Glioblastomas are characterized by fast uncontrolled growth leading to hypoxic areas and necrosis. Signalling from EGFR via mammalian target of rapamycin complex 1 (mTORC1) is a major driver of cell growth and proliferation and one of the most commonly altered signalling pathways in glioblastomas. Therefore, epidermal growth factor receptor and mTORC1 signalling are plausible therapeutic targets and clinical trials with inhibitors are in progress. However, we have previously shown that epidermal growth factor receptor and mTORC1 inhibition triggers metabolic changes leading to adverse effects under the conditions of the tumour microenvironment by protecting from hypoxia-induced cell death. We hypothesized that conversely mTORC1 activation sensitizes glioma cells to hypoxia-induced cell death. As a model for mTORC1 activation we used gene suppression of its physiological inhibitor TSC2 (TSC2sh). TSC2sh glioma cells showed increased sensitivity to hypoxia-induced cell death that was accompanied by an earlier ATP depletion and an increase in reactive oxygen species. There was no difference in extracellular glucose consumption but an altered intracellular metabolic profile with an increase of intermediates of the pentose phosphate pathway. Mechanistically, mTORC1 upregulated the first and rate limiting enzyme of the pentose phosphate pathway, G6PD. Furthermore, an increase in oxygen consumption in TSC2sh cells was detected. This appeared to be due to higher transcription rates of genes involved in mitochondrial respiratory function including PPARGC1A and PPARGC1B (also known as PGC-1α and -β). The finding that mTORC1 activation causes an increase in oxygen consumption and renders malignant glioma cells susceptible to hypoxia and nutrient deprivation could help identify glioblastoma patient cohorts more likely to benefit from hypoxia-inducing therapies such as the VEGFA-targeting antibody bevacizumab in future clinical evaluations.

(1) Background: The phase 2 Regorafenib in Relapsed Glioblastoma (REGOMA) trial indicated a survival benefit for patients with first recurrence of a glioblastoma when treated with the multikinase inhibitor regorafenib (REG) instead of lomustine. The aim of this retrospective study was to investigate REG penetration to cerebrospinal fluid (CSF), treatment efficacy, and effects on magnetic resonance imaging (MRI) in patients with recurrent high-grade gliomas. (2) Methods: Patients were characterized by histology, adverse events, steroid treatment, overall survival (OS), and MRI growth pattern. REG and its two active metabolites were quantified by liquid chromatography/tandem mass spectrometry in patients’ serum and CSF. (3) Results: 21 patients mainly with IDH-wildtype glioblastomas who had been treated with REG were retrospectively identified. Thirteen CFS samples collected from 3 patients of the cohort were available for pharmacokinetic testing. CSF levels of REG and its metabolites were significantly lower than in serum. Follow-up MRI was available in 19 patients and showed progressive disease (PD) in all but 2 patients. Two distinct MRI patterns were identified: 7 patients showed classic PD with progression of contrast enhancing lesions, whereas 11 patients showed a T2-dominant MRI pattern characterized by a marked reduction of contrast enhancement. Median OS was significantly better in patients with a T2-dominant growth pattern (10 vs. 27 weeks respectively, p = 0.003). Diffusion restrictions were observed in 13 patients. (4) Conclusion: REG and its metabolites were detectable in CSF. A distinct MRI pattern that might be associated with an improved OS was observed in half of the patient cohort. Treatment response in the total cohort was poor.

Glioblastomas (GBs) frequently display activation of the epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR). mTOR exists as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). In GBs, mTORC1 inhibitors such as rapamycin have performed poorly in clinical trials, and in vitro protect GB cells from nutrient and oxygen deprivation. Next generation ATP-competitive mTOR inhibitors with affinity for both mTOR complexes have been developed, but data exploring their effects on GB metabolism are scarce. In this study, we compared the ATP-competitive mTORC1/2 inhibitors torin2, INK-128 and NVP-Bez235 to the allosteric mTORC1 inhibitor rapamycin under conditions that mimic the glioma microenvironment. In addition to inhibiting mTORC2 signaling, INK-128 and NVP-Bez235 more effectively blocked mTORC1 signaling and prompted a stronger cell growth inhibition, partly by inducing cell cycle arrest. However, under hypoxic and nutrient-poor conditions mTORC1/2 inhibitors displayed even stronger cytoprotective effects than rapamycin by reducing oxygen and glucose consumption. Thus, therapies that arrest proliferation and inhibit anabolic metabolism must be expected to improve energy homeostasis of tumor cells. These results mandate caution when treating physiologically or therapeutically induced hypoxic GBs with mTOR inhibitors.

