Astrocytomas Of Different Grades Research Articles

Prognostic Value of Immunohistochemical Expression of MTAP and AKIP1 in IDH1 Mutant Astrocytoma.

Definite treatment for glioma is not exist, and with increased drug resistance, more effort should be paid to identify new prognostic biomarkers and molecular targets for therapy for glioma patients. The current study aimed to evaluate the immunohistochemical (IHC) expression of MTAP and A-Kinase Interacting Protein 1 (AKIP1) in astrocytoma and to investigate their association with the clinicopathological characters of these cases. Totally 66 cases of astrocytoma patients involved in this study. Cases underwent tumor resection and tissue sections were stained with MTAP, AKIP1 and IDH1 by IHC and evaluated in different grades of astrocytoma and their association with survival and response to therapy was investigated. High AKIP1 expression was positively correlated with treatment resistance and progressive disease. Positive IDH and retained MTAP expressions had shown better treatment response rather than negative IDH and lost MTAP. High AKIP, negative IDH and loss of MTAP expressions were significantly associated with poor survival outcome. Irrespective to grade and IDH status, the loss of MTAP immunoreactivity and high AKIP1 expression are predictive factors in astrocytoma, and they may be used as a biomarker for guiding astrocytoma management and prognosis surveillance.

Role of Wilms’ tumor 1 in diagnosis and grading of astrocytomas

Background: Wilms’ tumor 1 (WT1) mutation has recently been detected in gliomas. Growing data indicate that WT1 mutation plays a causal role in gliomagenesis and is overexpressed in most glioblastomas. Emerging immunotherapy targeting WT1 has shown to be effective in resistant glioblastomas in clinical trials. WT1 expression and its potential utility in various grades of astrocytomas is still unclear and needs further elucidation. The evaluation of WT1 can be done by molecular or immunohistochemical methods. As immunohistochemistry is easier with wider routine use, immunoexpression of this biomarker was studied. Aims and Objectives: This study aimed to evaluate WT1 immunoexpression across different histological grades of astrocytomas and differentiate low-grade astrocytomas from reactive astrogliosis. Materials and Methods: This was an observational prospective study on 54 cases of astrocytomas. Results: In the present study a total of 54 cases have been diagnosed as astrocytomas. Grade IV was the most common (31%) followed by grade II (44%). WT1 score correlated with histological tumor grades (P<0.001) with a higher score in a higher grade. It was also observed that different tumor grades depicted two distinct expression patterns. WT1 score and pattern were valuable in differentiating high- and low-grade astrocytomas. Conclusion: This study supports the oncogenic role of WT1 in astrocytomas. WT1 was found to be valuable in distinguishing different grades of astrocytomas. The scoring and distinctive patterns of WT1 can aid in distinguishing high-grade and low-grade astrocytomas. However, one needs to be careful in pilocytic astrocytomas in which there is a mixed expression. WT1, therefore, appears to be an attractive immunohistochemistry (IHC) marker to be used concomitantly with other IHC markers in astrocytomas. In addition, the frequent expression of WT1 in astrocytomas supports its promising role in immunotherapy and its potential to guide patient selection for targeted immunotherapy.

IRX1 is a novel gene, overexpressed in high-grade IDH-mutant astrocytomas.

IDH-mutant astrocytomas include CNS WHO grade 2 (A2), grade 3 (A3) and grade 4 (A4), of which A3 and A4 are high-grade. A3 has a heterogenous clinical outcome that cannot be explained entirely by the existing molecular biomarkers. We comprehensively studied the transcriptome profile of A3 to determine clinical significance. TCGA mRNA-sequencing data of A3 was analyzed to derive differentially expressed genes (DEG), which were short-listed using various approaches. mRNA expression of the short-listed genes was validated using NanoString platform on a uniformly treated and molecularly characterized A3 cohort. Protein expression of one prognostically significant gene, Iroquois-class homeodomain (IRX1) was assessed by immunohistochemistry and correlated with patient survival and tumor recurrence. IRX1 expression was also studied in different grades of astrocytoma. Since DNA methyltransferase 3 alpha (DNMT3A) influences IRX1 expression, its mutations were evaluated in a subset of tumors. TCGA analysis identified 96 DEG in A3 tumours. 57 genes were short-listed and finally narrowed down to 14 genes. mRNA values of 12/14 genes validated in our cohort. On multiple-variable analysis, IRX1 was the most prognostically relevant gene, with respect to progression free survival of patients. Further, IRX1 immunoexpression was significantly higher in A3 and A4 when compared to A2 and glioblastoma. Higher IRX1 immunoexpression correlated with poor prognosis in patients with A3 tumours. Also, a higher IRX1 expression was associated with DNMT3A mutation. Our study identifies IRX1 as a novel biomarker overexpressed in high-grade IDH-mutant astrocytomas with prognostic significance in A3. DNMT3A mutation probably modulates IRX1 expression.

