RNA sequencing of glioblastoma tissue slice cultures reveals the effects of treatment at the transcriptional level.

One of the major challenges in cancer research is finding models that closely resemble tumors within patients. Human tissue slice cultures are a promising approach to provide a model of the patient's tumor biology ex vivo. Recently, it was shown that these slices can be successfully analyzed by whole transcriptome sequencing as well as automated histochemistry, increasing their usability as preclinical model. Glioblastoma multiforme (GBM) is a highly malignant brain tumor with poor prognosis and little is known about its genetic background and heterogeneity regarding therapy success. In this study, tissue from the tumors of 25 patients with primary GBM was processed into slice cultures and treated with standard therapy (irradiation and temozolomide). Total RNA sequencing and automated histochemistry were performed to enable analysis of treatment effects at a transcriptional and histological level. Slice cultures from long-term survivors (overall survival [OS] > 24 months) exhibited more apoptosis than cultures from patients with shorter OS. Proliferation within these slices was slightly increased in contrast to other groups, but not significantly. Among all samples, 58 protein-coding genes were upregulated and 32 downregulated in treated vs. untreated slice cultures. In general, an upregulation of DNA damage-related and cell cycle checkpoint genes as well as enrichment of genotoxicity pathways and p53-dependent signaling was found after treatment. Overall, the current study reproduces knowledge from former studies regarding the feasibility of transcriptomic analyses and automated histology in tissue slice cultures. We further demonstrate that the experimental data merge with the clinical follow-up of the patients, which improves the applicability of our model system.

Salvage Radiation Therapy for Patients With Relapsing Glioblastoma Multiforme and the Role of Slow Fractionation.

BACKGROUND: Salvage radiation therapy (SRT) can be offered to patients with relapsing glioblastoma multiforme (GBM). Here we report our experience with a schedule extending the treatment time of SRT with the aim to prolong the cytotoxic effect of ionizing radiation while minimizing the cytotoxic hazards for the surrounding brain. METHODS AND PATIENTS: From 2009 until 2017, 124 of 218 patients received radical resection, adjuvant chemo-radiation with photons and temozolomide (TMZ) followed by adjuvant TMZ. Re-irradiation was performed in 26 patients due to local relapse. Treatment schedules varied. Survival and molecular markers were assessed. RESULTS: The median survival was respectively 12 months (9-14.5) of the 124 patients treated with tri-modal therapy and 19.2 months (14.9-24.6) for the 26 patients retreated with SRT (p=0.038). Patients who received daily fractions of 1,6 to 1,65 Gy to a total dose of >40 Gy had a median survival time of 24,6 months compared to patients treated with higher daily doses or a total dose of <40 Gy (p= 0.039), consistent with the observation that patients treated with 21-28 fractions had a median survival of 21,9 months compared to 15,8 months of patients who received 5-20 fractions (p=.0.05). Patients with Ki-67 expression of >30% seemed to perform better than patients with expression levels of ≤20% (p=0.03). MGMT methylation status, TERT promoter or ATRX mutations, overexpression of p53, p16, PD-L1, and EGFR were not prognostic. CONCLUSIONS: Re-irradiation of relapsing GBM is a highly valid treatment option. Our observation challenges hypofractionated stereotactic radiotherapy for retreatment and controlled trials on the fractionation dose for SRT are needed. Robust predictive molecular markers could be beneficial in the selection of patients for SRT.

Atypical Teratoid/Rhabdoid Tumor of the Pineal Region in a Young Adult Male Patient: Case Report and Review of the Literature.

Atypical teratoid/rhabdoid tumor (AT/RT) represents a highly malignant and aggressive embryonal neoplasm of the central nervous system (CNS). Histologically it consists of rhabdoid cells with a varying combination of neuroectodermal, epithelial, and mesenchymal tissue. Histologic features and the poor clinical outcome indicate that this tumor corresponds to World Health Organization grade IV. AT/RT mainly occurs in children < 3 years of age and has only been rarely described in adults. It often arises from the posterior fossa of infants and has the tendency to spread through the subarachnoid space. In contrast to pediatric cases, most of the AT/RTs in adults are located in the cerebral hemispheres; infratentorial appearance or localization in the spinal cord is relatively uncommon. Likewise there are only a few reports of AT/RT in the pineal region. We present another interesting case of this tumor occurring in the pineal region of a 19-year-old man and discuss our case in the context of the current literature. The patient is alive 18 months since his initial diagnosis without any signs of tumor relapse.

Distributive shock, cardiac arrhythmias and multiple organ failure following surgery of a fourth ventricular epidermoid.

A 33-years-old male patient presented with cardiac arrhythmias, acute shock and multiple organ dysfunction after the surgical removal of a massive epidermoid posterior to the brainstem. The patient initially presented with paraesthesia along the right C6 dermatome due to a big tumour at the brain stem. Surgical removal was performed without adverse events and he was transferred to our intensive care unit (ICU) immediately after the operation. Though initially showing a stable postsurgical course he developed cardiac arrhythmias and a state of acute distributive shock with consecutive multi organ failure. Extensive diagnostic measures could not identify a specific cause for this rapid deterioration. However, under carefully monitored symptomatic therapy the patient improved quickly, was extubated 72 h after admission and discharged from the ICU 6 days later. The follow-up did not show any persisting neurological deficits and no evidence of a residual tumour in the MRI-study.

Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.

BACKGROUND: Glioblastoma multiforme is the most common lethal brain tumor in human adults, with no major therapeutic breakthroughs in recent decades. Research is based mostly on human tumor cell lines deprived of their organotypic environment or inserted into immune-deficient animals required for graft survival. Here, we describe how glioblastoma specimens obtained from surgical biopsy material can be sectioned and transferred into cultures within minutes. METHODS: Slices were kept in 6-well plates, allowing direct observation, application of temozolomide, and irradiation. At the end of experiments, slice cultures were processed for histological analysis including hematoxylin-eosin staining, detection of proliferation (Ki67), apoptosis/cell death (cleaved caspase 3, propidium iodide), DNA double-strand breaks (γH2AX), and neural subpopulations. First clinical trials employed irradiation with the heavy ion carbon for the treatment of glioblastoma patients, but the biological effects and most effective dose regimens remain to be established. Therefore, we developed an approach to expose glioblastoma slice cultures to (12)C and X-rays. RESULTS: We found preservation of the individual histopathology over at least 16 days. Treatments resulted in activation of caspase 3, inhibition of proliferation, and cell loss. Irradiation induced γH2AX. In line with clinical observations, individual tumors differed significantly in their susceptibility to temozolomide (0.4%-2.5% apoptosis and 1%-15% cell loss). CONCLUSION: Glioblastoma multiforme slice cultures provide a unique tool to explore susceptibility of individual tumors for specific therapies including heavy ions, thus potentially allowing more personalized treatments plus exploration of mechanisms of (and strategies to overcome) tumor resistance.

Hedgehog signaling in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant brain tumor in adults with a median survival of 14.6 mo under the best available treatment. New treatment strategies are therefore urgently required, for which a profound understanding of tumor biology is necessary. Much effort has been devoted to tumor-specific aberrant signaling processes. Recently it was discovered that the transcription factor Gli1, which is activated by hedgehog signaling, is a highly predictive marker in GBM, as determined by immunohistochemistry. To determine whether GBM cells have transcriptionally active Gli1, we performed experiments with reporter genes with cells isolated from surgically removed human tumors and cell lines. We also determined whether the hedgehog signaling inhibitor cyclopamine influences reporter gene expression and cell viability, and we determined the expression of Gli1, SHH and Patched1 by quantitative real-time RT-PCR. Reporter gene analysis of nine cultures and four cell lines demonstrated a significantly enhanced transcriptional activity in six tumor cell cultures and all cell lines. Analysis of cell viability in the presence of cyclopamine revealed a response of all cell cultures with the exception of one primary culture and one cell line, but only one cell line responded to cyclopamine with reduced hedgehog signaling activity. This indicates that the toxicity of cyclopamine toward GBM cells is independent from hedgehog signaling. Since no correlation between hedgehog activity and SHH, Gli1 and Patched1 mRNA levels was observed we conclude that other mechanisms aside from transcriptional regulation of these factors are responsible for hedgehog activity in tumor cells derived from GBM.

Search for genetic variants in the ryanodine receptor 1 gene in patients with symptomatic cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

BACKGROUND: Cerebral vasospasm is one of the most serious complications after subarachnoid hemorrhage (SAH). The cerebral artery diameter is regulated by complex physiological mechanisms. Among them the regulation of intracellular calcium homeostasis seems to play a crucial role. Recent data suggest that ryanodine receptors (RYRs) are involved in regulating the luminal calcium concentration in vascular smooth muscle cells. In this gene association investigation, we studied the question as to whether variants in the gene for the ryanodine receptors subtype 1 (RYR1) are associated with symptomatic cerebral vasospasm following SAH. METHODS: After informed consent genomic DNA analysis was performed from a whole blood sample in 46 patients suffering from aneurysmal SAH. 16 Patients were affected by symptomatic vasospasm. The RYR1 gene was screened for possible genetic variants by means of direct sequencing. The association of these variants was correlated to the development of symptomatic vasospasm, which was confirmed by clinical examination combined with cerebral angiography, transcranial doppler sonography, or CT scan. RESULTS: Three different genetic RYR1 variants (c.5360C>T, c.6178G>T, and c.7244G>A) were identified in the study. The G/T genotype of RYR1 c.6178G>T was associated with an increased risk for development of symptomatic vasospasm (odds ratio 6.4; 95% CI 1.1-37.8; P = 0.04). CONCLUSION: Our pilot study suggests that RYRs are involved in the complex pathophysiology of vasospasm development following SAH. The potential role of RYR1 as a biomarker for prediction of cerebral vasospasm after SAH has to be confirmed in a larger clinical trial.

Identification of factors involved in the anti-tumor activity of carnosine on glioblastomas using a proteomics approach.

