Almuth F. Kessler

TERT Promoter Mutations and Risk of Recurrence in Meningioma

The World Health Organization (WHO) classification and grading system attempts to predict the clinical course of meningiomas based on morphological parameters. However, because of high interobserver variation of some criteria, more reliable prognostic markers are required. Here, we assessed the TERT promoter for mutations in the hotspot regions C228T and C250T in meningioma samples from 252 patients. Mutations were detected in 16 samples (6.4% across the cohort, 1.7%, 5.7%, and 20.0% of WHO grade I, II, and III cases, respectively). Data were analyzed by t test, Fishers exact test, log-rank test, and Cox proportional hazard model. All statistical tests were two-sided. Within a mean follow-up time in surviving patients of 68.1 months, TERT promoter mutations were statistically significantly associated with shorter time to progression (P < .001). Median time to progression among mutant cases was 10.1 months compared with 179.0 months among wild-type cases. Our results indicate that the inclusion of molecular data (ie, analysis of TERT promoter status) into a histologically and genetically integrated classification and grading system for meningiomas increases prognostic power. Consequently, we propose to incorporate the assessment of TERT promoter status in upcoming grading schemes for meningioma.

Effects of the Selective MPS1 Inhibitor MPS1-IN-3 on Glioblastoma Sensitivity to Antimitotic Drugs

BACKGROUND Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest. METHODS We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3-7 mice/group). MPS1 expression levels were examined in relation to patient survival. RESULTS Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity. CONCLUSIONS Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drugs.

Comparison of the Amino Acid Tracers 18F-FET and 18F-DOPA in High-Grade Glioma Patients

High-grade gliomas (HGGs) are the most common malignant primary tumors of the central nervous system. PET probes of amino acid transport such as O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET), 3,4-dihydroxy-6-18F-fluoro-l-phenylalanine (18F-DOPA), and 11C-methionine (11C-MET) detect primary and recurrent tumors with a high accuracy. 18F-FET is predominantly used in Europe, whereas amino acid transport imaging is infrequently done in the United States. The aim of this study was to determine whether 18F-FET and 18F-DOPA PET/CT provide comparable information in HGG. Methods: Thirty 18F-FET and 18F-DOPA PET/CT scans were obtained before surgery or biopsy in 27 patients with high clinical suspicion for primary or recurrent HGG (5 primary, 22 recurrent tumors). 18F-FET and 18F-DOPA PET/CT images were compared visually and semiquantitatively (maximum standardized uptake value [SUVmax], mean SUV [SUVmean]). Background (SUVmax and SUVmean) and tumor-to-background ratios (TBRs) were calculated for both PET probes. The degree of 18F-DOPA uptake in the basal ganglia (SUVmean) was also assessed. Results: Visual analysis revealed no difference in tumor uptake pattern between the 2 PET probes. The SUVmean and SUVmax for 18F-FET were higher than those of 18F-DOPA (4.0 ± 2.0 and 4.9 ± 2.3 vs. 3.5 ± 1.6 and 4.3 ± 2.0, respectively; all P < 0.001). TBRs for SUVmean but not for SUVmax were significantly higher for 18F-FET than 18F-DOPA (TBR SUVmean: 3.8 ± 1.7 vs. 3.4 ± 1.2, P = 0.004; TBR SUVmax: 3.3 ± 1.6 and 3.0 ± 1.1, respectively; P = 0.086). 18F-DOPA uptake by the basal ganglia was present (SUVmean, 2.6 ± 0.7) but did not affect tumor visualization. Conclusion: Whereas visual analysis revealed no significant differences in uptake pattern for 18F-FET and 18F-DOPA in patients with primary or recurrent HGG, both SUVs and TBRs for SUVmean were significantly higher for 18F-FET. However, regarding tumor delineation, both tracers performed equally well and seem equally feasible for imaging of primary and recurrent HGG. These findings suggest that both PET probes can be used based on availability in multicenter trials.

