Overexpression of cytosolic, plasma membrane bound and extracellular heat shock protein 70 (Hsp70) in primary glioblastomas.

A unique feature in several non-CNS-tumors is the overexpression of heat shock protein 70 (Hsp70, HSPA1A) in the cytosol, but also its unusual plasma membrane expression and release. Although in gliomas, cytosolic Hsp70 levels are not associated with histological grading, the role of membrane bound and released Hsp70 is still completely unknown. Membrane bound as well as cytosolic Hsp70 can be detected in viable tumor cells with the monoclonal antibody (mAb) cmHsp70.1. Herein, we analysed membrane bound Hsp70 levels in primary and secondary gliomas of different grades and on isolated glioma subpopulations (endothelial cells, CD133-positive cells, primary cultures) by immunohistochemistry and flow cytometry using cmHsp70.1 mAb. Extracellular Hsp70 was determined by a commercial Hsp70 sandwich ELISA (R&D) in plasma samples of glioblastoma patients and healthy volunteers. We found an overexpression of Hsp70 in primary glioblastomas compared to low-grade, anaplastic, or secondary gliomas as determined by immunohistochemistry. Especially in flow cytometry, a strong plasma membrane Hsp70 expression was only observed in primary but not secondary glioblastomas. Within the heterogeneous tumor mass, CD133-positive tumor-initiating and primary glioblastoma cells showed a high membrane Hsp70 expression density, whereas endothelial cells, isolated from glioblastoma tissues only showed a weak staining pattern. Also in plasma samples, secreted Hsp70 protein was significantly increased in patients harbouring primary glioblastomas compared to those with secondary and low grade glioblastomas. Taken together, we show for the first time that cytosolic, membrane bound and extracellular Hsp70 is uniquely overexpressed in primary glioblastomas.

Dynamic 18F-FET PET in suspected WHO grade II gliomas defines distinct biological subgroups with different clinical courses.

In suspected grade II gliomas, three distinct patterns of time-activity curves (TAC) on O-(2-[(18)F]fluoroethyl)-1-tyrosine ((18)F-FET) positron emission tomography (PET) have been delineated (i) increasing TAC homogeneously throughout the tumor, and decreasing TAC, (ii) either homogeneously throughout the tumor or (iii) only focally within otherwise increasing TAC patterns. Increasing TAC was associated with low-grade histology and decreasing TAC with high-grade histology. This prospective study analyzed whether these patterns correlate with distinct biological tumor subtypes and differential outcome. (18)F-FET PET-guided biopsies were used for stepwise histopathological evaluation. Molecular-genetic evaluation included O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation, isocitrate dehydrogenase (IDH1/2) mutational and 1p/19q codeletion status. Progression-free survival (PFS) was estimated with the Kaplan-Meier method. Prognostic factors were obtained from multivariate regression models. 98 adult patients were included. Homogeneous increasing, focal decreasing and homogeneous decreasing TAC were seen in 51, 19 and 28 patients. The corresponding 1-year (2-years) PFS were 92% (85%), 89% (51%) and 50% (28%; p = 0.002). IDH1/2 mutations were more frequent in tumors with homogeneous increasing (90%) and focal decreasing (79%) TAC, but were rare in those exhibiting homogeneous decreasing TAC (25%; p < 0.001). Overall, TAC patterns, IDH1/2 mutational and 1p/19q codeletion status were powerful and independent prognostic factors. Dynamic (18)F-FET PET might be an important and independent imaging biomarker for patients with suspected WHO grade II gliomas and offers perspectives for stratified diagnostic and therapeutic strategies. Tumors with focal decreasing TAC need highly targeted surgical interventions to avoid undergrading and undertreatment.

Molecular stereotactic biopsy technique improves diagnostic accuracy and enables personalized treatment strategies in glioma patients.

