Krüppel-like factor 8 (KLF8) is expressed in gliomas of different WHO grades and is essential for tumor cell proliferation.

Krüppel-like factor 8 (KLF8) has only recently been identified to be involved in tumor cell proliferation and invasion of several different tumor entities like renal cell carcinoma, hepatocellular carcinoma and breast cancer. In the present study, we show for the first time the expression of KLF8 in gliomas of different WHO grades and its functional impact on glioma cell proliferation. In order to get information about KLF8-mRNA regulation qPCR was performed and did not reveal any significant difference in samples (n = 10 each) of non-neoplastic brain (NNB), low-grade gliomas (LGG, WHO°II) and glioblastomas (GBM, WHO°IV). Immunohistochemistry of tissue samples (n = 7 LGG, 11 AA and 12 GBM) did not show any significant difference in the fraction of KLF8-immunopositive cells of all analyzed cells in LGG (87%), AA (80%) or GBM (89%). Tissue samples from cerebral breast cancer metastasis, meningiomas but also non-neoplastic brain demonstrated comparable relative cell counts as well. Moreover, there was no correlation between KLF8 expression and the expression pattern of the assumed proliferation marker Ki67, which showed high variability between different tumor grade (9% (LGG), 6% (AA) and 15% (GBM) of Ki67-immunopositive cells). Densitometric analysis of Western blotting revealed that the relative amount of KLF8-protein did also not differ between the highly aggressive and proliferative GBM (1.05) compared to LGG (0.93; p<0.05, studens t-test). As demonstrated for some other non-glial cancer entities, KLF8-knockdown by shRNA in U87-MG cells confirmed its functional relevance, leading to an almost complete loss of tumor cell proliferation. Selective blocking of KLF8 might represent a novel anti-proliferative treatment strategy for malignant gliomas. Yet, its simultaneous expression in non-proliferating tissues could hamper this approach

O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

Background: We analyzed prospectively whether MGMT (O(6)-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. 12 Hide Figures Abstract Introduction Methods Results Discussion Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings: Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance: MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined

MiRNA expression patterns predict survival in glioblastoma

<p>Abstract</p> <p>Background</p> <p>In order to define new prognostic subgroups in patients with glioblastoma a miRNA screen (> 1000 miRNAs) from paraffin tissues followed by a bio-mathematical analysis was performed.</p> <p>Methods</p> <p>35 glioblastoma patients treated between 7/2005 - 8/2008 at a single institution with surgery and postoperative radio(chemo)therapy were included in this retrospective analysis. For microarray analysis the febit biochip "Geniom^®Biochip MPEA homo-sapiens" was used. Total RNA was isolated from FFPE tissue sections and 1100 different miRNAs were analyzed.</p> <p>Results</p> <p>It was possible to define a distinct miRNA expression pattern allowing for a separation of distinct prognostic subgroups. The defined miRNA pattern was significantly associated with early death versus long-term survival (split at 450 days) (p = 0.01). The pattern and the prognostic power were both independent of the MGMT status.</p> <p>Conclusions</p> <p>At present, this is the first dataset defining a prognostic role of miRNA expression patterns in patients with glioblastoma. Having defined such a pattern, a prospective validation of this observation is required.</p

Report about Four Novel Mutations in the Prion Protein Gene

Background/Aims: Since detection of the prion protein gene (PRNP) morethan 30 mutations have been discovered. Some have only been found insingle case reports without known intrafamilial accumulation orneuropathological proof so that the causal connection between mutationand disease could not be proved. Those patients often present atypicalclinical phenotypes, and it is not unusual that they are classified asdiseases other than Creutzfeldt-Jakob disease (CJD). Methods: Cases ofsuspected CJD have been reported to the national reference center forprion diseases. Clinical and diagnostic data were collected, and aclassification of definite, possible or probable prion disease was made.Molecular analysis of PRNP was performed by capillary sequencing.Results: We have described 4 cases with atypical clinical and diagnosticfindings and unknown mutations in PRNP so far. Conclusion: Threepatients fulfilled the criteria of probable CJD, and 1 patient fulfilledthe criteria of possible CJD but the clinical picture in none of thepatients was typical CJD; hence, it remained questionable whether themutations were causal of the disease

Predominant influence of MGMT methylation in non-resectable glioblastoma after radiotherapy plus temozolomide

Patients with non-resectable glioblastoma generally exhibit a poor prognosis, even after radiotherapy plus concomitant and adjuvant temozolomide (XRT/TMZ→TMZ). Unfortunately, no data are available concerning the predictive value of O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation for this important subpopulation. For clarification, a prospective study was conducted

Analyses of least cost paths for determining effects of habitat types on landscape permeability: wolves in Poland

Determining ecological corridors is crucial for conservation efforts in fragmented habitats. Commonly employed least cost path (LCP) analysis relies on the underlying cost matrix. By using Ecological Niche Factor Analysis, we minimized the problems connected with subjective cost assessment or the use of presence/absence data. We used data on the wolf presence/absence in Poland to identify LCPs connecting patches of suitable wolf habitat, factors that influence patch occupancy, and compare LCPs between different genetic subpopulations. We found that a lower proportion of cities and roads surrounds the most densely populated patches. Least cost paths between areas where little dispersal takes place (i.e., leading to unpopulated patches or between different genetic subpopulations) ran through a higher proportion of roads and human settlements. They also crossed larger maximal distances over deforested areas. We propose that, apart from supplying the basis for direct conservation efforts, LCPs can be used to determine what factors might facilitate or hinder dispersal by comparing different subsets of LCPs. The methods employed can be widely applicable to gain more in-depth information on potential dispersal barriers for large carnivores

Brain-derived proteins in the CSF, do they correlate with brain pathology in CJD?