Human glioblastoma multiforme is the most malignant brain tumor in adults and is difficult to treat. Recently, it was demonstrated that the dipeptide carnosine inhibits tumor growth, but the main molecular targets are not known. Therefore, a proteomics study with glioblastoma cells treated with carnosine was performed. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight detected 31 proteins expressed differentially under the influence of carnosine. Finally, peptide mass fingerprinting identified the "BCL2-associated athanogene 2" and the "von Hippel-Lindau binding protein 1" among other proteins, linking the action of carnosine to protein folding and HIF-1α signalling.

Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model.

BACKGROUND: It was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy. RESULTS: A mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu), were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 microl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p < 0.0003) than untreated animals, confirming that carnosine affects proliferation in vivo. CONCLUSION: As a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.

Carnosine inhibits ATP production in cells from malignant glioma.

OBJECTIVES: Recently, it was revealed that carnosine inhibits growth of cells isolated from human malignant glioma. In order to understand how this effect is mediated, experiments were performed that addressed a possible influence of carnosine on energy metabolism. METHODS: Cells from the glioma line T98G and primary cultured cells from human malignant glioma were cultivated in the presence of carnosine and inhibitors of cellular energy metabolism. As a specific inhibitor for anaerobic glycolysis, oxamate, and as an inhibitor for mitochondrial oxidative phosphorylation, potassium cyanide, were used, and the influence on ATP production was determined using cell-based assays. RESULTS: The experiments identified glycolysis as crucial for ATP production in gliomas. In addition, ATP production by mitochondrial activity did not significantly contribute to ATP production and carnosine was identified to be an inhibitor of the vital anaerobic glycolysis. DISCUSSION: Carnosine might be considered as a potential drug for the treatment of malignant glioma or other tumors since it inhibits the glycolytic energy metabolism that is crucial for cancer cells and malignant gliomas as shown in the current study. This is especially interesting since the dipeptide is a naturally occurring substance that should be well tolerated.

Intracranial dural arteriovenous fistula associated with progressive cervical myelopathy and normal venous drainage of the thoracolumbar cord: case report and review of the literature.

BACKGROUND: Intracranial dural arteriovenous malformations draining into the perimedullary venous system are rare lesions. In these cases, the selective spinal catheterization of all vessels with potential of causing that malformation was negative, and additional cerebral angiography usually reveals the fistula. Because of venous congestion of the cord caused by the DAVF, a delayed drainage or stagnation of contrast material in the artery of Adamkiewicz was considered as a compelling angiographic disorder so far. CASE DESCRIPTION: We report about a 58-year-old patient with a DAVF of the right posterior fossa draining into the cervical and upper thoracic plexus of medullary veins, followed by progressive cervical myelopathy and a normal venous drainage of the artery of Adamkiewicz. Because of the failing endovascular treatment option, the neurosurgical intervention was performed. The fistula was explored and clipped without any complications. Immediately after operation, the patient reported an improvement of his neurological deficits. CONCLUSION: About 38 cases of intracranial DAVFs draining into the perimedullary venous system are reported, but to our knowledge, this is the second one with a normal drainage of the artery of Adamkiewicz. The pathophysiological mechanisms, diagnostic procedures, and treatment modalities are discussed.

Intraoperative MRI to guide the resection of primary supratentorial glioblastoma multiforme--a quantitative radiological analysis.

Patients with supratentorial high-grade glioma underwent surgery within a vertically open 0.5-T magnetic resonance (MR) system to evaluate the efficacy of intraoperative MR guidance in achieving gross-total resection. For 31 patients, preoperative clinical data and MR findings were consistent with the putative diagnosis of a high-grade glioma, in 23 cases in eloquent regions. Tumor resections were carried out within a 0.5-T MR SIGNA SP/i (GE Medical Systems, USA). The resection of the lesion was carried out using fully MR compatible neurosurgical equipment and was stopped at the point when the operation was considered complete by the surgeon viewing the operation field with the microscope. We repeated imaging to determine the residual tumor volume only visible with MRI. Areas of tissue that were abnormal on these images were localized in the bed of resection by using interactive MR guidance. The procedure of resection, imaging control and interactive image guidance was repeated where necessary. Almost all tissue with abnormal characteristics was resected, with the exception of tissue localized in eloquent brain areas. The diagnosis of glioblastoma was confirmed in all 31 cases. When comparing the tumor volume before resection and at the point where the neurosurgeon would otherwise have terminated surgery ("first control"), residual tumor tissue was detectable in 29/31 patients; the mean residual tumor volume was 30.7 +/- 24%. After repeated resections under interactive image guidance the mean residual tumor volume was 15.1%. At this step we found tumor remnants only in 20/31 patients. The perioperative morbidity (12.9%) was low. Twenty-seven patients underwent sufficient postoperative radiotherapy. We found a significant difference (log(rank)p = 0.0037) in the mean survival times of the two groups with complete resection (n = 10, median survival time 537 days) and incomplete resection (n = 17, median survival time 237 days). The resection of primary glioblastoma multiforme under intraoperative MR guidance as demonstrated is a possibility to achieve a more complete removal of the tumor than with conventional techniques. In our small but homogeneous patient group we found an increase in the median survival time in patients with MRI for complete tumor resection, and the overall surgical morbidity was low.