Pilocytic Astrocytoma Arising in Ovarian Mature Cystic Teratoma: An Unusual Neuroectodermal Malignant Transformation Occurring in Pregnant Female

Mature cystic teratomas are usually benign, however malignant transformation occurs rarely. It is identified in only 0.17-2% of cases. Malignant cells can arise from cell of any type, but most common malignant transformation found to be squamous cell carcinoma (80% cases) followed by adenocarcinoma. While neural tissue is identified in nearly 80% cases of mature cystic teratomas, neuroectodermal tissue with malignant transformation is the most exceptional event. There is minimal data available on primary neuroectodermal tumours of ovary, majority are astrocytoma of different grades, only few cases were reported in the literature so far. Authors report a extremely rare and unique case of pilocytic astrocytoma arising from a mature cystic teratoma in a pregnant female. A 33-year-old pregnant female in her first trimester came for regular antenatal visit, Ultrasound (USG) abdomen and pelvis single live intrauterine gestation corresponding to 10 weeks and incidental complex right ovarian cyst likely to be mature cystic teratoma. Patient underwent right ovarian cystectomy five months after normal vaginal delivery and provisional diagnosis was given as complex right ovarian dermoid cyst. Final detailed histopathological examination revealed a tumour with glial tissue within the cyst, reported as pilocytic astrocytoma {World Health Organization (WHO) grade 1} arising in mature cystic teratoma. To the best of authors’ knowledge this is the third case showing pilocytic astrocytoma component in ovarian teratoma and the first case of this entity occurring in a pregnant female. Authors present an unusual case where radiology gave preliminary diagnosis however, extensive histopathological examination, histochemistry and immunohistochemistry helped in definite diagnosis.

Immunohistochemical Expression of PD-L1 and IDH1 with Detection of MGMT Promoter Methylation in Astrocytoma

Programmed death ligand 1 (PD-L1) expression was suggested as a poor prognostic predictor for glioblastoma. While isocitrate dehydrogenase (IDH) has been linked to enhanced overall survival in glioma cells. In glioblastoma patients receiving treatment with alkylating drugs, the methylguanine-DNA methyltransferase (MGMT) promoter's methylation status has been discovered as a potent and distinct predictor of good survival. In this study, we aimed to investigate the expression rate of PD-L1, IDH1, and MGMT methylation in patients with different grades of astrocytoma. The present retrospective study retrieved the data and archived paraffin blocks of 60 cases of astrocytoma. Immunohistochemical evaluation was done to assess the expressions of PD-L1 and IDH1, Methylation-specific-PCR was used to investigate the MGMT promoter. This study included astrocytoma grade II 18% (11/60), grade III 22% (13/60), grade IV 60% (36 cases). PD-L1 expression was detected in 82% of all studied cases (49/60) while IDH1 mutant astrocytoma were 73% (44/60) & methylation was reported in 58.3% (35 cases). High grade astrocytoma showed highrer expression of PD-L1 & IDH1 but with insignificant correlation (p=0.989). There is a relatively high expression of PD-L1 and IDH1 in patients with astrocytoma. More than half of the patients presented with MGMT promoter methylation. Further studies with larger sample size are required to investigate the association between these biomarkers and characteristics of patients with astrocytoma.

