Differential analysis of glioblastoma multiforme proteome by a 2D-DIGE approach

<p>Abstract</p> <p>Background</p> <p>Genomics, transcriptomics and proteomics of glioblastoma multiforme (GBM) have recently emerged as possible tools to discover therapeutic targets and biomarkers for new therapies including immunotherapy. It is well known that macroscopically complete surgical excision, radiotherapy and chemotherapy have therapeutic limitations to improve survival in these patients. In this study, we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify proteins that may serve as brain tumor antigens in new therapeutic assays. Five samples of patients presenting a GBM and five samples of microscopically normal brain tissues derived from brain epileptic surgery specimen were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis) with an internal pool sample on each gel. Five gels were matched and compared with DIA (Difference In-gel Analysis) software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained.</p> <p>Results</p> <p>From 51 protein-spots significantly up-regulated in GBM samples, mass spectrometry (MS) identified twenty-two proteins. The differential expression of a selected protein set was first validated by western-blotting, then tested on large cohorts of GBM specimens and non-tumor tissues, using immunohistochemistry and real-time RT-PCR.</p> <p>Conclusions</p> <p>Our results confirmed the importance of previously described proteins in glioma pathology and their potential usefulness as biological markers but also revealed some new interesting targets for future therapies.</p

DNA methylation in glioblastoma: impact on gene expression and clinical outcome

International audienceBACKGROUND: Changes in promoter DNA methylation pattern of genes involved in key biological pathways have been reported in glioblastoma. Genome-wide assessments of DNA methylation levels are now required to decipher the epigenetic events involved in the aggressive phenotype of glioblastoma, and to guide new treatment strategies. RESULTS: We performed a whole-genome integrative analysis of methylation and gene expression profiles in 40 newly diagnosed glioblastoma patients. We also screened for associations between the level of methylation of CpG sites and overall survival in a cohort of 50 patients uniformly treated by surgery, radiotherapy and chemotherapy with concomitant and adjuvant temozolomide (STUPP protocol). The methylation analysis identified 616 CpG sites differentially methylated between glioblastoma and control brain, a quarter of which was differentially expressed in a concordant way. Thirteen of the genes with concordant CpG sites displayed an inverse correlation between promoter methylation and expression level in glioblastomas: B3GNT5, FABP7, ZNF217, BST2, OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14. Survival analysis identified six CpG sites associated with overall survival. SOX10 promoter methylation status (two CpG sites) stratified patients similarly to MGMT status, but with a higher Area Under the Curve (0.78 vs. 0.71, p-value < 5e-04). The methylation status of the FNDC3B, TBX3, DGKI, and FSD1 promoters identified patients with MGMT-methylated tumors that did not respond to STUPP treatment (p-value < 1e-04). CONCLUSIONS: This study provides the first genome-wide integrative analysis of DNA methylation and gene expression profiles obtained from the same GBM cohort. We also present a methylome-based survival analysis for one of the largest uniformly treated GBM cohort ever studied, for more than 27,000 CpG sites. We have identified genes whose expression may be tightly regulated by epigenetic mechanisms and markers that may guide treatment decisions

Intraneural ganglion cyst of the tibial nerve.: Intraneural ganglion cyst of the tibial nerve

Intraneural ganglion cyst of the tibial nerve is very rare. To date, only 5 cases of this entity in the popliteal fossa have been reported. We report a new case and review the previously reported cases. A 40-year-old man experienced a mild vague pain in the medial half of his right foot for 3 years. Magnetic resonance imaging scan demonstrated a soft-tissue mass along the right tibial nerve. At surgery, an intraneural ganglion cyst was evacuated. After 12 months, the patient was pain-free with no signs of recurrence. Trauma might be a contributing factor to the development of intraneural ganglion cysts. Application of microsurgical techniques is encouraged

Hearing loss attributable to a cerebellopontine-angle arachnoid cyst in a child

International audienceAlthough hearing loss in newborns and infants is predominantly due to malformations and infections, there are other situations which may compromise hearing quality in later stages, including posterior-fossa arachnoid cysts (ACs). We report the case of an 8-year-old girl who presented with hearing loss linked to a pontocerebellar-angle AC which had been diagnosed and treated when she was 14 months old. The pathophysiology of this late AC complication is discussed. This case reminds us that a close follow-up with audiologic monitoring and/or brain stem auditory evoked response is necessary in children with posterior-fossa AC because modern neuroradiological imaging methods do not inform about cerebral and nerve functions, although they provide excellent morphological details of ACs and have improved the ease and accuracy of their early diagnosis. Therefore, surgery should be performed before complete hearing loss occurs; however, in hearing-impaired patients, it remains unclear which surgical treatment is most appropriate

Integration of Genomic Alterations and Expression Profiling in Glioblastoma Multiforme.

International audiencePurpose: Glioblastomas (GBM) are highly malignant and heterogeneous gliomas with very poor prognosis. The biological and molecular characterization of these tumors is still challenging and impacts their therapeutic management. Previous genomic surveys have revealed the highly rearranged nature of GBM genome and transcriptome. However, the impacts of tumor DNA aberrations on gene expression remain unclear. Methods: We investigated copy number alterations (CNA) and gene expression to identify causal genetic events in GBM. High-resolution maps of somatic chromosomal alterations were obtained for 20 GBM. Gene expression profiling was carried out on the same tumor samples, and compared to those obtained on nonneoplastic brain samples. Concordance between CNA and gene expression was identified by two complementary approaches (correlated or targeted probes). The resulting GBM signature was validated with an independent microarray data set of 81 GBM and 23 normal brains. Results: Loci targeted for high-priority minimal common regions (MCR) of recurrent CNA were defined and combined with gene expression profiles performed on the same tumor samples. Genes with concordant changes in CNA and expression levels were defined as over/underexpressed genes located in amplified/deleted regions, or as MCR genes with expression correlated to the corresponding genomic state. After validation, we found that the expression of 318 genes was significantly affected by CNA. Associated enriched GO process annotations were related to cell cycle disorder, cellular adhesion and angiogenesis. The gene signature included well-known GBM genes such as EGFR, PDGFA, and p16INK4 but also novel candidate genes. Two tumor suppressor genes PCDH9 and STARD13, involved in tumor invasiveness and resistance to etoposide, were validated by qPCR in an independent set of 57 glioblastoma. Conclusion: This study shows the power of combining genomic alterations and gene expression to identify robust transcriptome signature and putative tumor biomarkers in GBM

SAH incidence in Martinique compared to other regions.

