Astrocytomas Of Different Grades Research Articles

67P - Association between altered expression of key enzymes involved in kynurenine pathway with clinical outcome in patients with different grades of astrocytoma

67P - Association between altered expression of key enzymes involved in kynurenine pathway with clinical outcome in patients with different grades of astrocytoma

Alterations of the levels of primary antioxidant enzymes in different grades of human astrocytoma tissues

Malignant astrocytoma is the most commonly occurring brain tumour in humans. Oxidative stress is implicated in the development of cancers. Superoxide dismutase 2 (SOD2) was found to exert tumour suppressive effect in basic research, but increased SOD2 protein level was associated with higher aggressiveness of human astrocytomas. However, studies reporting alterations of antioxidant enzymes in human astrocytomas often employed less accurate methods or included different types of tumours. Here we analysed the mRNA levels, activities, and protein levels of primary antioxidant enzymes in control brain tissues and various grades of astrocytomas obtained from 40 patients. SOD1 expression, SOD1 activity, and SOD1 protein level were lower in Grade IV astrocytomas. SOD2 expression was lower in low-grade (Grades I and II) and Grade III astrocytomas than in controls, but SOD2 expression and SOD2 protein level were higher in Grade IV astrocytomas than in Grade III astrocytomas. Although there was no change in SOD2 activity and a lower activity of citrate synthase (CS), the MnSOD:CS ratio increased in Grade IV astrocytomas compared with controls and low-grade astrocytomas. Furthermore, SOD1 activity, CS activity, SOD1 expression, GPX4 expression, and GPX4 protein level were inversely correlated with the malignancy, whereas catalase activity, catalase protein, SOD2 protein level, and the SOD2:CS ratio were positively correlated with the degree of malignancy. Lower SOD2:CS ratio was associated with poor outcomes for Grade IV astrocytomas. This is the first study to quantify changes of various primary antioxidant enzymes in different grades of astrocytomas at different levels concurrently in human astrocytomas.

ACTB and SDHA Are Suitable Endogenous Reference Genes for Gene Expression Studies in Human Astrocytomas Using Quantitative RT-PCR.

Background:Quantitative real-time reverse-transcription polymerase chain reaction is frequently used as research tool in experimental oncology. There are some studies of valid endogenous control genes in the field of human glioma research, which, however, only focus on the comparison between normal brain with tumor tissue and malignant transformation toward secondary glioblastomas. Aim of this study was to validate a more general reference gene also suitable for pre- and posttreatment analysis and other evaluations (eg, primary vs secondary glioblastoma).Methods:This quantitative polymerase chain reaction analysis was performed to test a panel of the 6 most suitable reference genes from other studies representing different physiological pathways (ACTB, GAPDH, POLR2A, RPL13A, SDHA, and TBP) in all common glioma groups, namely: diffuse astrocytoma World Health Organization II, anaplastic astrocytoma World Health Organization III, secondary glioblastoma World Health Organization IV with and without chemotherapy, primary glioblastoma, recurrent glioblastoma, and gliomas before and after radiation. Expression stability was tested during the longitudinal course of the disease in 8 single patients.Results:Evaluation of the expression levels of the 6 target genes showed that ACTB, GAPDH, and RPL13A show higher expression compared to SDHA, POLR2A, and TBP. ACTB, GAPDH, and RPL13A showed different expression levels between astrozytoma grade II and primary glioblastoma. Except for this difference, the candidate genes were not differentially expressed between primary and secondary glioblastomas and between the World Health Organization tumor grades. Furthermore, they remained stable before and after radiotherapy and/or chemotherapy. Therefore, they are adequate references for glioblastoma gene expression studies. The comparison of all tested genes resulted in SDHA and ACTB as most stable reference genes determined by the NormFinder software. Our data revealed lowest intragroup variation in the SDHA, highest in the RPL13A gene.Conclusions:All tested genes may be recommended as universal reference genes for data normalization in gene expression studies under different treatment regimens both in primary glioblastomas and astrocytomas of different grades (World Health Organization grades II-IV), respectively. In summary, ACTB and SDHA exhibited the best stability values and showed the lowest intergroup expression variability.