BACKGROUND: In gliomas molecular biomarkers are increasingly gaining diagnostic, prognostic and predictive significance. Determination of biomarker status after biopsy is important as not all patients are eligible for open tumor resection. We developed and validated prospectively (6/10-12/11) a protocol allowing for both reliable determination of multiple biomarkers and representative histological diagnoses from small-sized biopsies. METHODS: All molecular stereotactic biopsies were performed according to a detailed workflow. The selection of specimens best suited for molecular analyses was intra-operatively guided by the attending neuropathologist. Postoperative screening was done by methylation specific PCR using two distinct cryopreserved specimens to test for reproducibility of the findings and to rule out contamination. The DNA of a single best-suited specimen (1 mm(3)) was subjected to detailed molecular analysis (MGMT promoter methylation, IDH1/2 mutational status, LOH 1p and/or 19q). RESULTS: 159 consecutively enrolled untreated gliomas were analyzed (94 glioblastomas, 2 gliosarcomas, 24 anaplastic astrocytomas, 10 oligo-tumors grade II/III, 20 grade II astrocytomas and 9 pilocytic astrocytomas). Transient morbidity was 2 %. Overall, the drop-out rate due to tissue contamination was 0.4 %. Median time from biopsy to histological and molecular genetic analyses was 3 and 5 days, respectively. Distributions of the respective biomarker status for tumor subgroups were consistent with the literature. The final histological diagnosis was changed/modified in 5/159 patients according to molecular findings. Treatment after molecular biopsy was highly personalized. CONCLUSIONS: Molecular stereotactic biopsy is feasible and safe, can be implemented in daily clinical practice, improves diagnostic precision and enables personalized treatment.

[18F]fluoroethyltyrosine-positron emission tomography-based therapy monitoring after stereotactic iodine-125 brachytherapy in patients with recurrent high-grade glioma.

Therapy monitoring of glioma after stereotactic iodine-125 brachytherapy (SBT) remains challenging because posttherapeutic changes in magnetic resonance imaging can mimic tumor progression. We evaluated the prognostic value of serial [18F]fluoroethyltyrosine (FET)-positron emission tomographic (PET) scans for therapy monitoring of high-grade glioma (HGG) after SBT. Thirty-three patients with recurrent HGG were included. Serial FET-PET scans were performed prior to therapeutic intervention and at 3-month intervals during the first year after SBT. FET-PET evaluation was performed by both conventional data analysis and kinetic analysis. Prognostic factors were obtained from proportional hazard models. Median local progression-free survival (LPFS) was 11.1 months. Maximal standardized background uptake value (SUVmax/BG) and biologic tumor volume (BTV) differentiated accurately between therapeutic effects and local tumor progression at the 6-month and subsequent examinations. Increasing uptake kinetics at baseline (p < .05) and during follow-up (p < .01) were stringently associated with a longer LPFS. Early increase in FET uptake after SBT is not unequivocally associated with tumor progression; it might be induced by reactive changes and could easily lead to a misclassification of the tumor status (pseudoprogression). Six months after SBT (or later), however, increased SUVmax/BG and BTV values are associated with a worse prognosis. Multivariate analysis stresses the prognostic importance of dynamic studies.

Atypical parkinsonism due to a D202N Gerstmann-Sträussler-Scheinker prion protein mutation: first in vivo diagnosed case.

BACKGROUND: Parkinsonism with dopa-sensitivity and a correlating DaTSCAN turned out to be due to a D202N mutation which is associated with the Gerstmann-Sträussler-Scheinker (GSS) disease. METHODS/RESULTS: We report a 51-year old female who presented with left-dominant parkinsonism and a positive DaTSCAN. She was diagnosed with idiopathic Parkinson's syndrome. Dopaminergic medication reduced her symptoms. In addition, punding-like behavior, deficits in organizing daily life and abnormal sleep behavior were reported. Neuropsychological testing, EEG, polysomnography as well as PET imaging with fluorodexyglucose (FDG), [F-18]-desmethoxyfallypride (DMFP), and [C-11]-6-OH-BTA-1 (PIB) were not diagnostic. Cerebral spinal fluid analysis revealed no 14-3-3 protein, but elevated neuron-specific enolase (NSE) and S100-beta and a very low phospho-tau/total-tau ratio. Analysis of the prion gene disclosed the rare D202N mutation. CONCLUSIONS: The D202N prion mutation has been associated with GSS pathology and up to now was only reported post mortem. Our patient is the very first case diagnosed in vivo.

Report about four novel mutations in the prion protein gene.

BACKGROUND/AIMS: Since detection of the prion protein gene (PRNP) more than 30 mutations have been discovered. Some have only been found in single case reports without known intrafamilial accumulation or neuropathological proof so that the causal connection between mutation and disease could not be proved. Those patients often present atypical clinical phenotypes, and it is not unusual that they are classified as diseases other than Creutzfeldt-Jakob disease (CJD). METHODS: Cases of suspected CJD have been reported to the national reference center for prion diseases. Clinical and diagnostic data were collected, and a classification 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 diagnostic findings and unknown mutations in PRNP so far. CONCLUSION: Three patients fulfilled the criteria of probable CJD, and 1 patient fulfilled the criteria of possible CJD but the clinical picture in none of the patients was typical CJD; hence, it remained questionable whether the mutations were causal of the disease.

Magnetic resonance imaging in E200K and V210I mutations of the prion protein gene.

Magnetic resonance imaging (MRI), with diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR), is a useful diagnostic investigation for Creutzfeldt-Jakob disease (CJD). Amendment of the diagnostic criteria for sporadic CJD (sCJD) to include defined MRI alterations has just recently been proposed. We analyzed MRI scans with FLAIR and/or DWI available of 29 patients with the E200K or the V210I mutation, and a control group of 29 sCJD patients to compare the MRI lesion profile and evaluate the applicability of new MRI criteria to genetic CJD patients. FLAIR images of all 29 patients and controls and DWI images of 15 genetic CJD and 13 sCJD were rated by 2 neuroradiologists blinded to diagnosis and genetic testing. In genetic CJD (gCJD) patients, hyperintensities on FLAIR could be found in the putamen in 55% and the caudate nucleus in 66% and on DWI in 33% and 60%, respectively. Lesion profile and frequency of hyperintensities did not differ significantly from the findings in sCJD. New diagnostic MRI criteria yield similar sensitivity for gCJD and sCJD, being 79% and 72%, respectively. MRI provides useful information in E200K and V210I gCJD patients. The alterations strongly resembled those seen in sCJD, once again demonstrating the similarity of the clinical syndrome in patients with these particular mutations.

Low values of 5-hydroxymethylcytosine (5hmC), the "sixth base," are associated with anaplasia in human brain tumors.

5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5 mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5 hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC measurements were unrelated to 5 mC values. We find that the number of 5 hmC positive cells and the amount of 5 hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.

IDH1 mutations in grade II astrocytomas are associated with unfavorable progression-free survival and prolonged postrecurrence survival.

BACKGROUND: The favorable prognostic impact of mutations in the IDH1 gene is well documented for malignant gliomas; its influence on World Health Organization (WHO) grade II astrocytomas, however, is still under debate. METHODS: A previously published database of 127 predominantly surgically treated patients harboring WHO grade II astrocytomas was revisited. Patients were screened for TP53 mutations (sequencing analysis), IDH1 mutations (pyrosequencing), and MGMT promoter methylation (methylation-specific polymerase chain reaction and bisulfite sequencing). Endpoints were overall survival, progression-free survival (PFS), time to malignant transformation, and postrecurrence survival. Radiotherapy was usually withheld until tumor progression/malignant transformation occurred. RESULTS: IDH1 mutations, TP53 mutations, and methylated MGMT promoters were seen in 78.1%, 51.2%, and 80.0% of the analyzed tumors, respectively. IDH1 mutations, which were significantly associated with TP53 mutations and/or MGMT promoter methylation (P < .001), resulted in shortened PFS (median, 47 vs 84 months; P = .004); postrecurrence survival, however, was significantly increased in those patients undergoing malignant transformation (median, 49 vs 13.5 months; P = .006). Overall survival was not affected by IDH1. A similar pattern of influence was seen for MGMT promoter methylation. Methylated tumors did significantly worse (better) in terms of PFS (postrecurrence survival); a low number of unmethylated tumors, however, limited the power of this analysis. Conversely, TP53 mutations were stringently associated with a worse prognosis throughout the course of the disease. CONCLUSIONS: IDH1 mutations are associated with a Janus headlike phenomenon; unfavorable prognostic influence on PFS turns into favorable impact on postrecurrence survival. A similar pattern of influence might exist for MGMT methylation.

