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Background: Sox11 is a transcription factor expressed in foetal and neoplastic brain tissue, including gliomas. It has been shown to suppress the tumourigenicity of glioma stem cells in vivo, thereby being hypothesised to function as a tumour suppressor., Methods: We investigated the expression of Sox11 in 132 diffuse astrocytomas in relation to the regulator cell marker nestin, c-Met and IDH1-R132H, which have shown to be differentially expressed among the molecular subgroups of malignant gliomas, as well as to an inducer of astrocytic differentiation, that is, signal transducer and activator of transcription (p-STAT-3), clinicopathological features and survival., Results: Sox11 immunoreactivity was identified in all tumours irrespective of grade, but being correlated with p-STAT-3. Three out of seven cases showed partial Sox11 promoter methylation. In >50% of our cases neoplastic cells coexpressed Sox11 and nestin, a finding further confirmed in primary glioblastoma cell cultures. Furthermore, nestin, c-Met and IDH1-R132H expression differed among grade categories. Cluster analysis identified four groups of patients according to c-Met, nestin and IDH1-R132H expression. The c-Met/nestin high-expressor group displayed a higher Sox11 expression. Sox11 expression was an indicator of favourable prognosis in glioblastomas, which remained in multivariate analysis and validated in an independent set of 72 cases. The c-Met/nestin high-expressor group was marginally with shorter survival in univariate analysis., Conclusions: We highlight the importance of Sox11 expression as a favourable prognosticator in glioblastomas. c-Met/nestin/IDH1-R132H expression phenotypes recapitulate the molecular subgroups of malignant glioma.

Aims: To investigate the significance of the mammalian target of rapamycin (mTOR) pathway in astrocytic tumours, published information in this context being limited, especially regarding phosphorylated 4E-binding protein (p-4E-BP) 1., Methods and Results: Paraffin-embedded tissue from 111 patients with astroglial tumours (grades II-IV) was investigated for the association of phosphorylated mTOR (p-mTOR) signalling components with phosphorylated extracellular signal-related kinase 1/2 (p-ERK1/2) and phosphorylated AKT (p-AKT) expression, clinicopathological features, angiogenesis, isocitrate dehydrogenase 1 (IDH1)-R132H, and survival. Expression was also quantified by western blot analysis in 12 cases and in three primary glioma cell cultures following rapamycin treatment. p-mTOR expression correlated with p-4E-BP1 expression and marginally with p-p70S6K expression. p-4E-BP1 expression increased with tumour grade. Rapamycin induced a decline in phosphorylation levels of all three proteins. Nuclear p-AKT and cytoplasmic p-ERK1/2 immunoexpression correlated with p-4E-BP1 expression, whereas cytoplasmic p-AKT expression correlated with p-p70S6K expression. All three proteins were associated with increased angiogenesis but not with IDH1-R132H expression status. p-mTOR adversely affected overall and disease-free survival in univariate analysis. In multivariate survival analysis, the presence of p-4E-BP1 predicted shortened overall survival in the entire cohort and glioblastomas., Conclusions: mTOR signalling components are differentially involved in the acquisition of a more aggressive and angiogenic phenotype in astrocytic tumours. Moreover, p-4E-BP1 emerges as a novel prognostic marker, which might aid in the selection of patients who are more likely to benefit from therapy with mTOR inhibitors., (© 2012 Blackwell Publishing Ltd.)

The aim was to expand recently published information regarding the significance of the interleukin (IL)-8/p-STAT-3 (signal transducer and activator of transcription) pathway in astrocytomas, focusing on the IL-8 receptor, chemokine (C-X-C motif) receptor 2 (CXCR2), and the STAT-3 inhibitor SOCS-3 (suppressors of cytokine signaling). A total of 91 paraffin-embedded human astrocytoma tissues (grades II-IV) were investigated for the association of SOCS-3 and CXCR2 expression with clinicopathologic and morphometric microvascular characteristics, vascular endothelial growth factor (VEGF), IL-8 and p-STAT-3 expression and patient survival. Peripheral IL-8 secretion levels were assessed by enzyme-linked immunosorbent spot (ELISPOT). SOCS-3, p-STAT-3 and CXCR2 protein levels were also quantified by Western immunoblotting in six cases, and the protein levels of SOCS-3 and CXCR2 were correlated with the immunohistochemical expression of the respective proteins. All CXCR2-positive cases by Western immunoblotting displayed increased peripheral IL-8 secretion levels. Treatment of primary glioblastoma cell cultures with exogenous IL-8 enhanced proliferation, and this effect was inhibited by treatment with a neutralizing anti-CXCR2 antibody. SOCS-3 and CXCR2 were expressed by neoplastic astrocytes in 92.4% and 48.78% of cases, respectively, with their levels increasing with histological grade and extent of necrosis. VEGF expression and microvessel density, CXCR2 and IL-8 levels were interrelated. SOCS-3 and p-STAT-3 were co-expressed in 85.7% of cases, although they were not interrelated. In univariate survival analysis, increased SOCS-3 expression and the presence of CXCR2 adversely affected survival, whereas in multivariate analysis, only CXCR2 remained significant. The prognostic significance of CXCR2 was validated in an independent set of 63 patients. Our data implicate IL-8/CXCR2 signaling pathway in the progression and regulation of angiogenesis in astrocytomas and provide a rationale for CXCR2 therapeutic exploitation in these tumors.