Expression of P53 and Ki-67 in different grades of astrocytomas

Expression of P53 and Ki-67 in different grades of astrocytomas

Intratumor morphologic and molecular genetic heterogeneity in astrocytomas of different grade of malignancy in the material from the first operation

Introduction. Intratumor heterogeneity is one of the key reasons for unfavourable prognosis in malignant tumors. Astrocytic tumors are known to develop therapy resistance inevitably during the course of disease. One of possible reason is tumor heterogeneity. Purpose. The aim of this work was to assess the intratumor morphologic and molecular heterogeneity in diffuse astrocytoma, anaplastic astrocytomas and primary glioblastomas. Material and methods. We conducted morphologic (n=22) and molecular-genetic (n=8) analysis of surgical specimens obtained from primarily operated glioblastoma giv (gb), anaplastic astrocytomas giii (aa) and diffuse astrocytoma gii (da) patients aged 18 years and older in whom total or subtotal tumor resection was performed. Tissue sampling for the analysis was performed from 5 equidistant areas of each tumor. Morphologic diagnosis was established according to who classification of central nervous system tumors (2007/2016). Mgmt, c-kit, top2a, pdgfr-α, ercc1, vegf genes mrnaexpression was assessed by rt-pcr. Idh1 and idh2 mutational status was evaluated by allele-specific pcr. Results. Morphologic heterogeneity was evident in 72,7 % tumors (16/22) overall. Heterogeneity was observed in 68,8 % (11/16) of gb, 80 % (4/5) of aa and in the only case of da. In 50 % of cases at least 3 different morphologic variants were seen in different areas of the tumor. This morphologic heterogeneity presented as the combination of different grades of anaplasia (gii – giv) in one tumor. Molecular profile was assessed in 48 expression analysis of genes: mgmt, c-kit, top2a, pdgfr-α, ercc1, vegf from 8 patients. Intratumoral molecular heterogeneity was revealed in 41,7 % of cases (20/48). Conclusion. The presence of intratumoral heterogeneity should be taken into account during surgery for adequate tumor sampling for histologic and molecular analysis which is critical for proper assessment of prognosis and following treatment planning.

Glutaminolysis dynamics during astrocytoma progression correlates with tumor aggressiveness

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

Application of EGFR, ki 67 and GFAP as a panel for the diagnosis and grading of astrocytomas

Background: Astrocytic tumors are the most common primary central nervous system tumors. WHO grading scheme is based on the presence or absence of the following four histological parameters using haematoxylin and eosin (H&E) staining: cellular atypia (WHO grade II), mitotic figures (WHO grade III), endothelial proliferation, and/or necrosis (WHO grade IV). Immunohistochemistry (IHC) has become an essential tool in diagnosis of brain tumors and helps in predicting the prognosis of certain brain including astrocytomas. Aim: Study the immunohistochemical expressions of (Epidermal growth factor receptor (EGFR), Ki67 and Glial fibrillary acidic protein (GFAP)) in different grades of astrocytomas. Materials and Methods: This study included 44 cases of astrocytomas classified as: 3 pilocytic astrocytomas, 6 diffuse low grade astrocytomas, 15 anaplastic astrocytomas and 20 glioblastoma multiforms. Immunohistochemical staining of all cases using EGFR, Ki67 and GFAP was done. Results: EGFR showed positive membranous and cytoplasmic expression in 33 (75%) cases, GFAP positive staining was detected in 38 (86.4%) of studied cases while all astrocytomas of different grades showed ki67 positivity with variable degrees with the mean ± SEM of Ki67 LI 17.76% ± 2.46. There was statistically significant relation between EGFR, Ki 67 and GFAP expression and tumor grade. Conclusions: EGFR, Ki 67 and GFAP should be used in combination as a panel side by side with established histological criteria of malignancy and WHO grading system for accurate diagnosis and grading of astrocytic tumors.

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

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

Clinical Evaluation of Proline, Glutamic acid, and Leucine-Rich Protein 1 Expression in Astrocytomas and Correlations with the Proliferation Marker Ki-67

Malignant astrocytomas presenting in humans of any age group are a challenge to diagnose and treat. Hence, there is a quest for new markers to ascertain their grades and predict disease outcomes. Proline, glutamic acid, and leucine-rich protein 1 (PELP1), a nuclear receptor co-regulator, is an oncogene found in various cancers. We postulate that by screening for PELP1, its correlation with survival outcomes of patients across various grades can indicate a plausible novel diagnostic marker and a potential therapeutic target in gliomas. Immunostaining of 100 cases of astrocytomas for PELP1 was performed on paraffin-embedded sections. Results showed that PELP1 expression increases with higher grades; the mean H-score of PELP1 in grade-I astrocytomas was determined to be 112.3, whereas in grade-IV it was 235.1 (P value = 0.0001). Survival analysis of patients with H-score of 200-300 was only 8.8% and 68.8% in patients with scores of 0-100. PELP1 expression in high-grade astrocytomas is an important factor in determining the outcomes. Graphical abstract Evaluation of molecular expression of PELP1 along with Ki-67 LI signifies a linear increase in its expression pattern among different grades of astrocytomas from low- to high-grade tumors, which can serve as a potential prognostic molecular marker in differentiating various types of astrocytomas in humans.