68 Ga-Pentixafor-PET/CT for Imaging of Chemokine Receptor 4 Expression in Glioblastoma

Chemokine receptor-4 (CXCR4) has been reported to be overexpressed in glioblastoma (GBM) and to be associated with poor survival. This study investigated the feasibility of non-invasive CXCR4-directed imaging with positron emission tomography/computed tomography (PET/CT) using the radiolabelled chemokine receptor ligand 68Ga-Pentixafor. 15 patients with clinical suspicion on primary or recurrent glioblastoma (13 primary, 2 recurrent tumors) underwent 68Ga-Pentixafor-PET/CT for assessment of CXCR4 expression prior to surgery. O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) PET/CT images were available in 11/15 cases and were compared visually and semi-quantitatively (SUVmax, SUVmean). Tumor-to-background ratios (TBR) were calculated for both PET probes. 68Ga-Pentixafor-PET/CT results were also compared to histological CXCR4 expression on neuronavigated surgical samples. 68Ga-Pentixafor-PET/CT was visually positive in 13/15 cases with SUVmean and SUVmax of 3.0±1.5 and 3.9±2.0 respectively. Respective values for 18F-FET were 4.4±2.0 (SUVmean) and 5.3±2.3 (SUVmax). TBR for SUVmean and SUVmax were higher for 68Ga-Pentixafor than for 18F-FET (SUVmean 154.0±90.7 vs. 4.1±1.3; SUVmax 70.3±44.0 and 3.8±1.2, p<0.01), respectively. Histological analysis confirmed CXCR4 expression in tumor areas with high 68Ga-Pentixafor uptake; regions of the same tumor without apparent 68Ga-Pentixafor uptake showed no or low receptor expression. In this pilot study, 68Ga-Pentixafor retention has been observed in the vast majority of glioblastoma lesions and served as readout for non-invasive determination of CXCR4 expression. Given the paramount importance of the CXCR4/SDF-1 axis in tumor biology, 68Ga-Pentixafor-PET/CT might prove a useful tool for sensitive, non-invasive in-vivo quantification of CXCR4 as well as selection of patients who might benefit from CXCR4-directed therapy.

The cohesin-interacting protein, precocious dissociation of sisters 5A/sister chromatid cohesion protein 112, is up-regulated in human astrocytic tumors.

Glioblastoma multiforme (GBM) is the most prevalent, highly malignant, invasive and difficult-to-treat primary brain tumor in adults. At the genetic level, it is characterized by a high degree of chromosomal instability and aneuploidy. It has been shown that defects in the mitotic spindle checkpoint could lead to the development of aneuploidy as well as tumorigenesis. Additional proteins regulating sister chromatid cohesion could also be involved in maintaining the fidelity of chromosome segregation. One such protein is the precocious dissociation of sisters 5A (Pds5A), also known as sister chromatid cohesion protein 112. It is a nuclear protein, expressed from the S right through to the mitotic phase. It is highly conserved from yeast to man and plays a role in the establishment, maintenance and dissolution of sister chromatid cohesion. The mutation of Pds5A orthologs in lower organisms results in chromosome missegregation, aneuploidy and DNA repair defects. It is considered that such defects can cause either cell death or contribute to the development of cancer cells. Indeed, altered expression levels of Pds5A have been observed in tumors of the breast, kidney, oesophagus, stomach, liver and colon. Malignant gliomas, however, have not been analysed so far. Herein, we report on the cloning of Rattus norvegicus Pds5A and on the analysis of its expression pattern in rat tissue. We also show that Pds5A is significantly overexpressed at both the mRNA and protein level and that this overexpression correlates positively with the WHO grade of human gliomas. However, functional assays show that the siRNA-mediated knockdown of Pds5A affects sister chromatid cohesion but does not influence mitotic checkpoint function or the proliferation and survival of GBM cells. Although the mechanism by which Pds5A functions in GBM cells remains unclear, its overexpression in high grade gliomas implies that it could play a pivotal role during the development and progression of astrocytic tumors.

