Arimappamagan Arivazhagan

Identification of Potential Serum Biomarkers of Glioblastoma: Serum Osteopontin Levels Correlate with Poor Prognosis

Background: The aim of this study is to identify serum biomarkers with classification and prognosis utility for astrocytoma, in particular glioblastoma (GBM). Methods: Our previous glioma microarray database was mined to identify genes that encode secreted or membrane-localized proteins. Subsequent analysis was done using significant analysis of microarrays, followed by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemical validation in tumor tissues, ELISA and Western blot validation in sera, and correlation with survival of GBM patients. Results: Significant analysis of microarrays identified 31 upregulated and 3 downregulated genes specifically in GBMs. RT-qPCR validation on an independent set of samples confirmed the GBM-specific differential expression of several genes, including three upregulated (CALU, CXCL9, and TIMP1) and two downregulated (GPX3 and TIMP3) novel genes. With respect to osteopontin (OPN), we show the GBM-specific upregulation by RT-qPCR and immunohistochemical staining of tumor tissues. Elevated serum OPN levels in GBM patients were also shown by ELISA and Western blot. GBM patients with high serum OPN levels had poorer survival than those with low serum OPN levels (median survival 9 versus 22 months respectively; P = 0.0001). Further, we also show high serum TIMP1 levels in GBM patients compared with grade II/III patients by ELISA and downregulation of serum GPX3 and TIMP3 proteins in GBMs compared with normal control by Western blot analysis. Conclusions: Several novel potential serum biomarkers of GBM are identified and validated. High serum OPN level is found as a poor prognostic indicator in GBMs. Impact: Identified serum biomarkers may have potential utility in astrocytoma classification and GBM prognosis. Cancer Epidemiol Biomarkers Prev; 19(6); 1409–22. ©2010 AACR.

Insulin growth factor-2 binding protein 3 (IGF2BP3) is a glioblastoma-specific marker that activates phosphatidylinositol 3-kinase/mitogen-activated protein kinase (PI3K/MAPK) pathways by modulating IGF-2.

Glioblastoma is the most common and malignant form of primary astrocytoma. Upon investigation of the insulin-like growth factor (IGF) pathway, we found the IGF2BP3/IMP3 transcript and protein to be up-regulated in GBMs but not in lower grade astrocytomas (p < 0.0001). IMP3 is an RNA binding protein known to bind to the 5′-untranslated region of IGF-2 mRNA, thereby activating its translation. Overexpression- and knockdown-based studies establish a role for IMP3 in promoting proliferation, anchorage-independent growth, invasion, and chemoresistance. IMP3 overexpressing B16F10 cells also showed increased tumor growth, angiogenesis, and metastasis, resulting in poor survival in a mouse model. Additionally, the infiltrating front, perivascular, and subpial regions in a majority of the GBMs stained positive for IMP3. Furthermore, two different murine glioma models were used to substantiate the above findings. In agreement with the translation activation functions of IMP3, we also found increased IGF-2 protein in the GBM tumor samples without a corresponding increase in its transcript levels. Also, in vitro IMP3 overexpression/knockdown modulated the IGF-2 protein levels without altering its transcript levels. Additionally, IGF-2 neutralization and supplementation studies established that the proproliferative effects of IMP3 were indeed mediated through IGF-2. Concordantly, PI3K and MAPK, the downstream effectors of IGF-2, are activated by IMP3 and are found to be essential for IMP3-induced cell proliferation. Thus, we have identified IMP3 as a GBM-specific proproliferative and proinvasive marker acting through IGF-2 resulting in the activation of oncogenic PI3K and MAPK pathways.

Grade-Specific Expression of Insulin-like Growth Factor–Binding Proteins-2, -3, and -5 in Astrocytomas: IGFBP-3 Emerges as a Strong Predictor of Survival in Patients with Newly Diagnosed Glioblastoma

Background: Insulin-like growth factor (IGF)–binding protein (IGFBP) isoforms have been implicated in the pathogenesis of human neoplasms including glioma. In view of this, we evaluated the expression of IGFBP isoforms (IGFBP-2, -3, and -5) during malignant progression of astrocytoma and their prognostic significance in glioblastoma. Methods: The expression of IGFBP isoforms was analyzed in diffusely infiltrating astrocytomas by real-time quantitative PCR (n = 203) and immunohistochemistry (n = 256). Statistical methods were used to assess their grade-specific expression pattern and mRNA-protein intercorrelation. Survival analyses were done on a uniformly treated, prospective cohort of adult patients with newly diagnosed glioblastoma (n = 136) by using Cox regression models. Results: The mean transcript levels of IGFBP-2 and -3 were significantly higher in glioblastomas (GBM) relative to anaplastic astrocytoma (AA), diffuse astrocytoma (DA), and controls whereas IGFBP-5 mRNA was higher in GBM relative to AA and controls (P < 0.05). By immunohistochemistry, the mean labeling index of all isoforms was significantly higher in GBM compared with AA, DA, and control (P < 0.05). A strong positive correlation was observed between their respective mRNA and protein expressions (P < 0.01). Multivariate analysis revealed IGFBP-3 expression (hazard ratio, 1.021; P = 0.030) and patient age (hazard ratio, 1.027; P = 0.007) to be associated with shorter survival in glioblastoma. Conclusions: This study shows the associations of IGFBP-2, -3, and -5 expression with increasing grades of malignancy in astrocytomas. IGFBP-3 is identified as a novel prognostic glioblastoma biomarker. The strong correlation between their mRNA and protein expression patterns suggests their role in the pathogenesis of these tumors. Impact: IGFBP isoforms have emerged as biomarkers with diagnostic and prognostic utility in astrocytomas. Cancer Epidemiol Biomarkers Prev; 19(6); 1399–408. ©2010 AACR.