<p>Figure legend: Results are compared with the meta-analysis of de Rooij <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155945#pone.0155945.ref001" target="_blank">1</a>]. All countries other than Japan, Finland and South and Central America (SCA) were pooled in a reference group (Reference countries). Number per 100 000 person-years, with corresponding 95% CI, are represented.</p

A Four-Gene Signature Associated with Clinical Outcome in High-Grade Gliomas.

International audienceMolecular studies of high-grade gliomas (HGGs) have highlighted the heterogeneity of these tumors, and have linked molecular signatures to their natural history and to differences in survival rates. The ability to identify such molecular subtypes of tumors is essential for guiding therapeutic advances. We report the development and validation of a robust risk-score model highly associated with the outcome of patients with newly diagnosed HGG. We considered a supervised approach to account for the WHO grade of malignity when deriving gene biomarkers associated with outcome. We performed a meta-analysis of HGGs microarray data sets (267 patients) to identify such biomarkers from a robust signature related to tumor aggressiveness. These biomarkers were used to construct a risk-score equation based on a Cox proportional hazards model. The model best associated with overall survival (OS) and with good discrimination (C-statistic) was based on the expression of 4 genes. Patients... were ranked according to their risk score and stratified into 2 groups. Low-risk score patients had a median OS longer than high-risk score patients (46.6 vs 11.7 months, P , .001). These results were validated on an independent microarray study of 59 patients. We performed RT-qPCR validation on an independent set of HGGs (194 patients) and compared the performances of our risk-score model with the prognostic value of currently admitted clinical and molecular risk factors. Two multivariate models were built, including age, treatment, grade, RTOG RPA classes, MGMT methylation status, and IDH1 mutational status; one with and one without the 4-gene expression risk score. These models were used to estimate the prognostic value of the gene expression risk score for 176 patients with complete data for all variables and for a subset of 105 patients treated with temozolomide chemoradiation. This analysis confirmed that both the mutations of IDH1 and the presence of MGMT promoter methylation were associated with a survival benefit (P , .01 in the whole cohort and P , .05 in the subset). It also showed that the 4-gene risk score was strongly associated with OS in these two groups, independently from clinical and molecular risk factors (P , .01). Each time, the model discrimination improved significantly with the addition of the 4-gene risk score (0.816 vs 0.846, P , .001 and 0.792 vs 0.822, P , .001, respectively), showing that it added beyond standard clinical parameters and beyond both the MGMT methylation status and the IDH1 mutational status. One explanation for the association between the 4-gene signature and clinical outcome is that it may detect the molecular fingerprints inherent to glioma aggressiveness. These results suggest the importance of this 4-gene signature as a stratification factor for future comparative therapeutic trials, though it needs to be further investigated in a prospective clinical study

Immune genes are associated with human glioblastoma pathology and patient survival

International audienceBackground: Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults. Several recent transcriptomic studies in GBM have identified different signatures involving immune genes associated with GBM pathology, overall survival (OS) or response to treatment. Methods: In order to clarify the immune signatures found in GBM, we performed a co-expression network analysis that grouped 791 immune-associated genes (IA genes) in large clusters using a combined dataset of 161 GBM specimens from published databases. We next studied IA genes associated with patient survival using 3 different statistical methods. We then developed a 6-IA gene risk predictor which stratified patients into two groups with statistically significantly different survivals. We validated this risk predictor on two other Affymetrix data series, on a local Agilent data series, and using RT-Q-PCR on a local series of GBM patients treated by standard chemo-radiation therapy. Results: The co-expression network analysis of the immune genes disclosed 6 powerful modules identifying innate immune system and natural killer cells, myeloid cells and cytokine signatures. Two of these modules were significantly enriched in genes associated with OS. We also found 108 IA genes linked to the immune system significantly associated with OS in GBM patients. The 6-IA gene risk predictor successfully distinguished two groups of GBM patients with significantly different survival (OS low risk: 22.3 months versus high risk: 7.3 months; p < 0.001). Patients with significantly different OS could even be identified among those with known good prognosis (methylated MGMT promoter-bearing tumor) using Agilent (OS 25 versus 8.1 months; p < 0.01) and RT-PCR (OS 21.8 versus 13.9 months; p < 0.05) technologies. Interestingly, the 6-IA gene risk could also distinguish proneural GBM subtypes. Conclusions: This study demonstrates the immune signatures found in previous GBM genomic analyses and suggests the involvement of immune cells in GBM biology. The robust 6-IA gene risk predictor should be helpful in establishing prognosis in GBM patients, in particular in those with a proneural GBM subtype, and even in the well-known good prognosis group of patients with methylated MGMT promoter-bearing tumors

Number of spontaneous subarachnoid hemorrhages (SAH) recorded annually in Martinique between January 1st 2007 and December 31st 2013.

<p>Number of spontaneous subarachnoid hemorrhages (SAH) recorded annually in Martinique between January 1st 2007 and December 31st 2013.</p