Although pERK and pAKT are reportedly activated in various neoplasms, little information is available about their significance in astrocytomas. Paraffin-embedded tissue from 82 patients with diffuse infiltrating astrocytomas (grades II to IV) was investigated for the association of pERK and pAKT activation with clinicopathological features, vascular endothelial growth factor (VEGF), isocitrate dehydrogenase 1 and microvascular parameters. Nuclear pERK labelling index (LI) increased with increasing cytoplasmic pERK LI and nuclear and cytoplasmic pAKT LI (p = 0.0019, p = 0.0260 and p = 0.0012, respectively). Accordingly, cytoplasmic pERK increased with increasing levels of nuclear (p = 0.0001) and marginally with cytoplasmic pAKT LI (p = 0.0526). Nuclear and cytoplasmic pERK LI and nuclear pAKT LI were positively correlated with tumour histological grade (p = 0.0040, p = 0.0238 for pERK and p = 0.0004 for pAKT, respectively). VEGF expression was correlated with nuclear pERK (p = 0.0099) and nuclear pAKT LI (p = 0.0002). Interestingly, pERK cytoplasmic LI increased with microvessel calibre (p = 0.0287), whereas pAKT nuclear LI was marginally related to microvessel density (p = 0.0685). The presence of IDH1-R132H was related only to histological grade and lower microvessel calibre. Multivariate survival analysis in the entire cohort selected cytoplasmic pAKT LI (p = 0.045), histological grade, microvessel calibre (p = 0.028), patients' age, gender and surgical excision as independent predictors of survival. Moreover, in glioblastomas, pERK nuclear LI emerged as a favourable prognosticator in the presence of IDH1-R132H. pERK and pAKT in astrocytomas are interrelated and associated with tumour grade and angiogenesis. Moreover, the importance of cytoplasmic pAKT immunoexpression in patients' prognosis and nuclear pERK immunoexpression in glioblastomas is confirmed.

Objective: The analysis of the presence of PIK3CA and B-RAF gene mutations in relation to ERK and AKT activation in diffusely infiltrating astrocytomas, in order to determine their potential role in tumor aggressiveness., Methods: Polymerase chain reaction-single strand confirmation polymorphism (PCR-SSCP) and sequencing analysis were used for PIK3CA and B-RAF gene mutation detection. pERK and pAKT expression were examined by immunohistochemistry., Results: PIK3CA mutations were found in 2 (3%) cases of glioblastomas whereas none of these cases displayed mutations in exon 15 of B-RAF gene. Neither low grade astrocytomas nor anaplastic astrocytomas revealed any mutations in these genes. Nuclear and cytoplasmic pERK immunoreactivity was displayed in 100% and 82% of cases, respectively. pERK nuclear expression was positively correlated with pERK cytoplasmic expression (p = 0.0067). Moreover, pERK nuclear expression increased in parallel with tumor grade (II, III v/s IV, p = 0.0262). Nuclear and cytoplasmic pAKT immunoreactivity was displayed in 97% and 100% of cases, respectively. Similarly, pAKT nuclear expression was positively correlated with pAKT cytoplasmic expression (p = 0.0074). pAKT cytoplasmic expression increased with increasing tumor grade (II,III v/s IV, p = 0.0930), although the latter relationship was of marginal significance. pAKT cytoplasmic expression was also positively correlated with pERK nuclear expression (p = 0.0156)., Conclusions: Our study reports the low frequency of PIK3CA and B-RAF mutations in astrocytomas, despite the presence of activated ERK and AKT proteins. Moreover, the correlation of pERK nuclear and pAKT cytoplasmic expression with tumor grade suggests the possible crucial role of the activation of these proteins in human gliomagenesis.