Abstract 3739: Understanding the link between glutamine metabolism and angiogenesis in astrocytoma

Abstract Angiogenic switch is a hallmark for tumor growth, and strategies to inhibit this process have been pursued for cancer therapy, but with limited long-term efficacy. Recently, targeting endothelial cell (EC) metabolism emerged as an alternative to regulate angiogenesis. In fact, glutamine metabolism is essential for EC sprouting in vitro, and the consumption/secretion rate measurements revealed that proliferating ECs consumed glutamine more than any other amino acid. Glutamine is transported into the cells and converted into α-ketoglutarate to enter the tricarboxylic acid cycle through glutaminolysis, which is upregulated in cancer. Glutaminase (coded by GLS), the main regulator of this pathway, presents two isoforms: GLSiso1 and GLSiso2. We have analyzed the expression level of both GLS isoforms in different grades of astrocytoma, with a gradual increase of their expressions in parallel to the malignancy. GLSiso1 was highly expressed in normal brain samples, in contrast to low GLSiso2 expression, indicating GLSiso2 as a more eligible therapeutic target. GLSiso2-specific transcript inhibition by siRNA in GBM U87MG cells lead to significant decrease of tumor cell proliferation, and a temozolomide-sensitizing effect, relative to NTC-controls. The RNASeq transcriptome analysis of these GLSiso2-silenced cells revealed a differential expression of genes related to blood vessel development, extracellular matrix organization, angiogenesis and tube development with FDR of 4.95e-04, 5.25e-06, 5.92e-05, 8.6e-06 and 1.0e-04, respectively. The immunohistochemistry analysis in human astrocytoma FFPE samples demonstrated GLS positivity on tumor cells and blood vessels, with higher protein expression in more malignant astrocytoma. Such observation corroborated the transcriptomic finding of GLSiso2 participation in the EC compartment. Moreover, the system biology analysis revealed an interesting net among GLS, TP53, ITB1, TGFBI, TGFBR1/2, COL1A1/2, COL5A2, LUM, ENG, ANGPT1 connecting genes related to metabolism, extracellular matrix and angiogenesis. In the search for the most suitable EC line to study the impact of GLS isoforms on the vessel compartment, we observed that HUVEC immortalized umbilical EC line presented higher expression of GLSiso2 than GLSiso1. Chemical blockade of the total GLS by BPTES and 968 was tested in HUVEC cells and a significant proliferation and migration decrease were observed. Further GLSiso2 specific silencing of ECs might clarify the crosstalk between tumor cells and ECs, and will allow the identification of key targets in their metabolic reprogramming during tumor growth. Citation Format: Yollanda E. Franco, Roseli S. Soares, Marina T. Lima, Antonio M. Lerario, Sueli M. Shinjo, Suely K. Marie. Understanding the link between glutamine metabolism and angiogenesis in astrocytoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3739.

Abstract 2596: Toll like receptor 4 as a potential DNA repair modulator in U87MG-GBM cells