Optical needle endoscope for safe and precise stereotactically guided biopsy sampling in neurosurgery.

Proper treatment of deep seated brain tumors requires correct histological diagnosis which unambiguously necessitates biopsy sampling. Stereotactically guided sampling of biopsies is widely used but bears the danger of incorrect sampling locations and damage to intracerebral blood vessels. Here, we present a minimally invasive contact endoscopic probe that can be inserted into the tissue inside a standard biopsy needle and allows for fluorescence detection of both tumorous tissue and intracerebral blood vessels. Outer diameter of our contact probe is smaller than 1.5 mm, field-of-view in the range of several hundred microns; the optical design allows for simultaneous detection and visualization of tissue autofluorescence and selective fluorescence signals from deep seated brain tumors and vasculature as shown on in vivo animal models. We demonstrate the tumor detection capability during stereotactic needle insertion in a clinical pilot trial. Using our probe, we expect stereotactic interventions to become safer and more precise and the technology might ultimately be used also for various other kinds of applications.

MRI-suspected low-grade glioma: is there a need to perform dynamic FET PET?

PURPOSE: Since differentiation between low-grade glioma (LGG) and high-grade glioma (HGG) remains challenging according to MRI criteria alone, we investigated the discriminative value of additional dynamic FET PET in patients with MRI-suspected LGG. METHODS: Included in this retrospective study were 127 patients with newly diagnosed MRI-suspected LGG and dynamic FET PET prior to histopathological assessment. FET PET lesions were visually classified as having reduced, normal, or increased tracer uptake. Maximal tumour uptake scaled to the mean background uptake (SUV(max)/BG), mean tumour uptake (SUV(mean)/BG), biological tumour volume and kinetics were evaluated and correlated with individual histopathological findings. RESULTS: Histopathological analysis revealed 71 patients with LGG, 47 patients with HGG (including 5 glioblastoma multiforme), 2 patients with low-grade ganglioglioma and 7 patients with non-neoplastic lesions. Of the 127 patients, 97 had lesions with increased FET uptake, of which 93 were neoplastic. Increased uptake was found in 49/71 LGG (69 %) and 42/47 HGG (89 %). None of the conventional uptake parameters differed significantly between the HGG and LGG groups. Kinetic analysis reliably identified HGG (sensitivity 95 %, specificity 72 %, PPV 74 %, NPV 95 %). Normal tracer uptake was observed in 19 patients (15 with LGG, 1 with HGG and 3 with non-neoplastic lesions) and reduced uptake in 11 patients (7 with LGG and 4 with HGG). CONCLUSION: Among the MRI-suspected LGG, kinetic but not conventional analysis of FET uptake enabled remarkably high sensitivity for detection of HGG. This held true even for lesions with low or diffuse tracer uptake. Lesions with reduced tracer uptake must be interpreted with caution, as they can also harbour HGG tissue.

Re-irradiation in recurrent malignant glioma: prognostic value of [18F]FET-PET.

The aim of the present study is to determine new positron emission tomography (PET) imaging-related factors predictive of progression-free survival as well as survival in patients with recurrent malignant glioma (MG) prior to and after re-irradiation. Fifty-six patients with recurrent MG who underwent re-irradiation treatment and pretherapeutic dynamic [(18)F]-fluoroethyl-L-tyrosine (FET)-PET scan were retrospectively analyzed. The prognostic value of different parameters, such as biological tumor volume, maximal tumor uptake (SUV(max)/BG), mean tumor uptake (SUV(mean)/BG), as well as uptake kinetics, was evaluated. [(18)F]FET uptake kinetics was classified according to a five-point rating as category G(1-2) (strongly/mainly increasing kinetics), G(3) (mixed 1:1), or G(4-5) (mainly/strongly decreasing kinetics). Patients within the pretherapeutic kinetic group G(4-5) had significantly worse survival than the other two groups (p = 0.01). Multivariate analysis revealed that histologic grade, Karnofsky Performance Score (KPS), and kinetic group were independent significant predictors for survival after re-irradiation. The uptake kinetics of [(18)F]FET-PET is an independent determinant of overall and to a lesser extent also progression-free survival. Thus, [(18)F]FET-PET kinetics may provide valuable additional prognostic information for treatment decisions.

Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas.

Epigenetic silencing of the O(6) -methylguanine-DNA methyltransferase (MGMT) gene promoter is associated with prolonged survival in glioblastoma patients treated with temozolomide (TMZ). We investigated whether glioblastoma recurrence is associated with changes in the promoter methylation status and the expression of MGMT and the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 in pairs of primary and recurrent glioblastomas of 80 patients, including 64 patients treated with radiotherapy and TMZ after the first operation. Among the primary tumors, the MGMT promoter was methylated in 31 patients and unmethylated in 49 patients. In 71 patients (89%), the MGMT promoter methylation status of the primary tumor was retained at recurrence. MGMT promoter methylation, but not MGMT protein expression, was associated with longer progression-free survival, overall survival and postrecurrence survival (PRS). Moreover, PRS was increased under salvage chemotherapy. Investigation of primary and recurrent glioblastomas of 43 patients did not identify promoter methylation in any of the four MMR genes. However, recurrent glioblastomas demonstrated significantly lower MSH2, MSH6 and PMS2 protein expression as detected by immunohistochemistry. In conclusion, reduced expression of MMR proteins, but not changes in MGMT promoter methylation, is characteristic of glioblastomas recurring after the current standards of care.

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

BACKGROUND: 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. METHODS: Adult patients with a non-resectable glioblastoma were included. A molecular stereotactic biopsy technique was used for tumour characterisation combining histopathological diagnosis with small sample size adjusted methylation-specific PCR (MSP) and sodium bisulfite sequencing. Treatment included XRT (60 Gy in 30 fractions)/TMZ (daily dose of 75 mg/m(2))→TMZ (150-200 mg/m(2) per day for 5 days of every 28-day cycle). The primary end point was progression-free survival (PFS). Secondary endpoints were overall survival (OS) and treatment response (TR). Patients were categorised in the Radiation Therapy Oncology Group (RTOG)-recursive partitioning analysis (RPA) Classes III (N=4), IV (N=12), V (N=28) and VI (N=12). RESULTS AND DISCUSSION: The success rates of MSP and sequence analyses were 100%. The MGMT promoter was methylated in 30/56 tumours, which was associated with an increased PFS (median 56 versus 20 weeks; hazard ratio 0.15; range 0.07 to 0.33; p<0.0001), higher frequency of TR (93.3% vs 46.2%; p=0.0008) and increased OS (median 104 vs 28 weeks; hazard ratio 0.18; range 0.08 to 0.38; p<0.0001). The transient perioperative morbidity was 1.8%. CONCLUSION: MGMT promoter methylation has a predominant favourable influence even for the important subpopulation with non-resectable glioblastoma. The molecular stereotactic biopsy technique is safe and effective for predictive evaluation and helps to avoid both over- and undertreatment.

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.

Evidence for an association of prion protein and sphingolipid-mediated signaling.