Correlation between epidermal growth factor receptor and MIB-1 index in different grades of astrocytomas

Correlation between epidermal growth factor receptor and MIB-1 index in different grades of astrocytomas

CD74 as a Potential Prognostic Marker in Glioblastoma

CD74 is a cell membrane receptor for macrophage migration inhibitory factor (MIF) that is believed to play a role in tumor progression and metastasis. Astrocytoma is the most common type of brain cancers, which are classified by the World Health Organization (WHO) into four grades according to the degree of malignancy. In this study, we investigated MIF and CD74 gene expression levels in a large cohort of publicly available RNA sequencing data and evaluated the correlation between their protein expression with clinicopathological features as well as prognosis in an independent cohort of astrocytoma samples. Seventy-three patients with different grades of astrocytoma (WHO grade II diffuse astrocytoma, grade III anaplastic astrocytoma, and grade IV glioblastoma) were used for protein expression in this study. The expression of CD74 was assayed by immunohistochemistry and was significantly higher in high-grade glioblastoma than grade II and grade III astrocytoma (P < 0.004). Upregulated CD74 expression was frequently found in older patients (≥ 62 years). The expression of CD74 is correlated with overall survival (P = 0.05). Multivariate analysis shows that the CD74 expression is associated with poor prognosis in the presence of MIF expression (P = 0.039) and that CD74 is not an independent prognostic marker in glioblastoma. However, similar studies on larger sample size are required to make a definite conclusion on the role of CD74 in prognosis in glioblastoma.

Evaluation of Akt and RICTOR Expression Levels in Astrocytomas of All Grades.

The mammalian target of rapamycin (mTOR) binds to several protein partners and forms two complexes, termed mTOR complexes 1 and 2 (mTORC1/2), that differ in components, substrates, and regulation. mTORC2 contains the protein Rapamycin-insensitive companion of mTOR (RICTOR); phosphorylates kinases of the AGC family, such as Akt; and controls the cytoskeleton. Even though the regulation of mTORC2 activity remains poorly understood, the hyperactivation of this signaling pathway has been shown to contribute to the oncogenic properties of gliomas in experimental models. In this work, we evaluated expression and phosphorylation of Akt, and expression of RICTOR and Ki-67 in 195 human astrocytomas of different grades (38 cases of grade I, 49 grade II, 15 grade III, and 93 grade IV) and 30 normal brains. Expression and phosphorylation of Akt increased with histological grade and correlated with a worse overall survival in glioblastomas (GBMs). RICTOR was overexpressed in grade I and II astrocytomas and demonstrated a shift to nuclear localization in GBMs. Nuclear RICTOR was associated to increased proliferation in GBMs. Our results point to an increase in total and phosphorylated Akt in high-grade gliomas and to a possible role of RICTOR in proliferations of high-grade GBM cells.

Ki-67 immunostaining in astrocytomas: Association with histopathological grade - A South Indian study.

Background:Astrocytomas are the most common primary tumor of the central nervous system. The distinction between different tumor grades can be tested despite criteria given by the World Health Organization (WHO). Ki-67 is a potent biological marker used in grading of astrocytomas, which estimates growth of the neoplasm quantitatively and will help in predicting prognosis accurately.Objectives:The aim of this was to study the proliferative activity using Ki-67 immunostaining and to assess the relationship of Ki-67 staining with the histopathological grading of astrocytomas.Patients and Methods:Thirty cases of histologically proven astrocytomas were studied. The histopathological grade was assessed using the 2007 WHO criteria. Immunohistochemistry for Ki-67 was done on paraffin-embedded wax sections. P < 0.05 was considered statistically significant.Results:Thirty cases of astrocytomas studied showed a male preponderance (M:F = 1.72:1) with a mean age of 48.1 years. Of these, Grade I, (n = 1, 3.33%), Grade II, (n = 7, 23.3%), Grade III (n = 6, 20%), and Grade IV (n = 16, 53.3%) astrocytomas were analyzed. The mean Ki-67 labeling index (LI) in Grades I, II, III, and IV was 0.02, 0.81, 9.14, and 17.81, respectively. Statistically significant difference was seen in the Ki-67 LI of low-grade (Grade II) and high-grade astrocytomas (Grades III and IV). There was concordance between histopathological grading and Ki-67 LI in 27 (90%) and discordance in 3 (10%) cases.Conclusion:Ki-67 LI varies considerably in different grades of astrocytomas and considerable overlaps can be observed between them. It can be of great help in situations where there is a lack of correlation between clinical parameters and histopathological diagnosis. Determination of Ki-67 LI should constitute a part of routine investigations in patients with astrocytomas.

Overexpression of vascular adhesion protein-1 is associated with poor prognosis of astrocytomas.