Abstract Toll like receptor 4 (TLR4) has a dual role in cellular fate, depending on the stimuli and microenvironment context. Its activation may promotes cell survival and proliferative phenotype via MyD88 with NFkB activation, or leads to cell death via TRIF. Glioblastoma (GBM), grade IV astrocytoma, is the most common and aggressive primary malignancy in the brain. Surgical tumor cytoreduction followed by radiotherapy and adjunct chemotherapy with Temozolomide (TMZ) have been considered the standard of care for GBM. And, in spite of several combinatory rescue therapeutic trials, the median overall survival of GBM patients continue to be approximately 15 months. In this context, new therapeutic strategies are urgent, and immune modulation has demonstrated optimistic results in several tumor types. Therefore, the aim of this study was to analyze the role of TLR4 in human astrocytomas. First, we evaluated TLR4 expression in 140 human astrocytoma of different grades of malignancy, by qRT-PCR. TLR4 molecular signaling pathways were investigated through LPS stimuli in U87MG-GBM cell line. In astrocytoma samples, TLR4 was up-regulated when compared to brain non-neoplastic samples (p<0.05, Kruskall Wallis and Dunn tests), with increased expression in Mesenchymal compare to Classical and Proneural subtypes. U87MG cells stimulation with LPS (10ug/ml) lead to NF-kB (p65) nuclear translocation after 12hr through western blot and immunofluorescence, which was unexpected as such translocation through the MyD88/TRAF6 canonical pathway occurs within 30min to 2hr after LPS stimulation. TRIF, IL1B, RIPK1/3 expressions were increased after LPS stimulation, suggesting an activation of inflammasome and ripoptosome pathways. Moreover, the combined LPS treatment with TMZ showed a higher apoptotic rate after 48hr when compared to TMZ alone. However, the transcriptomic analysis by RNASeq of U87MG cells after LPS stimulation showed an upregulation of DNA repair genes, particularly MBD4 and PLAUR. Both genes are upregulated in mesenchymal GBM subtype in the TCGA RNASeq dataset, and they present correlated expression with TLR4 (r=0.406, p=0.004 and r=0.338, p=0.019 by Spearman's test). Our results suggest that the anti-tumoral effect by TLR4 pathway activation by enhancement of tumor apoptosis might be more efficient with combined inhibition of DNA repair cascade. In fact, OSI-296 and BKM-120 have shown to decrease MBD4 expression, and increased overall survival was observed in a phase II clinical trial for solid tumor. Citation Format: Isabele F. Moretti, Antonio Lerario, Sueli M. Oba-Shinjo, Suely K. Marie, Marina Trombetta Lima. Toll like receptor 4 as a potential DNA repair modulator in U87MG-GBM cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2596.

Immunohistochemical Expression of Epidermal Growth Factor Receptor in Astrocytic Tumors in Iraqi Patients.

BACKGROUND:Diffuse astrocytomas constitute the largest group of primary malignant human intracranial tumours. They are classified by the World Health Organization (WHO) into three histological malignancy grades: diffuse astrocytomas (grade II), anaplastic astrocytomas (grade III) and glioblastoma (grade IV) based on histopathological features such as cellular atypia, mitotic activity, necrosis and microvascular proliferation. Epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane tyrosine kinase receptor expressed in a variety of normal and malignant cells regulating critical cellular processes. When activated, epidermal growth factor receptor (EGFR) triggers several signalling cascades leading to increased proliferation and angiogenesis and decreased apoptosis and hence associated with aggressive progression of the tumour. Epidermal growth factor receptor (EGFR) level is known to be a strong indicator associated with the aggressive behaviour of the tumour and acts as a prognostic factor for evaluating the survival rate.AIM:To evaluate the expression of epidermal growth factor receptor (EGFR) in different grades of astrocytoma.MATERIAL AND METHODS:formalin-fixed paraffin-embedded astrocytic tumours of 44 patients were collected from the archival material of pathology department of Ghazi Al Hariri Teaching Hospital during the period from June to December 2018. Hematoxylin and eosin-stained sections were used to characterise the tumours histologically based on cellularity, nuclear hyperchromasia, polymorphism, mitotic activity, vascular proliferation and necrosis with or without pseudopallisading of tumour cells. Diagnosis and grading of astrocytic tumours in this study were made according to WHO criteria (2016). Using a monoclonal antibody to the epidermal growth factor receptor (EGFR) and immunohistochemical analysis, the expression and distribution of epidermal growth factor receptor in astrocytic tumours were examined.RESULTS:The study included 1 case pilocytic astrocytoma (grade I), 20 cases diffuse astrocytoma (grade II), 5 cases anaplastic astrocytoma (grade III) and 18 cases of glioblastoma (grade IV). Expression of EGFR was found in 38.88% of the glioblastoma samples (grade IV). However, none of the astrocytomas of WHO grades I, II and III showed immunoreactivity for EGFR protein. Different patterns of immunoreactive cells and significant intratumor heterogeneity of EGFR expression were observed in glioblastomas.CONCLUSION:The immunohistochemical expression of Epidermal growth factor receptor (EGFR) was restricted only to high-grade astrocytic tumours, namely glioblastoma, thus may use to predict glioblastoma.

Diagnostic Accuracy and Validity of Magnetic Resonance Imaging in the Detection of Speci!c Type of Brain Tumour

Magnetic Resonance Imaging (MRI) is a widely accessible imaging technique for the detection of brain tumours and cancer, which are further confirmed by histopathological examination. Accurate detection of the tumours and its extent is very difficult. The present study attempted to evaluate the convenience of MRI in detection of different grades of astrocytomas, which are the most commonly occurring brain tumours. This cross-sectional study was conducted at the Department of Radiology and Imaging with the collaboration of Department of Neurosurgery and Department of Pathology at Sir Salimullah Medical College (SSMC & MH), Dhaka from January 2013 to December 2013 for a period of one year. The study population was all the diagnosed cases of intracranial astrocytoma patients regardless of their age and sex. The studied included 48 brain tumour (astrocytoma) patients, ages between 13 and 69 years old. All cases having no contraindication for MRI underwent MR examination followed by histopathological examination of the postoperative resected tissues. The findings of the MRI and histopathological examination were compared to find out the test validity of the MRI findings of the different grades of astrocytoma’s. The highest sensitivity was found in grade III astrocytoma (90.5%) followed by grade II (85.7%) grade IV (75.0%) and grade I (60.0%). The highest specificity was found in grade I astrocytoma (97.7%) followed by Grade III (96.3%), grade IV (92.5%) and grade II (91.5%). The highest accuracy was found in both grade I astrocytoma (93.7%) and grade III (93.7%) followed by grade II (92.5%) and grade IV (89.6%). As per the study findings it can be concluded that,MRI has a high diagnostic accuracy and validity for the detection of different grades of astrocytoma.
 Bangladesh Med J. 2017 Sep; 46 (3): 85-89

Abstract 5175: Expression profile and role of LOXL3 in astrocytomas

Abstract Astrocytomas are the most common primary central nervous system tumors in adults. The WHO classification based on histologial characteristics according consider four malignant grades, glioblastoma (GBM) being the most malignant (grade IV). Molecular classification of GBM is based on the integration of somatic mutations, DNA methylation and specific transcript/protein expression and consider four subtypes: proneural, classic, mesenchymal and neural. Lysyl oxidase family is composed of five members (LOX, LOXL1, LOXL2, LOXL3 and LOXL4) which are enzymes responsible for catalyzing lysine-derived cross-links of collagen and elastin. The aim of the present work was to analyze the gene expression of LOXL3 in astrocytomas of different grades and different GBM molecular subtypes, the impact of LOXL3 expression level on overall survival in GBM cases of our cohort. These data were also analyzed in the TCGA database to validate our findings. LOXL3expression increased with astrocytoma malignant grades, and proved to be a prognostic factor, as GBM patients with lower LOXL3 expression presented longer survival time than those with higher expression (p<0.041). Analogous data was obtained in the TCGA database, when considering temozolamide-treated GBM patients (p=0.049). Additionally, LOXL3 expression was higher in mesenchymal GBM subtype than in proneural and classic GBM subtypes in TCGA cohort (p<0.001). To further understand LOXL3 functional role in gliomagenesis, LOXL3 was silenced with two distinct siRNA sequences in U87MG, a mesenchymal subtype of GBM cell line. LOXL3 down regulation was confirmed at gene and protein expression levels. Functional assays with silenced LOXL3 demonstrated a decrease in the cell proliferation (p<0.05), and an increase in the apoptosis associated to temozolamide treatment (p<0.05). Immunofluorescence analysis in U87MG cells showed LOXL3 colocalized with mitochondria. Interestingly, such colocalization decreased when LOXL3 was downregulated, associated to a clear morphological alteration in mitochondria shape. A rough increase in mitochondria volume was observed, suggesting fused mitochondria. In addition, such condition was associated to a 23% decrease in mitochondrial DNA copy number. Altogether, our findings suggest that LOXL3 is upregulated in GBM, with impact in prognosis and might play a role in mitochondria dynamics and/or mitophagy, modulating apoptosis. Transcriptome analysis, currently in progress, will further clarify the associated players in this signaling pathway. Citation Format: Talita de Souza Laurentinho, Roseli da Silva Soares, Suely Kazue Marie, Sueli Mieko Oba-Shinjo. Expression profile and role of LOXL3 in astrocytomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5175.