Transcriptomic analysis of aggressive meningiomas identifies PTTG1 and LEPR as prognostic biomarkers independent of WHO grade

Meningiomas are frequent central nervous system tumors. Although most meningiomas are benign (WHO grade I) and curable by surgery, WHO grade II and III tumors remain therapeutically challenging due to frequent recurrence. Interestingly, relapse also occurs in some WHO grade I meningiomas. Hence, we investigated the transcriptional features defining aggressive (recurrent, malignantly progressing or WHO grade III) meningiomas in 144 cases. Meningiomas were categorized into non-recurrent (NR), recurrent (R), and tumors undergoing malignant progression (M) in addition to their WHO grade. Unsupervised transcriptomic analysis in 62 meningiomas revealed transcriptional profiles lining up according to WHO grade and clinical subgroup. Notably aggressive subgroups (R+M tumors and WHO grade III) shared a large set of differentially expressed genes (n=332; p<0.01, FC>1.25). In an independent multicenter validation set (n=82), differential expression of 10 genes between WHO grades was confirmed. Additionally, among WHO grade I tumors differential expression between NR and aggressive R+M tumors was affirmed for PTTG1, AURKB, ECT2, UBE2C and PRC1, while MN1 and LEPR discriminated between NR and R+M WHO grade II tumors. Univariate survival analysis revealed a significant association with progression-free survival for PTTG1, LEPR, MN1, ECT2, PRC1, COX10, UBE2C expression, while multivariate analysis identified a prediction for PTTG1 and LEPR mRNA expression independent of gender, WHO grade and extent of resection. Finally, stainings of PTTG1 and LEPR confirmed malignancy-associated protein expression changes. In conclusion, based on the so far largest study sample of WHO grade III and recurrent meningiomas we report a comprehensive transcriptional landscape and two prognostic markers.

Long-term tumor control of spinal dissemination of cerebellar glioblastoma multiforme by combined adjuvant bevacizumab antibody therapy: a case report

BackgroundGlioblastoma multiforme located in the posterior fossa is extremely rare with a frequency up to 3.4%. Compared with glioblastoma of the hemispheres the prognosis of infratentorial glioblastoma seems to be slightly better. Absence of brainstem invasion and low expression rates of epidermal growth factor receptor are described as factors for long-time survival due to the higher radiosensitivity of these tumors.Case presentationIn this case study, we report a German female patient with an exophytic glioblastoma multiforme arising from the cerebellar tonsil and a secondary spinal manifestation. Furthermore, the tumor showed no O (6)-Methylguanine-DNA methyltransferase promotor-hypermethylation and no isocitrate dehydrogenase 1 mutations. All these signs are accompanied by significantly shorter median overall survival. A long-term tumor control of the spinal metastases was achieved by a combined temozolomide/bevacizumab and irradiation therapy, as part of a standard care administered by the treating physician team.ConclusionTo our knowledge this is the first published case of a combined cerebellar exophytic glioblastoma with a subsequent solid spinal manifestation. Furthermore this case demonstrates a benefit undergoing this special adjuvant therapy regime in terms of overall survival. Due to the limited overall prognosis of the disease, spinal manifestations of glioma are rarely clinically relevant. The results of our instructive case, however, with a positive effect on both life quality and survival warrant treating future patients in the frame of a prospective clinical study.

High-Efficiency Transfection of Glioblastoma Cells and a Simple Spheroid Migration Assay

Despite international research efforts, patients with glioblastoma multiforme (GBM)-the most common malignant brain tumors in adults-exhibit a very unfavorable prognosis. Their aggressive local growth pattern and increased invasiveness, due to a high motility of the tumor cells, hamper treatment. However, the molecular mechanisms regulating glioblastoma cell migration are still elusive. Here, we describe the combination of a highly efficient cell transfection by Nucleofection® technology and the generation of spheroids from these transfected glioblastoma cell lines. Nucleofection allows the manipulation of protein expression by overexpression and siRNA mediated protein knockdown. Transfection efficiencies >70% can be achieved with some GBM cell lines. Transfected neurospheres then can be used for migration assays (as described here in detail) and a multitude of other functional assays. In comparison to monolayer cultures, the advantage of spheroids is their resemblance with organized tissue in combination with the accuracy of in vitro methodology and marked experimental flexibility.