Vascular adhesion protein-1 (VAP-1) is one of the endothelial adhesion molecules that is believed to play a role in tumor progression and metastasis, supporting cancer cell extravasation. Very few studies have been performed on analyzing the contribution of VAP-1 in brain tumor. Astrocytomas are the most common type of brain tumors, which are classified by World Health Organization (WHO) into four grades according to the degree of malignancy. This study was designed to investigate VAP-1 expression level in different astrocytoma grades and its correlation with clinicopathological features as well as prognosis of astrocytoma patients. Eighty-seven patients with different grades of astrocytoma (WHO Grade I-Grade IV) were enrolled in this study. The expression of VAP-1 was assayed by immunohistochemistry. The correlation between VAP-1 expression and clinicopathological features was evaluated by Chi-square test, and overall survival was analyzed by Kaplan-Meier method. Cox regression analysis was applied to analyze the independent influence of each parameter on overall survival. The expression level of VAP-1 was significantly higher in diffuse astrocytoma than those of pilocytic astrocytoma (p<0.0001). In the subgroup analysis, upregulated VAP-1 expression was frequently found in older age patients (≥50years). The VAP-1 expression was found to be significantly correlated with the overall survival (p=0.0002). There was a statistical correlation between VAP-1(high) tumors in diffuse astrocytoma and VAP-1(low) tumors in pilocytic astrocytoma (p<0.0001). Multivariate Cox analysis indicated VAP-1 was an independent predictive marker for poorer prognosis (p=0.0036). Therefore, VAP-1 could be a promising prognostic biomarker in astrocytoma.

Expression of DNA methyltransferases 1 and 3B correlates with EZH2 and this 3-marker epigenetic signature predicts outcome in glioblastomas

This study aims to analyze expression of EZH2 and DNA-methyltransferases (DNMT1, 3A and 3B) in astrocytic tumors and investigate their link as well as their correlation with survival, especially in GBMs. Expression of EZH2 and DNMTs (DNMT1, DNMT3A and DNMT3B) in different grades of astrocytomas (n=93) was assessed by qRT-PCR and immunohistochemistry. GBM-U87MG cell line was used for functional studies. Strong immunopositivity (LI≥25%) for EZH2, DNMT1 and DNMT3B was detected in 52%, 56% and 64% cases of GBMs respectively, which was significantly higher as compared to Grade II/III cases. Similarly, their median fold change of mRNA expression was also significantly higher in GBMs. There was also a significant positive correlation between DNMT1/DNMT3B and EZH2 mRNA and protein expression, which was in concordance with TCGA data set. Inhibition of DNMTs in cell line by Azacytidine resulted in down-regulation of EZH2, while knock-down of EZH2 by siRNA was not associated with any significant alteration of DNMTs, indicating that EZH2 expression in GBMs is possibly regulated by DNMTs, but not the reverse. Strong immunopositivity for EZH2, DNMT1 and DNMT3B were individually associated with significantly shorter survival and showed no correlation with IDH1 mutation status. In addition, the combination of these 3 markers represented an independent prognostic signature with cases having weak/negative expression of all 3 markers being associated with best prognosis. For the first time, the present study describes an epigenetic prognostic signature in GBMs based on immunohistochemical expression of EZH2, DNMT1 and 3B which can be used easily in routine neuropathology practice.

Isolation and characterization of novel RECK tumor suppressor gene splice variants.

Glioblastoma multiforme is the most common and lethal of the central nervous system glial-derived tumors. RECK suppresses tumor invasion by negatively regulating at least three members of the matrix metalloproteinase family: MMP-9, MMP-2, and MT1-MMP. A positive correlation has been observed between the abundance of RECK expression in tumor samples and a more favorable prognosis for patients with several types of tumors. In the present study, novel alternatively spliced variants of the RECK gene: RECK-B and RECK-I were isolated by RT-PCR and sequenced. The expression levels and profiles of these alternative RECK transcripts, as well as canonical RECK were determined in tissue samples of malignant astrocytomas of different grades and in a normal tissue RNA panel by qRT-PCR. Our results show that higher canonical RECK expression, accompanied by a higher canonical to alternative transcript expression ratio, positively correlates with higher overall survival rate after chemotherapeutic treatment of GBM patients. U87MG and T98G cells over-expressing the RECK-B alternative variant display higher anchorage-independent clonal growth and do not display modulation of, respectively, MMP-2 and MMP-9 expression. Our findings suggest that RECK transcript variants might have opposite roles in GBM biology and the ratio of their expression levels may be informative for the prognostic outcome of GBM patients.