Abstract 3586: Silencing of GLSiso2 (GAC) decreases cell proliferation and induces cell death in glioblastoma cell line

Abstract The metabolic reprogramming is currently recognized as one of the hallmarks of cancer. Tumor cells have unusual metabolic activity in comparison to normal cells, and ability to reprogram their metabolic machinery to meet their biosynthetic and bioenergetic needs. Factors such as hypoxia, mutation in oncogenes and changes in signaling pathways may induce an upregulation of anabolic processes and suppression of catabolic pathways that leads to the maintenance of bioenergetics, redox status, cell signaling, and biosynthesis, which support rapid cell proliferation. Tumor cells can use a variety of alternative energy production pathways depending on the availability of nutrients. Glutaminolysis, the process where glutamine is transported into the cells and converted into α-ketoglutarate (α-KG) to enter into the tricarboxylic acid (TCA) cycle, is regulated positively in cancer. Glutaminase (GLS) is the main regulator of this pathway and presents two forms in humans: kidney-type glutaminase (KGA or GAC) and liver-type GLS2 (LGA or GAB), both with distinct tissue distribution, regulation and functions. The GLS presents two isoforms: GLSiso1 and GLSiso2, and their regulation are not yet well understood. The aim of this study was to analyze the expression of GLS in astrocytoma of different malignant grades. Additionally, in vitro assays were performed in a glioblastoma cell line (U87MG) to access GLSiso2 functions in astrocytoma. Differential expression levels of both isoforms were detected in 173 astrocytomas of our group cohort. A gradual increase of both isoforms expression levels was observed in parallel to the increase of the tumor malignancy. However, high expression level of GLSiso1 was detected in normal brain samples, while very low expression of GLSiso2. Hence, the isoform 2 is more eligible and relevant as a therapeutic target. To analyze the role of GLSiso2 in gliomagenesis, the isoform-specific transcript expression was inhibited by the interfering RNA method in U87MG cells. The efficiency of silencing was confirmed at gene and protein levels by quantitative real time PCR and western blotting, respectively. Additionally, GLSiso2 silenced cells presented significant decrease of cell proliferation, a temozolamide-senzitizing effect and an increase of cell death, when compared to control cells. Total GLS protein distribution was analyzed in different grades of astrocytoma through immunohistochemistry. The higher the malignant grade of astrocytomas, the higher the expression of GLS observed in tumor cell cytoplasm. Interestingly GLS was also expressed in endothelium, with higher expression in GBM cases compared to lower grades of astrocytoma and non-neoplastic brain tissues. Further analysis of mechanistic role of GLS isoforms in the endothelium cell compartment might better clarify a possible complementary effect in the metabolic reprogramming over the tumor growth. Citation Format: Yollanda Moreira Franco, Roseli Silva Soares, Sueli M. Oba-Shinjo, Suely Kazue Nagahashi Marie. Silencing of GLSiso2 (GAC) decreases cell proliferation and induces cell death in glioblastoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3586.

Comparison of Test Validity of Magnetic Resonance Imaging in the Diagnosis of Diffierent Grades of Astrocytoma

Accurate detection of astrocytomas is very difficult. The purpose of the present study was to evaluate the usefulness of MRI in detection of intracranial astrocytoma. This cross-sectional study was conducted at the Department of Radiology and Imaging with the collaboration of Department of Neurosurgery and Department of Pathology at Sir Salimullah Medical College (SSMC & MH), Dhaka from January 2013 to December 2013 for a period of one year. All the clinically suspected and CT scan diagnosed cases of intracranial astrocytoma patients of any age of both sexes were included as study population. All cases having no contraindication for MRI underwent MR examination. MR imaging was obtained with 0.5 Tesla machine (SIEMENS). The postoperative resected tissues were examined histopathological in the respective department. Then the collected reports were compared with findings of MRI. The sample size of the present study was 48 astrocytoma patients. The highest sensitivity was found in grade III astrocytoma (90.5%) followed by grade II (85.7%) grade IV (75.0%) and grade I (60.0%). The highest specificity was found in grade I astrocytoma (97.7%) followed by Grade III (96.3%), grade IV (92.5%) and grade II (91.5%). The highest accuracy was found in both grade I astrocytoma (93.7%) and grade III (93.7%) followed by grade II (92.5%) and grade IV (89.6%). In the conclusion, MRI is an effective tool for the diagnosis of astrocytoma. MRI has a high diagnostic validity for the detection of different grades of astrocytoma.
 Bangladesh Med J. 2017 May; 46 (2): 48-52