Proteomic identification of Haptoglobin α2 as a glioblastoma serum biomarker: implications in cancer cell migration and tumor growth

Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin α2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = <0.0001 and <0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.

Pediatric Intracranial Meningiomas – Do They Differ from Their Counterparts in Adults?

Aim: Meningiomas are very rare in children comprising only 0.44.1% of pediatric age tumors and only 1.5–1.8% of all intracranial neoplasms. We analyzed the clinical, pathological and management profile of these rare tumors and elucidated their differences from meningiomas in adults. Methods: From 1990 to 2005, 33 patients belonging to the pediatric age group with intracranial meningiomas were treated in NIMHANS. Results: There were 19 male and 14 female children. The duration of symptoms ranged from 1 to 60 months. The study had a mean follow-up of 23.4 months. The commonest presenting symptoms were headache (90.9%) and visual disturbances (51.5%). Three patients had associated neurofibromatosis. Calcification was noticed in 22% of the tumors. Intraventricular meningiomas were the commonest (24.2%). Gross total or near total resection was possible in 22 out of 33 patients. Nine patients had reversible postoperative motor deficits. Fibrous meningioma was the commonest histological subtype (24.2%). Five patients had atypical or anaplastic subtypes. Adjuvant radiotherapy was administered in 4 patients. Six patients had recurrences and underwent surgery. Ten patients required either staged procedures or multiple surgeries for the recurrence/residual tumors. Conclusion: Pediatric meningiomas are rare tumors and differ from those in adults by their male predominance, atypical locations, higher rates of malignant subtypes, recurrence and association with neurofibromatosis. Surgical management is challenging because of atypical location.

A DNA Methylation Prognostic Signature of Glioblastoma: Identification of NPTX2-PTEN-NF-κB Nexus

Glioblastoma (GBM) is the most common, malignant adult primary tumor with dismal patient survival, yet the molecular determinants of patient survival are poorly characterized. Global methylation profile of GBM samples (our cohort; n = 44) using high-resolution methylation microarrays was carried out. Cox regression analysis identified a 9-gene methylation signature that predicted survival in GBM patients. A risk-score derived from methylation signature predicted survival in univariate analysis in our and The Cancer Genome Atlas (TCGA) cohort. Multivariate analysis identified methylation risk score as an independent survival predictor in TCGA cohort. Methylation risk score stratified the patients into low-risk and high-risk groups with significant survival difference. Network analysis revealed an activated NF-κB pathway association with high-risk group. NF-κB inhibition reversed glioma chemoresistance, and RNA interference studies identified interleukin-6 and intercellular adhesion molecule-1 as key NF-κB targets in imparting chemoresistance. Promoter hypermethylation of neuronal pentraxin II (NPTX2), a risky methylated gene, was confirmed by bisulfite sequencing in GBMs. GBMs and glioma cell lines had low levels of NPTX2 transcripts, which could be reversed upon methylation inhibitor treatment. NPTX2 overexpression induced apoptosis, inhibited proliferation and anchorage-independent growth, and rendered glioma cells chemosensitive. Furthermore, NPTX2 repressed NF-κB activity by inhibiting AKT through a p53-PTEN-dependent pathway, thus explaining the hypermethylation and downregulation of NPTX2 in NF-κB-activated high-risk GBMs. Taken together, a 9-gene methylation signature was identified as an independent GBM prognosticator and could be used for GBM risk stratification. Prosurvival NF-κB pathway activation characterized high-risk patients with poor prognosis, indicating it to be a therapeutic target.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Background: Glioblastoma is highly aggressive and incurable by current treatment modalities. Results: MCSF is regulated by the SYK-PI3K-NFκB pathway in glioma and induces secretion of IGFBP1 from microglia to promote angiogenesis. Conclusion: Microglial IGFBP1 is a key mediator of MCSF-induced angiogenesis. Significance: IGFBP1 is a potential target for glioblastoma therapy. Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NFκB pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NFκB-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