Abstract 3047: Mitochondrial DNA copy variation and TFAM expression in astrocytoma

Abstract Mitochondrial dysfunction plays a role in determining the phenotype through bioenergetic depletion and increased production of ROS in several diseases, including cancer. We have previously demonstrated a relevant reduction of mitochondrial DNA (mtDNA) copy number in astrocytoma of different grades of malignancy, predominantly in grade IV-glioblastoma (GBM). We observed a stepwise increase of TFAM in parallel to the increase of malignancy, and TFAM expression was higher in GBM patients with overall survival longer than 24 months than less than 12 months. TFAM is codified in the nucleus and transported into mitochondria, where is essential for mtDNA replication, repair and protection by forming nucleoid complexes. To further understand the role of TFAM in astrocytoma progression we performed TFAM immunohistochestry in human astrocytoma samples, confocal analysis in GBM cell lines, in vitro functional assays after silencing TFAM by siRNA in two GBM cell lines (U87MG, A172), and high throughput RNA-Seq in Illumina platform for network analysis by Metacore. TFAM protein was detected in cytoplasm in granular pattern of low grade astrocytomas, and also in nuclei of malignant atsrocytomas. Such localization was also confirmed by confocal analysis in two GBM cell lines. TFAM-siRNA transfection decreased TFAM expression level &amp;gt;80% in D2, which persisted up to D7 with evident decrease of mtDNA copy number after 4 days, and maintained until 7 days. Interestingly, a decrease in migration but not in proliferation was observed in both cell lines after TFAM knockdown. Comparison of RNA-Seq of TFAM-siRNA to NTC- siRNA, with 20 million of reads in each experiment performed in duplicate, resulted in 1000 genes differentially expressed and among them 315 were detected in both cell lines. These genes were in pathways related to cell adhesion (integrin-mediated: p = 5.79e-5, histamine H1 receptor signaling in the interruption of cell barrier integrity: p = 3.98e-5, chemokines-mediated: p = 1.22e-5) and ECM remodeling (p = 9.17e-5), supporting the results of in vitro migration assay. 82 genes were downregulated together with TFAM in both cell lines. These genes were in pathways related to immune response IL15 signaling via JAK-STAT cascade involving MyD88-independent toll-like receptor signaling pathway involving ADAM17 and STAT2 (p = 1.89e-5), regulation of cytoskeleton proteins (p = 1.65e-2), hypoxia-induced EMT in cancer and fibrosis (p = 3.37e-2), polyamine and arginine metabolism (p = 2.73e-2, p = 4.65e-2) and as expected, regulation of apoptotic process (p = 9.50e-21). The role of TFAM in cytoskeleton and ECM remodeling may worth additional studies. Also, TFAM in extracellular space, as released by necrotic cells, may potentiate mitochondrial N-formyl peptide-induced secretion of chemokines and activate inflammatory responses, which may open another strategy to further understand TFAM role in tumorigeniesis. Citation Format: Suely K. Marie, Roseli Silva, Antonio Lerario, Miyuki Uno, Sueli Mieko Oba-Shinjo. Mitochondrial DNA copy variation and TFAM expression in astrocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2015-3047

Expression profile of parkin isoforms in human gliomas.

Mutations of parkin gene are not restricted to familial forms of Parkinsonism but they also occur in a wide variety of malignancies including gliomas. Parkin over-expression reduces glioma cells proliferation and analysis of its expression is predictive for the survival outcome of patients with glioma. To date have been identified 21 parkin alternative splice variants. However, most of the studies have focused their attention exclusively on full-length protein. In the present study, the expression profile of parkin isoforms in different grades of astrocytomas was analyzed for the first time, in order to evaluate their involvement in this malignancy. Furthermore, to investigate their role in cellular processes, their expression in three glioblastoma cell lines was analyzed following treatment with the proteasome inhibitor MG132, or induction of mitophagy with CCCP, or after serum deprivation. Results suggested that H20, H1 and H5 isoforms are always expressed in tumors both in vivo and in vitro models. Therefore, these isoforms might be used as specific biomarkers to develop a prognostic tool for brain tumors.

LOX expression and functional analysis in astrocytomas and impact of IDH1 mutation.