The physiological function of the cellular prion protein (PrP(c)) is unclear. PrP(c) associates with lipid rafts, highly glycolipid-rich membrane domains containing a large variety of signaling molecules, e.g., sphingolipids (SL). In this study, we investigated possible connections between PrP(c) and sphingolipid-associated signaling pathways. Using PrP(c)-wt and PrP(c)-k.o. hippocampal cell lines and mouse brains we showed higher activity of neutral and acid sphingomyelinase (SMase) in PrP(c)-k.o.-groups, while ceramide and sphingomyelin-levels were unchanged. Furthermore, despite lower basal expression levels of sphingosine kinase (SphK) in PrP(c)-k.o.-groups, the levels of its metabolite sphingosine-1-phosphate were increased, whereas S1P(3)-receptor expression was higher in PrP(c)-wt-groups again. In addition, we detected enhanced activity of phospholipase D1, an enzyme that seems to be suitable to act as a connector between the S1P(3) receptor and continuative signaling. Finally, evidence for an impact on downstream signaling cascades, especially activation of the PI3K/Akt pathway, was found. In summary, our data suggest that PrP(c) is involved in sphingolipid-associated signaling, modulating pathways that exert anti-apoptotic functions, hence indicating that PrP(c) plays a role in neuroprotection.

MGMT promoter methylation is not correlated with integrin expression in malignant gliomas: clarifying recent clinical trial results.

Integrin alpha-v-beta-3 (αvß3) is important for invasive tumor growth and angiogenesis in glioblastomas (GBM). However, recent clinical trials on inhibition of this integrin led to ambiguous results whether patients with methylated or unmethylated 6O-methylguanine methyltransferase (MGMT) promoter might profit from this kind of therapy. Therefore, we addressed the still unanswered question about a possible correlation between integrin αvß3 expression and MGMT promoter methylation in GBM. For this purpose, tumor samples from newly diagnosed and untreated GBM patients with methylated (n = 22) or unmethylated (n = 17) MGMT promoter were simultaneously analyzed for integrin αvß3 expression by an automated immunohistochemical staining platform. Interestingly, subsequent semi-quantitative analysis by a special imaging software did not show any difference in integrin expression between patients with methylated or unmethylated MGMT promoter status. Moreover, further analysis of the integrin subunits via ELISA from histologic sections revealed that there is no difference in integrin subunit expression between these patients. Hence, our results are important for designing future clinical trials with respect to treatment stratification, while it still has to be identified which other molecular factors determine differential responses to targeted anti-integrin αvß3 treatment.

Outcome in unresectable glioblastoma: MGMT promoter methylation makes the difference.

In 2011, we reported a predominant prognostic/predictive role of MGMT promoter methylation status on progression-free survival (PFS) in unresectable glioblastoma patients undergoing upfront radiotherapy plus concomitant and maintenance temozolomide (RTX/TMZ â†’ TMZ). We, here, present the final results of this prospective study focussing on the prognostic/predictive value of MGMT promoter methylation status for death risk stratification. Overall, 56 adult patients with unresectable, biopsy proven glioblastoma were prospectively assigned to upfront RTX/TMZ â†’ TMZ treatment between March 2006 and August 2008. Last follow-up was performed in June 2016. MGMT promoter methylation was determined using methylation-specific PCR (MSP) and sodium bisulfite sequencing. Analyses were done by intention to treat. Prognostic factors were obtained from proportional hazard models. At the time of the final analysis 55 patients showed progressive disease and 53 patients had died. MGMT promoter was methylated (unmethylated) in 30 (26) patients. Methylation of the MGMT promoter was the strongest favorable predictor for overall survival (OS, median: 20.3 vs. 7.3 months, p < 0.001, HR 0.30, 95% CI 0.16-0.55), and PFS (median: 15.0 vs. 6.1 months, p < 0.001, HR 0.31, 95% CI 0.17-0.57) and was also associated with higher frequencies of treatment response and prolonged post-recurrence survival (PRS, median: 4.5 vs. 1.4 months, p < 0.002, HR 0.39, 95% CI 0.21-0.71). Knowledge of MGMT promoter methylation status is essential for patients' counseling, prognostic evaluation, and for the design of future trials dealing with unresectable glioblastomas.

Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.