Quantitative Imaging of D-2-Hydroxyglutarate in Selected Histological Tissue Areas by a Novel Bioluminescence Technique

Patients with malignant gliomas have a poor prognosis with average survival of less than 1 year. Whereas in other tumor entities the characteristics of tumor metabolism are successfully used for therapeutic approaches, such developments are very rare in brain tumors, notably in gliomas. One metabolic feature characteristic of gliomas, in particular diffuse astrocytomas and oligodendroglial tumors, is the variable content of D-2-hydroxyglutarate (D2HG), a metabolite that was discovered first in this tumor entity. D2HG is generated in large amounts due to various "gain-of-function" mutations in the isocitrate dehydrogenases IDH1 and 1DH2. Meanwhile, D2HG has been detected in several other tumor entities, including intrahepatic bile-duct cancer, chondrosarcoma, acute myeloid leukemia, and angioimmunoblastic T-cell lymphoma. D2HG is barely detectable in healthy tissue (<0.1 mM), but its concentration increases up to 35 mM in malignant tumor tissues. Consequently, the "oncometabolite" D2HG has gained increasing interest in the field of tumor metabolism. To facilitate its quantitative measurement without loss of spatial resolution at a microscopical level, we have developed a novel bioluminescence assay for determining D2HG in sections of snap-frozen tissue. The assay was verified independently by photometric tests and liquid chromatography/mass spectrometry. The novel technique allows the microscopically resolved determination of D2HG in a concentration range of 0-10 mu mol/g tissue (wet weight). In combination with the already established bioluminescence imaging techniques for ATP, glucose, pyruvate, and lactate, the novel D2HG assay enables a comparative characterization of the metabolic profile of individual tumors in a further dimension

Molecular dissection of the valproic acid effects on glioma cells

Many glioblastoma patients suffer from seizures why they are treated with antiepileptic agents. Valproic acid (VPA) is a histone deacetylase inhibitor that apart from its anticonvulsive effects in some retrospective studies has been suggested to lead to a superior outcome of glioblastoma patients. However, the exact molecular effects of VPA treatment on glioblastoma cells have not yet been deciphered. We treated glioblastoma cells with VPA, recorded the functional effects of this treatment and performed a global and unbiased next generation sequencing study on the chromatin (ChIP) and RNA level. 1) VPA treatment clearly sensitized glioma cells to temozolomide: A protruding VPA-induced molecular feature in this context was the transcriptional upregulation/reexpression of numerous solute carrier (SLC) transporters that was also reflected by euchromatinization on the histone level and a reexpression of SLC transporters in human biopsy samples after VPA treatment. DNA repair genes were adversely reduced. 2) VPA treatment, however, also reduced cell proliferation in temozolomide-naive cells: On the molecular level in this context we observed a transcriptional upregulation/reexpression and euchromatinization of several glioblastoma relevant tumor suppressor genes and a reduction of stemness markers, while transcriptional subtype classification (mesenchymal/proneural) remained unaltered. Taken together, these findings argue for both temozolomide-dependent and -independent effects of VPA. VPA might increase the uptake of temozolomide and simultaneously lead to a less malignant glioblastoma phenotype. From a mere molecular perspective these findings might indicate a surplus value of VPA in glioblastoma therapy and could therefore contribute an additional ratio for clinical decision making

Symptom burden and surgical outcome in non-skull base meningiomas

Purpose: Non-skull base meningiomas (NSBM) are a distinct entity and frequently present with focal neurological deficits. This study was designed to analyze functional and oncological outcome following microsurgical tumor resection in patients with NSBM. Patients and methods: An analysis of 300 patients that underwent NSBM resection between 2003 and 2013 was performed. Assessment reasures for functional outcome were Karnofsky Performance Scale (KPS), Medical Research Council - Neurological Performance Scale (MRC-NPS), and improvement rates of focal deficits and seizures. The extent of resection; recurrence-free survival (RFS) and tumor-specific survival (TSS) were also determined. Results: Impaired KPS and MRC-NPS were present in 73.3% and 45.7%, respectively. Focal neurological deficits were recorded in 123 patients (41.0%), with hemiparesis (21.7%) and aphasia (9.3%) the most prevalent form of impairment. Most meningiomas were localized at the convexity (64.0%), followed by falcine tumors (20.3%). Both KPI and MRC-NPS scores were significantly improved by surgical resection. Postoperative improvement rates of 96.6%, 89.3%, 72.3%, 57.9%, and 27.3% were observed for aphasia, epilepsy, hemiparesis, cranial nerve, and visual field deficits, respectively. Long-term improvement was achieved in 83.2%, 89.3%, 80.0%, 68.4% and 54.6% of patients, respectively. Gross total resection (GTR) over subtotal resection (STR) significantly improved preoperative seizures and visual field deficits and correlated with reduced risk of new postoperative hemiparesis. Poor Simpson grade was the only significant prognostic factor in multivariate analysis for long-term functional deficit, which occurred in 7.3%. Median RFS was 45.9 months (6.0 - 151.5 months), while median TSS was 53.7 months (3.1 – 153.2 months). Both WHO grade (p= 0.001) and Simpson classification (p= 0.014 and p= 0.031) were independent significant prognostic factors for decreased RFS and TSS by multivariate analysis, respectively. Furthermore, tumor diameter > 50 mm (p= 0.039) significantly correlated with decreased TSS in multivariate analysis. Conclusion: Surgical resection significantly and stably improves neurological deficits in patients with NSBM