Lysyl oxidase (LOX) is involved in vital biological processes such as cell motility, cell signaling and gene regulation. Deregulation of this protein can contribute to tumor formation and progression. Although it is known that LOX is involved in invasion, proliferation and tumor migration in other types of tumors, studies of LOX in astrocytomas of different grades are scarce. The purpose of our study was to characterize LOX, BMP1 and HIF1A expression by real-time PCR in astrocytomas with WHO grades I to IV compared to non-neoplastic brain tissue. IDH1 mutational status was determined by PCR and sequencing. LOX protein expression was also analyzed by immunohistochemistry. LOX functional analyses were performed using siRNA knockdown and the specific inhibitor BAPN in two glioblastoma cell lines. The expression levels of LOX, BMP1 and HIF1A were correlated and analyzed according to IDH1 mutation status and to the clinical end-point of overall survival of glioblastoma patients. The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas. LOX protein expression also increased according to the degree of malignancy, with localization in the cytoplasm and nucleus and staining observed in endothelial cells. Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases. LOX knockdown and inhibition by BAPN in U87MG and A172 cell lines affected migration, invasion and soft agar colony formation. Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas. Furthermore, LOX expression is influenced by IDH1 mutational status. This work provides new insights for researchers aiming to design targeted therapies to control astrocytomas.

CRABP-II- and FABP5-independent responsiveness of human glioblastoma cells to all-trans retinoic acid.

Glioblastomas respond differently to all-trans retinoic acid (RA) for unknown reasons. Because CRABP-II and FABP5 mediate RA intracellular signaling respectively and lead to distinct biological consequences, their expression patterns in different grades of astrocytomas and the glioblastoma cells lines LN18, LN428 and U251 were examined to identify potential correlations with RA sensitivities. The response of glioblastoma cells to RA, decitabine or the FABP5 competitive inhibitor, BMS309403, was analyzed. CRABP-II and FABP5 were expressed to varying degrees by the 84-astrocytoma cases examined. Treatment of LN428, U251 and LN18 cells with RA failed to suppress their growth; however, U251 proliferation was inhibited by decitabine. The combination of decitabine and RA suppressed the growth of all three cell lines and induced significant apoptosis of LN428 and U251 cells. Both CRABP-II and FABP5 were transcribed in the three cell lines but FABP5 proteins were undetectable in U251 cells. The ratio of CRABP-II to FABP5 was not altered after RA, decitabine or RA and decitabine treatment and the resistance of cells to RA was not reversed by BMS309403 treatment. In conclusion, CRABP-II and FABP5 expression patterns are neither related to the tumor grades nor correlated with RA sensitivity. Additional molecular factors may be present that determines the sensitivity of glioblastoma cells to RA. Dicitabine may improve the sensitivity of glioblastoma cells to RA, however, its underlying mechanism and its in vivo feasibility need to be investigated.

HDACs expression in glioblastoma: an immunohistochemical study

Glioblastoma is the most common and lethal primary malignant brain tumor. Although standard treatments have been improving, the clinical outcome remains unacceptably poor. Several genetic alterations are supposed to be involved in the eti- ology of different grades of astrocytoma, including epimutations. Histone deacety - lases (HDACs) are involved in the post-translational modification on the lysines of histone tails. For this reason HDACs are recognized as promising targets for cancer treatment (1). In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global HDAC expression in gliomas and its possible correlation to the use of HDACis (2). Aim of our study was to evaluate with an immunohistochemi - cal and immunoblotting analyses the expression of different classes of HDACs (Class I: HDAC 1-2-3-8; class II: HDAC 4-6) in microdissected glioblastoma. Tumor sam - ples were taken from 14 patients (n.8 men and n.6 women) ranging in age from 43 to 74 years. HDAC1 and HDAC3 expression was not significantly different between the two proteins and was predominantely located at cytoplasmic level of cancer cells with different intensity of immureaction from mild to moderate whereas HDAC2 staining was localized to the nucleous of neoplastic cells. The pattern of HDAC4 immureactivity was always cytoplasmatic and showed a marked and diffuse increase of immunostaining in neoplastic areas. HDAC8 was always absent in cancer cells and the only positivity was located in the endothelial cells of the vessels. HDAC6 was often absent and, if present, showed a very low cytoplasmic immunopositivity in cancer cells. HDAC1, HDAC2 and HDAC3 levels were not significantly different in immunoblotting results; HDAC4 showed a marked increase while HDAC6 and HDAC8 expression was poor, confirming the IHC data. These previous results dem - onstrate a different pattern of HDAC expression and could suggest a more addressed therapeutical use of HDACis in glioblastoma.