IDH1 Mutations in Diffusely Infiltrating Astrocytomas Grade Specificity, Association With Protein Expression, and Clinical Relevance

IDH1 mutations are frequent genetic alterations in low-grade diffuse gliomas and secondary glioblastoma (GBM). To validate mutation frequency, IDH1 gene at codon 132 was sequenced in 74 diffusely infiltrating astrocytomas: diffuse astrocytoma (DA; World Health Organization [WHO] grade II), anaplastic astrocytoma (AA; WHO grade III), and GBM (WHO grade IV). All cases were immunostained with IDH1-R132H monoclonal antibody. Mutational status was correlated with mutant protein expression, patient age, duration of symptoms, and prognosis of patients with GBM. We detected 31 (41.9%) heterozygous IDH1 mutations resulting in arginine-to-histidine substitution (R132H;CGT-CAT). All 12 DAs (100%), 13 of 14 AAs (92.9%), and 6 of 48 GBMs (12.5%) (5/6 [83.3%] secondary, and 1/42 [2.4%] primary) harbored IDH1 mutations. The correlation between mutational status and protein expression was significant (P < . 001). IDH1 mutation status, though not associated with prognosis of patients with GBM, showed significant association with younger age and longer duration of symptoms in the whole cohort (P < .001). Our study validates IDH1 mutant protein expression across various grades of astrocytoma, and demonstrates a high incidence of IDH1 mutations in DA, AA, and secondary GBM.

Homozygous 10q23/PTEN deletion and its impact on outcome in glioblastoma: A prospective translational study on a uniformly treated cohort of adult patients

Tumors from a prospective cohort of adult patients with newly diagnosed glioblastoma (n = 73), treated uniformly with radiochemotherapy, were examined for 10q23/PTEN deletion by fluorescence in situ hybridization (FISH). Statistical methods were employed to evaluate the degree of association between 10q23/PTEN deletion status and patient age. Survival analysis was performed using Kaplan‐Meier log‐rank test and multivariable Cox models to assess the prognostic value of 10q23/PTEN deletion. Interestingly, 10q23/PTEN homozygous deletion was frequent in patients >45 years of age (P = 0.034) and the median age of patients harboring PTEN homozygous deletions was significantly higher than those with the retained status (P = 0.019). 10q23/PTEN homozygous deletion was associated with shorter survival in the entire cohort as well in patients >45 years (P < 0.05), indicating that loss of 10q23/PTEN showed clinical importance in elderly patients. Our study highlights the independent prognostic/predictive value of 10q23/PTEN deletion status as identified by FISH, particularly in glioblastoma patients aged >45 years.

Methylation Silencing of ULK2, an Autophagy Gene, Is Essential for Astrocyte Transformation and Tumor Growth

Background: Autophagy, a catabolic degradation process, has been shown to promote and inhibit cell growth. Results: ULK2, an upstream autophagy initiator, is silenced by methylation in glioblastoma, and its ectopic expression inhibited astrocyte transformation and glioma cell growth through autophagy. Conclusion: ULK2 down-regulation is important for the astrocyte transformation and tumor growth. Significance: Autophagy inhibition is essential for glioma development. Glioblastoma (GBM) is the most aggressive type of brain tumor and shows very poor prognosis. Here, using genome-wide methylation analysis, we show that G-CIMP+ and G-CIMP-subtypes enrich distinct classes of biological processes. One of the hypermethylated genes in GBM, ULK2, an upstream autophagy inducer, was found to be down-regulated in GBM. Promoter hypermethylation of ULK2 was confirmed by bisulfite sequencing. GBM and glioma cell lines had low levels of ULK2 transcripts, which could be reversed upon methylation inhibitor treatment. ULK2 promoter methylation and transcript levels showed significant negative correlation. Ectopic overexpression of ULK2-induced autophagy, which further enhanced upon nutrient starvation or temozolomide chemotherapy. ULK2 also inhibited the growth of glioma cells, which required autophagy induction as kinase mutant of ULK2 failed to induce autophagy and inhibit growth. Furthermore, ULK2 induced autophagy and inhibited growth in Ras-transformed immortalized Baby Mouse Kidney (iBMK) ATG5+/+ but not in autophagy-deficient ATG5−/− cells. Growth inhibition due to ULK2 induced high levels of autophagy under starvation or chemotherapy utilized apoptotic cell death but not at low levels of autophagy. Growth inhibition by ULK2 also appears to involve catalase degradation and reactive oxygen species generation. ULK2 overexpression inhibited anchorage independent growth, inhibited astrocyte transformation in vitro and tumor growth in vivo. Of all autophagy genes, we found ULK2 and its homologue ULK1 were only down-regulated in all grades of glioma. Thus these results altogether suggest that inhibition of autophagy by ULK1/2 down-regulation is essential for glioma development.