diffuse gliomas (n=515) were profiled. Subgroup-differential splicing analysis and functional annotation revealed that spliced genes were significantly enriched in numerous cancer-related biological phenotypes and signaling pathways. Consensus clustering of AS events identified three robust clusters of patients with distinguished molecular and prognostic features. Based on 445 AS events correlated with prognosis, an 18-AS event signature was built with Cox proportional hazards model. Kaplan-Meier analysis found that the acquired signature could differentiate the outcome of low and high-risk cases. Moreover, the signature, significantly associated with clinical and molecular features, could serve as an independent prognostic factor for lower-grade diffuse gliomas. Further analysis found that cases with high and low-risk scores showed distinct patterns of genomic alterations. Conclusion: Our results indicate that AS events are able to discriminate molecular subtypes and have prognostic impact in lower-grade diffuse gliomas. Funding Statement: This work was supported by National Natural Science Foundation of China (NSFC)/Research Grants Council (RGC) Joint Research Scheme (81761168038), the National Key Research and Development Plan (No. 2016YFC0902500) and National Natural Science Foundation of China (81672479, 81773208). Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: All of these tissue samples and clinicopathologic information were collected with informed consent. The study was conducted in accordance with the Declaration of Helsinki. Study protocols were approved by the ethics committees of the participating institutions.

Glioma is the most common primary brain tumor with various genetic alterations; among which, IDH mutation is the most common mutation and plays an important role in glioma early development, especially in lower grade glioma (LGG, WHO II-III). Previous studies have found that IDH mutation is tightly associated with extensive methylation across whole genome in glioma. To further investigate the role of IDH, we obtained methylation data of 777 samples from CGGA (Chinese Glioma Genome Atlas) and TCGA (The Cancer Genome Atlas) with IDH mutation status available. A package compiled under R language called Tspair was used as the main analytic tool to find potential probes that were significantly affected by IDH mutation. As a result, we found one pair of probes, cg06940792 and cg26025891, which was capable of predicting IDH mutation status precisely. The hypermethylated probe was cg06940792, designed in the promoter region of MEGF10, while the hypomethylated probe was cg26025891, designed in the promoter region of PSTPIP1. Survival analysis proved that hypermethylation or low expression of MEGF10 indicated a favorable prognosis in 983 glioma samples. Moreover, gene ontology analysis demonstrated that MEGF10 was associated with cell migration, cell proliferation, and regulation of apoptosis in glioma. All findings above can be validated in three other independent cohorts. In a word, our results suggested that methylation level and mRNA expression of MEGF10 in glioma were not only correlated with IDH mutation but also associated with clinical outcome of patients, providing potential guide for future dissection of IDH role in glioma.

Background: This study identifies optimal radiomic machine-learning classifiers to differentiate glioblastomas (GBM) from solitary brain metastases (MET), the most common neoplasms in adults, preoperatively. Methods: Four hundred and twelve patients with solitary brain tumors (242 with GBM and 170 with solitary brain MET) were divided into training (n =227) and validation (n =185) sets. Extraction of radiomic features from preoperative magnetic resonance images of each patient was accomplished with PyRadiomics software. Twelve feature selection methods and seven classification methods were evaluated to construct optimal radiomic machine-learning classifiers in the training set that were subsequently evaluated in a validation set. The role of the classifiers in differential diagnosis was evaluated using the mean area under the curve (AUC) and relative standard deviation in percentile (RSD). Findings:In the training set, thirteen classifiers had favorable predictive performances (AUC≥0.95 and RSD ≤6). In the validation set, receiver operating characteristic (ROC) curve analysis revealed that support vector machines (SVM) least absolute shrinkage and selection operator (LASSO) (AUC, 0.90) classifiers had the highest prediction efficacy, followed by Adaboost (ADa) LASSO (AUC, 0.89), Multi-Layer Perceptron (MLP) LASSO (AUC, 0.87), and random forest (RF) LASSO (AUC, 0.87). Furthermore, the clinical performance of these radiomic machine-learning classifiers were superior to neuroradiologists in accuracy, sensitivity, and specificity. Interpretation: By employing radiomic machine-learning technology, optimal machine-learning classifiers were identified for differentiating GBM from solitary brain MET preoperatively, which could significantly augment therapeutic strategies. Funding Statement: We acknowledge financial support from the National Natural Science Foundation of China (No. 81601452), and Key laboratory of functional and clinical translational medicine, Fujian province university(JNYLC1808) Declaration of Interests: The author declares no competing interest. Ethics Approval Statement: This study was approved by the ethics committee of Beijing Tiantan Hospital.