Tissue thioredoxin reductase-1 expression in astrocytomas of different grades.

Thioredoxin (Trx) is a redox active protein that regulates several physiological and biochemical functions, such as growth, apoptosis and cellular defense. The function of Trx itself is regulated by thioredoxin reductase (TrxR). Studies performed in a variety of human primary tumors have shown that thioredoxin reductase 1 (TrxR1) is overexpressed in tumoral tissues compared with corresponding normal tissues. This study was designed to determine the expression of TrxR1 in astrocytoma tissues of different World Health Organization (WHO) grades (grade I-IV). The proliferative (Ki-67) and apoptotic indices of the specimens were also investigated for correlation analysis. Astrocytoma tissues were extracted from the histopathological specimens of 40 patients. These samples included seven histologically normal brain tissues that served as a control group and ten tumoral samples for each grade of astrocytoma (grade I-IV). The histologically normal brain tissues were obtained from the non-tumoral portions of the pathological specimens of grade I (2 cases), grade II (2 cases), grade III (2 cases) and grade IV (1 case) astrocytomas. TrxR1 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunostaining. The proliferative and apoptotic indices of the specimens were investigated by Ki-67 immunostaining and TUNEL assay, respectively. TrxR1 expression, as assessed by qRT-PCR, increased significantly with astrocytoma grade (p = 0.01). The immunostaining intensity of TrxR1 in grade IV astrocytomas was significantly greater than that in the control tissue and all other astrocytoma grades (p < 0.001). Similarly, immunostaining intensity of TrxR1 in the grade III astrocytomas was significantly greater than that in the control group and grade I astrocytomas (p < 0.001). All astrocytoma tissues showed more intense staining in ascending grades, but the differences between grade I and the control, grade II and the control, grades II and I, grades III and II were not statistically significant (p > 0.05). Ki-67 index values increased significant in accordance with grade progression (p = 0.01). The apoptotic index values were not significantly different in any group (p > 0.05); however, the differences between grade IV and the control and between grades IV and I were statistically significant (p < 0.05). Expression of TrxR1, as assessed by both qRT-PCR and immunostaining, correlated highly with both the astrocytoma grade and Ki-67 index.

IMMUNOHISTOCHEMICAL EXPRESSION OF CLUSTER OF DIFFERENTIATION-68(CD-68) AND KI-67 IN ASTROCYTOMAS

Astrocytoma is common primary malignant brain tumors which arise from immortalized astrocytes. In astrocytoma microglia density is higher than in normal brain, and microglia increase in number according to the grade of malignancyIntense CD68 staining was marginally significant prognosticator for shorter survival.Since proliferative activity is a reliable method to assess tumour biology. Ki-67/MIB-1 has proven to be of prognostic and diagnostic power in astrocytic tumours and shown to correlate with tumour grade. Aim: This study was conducted to detect immunohistochemical expression of CD-68 and Ki-67 in astrocytoma and assessing their relation to proliferative activity and prognosis of such tumors. Methods: Thirty representative cases; 30 cases of astrocytoma examined immunohistochemically using antibodies against CD-68 and Ki-67. Results: CD-68 was positive in 73.3% of astrocytoma cases and it was negative in 26.7%. LowKi-67LI was found in low grade astrocytoma and high Ki-67 LI was found in high grade astrocytoma. Conclusion: CD-68 and Ki-67 were the most useful IHC markers in the discrimination of different grades of astrocytoma and predict proliferative activity and prognosis of such tumors. Key words: Astrocytoma, Prognosis, CD-68, Ki-67.

CXCR7 and CXCR4 Expressions in Infiltrative Astrocytomas and Their Interactions with HIF1α Expression and IDH1 Mutation