The endogenous neuropeptide calcitonin gene-related peptide after spontaneous subarachnoid hemorrhage–A potential psychoactive prognostic serum biomarker of pain-associated neuropsychological symptoms

Background: The pronociceptive neuromediator calcitonin gene-related peptide (CGRP) is associated with pain transmission and modulation. After spontaneous subarachnoid hemorrhage (sSAH), the vasodilatory CGRP is excessively released into cerebrospinal fluid (CSF) and serum and modulates psycho-behavioral function. In CSF, the hypersecretion of CGRP subacutely after good-grade sSAH was significantly correlated with an impaired health-related quality of life (hrQoL). Now, we prospectively analyzed the treatment-specific differences in the secretion of endogenous CGRP into serum after good-grade sSAH and its impact on hrQoL. Methods: Twenty-six consecutive patients (f:m = 13:8; mean age 50.6 years) with good-grade sSAH were enrolled (drop out n = 5): n = 9 underwent endovascular aneurysm occlusion, n = 6 microsurgery, and n = 6 patients with perimesencephalic SAH received standardized intensive medical care. Plasma was drawn daily from day 1 to 10, at 3 weeks, and at the 6-month follow-up (FU). CGRP levels were determined with competitive enzyme immunoassay in duplicate serum samples. All patients underwent neuropsychological self-report assessment after the onset of sSAH (t1: day 11–35) and at the FU (t2). Results: During the first 10 days, the mean CGRP levels in serum (0.470 ± 0.10 ng/ml) were significantly lower than the previously analyzed mean CGRP values in CSF (0.662 ± 0.173; p = 0.0001). The mean serum CGRP levels within the first 10 days did not differ significantly from the values at 3 weeks (p = 0.304). At 6 months, the mean serum CGRP value (0.429 ± 0.121 ng/ml) was significantly lower compared to 3 weeks (p = 0.010) and compared to the first 10 days (p = 0.026). Higher mean serum CGRP levels at 3 weeks (p = 0.001) and at 6 months (p = 0.005) correlated with a significantly poorer performance in the item pain, and, at 3 weeks, with a higher symptom burden regarding somatoform syndrome (p = 0.001) at t2. Conclusion: Our study reveals the first insight into the serum levels of endogenous CGRP in good-grade sSAH patients with regard to hrQoL. In serum, upregulated CGRP levels at 3 weeks and 6 months seem to be associated with a poorer mid-term hrQoL in terms of pain. In migraineurs, CGRP receptor antagonists have proven clinical efficacy. Our findings corroborate the potential capacity of CGRP in pain processing

Clinical Benefits of Combining Different Visualization Modalities in Neurosurgery

The prevailing philosophy in oncologic neurosurgery, has shifted from maximally invasive resection to the preservation of neurologic function. The foundation of safe surgery is the multifaceted visualization of the target region and the surrounding eloquent tissue. Recent advancements in pre-operative and intraoperative visualization modalities have changed the face of modern neurosurgery. Metabolic and functional data can be integrated into intraoperative guidance software, and fluorescent dyes under dedicated filters can potentially visualize patterns of blood flow and better define tumor borders or isolated tumor foci. High definition endoscopes enable the depiction of tiny vessels and tumor extension to the ventricles or skull base. Fluorescein sodium-based confocal endomicroscopy, which is under scientific evaluation, may further enhance the neurosurgical armamentarium. We aim to present our institutional workup of combining different neuroimaging modalities for surgical neuro-oncological procedures. This institutional algorithm (IA) was the basis of the recent publication by Haj et al. describing outcome and survival data of consecutive patients with high grade glioma (HGG) before and after the introduction of our Neuro-Oncology Center

fMRI Retinotopic Mapping in Patients with Brain Tumors and Space-occupying Brain Lesions in the Area of the Occipital Lobe

Functional magnetic resonance imaging (fMRI) is a valuable tool in the clinical routine of neurosurgery when planning surgical interventions and assessing the risk of postoperative functional deficits. Here, we examined how the presence of a brain tumor or lesion in the area of the occipital lobe affects the results of fMRI retinotopic mapping. fMRI data were evaluated on a retrospectively selected sample of 12 patients with occipital brain tumors, 7 patients with brain lesions and 19 control subjects. Analyses of the cortical activation, percent signal change, cluster size of the activated voxels and functional connectivity were carried out using Statistical Parametric Mapping (SPM12) and the CONN and Marsbar toolboxes. We found similar but reduced patterns of cortical activation and functional connectivity between the two patient groups compared to a healthy control group. Here, we found that retinotopic organization was well-preserved in the patients and was comparable to that of the age-matched controls. The results also showed that, compared to the tumor patients, the lesion patients showed higher percent signal changes but lower values in the cluster sizes of the activated voxels in the calcarine fissure region. Our results suggest that the lesion patients exhibited results that were more similar to those of the control subjects in terms of the BOLD signal, whereas the extent of the activation was comparable to that of the tumor patients

