Shivayogi D. Shwetha

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.

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.

Prognostic significance of histomolecular subgroups of adult anaplastic (WHO Grade III) gliomas: applying the ‘integrated’ diagnosis approach

Aims Anaplastic gliomas (AGs; WHO Grade III) include anaplastic astrocytoma (AA), anaplastic oligodendroglioma (AO) and anaplastic oligoastrocytoma (AOA) and are known to have variable prognosis. Since biomarkers have a major impact on prognosis of gliomas, we compared the prognostic significance of the established biomarkers of AGs and the ‘histomolecular’ subgroups based on the proposed International Society of Neuropathology-Haarlem (‘ISN-Haarlem’) guidelines, with the current WHO 2007 classification. Methods The study was carried out on formalin-fixed paraffin-embedded (FFPE) tissues from 91 adult patients with AG. Clinical, histological and molecular parameters, including 1p/19q codeletion, isocitrate dehydrogenase gene (IDH1)-R132H positivity, α thalassemia/mental retardation syndrome X-linked gene (ATRX) expression and O6-methylguanine-DNA-methyltransferase gene (MGMT) promoter methylation (mMGMT), were correlated with overall survival (OS) and recurrence-free survival (RFS). Subsequently, following sequencing for rare IDH mutations, we derived three ‘histomolecular’ subgroups based on the ‘integrated’ diagnosis approach proposed by ‘ISN-Haarlem’ guidelines and correlated this with clinical outcome. Results Gross tumour resection, administration of radiochemotherapy, 1p/19q codeletion, IDH1-R132H positivity and mMGMT were associated with favourable OS and RFS (p≤0.001), while the WHO histological subgroups were prognostically not significant. The ISN ‘histomolecular’ subgroups prognosticated best with AOs (IDHmut, 1p/19q codeleted, ATRX predominantly retained) having the best survival, followed by the AAs (IDHmut, ATRX loss or retained, 1p19q non-codeleted) and AA IDH wild type group having the worst OS and RFS (p=<0.001 for OS). Conclusions Our study reiterates the prognostic significance of biomarkers, 1p/19q codeletion, IDH1-R132H positivity and mMGMT in AGs. Importantly, we show that the ‘histomolecular’ subgroups of AGs based on the ‘integrated’ diagnosis has a prognostic value, superior to the WHO histological classification.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1β in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n=27) and GBM (n=28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p<0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-FcγRIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1β (IL1β) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1β neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1β which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1β as a potential candidate for developing targeted therapy.

Integrative functional genomic analysis identifies epigenetically regulated fibromodulin as an essential gene for glioma cell migration

An integrative functional genomics study of multiple forms of data are vital for discovering molecular drivers of cancer development and progression. Here, we present an integrated genomic strategy utilizing DNA methylation and transcriptome profile data to discover epigenetically regulated genes implicated in cancer development and invasive progression. More specifically, this analysis identified fibromodulin (FMOD) as a glioblastoma (GBM) upregulated gene because of the loss of promoter methylation. Secreted FMOD promotes glioma cell migration through its ability to induce filamentous actin stress fiber formation. Treatment with cytochalasin D, an actin polymerization inhibitor, significantly reduced the FMOD-induced glioma cell migration. Small interfering RNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on integrin-FAK-Src-Rho-ROCK signaling pathway. FMOD lacking C-terminus LRR11 domain (ΔFMOD), which does not bind collagen type I, failed to induce integrin and promote glioma cell migration. Further, FMOD-induced integrin activation and migration was abrogated by a 9-mer wild-type peptide from the FMOD C-terminus. However, the same peptide with mutation in two residues essential for FMOD interaction with collagen type I failed to compete with FMOD, thus signifying the importance of collagen type I–FMOD interaction in integrin activation. Chromatin immunoprecipitation–PCR experiments revealed that transforming growth factor beta-1 (TGF-β1) regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but enrichment of Sma- and Mad-related protein-2 (SMAD2) and CBP. FMOD silencing inhibited the TGF-β1-mediated glioma cell migration significantly. In univariate and multivariate Cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, this study identified several epigenetically regulated alterations responsible for cancer development and progression. Specifically, we found that secreted FMOD as an important regulator of glioma cell migration downstream of TGF-β1 pathway and forms a potential basis for therapeutic intervention in GBM.