Background Chloride is the main anion in human body. A simple and accurate method for serum chloride measurement was developed using ion chromatography (IC) in China. Methods In the measurement, serum samples were diluted 500 times with water, filtered and injected into ion chromatography column. A mixed eluent (2 mmol/L CO32- + 12 mmol/L OH-) was used and peak area signal was collected. Five calibrators made from Standard Reference Material (SRM) 919b were used in the bracketing method. The IC method was applied as the comparative method and six ion selective electrode (ISE) measurement systems were evaluated using 60 individual patient serums. Results The IC method was proven to be accurate. The precision was 0.18% - 0.30%, the recovery was 99.66% - 100.60%, the bias was 0.19% to -0.06%, and the related expanded uncertainty was 0.775% (k = 2). The precisions of the ISE systems were smaller than the 0.9% tolerable CV except for the Beckman DXC (0.91% - 1.16%). In comparison, the results of linear regression analysis showed that the correlation coefficients were 0.9876 to 0.9979. For all systems, the range of mean biases was -5.96 - 1.48 mmol/L (-5.57% - 1.36%); the expected biases at the medical decision levels were -4.97% - 0.84% at 90 mmol/L and -6.02% - 1.76% at 120 mmol/L. All biases of the Beckman AU met the requirement of within ± 1.5%. Conclusions The IC measurement method is proven to be of high precision and trueness, and the quality of routine ISE measurement of serum chloride still needs significant improvement. The establishment of the IC method can improve the measurement quality and promote its standardization process in China.

Oligodendrogliomas (ODs) are the second most common malignant brain tumor and exhibit characteristic co-deletion of chromosomal arms 1p and 19q (co-deletion 1p/19q), which is associated with down-regulation of tumor suppressors. However, co-deletion 1p/19q indicates a favorable prognosis that cannot be explained by the down-regulation of tumor suppressors. In the present study, we determined that co-deletion 1p/19q was associated with reduced Ki-67 protein level based on analysis of 354 ODs. To identify genes associated with reduced Ki-67 and a favorable prognosis of codeletion 1p/19q, we analyzed 96 ODs with RNA-sequencing and 136 ODs and 4 normal brain tissue samples with RNA microarrays. We thus identified seven genes within chromosomal arms 1p/19q with significantly reduced expression in samples with co-deletion of 1p/19q compared to samples with intact 1p/19q. A significant positive correlation was observed between these candidate genes and Ki-67 expression based on analysis of mRNA expression in 305 gliomas and 5 normal brain tissue samples. Survival analysis confirmed the prognostic value of these candidate genes. This finding suggests that these genes within chromosomal arms 1p/19q are associated with low Ki-67 and a favorable prognosis in ODs with co-deletion 1p/19q and provides novel therapeutic targets.

The clinical prognosis of patients with glioma is determined by tumor grades, but tumors of different subtypes with equal malignancy grade usually have different prognosis that is largely determined by genetic abnormalities. Oligodendrogliomas (ODs) are the second most common type of gliomas. In this study, integrative analyses found that distribution of TCGA transcriptomic subtypes was associated with grade progression in ODs. To identify critical gene(s) associated with tumor grades and TCGA subtypes, we analyzed 34 normal brain tissue (NBT), 146 WHO grade II and 130 grade III ODs by microarray and RNA sequencing, and identified a co-expression network of six genes (AURKA, NDC80, CENPK, KIAA0101, TIMELESS and MELK) that was associated with tumor grades and TCGA subtypes as well as Ki-67 expression. Validation of the six genes was performed by qPCR in additional 28 ODs. Importantly, these genes also were validated in four high-grade recurrent gliomas and the initial lower-grade gliomas resected from the same patients. Finally, the RNA data on two genes with the highest discrimination potential (AURKA and NDC80) and Ki-67 were validated on an independent cohort (5 NBTs and 86 ODs) by immunohistochemistry. Knockdown of AURKA and NDC80 by siRNAs suppressed Ki-67 expression and proliferation of gliomas cells. Survival analysis showed that high expression of the six genes corporately indicated a poor survival outcome. Correlation and protein interaction analysis provided further evidence for this co-expression network. These data suggest that the co-expression of the six mitosis-regulating genes was associated with malignant progression and prognosis in ODs.

Six sigma provides an objective and quantitative methodology to describe the laboratory testing performance. In this study, we conducted a national trueness verification scheme with fresh frozen serum (FFS) for serum creatinine to evaluate its performance in China.Two different concentration levels of FFS, targeted with reference method, were sent to 98 laboratories in China. Imprecision and bias of the measurement procedure were calculated for each participant to further evaluate the sigma value. Quality goal index (QGI) analysis was used to investigate the reason of unacceptable performance for laboratories with σ3.Our study indicated that the sample with high concentration of creatinine had preferable sigma values. For the enzymatic method, 7.0% (5/71) to 45.1% (32/71) of the laboratories need to improve their measurement procedures (σ3). And for the Jaffe method, the percentages were from 11.5% (3/26) to 73.1% (19/26). QGI analysis suggested that most of the laboratories (62.5% for the enzymatic method and 68.4% for the Jaffe method) should make an effort to improve the trueness (QGI 1.2). Only 3.1-5.3% of the laboratories should improve both of the precision and trueness.Sigma metric analysis of the serum creatinine assays is disappointing, which was mainly due to the unacceptable analytical bias according to the QGI analysis. Further effort is needed to enhance the trueness of the creatinine measurement.

Anaplastic gliomas are characterized by variable clinical and genetic features, but there are few studies focusing on the substratification of anaplastic gliomas. To identify a more objective and applicable classification of anaplastic gliomas, we analyzed whole genome mRNA expression profiling of four independent datasets. Univariate Cox regression, linear risk score formula and receiver operating characteristic (ROC) curve were applied to derive a gene signature with best prognostic performance. The corresponding clinical and molecular information were further analyzed for interpretation of the different prognosis and the independence of the signature. Gene ontology (GO), Gene Set Variation Analysis (GSVA) and Gene Set Enrichment Analysis (GSEA) were performed for functional annotation of the differences. We found a three-gene signature, by applying which, the anaplastic gliomas could be divided into low risk and high risk groups. The two groups showed a high concordance with grade II and grade IV gliomas, respectively. The high risk group was more aggressive and complex. The three-gene signature showed diagnostic and prognostic value in anaplastic gliomas.

Although O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation status is an important marker for glioblastoma multiforme (GBM), there is considerable variability in the clinical outcome of patients with similar methylation profiles. The present study aimed to refine the prognostic and predictive value of MGMT promoter status in GBM by identifying a micro (mi)RNA risk signature. Data from The Cancer Genome Atlas was used for this study, with MGMT promoter-methylated samples randomly divided into training and internal validation sets. Data from The Chinese Glioma Genome Atlas was used for independent validation. A five miRNA-based risk signature was established for MGMT promoter-methylated GBM to distinguish cases as high- or low-risk with distinct prognoses, which was confirmed using internal and external validation sets. Importantly, the prognostic value of the signature was significant in different cohorts stratified by clinicopathologic factors and alkylating chemotherapy, and a multivariate Cox analysis found it to be an independent prognostic marker along with age and chemotherapy. Based on these three factors, we developed a quantitative model with greater accuracy for predicting the 1-year survival of patients with MGMT promoter-methylated GBM. These results indicate that the five-miRNA signature is an independent risk predictor for GBM with MGMT promoter methylation and can be used to identify patients at high risk of unfavorable outcome and resistant to alkylating chemotherapy, underscoring its potential for personalized GBM management.

// Xuan Zhou 1, 2, * , Yu Ren 3, * , Jing Zhang 4, * , Chuanbao Zhang 5, * , Kailiang Zhang 1, 6 , Lei Han 1 , Lingping Kong 2 , Jianwei Wei 1 , Luyue Chen 1 , Jingxuan Yang 6 , Qixue Wang 1 , Jianning Zhang 1 , Yuqi Yang 7 , Tao Jiang 5 , Min Li 2, 6 , Chunsheng Kang 1 1 Department of Neurosurgery, Tianjin Medical University General Hospital, Laboratory of Neuro-oncology, Tianjin Neurological Institute, Tianjin 300052, China 2 The Department of Otorhinolaryngology and Maxillofacial Oncology, Tianjin Medical University Cancer Institute & Hospital, Key Laboratory of Cancer Prevention and Therapy, Tianjin Cancer Institute, National Clinical Research Center of Cancer, Tianjin 300060, China 3 Tianjin Research Center of Basic Medical Science, Tianjin Medical University, Tianjin 300070, China 4 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China 5 Beijing Neurosurgical Institute, Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China 6 Department of Medicine, Department of Surgery, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104,USA 7 Department of Pharmacology, Tianjin Medical University, Tianjin 300070, China * These authors have contributed equally to this work Correspondence to: Chunsheng Kang, e-mail: kang97061@gmail.com Tao Jiang, e-mail: jiangtao369@sohu.com Min Li, e-mail: Min-Li@ouhsc.edu Keywords: HOTAIR, NLK (Nemo-like kinase), β-catenin, PRC2 (Polycomb repressive complex 2), Glioblastoma Received: December 01, 2014 Accepted: January 28, 2015 Published: March 21, 2015 ABSTRACT HOTAIR is a negative prognostic factor and is overexpressed in multiple human cancers including glioblastoma multiform (GBM). Survival analysis of Chinese Glioma Genome Atlas (CGGA) patient data indicated that high HOTAIR expression was associated with poor outcome in GBM patients. NLK (Nemo-like kinase), a negative regulator of the β-catenin pathway, was negatively correlated with HOTAIR expression. When the β-catenin pathway was inhibited, GBM cells became susceptible to cell cycle arrest and inhibition of invasion. Introduction of the HOTAIR 5’ domain in human glioma-derived astrocytoma induced β-catenin. An intracranial animal model was used to confirm that HOTAIR depletion inhibited GBM cell migration/invasion. In the orthotopic model, HOTAIR was required for GBM formation in vivo . In summary, HOTAIR is a potential therapeutic target in GBM.

Chromosomal instability is a hallmark of human cancers and is closely linked to tumorigenesis. The prognostic value of molecular signatures of chromosomal instability (CIN) has been validated in various cancers. However, few studies have examined the relationship between CIN and glioma. Histone deacetylases (HDACs) regulate chromosome structure and are linked to the loss of genomic integrity in cancer cells. In this study, the prognostic value of HDAC4 expression and its association with markers of CIN were investigated by analyzing data from our own and four other large sample databases. The results showed that HDAC4 expression is downregulated in high- as compared to low-grade glioma and is associated with a favorable clinical outcome. HDAC4 expression and CIN were closely related in glioma from both functional and statistical standpoints. Moreover, the predictive value of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status—a widely used glioma marker—was refined by HDAC4 expression level, which was significantly related to CIN in our study. In conclusion, we propose that HDAC4 expression, a prognostic and CIN marker, enhances the predictive value of MGMT promoter methylation status for identifying patients who will most benefit from radiochemotherapy. Electronic supplementary material The online version of this article (doi:10.1007/s11060-014-1709-6) contains supplementary material, which is available to authorized users.