The median survival time of patients with glioblastoma is still poor (14.6 month), partly due to a lack of effective treatment.We have observed that androgen receptor (AR) is amplified in glioblastomas at the DNA, RNA and protein levels. The AR gene was amplified in 27% of glioblastoma specimens from men (n=22) and of 38.2% from women (n=21). AR-RNA was overexpressed (>2.5 fold) in 93% (n=30), and AR-protein was induced (>two fold) in 56% of the glioblastomas samples (n=16). Thirty percent of the glioblastomas (n=21) also expressed a constitutively active AR-splice-variant (AR-V7/AR3) lacking the Ligand-Binding-Domain. Following these findings, we examined the effect of pharmacological inhibition of androgen receptor in vitro and in vivo, as well as of genetic silencing of the receptor in glioblastoma cell lines. AR antagonists, induced concentration-dependent death in three glioblastoma cell lines, as well as in two glioma initiating cell lines. Silencing of AR expression by siRNA induced cell death in the three tested glioblastoma cell lines. Enzalutamide given orally to nude mice bearing subcutaneous human glioma xenografts resulted in a 72% reduction in tumor volume (p=0.0027).The presence of AR-V7/AR3 in glioblastoma, together with the present data showing that genetic silencing of the full length AR in cell lines and pharmacological inhibition of AR, induce GBM cell death in vivo and in vitro, point to the important role of AR in GBM survival and render a potential therapeutic target for this devastating disease.

OBJECTIVE Bevacizumab is an antiangiogenic agent under investigation for use in patients with high-grade glioma. It produces a high rate of radiological response; however, this response should be interpreted with caution because it may reflect normalization of the tumor vasculature and not necessarily a true antitumor effect. The authors previously demonstrated that 4 hypoxia-mediated microRNAs (miRNA)-miR-210, miR-21, miR-10b, and miR-196b-are upregulated in glioma as compared with normal brain tissue. The authors hypothesized that the regulation and expression of these miRNAs would be altered in response to bevacizumab treatment. The object of this study was to perform longitudinal monitoring of circulating miRNA levels in patients undergoing bevacizumab treatment and to correlate it with tumor response. METHODS A total of 120 serum samples from 28 patients with high-grade glioma were prospectively collected prior to bevacizumab (n = 15) or temozolomide (TMZ; n = 13) treatment and then longitudinally during treatment. Quantification of the 4 miRNAs was evaluated by real-time polymerase chain reaction using total RNA extracted from the serum. At each time point, tumor response was assessed by Response Assessment in Neuro-Oncology criteria and by performing MRI using fluid attenuated inversion recovery (FLAIR) and contrast-enhanced images. RESULTS As compared with pretreatment levels, high levels of miR-10b and miR-21 were observed in the majority of patients throughout the bevacizumab treatment period. miR-10b and miR-21 levels correlated negatively and significantly with changes in enhancing tumor diameters (r = -0.648, p < 0.0001) in the bevacizumab group but not in the TMZ group. FLAIR images and the RANO assessment did not correlate with the sum quantification of these miRNAs in either group. CONCLUSIONS Circulating levels of miR-10b and miR-21 probably reflect the antiangiogenic effect of therapy, but their role as biomarkers for tumor response remains uncertain and requires further investigation.

BackgroundNeuromyelitis optica (NMO) is a chronic autoimmune disease of the central nervous system (CNS). The main immunological feature of the disease is the presence of autoantibodies to Aquaporin 4 (AQP4+), identified in about 82 % of cases. Currently, there are no reliable biomarkers for monitoring treatment response in patients with NMO. In an effort to identify biomarkers, we analyzed microRNAs (miRNAs) in the blood of rituximab-treated NMO patients before and after therapy.MethodsTotal RNA extracted from whole blood of nine rituximab-responsive NMO patients before and 6 months following treatment was subjected to small RNAseq analysis. The study included an additional group of seven untreated AQP4+ seropositive NMO patients and 15 healthy controls (HCs).ResultsFourteen miRNAs were up regulated and 32 were downregulated significantly in the blood of NMO patients following effective therapy with rituximab (all p < 0.05). Furthermore, we show that expression of 17 miRNAs was significantly higher and of 25 miRNAs was significantly lower in untreated NMO patients compared with HCs (all p < 0.05). Following rituximab treatment, the expression levels of 10 of the 17 miRNAs that show increased expression in NMO reverted to the levels seen in HCs. Six of these “normalized” miRNAs are known as brain-specific/enriched miRNAs.ConclusionsSpecific miRNA signatures in whole blood of patients with NMO might serve as biomarkers for therapy response. Furthermore, monitoring the levels of brain-specific/enriched miRNAs in the blood might reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0648-x) contains supplementary material, which is available to authorized users.