Manganese superoxide dismutase (MnSOD) is a malignant astrocytoma specific biomarker and associated with adverse prognosis in p53 expressing glioblastoma

BACKGROUND Manganese super oxide dismutase (MnSOD) has been previously identified as one of the top regulated genes associated with poor survival in glioblastoma (GBM) patients. In the current study we have evaluated the protein expression of MnSOD across various grades of astrocytoma, studied its influence on survival of GBM patients and following recurrence. METHODS The protein expression of MnSOD was analyzed on tumor tissue sections by immunohistochemistry on 30 diffuse astrocytomas (DA), 50 anaplastic astrocytomas (AA), 30 paired (primary and recurrent) GBM samples and 30 non-tumor brain tissues. The protein expression among the different grades of diffusely infiltrating astrocytoma (DIA) was evaluated by Kruskal-Wallis one-way ANOVA followed by post hoc test. Wilcoxon matched pair test was employed to assess MnSOD protein expression across 30 paired GBM samples (primary and recurrent). The prognostic impact of MnSOD protein expression individually and following stratification with p53 expression was evaluated in a cohort of 123 GBM patients. Both over-all survival (OS) and progression free survival (PFS) analysis were performed by employing Cox regression analysis and Kaplan-Meier survival analysis on GBM patients. RESULTS A significantly increased protein expression of MnSOD was observed among malignant astrocytomas (GBM and AA) in comparison with either DA or non-tumor brain tissues (p<0.05). Among the GBM cases it was noted that the IDH1 immunopositive tumors (R132H mutant protein; n=17) had a low MnSOD expression as opposed to IDH1 immunonegative tumors (n=106), which had high expression of MnSOD (p=0.0307). Further, a statistically significant increase (p=0.010) in extent of MnSOD protein expression was also noted in GBM tumors following recurrence. Protein expression of MnSOD was associated with both poor OS (HR: 1.021; p=0.011) and early PFS (HR: 1.022; p=0.006) on univariate analysis. Multivariate Cox regression analysis as well as Kaplan-Meier survival analysis demonstrated similar poor prognostic association. Stratification of GBM cases based on p53 expression status revealed a strong association of MnSOD with OS (HR: 1.042; p=0.002) and PFS (HR: 1.044; p=0.001) in p53 positive tumor tissue samples. Similar findings were noted on multivariate Cox regression analysis and K-M survival analysis, while no such association was noted in tumor tissues staining negative for p53 expression. CONCLUSIONS Our study shows an increased expression of MnSOD in anaplastic astrocytoma and GBM compared to low grade astrocytoma and control brain. An increase in MnSOD expression following GBM tumor recurrence strengthens its putative role in tumor aggressiveness. Further, MnSOD emerges as a poor prognostic biomarker in p53 expressing GBMs, rendering this molecule as a potential therapeutic target in such patients.

Granulomatous and lymphocytic hypophysitis - are they immunologically distinct?

Hypophysitis includes three histopathologically distinct entities – granulomatous, lymphocytic and xanthomatous forms. Etiopathogenesis and the immunological differences among these is not well characterized. This study aims to explore the immunopathogenesis of granulomatous and lymphocytic forms of hypophysitis. Demographic, clinical, endocrine function and radiological features of 33 histologically confirmed cases of hypophysitis were reviewed. Immunophenotyping of inflammatory component was performed in 13/33 cases. Visual disturbances (46%), headache (36%), polyuria/polydipsia (6%), menstrual disturbance (6%) and galactorrhoea (6%) were the frequent presenting symptoms. Endocrine abnormalities were noted in 11/18 cases evaluated (61%). Hypothyroidism was the most common endocrine abnormality (33.33%) followed by hyperprolactinaemia (22%) and hypocortisolism (16.66%). On neuroimaging, sellar mass with variable contrast enhancement was observed. On histology, granulomatous hypophysitis (GH) was more common (84.84%) than lymphocytic hypophysitis (LH) (15.15%). In GH, the infiltrate had almost equal proportions of CD3+ T cells and CD68+ histiocytes. Cytotoxic T cells (CD8+) predominated [CD4:CD8 < 1]. CD20+ B cell component ranged from <5% to 50%. Fibrosis, necrosis and giant cells accompanied GH. LH in contrast, had CD4+ T‐helper cell predominance [CD4: CD8 > 1]. CD68+ histiocytes constituted <20% and CD20+ B cells, 5–40% of the infiltrates. In conclusion, GH revealed cytotoxic T cell and histiocyte rich infiltrate in contrast to CD4+ T‐cell predominance in LH suggesting that the two forms have distinct immunological mechanisms in evolution, an autoimmune process in LH and type IV hypersensitivity response in GH.

Crooke's cell adenoma of the pituitary: a histological, immunocytochemical, and electron microscopic study of a rare case.

216 Neurology India | Mar-Apr 2014 | Vol 62 | Issue 2 recurrence attributed to partial resection due to the proximity of the lesion to critical neuronal and vascular structures. However, no recurrences have been reported to date in dural-based classical chondrosarcomas.[5-8] Mesenchymal and myxoid subtype indicates worse clinical behavior and poorer prognosis than conventional histology. About 50% of the mesenchymal and nearly all the myxoid variants showed recurrence.[3] Some authors recommend adjuvant radiation therapy to prevent recurrence, but it is diffi cult to tell if this mode of adjuvant therapy is effective because the few cases prohibit drawing any conclusions about such treatment.