The CXCR7, a new receptor for CXCL12 with higher affinity than CXCR4 has raised key issues on glioma cell migration. The aim of this study is to investigate the CXCR7 mRNA expression in diffuse astrocytomas tissues and to evaluate its interactions with CXCR4 and HIF1α expression and IDH1 mutation. CXCR7, CXCR4 and HIF1α mRNA expression were evaluated in 129 frozen samples of astrocytomas. IDH1 mutation status was analyzed with gene expressions, matched with clinicopathological parameters and overall survival time. Protein expression was analyzed by immunohistochemistry in different grades of astrocytoma and in glioma cell line (U87MG) by confocal microscopy. There was significant difference in the expression levels of the genes studied between astrocytomas and non-neoplasic (NN) controls (p < 0.001). AGII showed no significant correlation between CXCR7/HIF1α (p = 0.548); there was significant correlation between CXCR7/CXCR4 (p = 0.042) and CXCR7/IDH1 (p = 0.008). GBM showed significant correlations between CXCR7/CXCR4 (p = 0.002), CXCR7/IDH1 (p < 0.001) and CXCR7/HIF1α (p = 0.008). HIF1α overexpression was associated with higher expressions of CXCR7 (p = 0.01) and CXCR4 (p < 0.0001), while IDH1 mutation was associated with lower CXCR7 (p = 0.009) and CXCR4 (p = 0.0005) mRNA expressions. Protein expression increased with malignancy and in U87MG cell line was mainly localized in the cellular membrane. CXCR7 was overexpressed in astrocytoma and correlates with CXCR4 and IDH1 in AGII and CXCR4, IDH1 and HIF1α in GBM. Overexpression HIF1α was related with higher expressions of CXCR7 and CXCR4, otherwise IDH1 mutation related with lower expression of both genes. No association between CXCR7 and CXCR4 expression and survival data was related.

Expression of phosphatase and tensin homolog, epidermal growth factor receptor, and Ki-67 in astrocytoma: A prospective study in a tertiary care hospital

Background:Though the tumor grade is a key factor influencing the choice of therapies, particularly determining the use of adjuvant radiation and specific chemotherapy protocols, role of abnormality in phosphatase and tensin homolog (PTEN) expression and variation in epidermal growth factor receptor (EGFR) labeling index (LI) and Ki-67 LI in survival and clinical outcome have been studied by many researchers in the recent past.Aims:The aim was to evaluate the expression of PTEN, EGFR and Ki-67 in different grades of astrocytic tumors by means of immunohistochemistry and to judge their role in overall survival (OS).Materials and Methods:This study was conducted on 57 cases of different grades of astrocytomas. Expression of PTEN, EGFR and Ki-67 was assessed by immunohistochemistry on formalin fixed and paraffin-embedded sections and the OS was evaluated by Kaplan–Meier survival curves and log-rank test for 2.5 years from the date of primary resection.Results:Most of the tumors (59.6%; 34 cases out of 57) displayed WHO Grade IV features. Distribution of age, EGFR LI and Ki-67 LI expressed strong positive (≥0.5) correlation with the grade of tumors. However, the PTEN positivity was inversely related with the grade of the tumors. Lower WHO grades, lower values of Ki-67 LI, EGFR LI and PTEN positivity were associated with better survival.Conclusion:Expression of PTEN, EGFR LI and Ki-67 LI should be combined with the basic histopathological features including WHO grade to predict the prognosis and therapeutic outcome.

Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs

BackgroundThe genome is pervasively transcribed but most transcripts do not code for proteins, constituting non-protein-coding RNAs. Despite increasing numbers of functional reports of individual long non-coding RNAs (lncRNAs), assessing the extent of functionality among the non-coding transcriptional output of mammalian cells remains intricate. In the protein-coding world, transcripts differentially expressed in the context of processes essential for the survival of multicellular organisms have been instrumental in the discovery of functionally relevant proteins and their deregulation is frequently associated with diseases. We therefore systematically identified lncRNAs expressed differentially in response to oncologically relevant processes and cell-cycle, p53 and STAT3 pathways, using tiling arrays.ResultsWe found that up to 80% of the pathway-triggered transcriptional responses are non-coding. Among these we identified very large macroRNAs with pathway-specific expression patterns and demonstrated that these are likely continuous transcripts. MacroRNAs contain elements conserved in mammals and sauropsids, which in part exhibit conserved RNA secondary structure. Comparing evolutionary rates of a macroRNA to adjacent protein-coding genes suggests a local action of the transcript. Finally, in different grades of astrocytoma, a tumor disease unrelated to the initially used cell lines, macroRNAs are differentially expressed.ConclusionsIt has been shown previously that the majority of expressed non-ribosomal transcripts are non-coding. We now conclude that differential expression triggered by signaling pathways gives rise to a similar abundance of non-coding content. It is thus unlikely that the prevalence of non-coding transcripts in the cell is a trivial consequence of leaky or random transcription events.