Feasibility of the Combined Application of Navigated Probabilistic Fiber Tracking and Navigated Ultrasonography in Brain Tumor Surgery

BACKGROUND Surgical resection of intra-axial tumors is a challenging procedure because of indistinct tumor margins, infiltration, and displacement of white matter tracts surrounding the lesion. Hence, gross total tumor resection without causing new neurologic deficits is demanding, especially in tumor sites adjoining eloquent structures. Feasibility of the combination of navigated probabilistic fiber tracking to identify eloquent fiber pathways and navigated ultrasonography to control brain shift was tested. METHODS Eleven patients with lesions adjacent to eloquent white matter structures (pyramidal tract, optic radiation and arcuate fascicle) were preoperatively subjected to magnetic resonance imaging including diffusion-weighted imaging on a 3-T magnetic resonance system (Trio [Siemens, Erlangen, Germany]). Probabilistic fiber tracking was performed using the tools of the FMRIB Software Library (FSL). Results of probabilistic fiber tracking and high-resolution anatomic images were integrated into the neuronavigation system Stealth Station (Medtronic, Minneapolis, Minnesota, USA) together with the navigated ultrasonography (SonoNav [Medtronic]). RESULTS FSL-based probabilistic fiber tracking depicted the pyramidal tract, the optic radiation, and arcuate fascicle anatomically plausibly. Integration of the probabilistic fiber tracking into neuronavigation was technically feasible and allowed visualization of the reconstructed fiber pathways. Navigated ultrasonography controlled brain shift. CONCLUSIONS Integration of probabilistic fiber tracking and navigated ultrasonography into intraoperative neuronavigation facilitated anatomic orientation during glioma resection. FSL-based probabilistic fiber tracking integrated sophisticated fiber tracking algorithms, including modeling of crossing fibers. Combination with navigated ultrasonography provided a three-dimensional estimation of intraoperative brain shift and, therefore, improved the reliability of neuronavigation.

CXCR4: A new player in vestibular schwannoma pathogenesis

Background CXCR4 is a chemokine receptor that recruits blood stem cells and increases tumor cell growth and invasiveness. We examined CXCR4 expression in vestibular schwannomas (VS) from patients with and without neurofibromatosis type 2 (NF2) and correlated the levels with the patients’ clinical characteristics. The aim was to determine whether CXCR4 can be used as a prognostic marker and as a target for systemic therapy. Results Overall, CXCR4 mRNA levels were 4.6-fold higher in VS versus control; the levels were 4.9-fold higher in NF2 patients and 4.2-fold higher in sporadic VS patients. IHC and WB showed heterogeneous protein expression, and CXCR4 was expressed mainly in S100-positive Schwann cells. There was no correlation between the CXCR4 protein levels and tumor extension. However, there was a trend towards correlation between higher expression levels and greater hearing loss. Materials and Methods CXCR4 mRNA and protein levels were determined in VS samples (n = 60); of these, 30 samples were from patients with NF2. Healthy nerves from autopsies served as controls. CXCR4 mRNA levels were measured by PCR, and protein levels were measured by immunohistochemistry (IHC) and Western blotting (WB). Tumor extension and hearing loss were categorized according to the Hannover Classification as clinical parameters. Conclusions CXCR4 mRNA was overexpressed in VS relative to healthy vestibular nerves, and there was a trend towards higher CXCR4 expression levels being correlated with greater functional impairment. Thus, CXCR4 may be a prognostic marker of VS, and CXCR4 inhibition has potential as a systemic approach for the treatment of VS.