CXCR2-Blocking Has Context-Sensitive Effects on Rat Glioblastoma Cell Line Outgrowth (S635) in an Organotypic Rat Brain Slice Culture Depending on Microglia-Depletion (PLX5622) and Dexamethasone Treatment

In glioblastoma (GBM), the interplay of different immune cell subtypes, cytokines, and/or drugs shows high context-dependencies. Interrelations between the routinely applied dexamethasone (Dex) and microglia remain elusive. Here, we exploited rat organotypic brain slice co-cultures (OBSC) to examine the effects on a rat GBM cell line (S635) outgrowth resulting from the presence of Dex and pretreatment with the colony-stimulating factor receptor 1 (CSF1-R) inhibitor PLX5622: in native OBSC (without PLX5622-pretreatment), a diminished S635 spheroid outgrowth was observable, whereas Dex-treatment enhanced outgrowth in this condition compared to PLX5622-pretreated OBSC. Screening the supernatants of our model with a proteome profiler, we found that CXCL2 was differentially secreted in a Dex- and PLX5622-dependent fashion. To analyze causal interrelations, we interrupted the CXCL2/CXCR2-axis: in the native OBSC condition, CXCR2-blocking resulted in increased outgrowth, in combination with Dex, we found potentiated outgrowth. No effect was found in the PLX5622-pretreated. Our method allowed us to study the influence of three different factors—dexamethasone, PLX5622, and CXCL2—in a well-controlled, simplified, and straight-forward mechanistic manner, and at the same time in a more realistic ex vivo scenario compared to in vitro studies. In our model, we showed a GBM outgrowth enhancing synergism between CXCR2-blocking and Dex-treatment in the native condition, which was levelled by PLX5622-pretreatment

Combined Modulation of Tumor Metabolism by Metformin and Diclofenac in Glioma

Glioblastoma remains a fatal diagnosis. Previous research has shown that metformin, which is an inhibitor of complex I of the respiratory chain, may inhibit some brain tumor initiating cells (BTICs), albeit at dosages that are too high for clinical use. Here, we explored whether a combined treatment of metformin and diclofenac, which is a non-steroidal anti-inflammatory drug (NSAID) shown to inhibit glycolysis by interfering with lactate efflux, may lead to additive or even synergistic effects on BTICs (BTIC-8, -11, -13 and -18) and tumor cell lines (TCs, U87, and HTZ349). Therefore, we investigated the functional effects, including proliferation and migration, metabolic effects including oxygen consumption and extracellular lactate levels, and effects on the protein level, including signaling pathways. Functional investigation revealed synergistic anti-migratory and anti-proliferative effects of the combined treatment with metformin and diclofenac on BTICs and TCs. Signaling pathways did not sufficiently explain synergistic effects. However, we observed that metformin inhibited cellular oxygen consumption and increased extracellular lactate levels, indicating glycolytic rescue mechanisms. Combined treatment inhibited metformin-induced lactate increase. The combination of metformin and diclofenac may represent a promising new strategy in the treatment of glioblastoma. Combined treatment may reduce the effective doses of the single agents and prevent metabolic rescue mechanisms. Further studies are needed in order to determine possible side effects in humans

Does the distance to the cancer center affect psycho-oncological care and emergency visits of patients with IDH wild-type gliomas? A retrospective study

Background Malignant isocitrate dehydrogenase wild-type (IDHwt) gliomas impose a high symptomatic and psychological burden. Wide distances from patients’ homes to cancer centers may affect the delivery of psycho-oncological care. Here, we investigated, in a large brain tumor center with a rural outreach, the initiation of psycho-oncological care depending on spatial distance and impact of psycho-oncological care on emergency visits. Methods Electronic patient charts, the regional tumor registry, and interviews with the primary care physicians were used to investigate clinical data, psycho-oncological care, and emergency unit visits. Interrelations with socio-demographic, clinical, and treatment aspects were investigated using univariable and multivariable binary logistic regression analysis and the Pearson’s Chi-square test. Results Of 491, 229 adult patients of this retrospective cohort fulfilled the inclusion criteria for analysis. During the last three months of their lives, 48.9% received at least one psycho-oncological consultation, and 37.1% visited the emergency unit at least once. The distance from the cancer center did neither affect the initiation of psycho-oncological care nor the rate of emergency unit visits. Receiving psycho-oncological care did not correlate with the frequency of emergency unit visits in the last three months of life. Conclusion We conclude that the distance of IDHwt glioma patients’ homes from their cancer center, even in a rural area, does not significantly influence the rate of psycho-oncological care

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo