Pavel Fadrus

MiR-195, miR-196b, miR-181c, miR-21 expression levels and O-6-methylguanine-DNA methyltransferase methylation status are associated with clinical outcome in glioblastoma patients

Glioblastoma multiforme (GBM) is the most frequently occurring primary malignant brain tumor; patients with GBM often have a very poor prognosis and differing responses to treatment. Therefore, it is very important to find new biomarkers that can predict clinical outcomes and help in treatment decisions. MicroRNAs are small, non‐coding RNAs that function as post‐transcriptional regulators of gene expression and play a key role in the pathogenesis of GBM. In a group of 38 patients with primary GBM, we analyzed the expression of eight microRNAs (miR‐21, miR‐128a, miR‐181c, miR‐195, miR‐196a, miR‐196b, miR‐221, and miR‐222). In addition, we examined the methylation status of O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter by high‐resolution melting analysis, as this has been shown to be a predictive marker in GBM. MGMT methylation status correlated with progression‐free survival (P = 0.0201; log–rank test) as well as with overall survival (P = 0.0054; log–rank test). MiR‐195 (P = 0.0124; log–rank test) and miR‐196b (P = 0.0492; log–rank test) positively correlated with overall survival. Evaluation of miR‐181c in combination with miR‐21 predicted time to progression within 6 months of diagnosis with 92% sensitivity and 81% specificity (P < 0.0001). Our data confirmed that the methylation status of MGMT but also miR‐21, miR‐181c, miR‐195, and miR‐196b to be associated with survival of GBM patients. Above all, we suggest that the combination of miR‐181c and miR‐21 could be a very sensitive and specific test to identify patients at high risk of early progression after surgery. (Cancer Sci 2011; 102: 2186–2190)

Expression of immune-modulatory molecules HLA-G and HLA-E by tumor cells in glioblastomas: An unexpected prognostic significance?

The role of nonclassical human leukocyte antigens G and E (HLA‐G and HLA‐E) was originally thought to be restricted to the protection of the fetus from a maternal allorecognition. Now it is known that HLA‐G and HLA‐E exert multiple immunoregulatory functions. A prognostic significance of the expression of HLA‐G and HLA‐E by neoplastic cells in glioblastoma is not well characterized. In this study, we evaluated the expression of HLA‐G and HLA‐E by neoplastic cells in 39 cases of glioblastoma. We found the production of HLA‐G and HLA in a majority of cases. There was an unexpected positive correlation between the expression of HLA‐E and length of survival. We speculate that the expression of this molecule by neoplastic cells may represent a coincidental selective pro‐host advantage related to better response to subsequent therapeutic modalities. Mechanisms of glioblastoma cell pathophysiology and mechanisms of responses to therapeutic interventions in respect to the expression of these molecules deserves further study.

MicroRNAs involved in chemo- and radioresistance of high-grade gliomas

High-grade gliomas (HGGs) are malignant primary brain tumors of glial cell origin. Despite optimal course of treatment, including maximal surgical resection followed by adjuvant chemo- and/or radiotherapy, the prognosis still remains poor. The main reason is the commonly occurring chemo- and radioresistance of these tumors. In recent years, several signaling pathways, especially PI3K/AKT and ATM/CHK2/p53, have been linked to the resistance of gliomas. Moreover, additional studies have shown that these pathways are significantly regulated by microRNAs (miRNAs), short endogenous RNA molecules that modulate gene expression and control many biological processes including apoptosis, proliferation, cell cycle, invasivity, and angiogenesis. MiRNAs are not only highly deregulated in gliomas, their expression signatures have also been shown to predict prognosis and therapy response. Therefore, they present promising biomarkers and therapeutic targets that might overcome the resistance to treatment and improve prognosis of glioma patients. In this review, we summarize the current knowledge of the functional role of miRNAs in gliomas resistance to chemo- and radiotherapy.

Risk Score based on microRNA expression signature is independent prognostic classifier of glioblastoma patients

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. The prognosis of GBM patients varies considerably and the histopathological examination is not sufficient for individual risk estimation. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and were repeatedly proved to play important roles in pathogenesis of GBM. In our study, we performed global miRNA expression profiling of 58 glioblastoma tissue samples obtained during surgical resections and 10 non-tumor brain tissues. The subsequent analysis revealed 28 significantly deregulated miRNAs in GBM tissue, which were able to precisely classify all examined samples. Correlation with clinical data led to identification of six-miRNA signature significantly associated with progression free survival [hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.33-2.94, P < 0.001] and overa+ll survival (HR 2.86, 95% CI 1.91-4.29, P < 0.001). O(6)-methylguanine-DNA methyltransferase methylation status was evaluated as reference method and Risk Score based on six-miRNA signature indicated significant superiority in prediction of clinical outcome in GBM patients. Multivariate Cox analysis indicated that the Risk Score based on six-miRNA signature is an independent prognostic classifier of GBM patients. We suggest that the Risk Score presents promising prognostic algorithm with potential for individualized treatment decisions in clinical management of GBM patients.

Identification of microRNAs differentially expressed in glioblastoma stem-like cells and their association with patient survival

Glioblastoma stem-like cells (GSCs) are critical for the aggressiveness and progression of glioblastoma (GBM) and contribute to its resistance to adjuvant treatment. MicroRNAs (miRNAs) are small, non-coding RNAs controlling gene expression at the post-transcriptional level, which are known to be important regulators of the stem-like features. Moreover, miRNAs have been previously proved to be promising diagnostic biomarkers in several cancers including GBM. Using global expression analysis of miRNAs in 10 paired in-vitro as well as in-vivo characterized primary GSC and non-stem glioblastoma cultures, we identified a miRNA signature associated with the stem-like phenotype in GBM. 51 most deregulated miRNAs classified the cell cultures into GSC and non-stem cell clusters and identified a subgroup of GSC cultures with more pronounced stem-cell characteristics. The importance of the identified miRNA signature was further supported by demonstrating that a Risk Score based on the expression of seven miRNAs overexpressed in GSC predicted overall survival in GBM patients in the TCGA dataset independently of the IDH1 status. In summary, we identified miRNAs differentially expressed in GSCs and described their association with GBM patient survival. We propose that these miRNAs participate on GSC features and could represent helpful prognostic markers and potential therapeutic targets in GBM.

Cerebrospinal fluid microRNAs as diagnostic biomarkers in brain tumors

Abstract Cerebrospinal fluid (CSF) is a body fluid that has many important functions and is in direct contact with the extracellular environment of the central nervous system (CNS). CSF serves as both the communication channel allowing the distribution of various substances among the CNS cells and the storage facility for the waste products these cells release. For these reasons, CSF is a potential source of diagnostic biomarkers of many CNS diseases, including brain tumors. Recent studies have revealed that CSF also contains circulating microRNAs (miRNAs), short non-coding RNAs that have been described as biomarkers in many cancers. However, CSF miRNAs are difficult to detect, which is why researchers face major challenges, including technological difficulties in its detection and its lack of standardization. Therefore, this review aims (i) to highlight the potential of CSF miRNAs as diagnostic, prognostic and predictive biomarkers in brain tumors, and (ii) to summarize technological approaches for detection of CSF miRNAs.

Abstract 1959: Global microRNA expression analysis of Sox-2 positive and negative glioblastoma cells

Introduction: Glioblastoma multiforme (GBM) is the most frequent intracranial malignity of astrocytic origin in adults. This tumor is characterized by infaust prognosis, which is primarily caused by resistance to the therapy and early relapses relate to the presence of glioblastoma stem cells (GSCs). These cells form neurospheres in vitro and express markers of stemness such as Sox-2, Oct-4, and nestin. Targeting of GSCs could be a novel promising therapeutic approach leading to the overcome of therapy resistance and better prognosis of GBM patients. One of the approaches how to successfully regulate GSC is a targeted regulation of microRNAs (miRNAs). These small non-coding RNA molecules post-transcriptionally regulate an expression of more than 2/3 of all human genes that are also involved in stem cell associated signaling pathways. Moreover, deregulated expression of some miRNAs has been observed in many cancers including GBM. Cell lines and Methods: We have prepared eleven Sox-2 positive and negative paired primary GBM cell lines, which have been cultured under DMEM/F12 medium supplemented with bFGF, EGF, and B12 supplement and DMEM medium supplemented with 10% FBS, respectively. The global miRNA expression analysis was performed using GeneChip miRNA 4.0 Array (Affymetrix). Targeted regulation of miRNA levels have been carried out by the transient transfection of specific anti-miRNAs or miRNA mimics in GSC cell lines NCH 601 acquired from Interdisciplinary Center For Neurosciences (Heidelberg, Germany). The sphere formation assay was analyzed using IncuCyte ZOOM instrument (Essen BioScience). Sox-2 and nestin expressions were analyzed on both protein and transcriptional levels using combination of PAGE with Western blotting and real-time PCR, respectively. Results: Analysis of Sox-2 positive and negative paired GBM cell lines revealed 29 differentially expressed miRNAs, from which miR-93-3p, miR-95-5p, miR-106b-5p, miR-22-3p, and miR-3195 showed high significance (adjust. P value Conclusion: Taken together, our data suggest that miR-22-3p and miR-106b-5p are probably involved in GSC biology and, thus, should be promising molecular targets to overcome GBM therapeutic resistance. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved. Citation Format: Jiri Sana, Marek Vecera, Romana Butova, Pavel Fadrus, Leos Kren, Jaroslav Juracek, Robert Illiev, Jitka Mlcochova, Zuzana Ozanova, Petra Vychytilova, Ondrej Slaby. Global microRNA expression analysis of Sox-2 positive and negative glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1959.

Abstract 1496: MicroRNA signature associated with poor outcome of glioblastoma patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Glioblastoma multiforme (GBM) is the most frequently occurring primary malignant brain tumor with very poor prognosis, varying therapeutic response. Therefore, it is very important to find new biomarkers which can predict a clinical outcomes and help in treatment decisions. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and play key role in pathogenesis of wide range of cancer, including GBM. Aim of our study is to identify miRNA signature associated with the more aggressive phenotype of GBMs, which will enable sensitive prediction of clinical outcome in GBM patients under standard therapeutic protocols. Methods: We determined the expression profile of 667 miRNAs in tumor tissues of 58 patients with primary GBM with completed concomitant chemoradiotherapy with temozolomide according to standard Stupp protocol and 12 non-malignant brain tissues obtained from patients with drug-resistant anteromedial temporal lobe epilepsy. Methylation status of MGMT promoter (methylation-specific HRM), isocitrate dehydrogenase (IDH1) status (immunohistochemistry) and co-deletion of 1p/19q (FISH) was also evaluated. Results: We have confirmed frequencies of commonly used prognostic biomarkers in GBM patients (MGMT, IDH1, 1p/19q deletion), and observed significant correlation of MGMT methylation status, response to chemoradiotherapy with temozolomide and survival of GBM patients. We have identified specific signature of miRNAs diferentially expressed between GBM tissue and non-tumoral brain tissue from epilepsy surgeries (28 miRNAs, p<10-10). We have confirmed some previous observations (e.g. up-regulation of miR-21, miR-155, down-regulation of miR-128a, miR-23a) and also identified miRNAs deregulated in GBM tissue which were observed for the first time (e.g. miR-220, miR-328, miR-888, miR-504). More importantly we have found miRNA signature (miR-224, miR-31, miR-454, miR-672, miR-885-5p, miR-432) significantly associated short progression-free survival (p<10-6) and overall survival (p<10-6). Conclusions: These findings indicate that miRNAs play an important role in GBM pathogenesis and may serve not only as promising predictors of therapeutic outcome but also as potential therapeutic targets in GBM patients. This work was supported by Grant NT13549-4/2012, NT13860 4/2012, NT11214-4/2010 and NT13514-4/2012 of the Czech Ministry of Health, MH CZ - DRO (MMCI, 00209805). Citation Format: Jiri Sana, Radek Lakomy, Pavel Fadrus, Martin Smrcka, Pavel Slampa, Leos Kren, Marketa Hermanova, Marek Svoboda, Ondrej Slaby. MicroRNA signature associated with poor outcome of glioblastoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1496. doi:10.1158/1538-7445.AM2014-1496

P.147 Hydrocephalus with cerebrospinal fluid shunts during pregnancy: Report of two cases

Women with cerebrospinal fluid shunts are now reaching reproductive age. They are making independent decisions in regard to planning their families. Two cases of pregnant women with cerebrospinal fluid shunts are presented. Cooperation of neurosurgeons, obstetricians and other specialists is always necessary to take care of shunted pregnant women. These women are able to gave birth to a healthy child but complications can occur during pregnancy and delivery.

O.101 Laparoscopic versus laparotomic shunt placement in adults with hydrocephalus

Implantace ventrikuloperitonealniho shuntu je velmi castou operaci v lecbě hydrocefalus dospělých. Autoři ve svem sděleni srovnavaji laparotomickou a laparoskopickou techniku při implantaci ventrikuloperitonealniho shuntu. Komplikace peritonealni casti shuntu se vyskytuji castěji ve skupině pacientů operovaných klasickou laparotomickou technikou.