WT1 in astrocytomas: Comprehensive evaluation of immunohistochemical expression and its potential utility in different histological grades.

Wilms' tumor 1 (WT1) mutation has recently been detected in gliomas. Growing data indicate that WT1 mutation plays a causal role in gliomagenesis and is overexpressed in most glioblastomas. An emerging immunotherapy targeting WT1 has shown to be effective in resistant glioblastomas in clinical trials. WT1 expression and its potential utility in various grades of astrocytomas is still unclear and needs further elucidation. The evaluation of WT1 can be done by molecular or immunohistochemical methods. As immunohistochemistry is easier with wider routine use, immunoexpression of this biomarker was studied. The aim of this study was to characterize WT1 immunoexpression across different histological grades of astrocytomas to routinely aid in diagnosis and reproducibility and to assess the association between WT1 and immunomarker isocitrate dehydrogenase (IDH1). This was an observational prospective study on 79 cases of astrocytomas. Seventy-nine astrocytomas including 11 recurrent tumors were assessed for WT1 by immunohistochemistry. WT1 expression was detected in all astrocytomas (100%). The control group of reactive gliosis was negative. WT1 score correlated with histological tumor grades (P < 0.001) with higher score in higher grade. It was also observed that different tumor grades depicted two distinct expression patterns. WT1 score and pattern were valuable in differentiating high- and low-grade astrocytomas. This study supports the oncogenic role of WT1 in astrocytomas. WT1 was found to be valuable in distinguishing different grades of astrocytomas. WT1 can aid in differentiating neoplastic process from reactive gliosis, particularly in recurrent tumors. Higher expression in glioblastomas supports its immunotherapy potential.

IDH1 mutation decreases the invasiveness of glioma by downregulating the expression and activity of TAZ

Background and Aim: Gliomas carrying mutated isocitrate dehydrogenase 1 (IDH1) have an improved prognosis, but how this mutation improves survival is not known. In this study, we evaluated the correlation of expression of the gene transcriptional coactivator with PDZ-binding motif (TAZ) with IDH1 mutation in astrocytomas of different grades. Materials and Methods: We analyzed the expression of TAZ by immunohistochemistry in a cohort of 90 formalin-fixed paraffin-embedded human astrocytoma samples. A human glioblastoma cell line (U87) was transfected with mutated IDH1R132H; the expression and subcellular location of TAZ were analyzed by western blot assay, quantitative real-time polymerase chain reaction, and immunofluorescence staining. We detected activation of the Hippo signaling pathway by western blot. Octyl-2-hydroxyglutarate (Octyl-2-HG), an analog of 2-HG, was used to treat IDH1 wild-type U87 cells to determine its influence on the expression of TAZ. Cell viability assay, flow cytometry, Transwell migration assay, and scratch assay were used to analyze cell proliferation and invasive capacity. To verify that those changes were caused by the expression of TAZ, we did a rescue experiment by transfecting TAZ in the IDH1R132H cells. The study was approved by the Ethics Committee of Fudan University (approval No. 2016-Y013) on January 18, 2016. Results: TAZ expression was significantly lower in IDH1-mutated astrocytoma than the wild type in the same tumor grade. In IDH1-mutant cells, the nuclear TAZ location was decreased and the Hippo signaling pathway was activated as determined by TAZ phosphorylation and increased 14-3-3e expression. Treatment with Octyl-2-HG reduced the expression of TAZ. IDH1R132H cells showed decreased invasion proliferation compared with IDH1 wild-type cells. Overexpression of TAZ rescued cell migration and invasion capacity. Conclusion: In glioma, IDH1R132H mutation decreases TAZ expression and significantly reduces the invasive character of glioma cells.