Background: The clinical course of multiple sclerosis ranges from benign with little disease progression and minimal disability, to severe disease requiring intensive medical treatment. There are no reliable circulating biomarkers for predicting disease outcome. Co-inhibitory receptors regulate the termination of effective immune responses to infections while limiting autoimmunity and/or immunopathology. Based on this, we studied the potential of circulating co-inhibitory receptor levels as predictive biomarkers of multiple sclerosis outcome. Methods: Co-inhibitory receptor [TIGIT (T cell immunoreceptor with Ig and ITIM domains), TIM-3 (T-cell immunoglobulin and mucin domain–containing 3), LAG-3 (lymphocyte activation gene 3), PD-1 (programmed cell death 1), CTLA-4 (cytotoxic T-lymphocyte–associated protein 4)] expression levels in peripheral blood mononuclear cells (PBMCs) were measured using reverse transcription–PCR in 19 healthy controls and 57 patients with untreated multiple sclerosis. All patients were evaluated for disease outcome and paraclinical measures during the following 9–10 years [progression index, Expanded Disability Status Scale (EDSS) score, number of relapses, number of disease modifying therapies (DMTs), baseline brain magnetic resonance imaging T2 lesion volume, and oligoclonal bands (OCBs)]. Results: Patients had significantly lower TIGIT and LAG-3 levels than the controls ( P < 0.02 and P < 0.04, respectively). TIM-3 levels were significantly lower in patients with high vs. low disability index and in patients with SPMS diagnosis compared to patients who remained in the relapsing stage of the disease at final visit (both, P < 0.02). LAG-3 levels were significantly higher in patients with low disability index vs. non-low disability index multiple sclerosis ( P < 0.05). TIM-3 and LAG-3 expression levels correlated significantly with 1-year progression index ( r 2 = 0.076, P < 0.05; 0.087, P < 0.04, respectively) and EDSS score at final visit ( r 2 = 0.31, P < 0.04; 0.320.088, P < 0.04, respectively). Lower LAG-3 levels were associated with higher DMT switching ( r 2 = 0.67, P < 0.05). Compared to the paraclinical and clinical parameters alone, the combined data of the baseline co-inhibitory receptor expression levels and the paraclinical and clinical parameters were superior for predicting the patients that would progress to secondary progressive multiple sclerosis (SPMS). Interpretation: This is an initial exploration of the utility of CTLA-4, PD-1, TIM-3, LAG-3, and TIGIT expression levels as prognostic indicators in untreated, recently diagnosed multiple sclerosis. Our results support the ...

Background Glioblastoma (GBM) is associated with poor overall survival. Recently, we showed that androgen receptor (AR) protein is overexpressed in 56% of GBM specimens and AR antagonists induced dose-dependent death in several GBM cell lines and significantly reduced tumor growth and prolonged the lifespan of mice implanted with human GBM. 16β-18F-fluoro-5α-dihydrotestosterone ([18F]-FDHT) is a PET tracer used to detect AR expression in prostate and breast cancers. This study was aimed at exploring the ability of [18F]-FDHT-PET to detect AR expression in high-grade gliomas. Methods Twelve patients with suspected high-grade glioma underwent a regular workup and additional dynamic and static [ 18 F]-FDHT-PET/CT. Visual and quantitative analyses of [ 18 F]-FDHT kinetics in the tumor and normal brain were performed. Mean and maximum (max) standardized uptake values (SUVs) were determined in selected volumes of interest. The patients had surgery or biopsy after PET/CT. AR protein was analyzed in the tumor samples by western blot. Fold change in AR expression was calculated by densitometry analysis. Correlation between imaging and AR protein samples was determined. Results In six of the 12 patients, [ 18 F]-FDHT uptake was significantly higher in the tumor than in the normal brain. These patients also had increased AR protein expression within the tumor. Pearson correlation coefficient analysis for the tumor-to-control normal brain uptake ratio in terms of SUVmean versus AR protein expression, was positive and significant (R = 0.84; p = 0.002). Conclusion [ 18 F]-FDHT-PET/CT could identify increased AR expression in high-grade glioma.

The 54 microRNAs (miRNAs) within the DLK-DIO3 genomic region on chromosome 14q32.31 (cluster-14-miRNAs) are organized into sub-clusters 14A and 14B. These miRNAs are downregulated in glioblastomas and might have a tumor suppressive role. Any association between the expression levels of cluster-14-miRNAs with overall survival (OS) is undetermined. We randomly selected miR-433, belonging to sub-cluster 14A and miR-323a-3p and miR-369-3p, belonging to sub-cluster 14B, and assessed their role in glioblastomas in vitro and in vivo. We also determined the expression level of cluster-14-miRNAs in 27 patients with newly diagnosed glioblastoma, and analyzed the association between their level of expression and OS. Overexpression of miR-323a-3p and miR-369-3p, but not miR-433, in glioblastoma cells inhibited their proliferation and migration in vitro. Mice implanted with glioblastoma cells overexpressing miR323a-3p and miR369-3p, but not miR433, exhibited prolonged survival compared to controls (P = .003). Bioinformatics analysis identified 13 putative target genes of cluster-14-miRNAs, and real-time RT-PCR validated these findings. Pathway analysis of the putative target genes identified neuregulin as the most enriched pathway. The expression level of cluster-14-miRNAs correlated with patients' OS. The median OS was 8.5 months for patients with low expression levels and 52.7 months for patients with high expression levels (HR 0.34; 95 % CI 0.12-0.59, P = .003). The expression level of cluster-14-miRNAs correlates directly with OS, suggesting a role for this cluster in promoting aggressive behavior of glioblastoma, possibly through ErBb/neuregulin signaling.

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