BACKGROUND: Brain derived proteins such as 14-3-3, neuron-specific enolase (NSE), S 100b, tau, phosphorylated tau and Aβ(1–42 )were found to be altered in the cerebrospinal fluid (CSF) in Creutzfeldt-Jakob disease (CJD) patients. The pathogenic mechanisms leading to these abnormalities are not known, but a relation to rapid neuronal damage is assumed. No systematic analysis on brain-derived proteins in the CSF and neuropathological lesion profiles has been performed. METHODS: CSF protein levels of brain-derived proteins and the degree of spongiform changes, neuronal loss and gliosis in various brain areas were analyzed in 57 CJD patients. RESULTS: We observed three different patterns of CSF alteration associated with the degree of cortical and subcortical changes. NSE levels increased with lesion severity of subcortical areas. Tau and 14-3-3 levels increased with minor pathological changes, a negative correlation was observed with severity of cortical lesions. Levels of the physiological form of the prion protein (PrP(c)) and Aβ(1–42 )levels correlated negatively with cortical pathology, most clearly with temporal and occipital lesions. CONCLUSION: Our results indicate that the alteration of levels of brain-derived proteins in the CSF does not only reflect the degree of neuronal damage, but it is also modified by the localization on the brain pathology. Brain specific lesion patterns have to be considered when analyzing CSF neuronal proteins

The number of methylated CpG sites within the MGMT promoter region linearly correlates with outcome in glioblastoma receiving alkylating agents

MGMT-promoter methylation is associated with favorable outcome in glioblastoma. The aim of this study was to determine whether the absolute number of methylated Cytosine-Guanine-dinucleotide-(CpG-)sites within the DMR-2 island of the MGMT-promoter may correlate with outcome in a qualitative or quantitative fashion. In a cohort of newly diagnosed glioblastoma patients treated with stereotactic biopsy or open tumor resection plus concomitant chemoradiotherapy, we assessed MGMT-promoter methylation by methylation-specific polymerase-chain-reaction (MSP). Methylation of the CpG-sites 74-98 within the MGMT-promoter region was additionally analysed by Sanger sequencing, and the total number of methylated CpG-sites was correlated with outcome using proportional hazards models. 215 patients with glioblastoma were identified and stratified per MSP (positive: 53%, negative: 47%). Among MSP-positive tumors, hierarchical clustering identified three subgroups with different methylation rates (median: 80% vs. 52% vs. 47%), indicating a site-dependent methylation propagation. The methylation status of a given CpG-site indicated a neighborhood-dependent methylation propagation. Survival was linearly associated with the cumulative number of methylated CpG-sites. This was particularly true in patients who received at least one adjuvant cycle of temozolomide. Notably, all CpG-sites analyzed contributed similarly to effect size; this enabled a further predictive substratification of MSP-positive tumors with median OS ranging from as low as 17.1~months (< 18 methylated CpG-sites) to as high as 26.2~months (≥ 18 methylated CpG-sites) in the overall cohort. All in all, total number of methylated CpG-sites may correlate with outcome in a linear fashion. Such analysis may therefore add further predictive value to conventional methods of determining the MGMT-promoter status

Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice

Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrP(Sc), the infectious and protease-resistant form of the cellular prion protein (PrP(C)). We generated lentivectors expressing PrP(C)-specific short hairpin RNAs (shRNAs) that efficiently silenced expression of the prion protein gene (Prnp) in primary neuronal cells. Treatment of scrapie-infected neuronal cells with these lentivectors resulted in an efficient and stable suppression of PrP(Sc) accumulation. After intracranial injection, lentiviral shRNA reduced PrP(C) expression in transgenic mice carrying multiple copies of Prnp. To test the therapeutic potential of lentiviral shRNA, we used what we believe to be a novel approach in which the clinical situation was mimicked. We generated chimeric mice derived from lentivector-transduced embryonic stem cells. Depending on the degree of chimerism, these animals carried the lentiviral shRNAs in a certain percentage of brain cells and expressed reduced levels of PrP(C). Importantly, in highly chimeric mice, survival after scrapie infection was significantly extended. Taken together, these data suggest that lentivector-mediated RNA interference could be an approach for the treatment of prion disease

Report about Four Novel Mutations in the Prion Protein Gene

Background/Aims: Since detection of the prion protein gene (PRNP) morethan 30 mutations have been discovered. Some have only been found insingle case reports without known intrafamilial accumulation orneuropathological proof so that the causal connection between mutationand disease could not be proved. Those patients often present atypicalclinical phenotypes, and it is not unusual that they are classified asdiseases other than Creutzfeldt-Jakob disease (CJD). Methods: Cases ofsuspected CJD have been reported to the national reference center forprion diseases. Clinical and diagnostic data were collected, and aclassification of definite, possible or probable prion disease was made.Molecular analysis of PRNP was performed by capillary sequencing.Results: We have described 4 cases with atypical clinical and diagnosticfindings and unknown mutations in PRNP so far. Conclusion: Threepatients fulfilled the criteria of probable CJD, and 1 patient fulfilledthe criteria of possible CJD but the clinical picture in none of thepatients was typical CJD; hence, it remained questionable whether themutations were causal of the disease