Background DNA methylation-based tumor classification allows an enhanced distinction into subgroups of glioblastoma. However, the clinical benefit of DNA methylation-based stratification of glioblastomas remains inconclusive. Methods Multicentric cohort study including 430 patients with newly diagnosed glioblastoma subjected to global DNA methylation profiling. Outcome measures included overall survival (OS), progression-free survival (PFS), prognostic relevance of EOR and MGMT promoter methylation status as well as surgical benefit for recurrent glioblastoma. Results 345 patients (80.2%) fulfilled the inclusion criteria. DNA methylation subclasses RTK I, RTK II, and mesenchymal (MES) revealed no significant survival differences (p = 0.06). 305 Patients receiving combined adjuvant therapy (RTK I: Ref.; RTK II: HR 0.9 [95% CI, 0.64-1.28]; p = 0.56; MES: 0.69 [0.47-1.02]; p = 0.06). RTK I (GTR/near GTR: Ref.; PR: HR 2.87 [95% CI, 1.36-6.08]; p < 0.01) or RTK II (GTR/near GTR: Ref.; PR: HR 5.09 [95% CI, 2.80-9.26]; p < 0.01) tumors who underwent gross-total resection (GTR) or near GTR had a longer OS and PFS than partially resected patients. The MES subclass showed no survival benefit for a maximized EOR (GTR/near GTR: Ref.; PR: HR 1.45 [95% CI, 0.68-3.09]; p = 0.33). Therapy response-predictive value of MGMT promoter methylation was evident for RTK I (HR 0.37 [95% CI, 0.19-0.71]; p < 0.01) and RTK II (HR 0.56 [95% CI, 0.34-0.91]; p = 0.02) but not the MES subclass (HR 0.52 [95% CI, 0.27-1.02]; p = 0.06). For local recurrence (n=112), re-resection conveyed a progression-to-overall survival (POS) benefit (p < 0.01), which was evident in RTK I (p = 0.03) and RTK II (p < 0.01) tumors, but not in MES tumors (p = 0.33). Conclusion We demonstrate a survival benefit from maximized EOR for newly diagnosed and recurrent glioblastomas of the RTK I and RTK II but not the MES subclass. Hence, it needs to be debated whether the MES subclass should be treated with maximal surgical resection, especially when located in eloquent areas and at time of recurrence.

No abstract

Purpose Molecular diagnostics including next generation gene sequencing are increasingly used to determine options for individualized therapies in brain tumor patients. We aimed to evaluate the decision-making process of molecular targeted therapies and analyze data on tolerability as well as signals for efficacy. Methods Via retrospective analysis, we identified primary brain tumor patients who were treated off-label with a targeted therapy at the University Hospital Frankfurt, Goethe University. We analyzed which types of molecular alterations were utilized to guide molecular off-label therapies and the diagnostic procedures for their assessment during the period from 2008 to 2021. Data on tolerability and outcomes were collected. Results 413 off-label therapies were identified with an increasing annual number for the interval after 2016. 37 interventions (9%) were targeted therapies based on molecular markers. Glioma and meningioma were the most frequent entities treated with molecular matched targeted therapies. Rare entities comprised e.g. medulloblastoma and papillary craniopharyngeoma. Molecular targeted approaches included checkpoint inhibitors, inhibitors of mTOR, FGFR, ALK, MET, ROS1, PIK3CA, CDK4/6, BRAF/MEK and PARP. Responses in the first follow-up MRI were partial response (13.5%), stable disease (29.7%) and progressive disease (46.0%). There were no new safety signals. Adverse events with fatal outcome (CTCAE grade 5) were not observed. Only, two patients discontinued treatment due to side effects. Median progression-free and overall survival were 9.1/18 months in patients with at least stable disease, and 1.8/3.6 months in those with progressive disease at the first follow-up MRI. Conclusion A broad range of actionable alterations was targeted with available molecular therapeutics. However, efficacy was largely observed in entities with paradigmatic oncogenic drivers, in particular with BRAF mutations. Further research on biomarker-informed molecular matched therapies is urgently necessary.

Background: The epidermal growth factor receptor (EGFR) signaling pathway is genetically activated in approximately 50% of glioblastomas (GBs). Its inhibition has been explored clinically but produced disappointing results, potentially due to metabolic effects that protect GB cells against nutrient deprivation and hypoxia. Here, we hypothesized that EGFR activation could disable metabolic adaptation and define a GB cell population sensitive to starvation. Methods: Using genetically engineered GB cells to model different types of EGFR activation, we analyzed changes in metabolism and cell survival under conditions of the tumor microenvironment. Results: We found that expression of mutant EGFRvIII as well as EGF stimulation of EGFR-overexpressing cells impaired physiological adaptation to starvation and rendered cells sensitive to hypoxia-induced cell death. This was preceded by adenosine triphosphate (ATP) depletion and an increase in glycolysis. Furthermore, EGFRvIII mutant cells had higher levels of mitochondrial superoxides potentially due to decreased metabolic flux into the serine synthesis pathway which was associated with a decrease in the NADPH/NADP+ ratio. Conclusions: The finding that EGFR activation renders GB cells susceptible to starvation could help to identify a subgroup of patients more likely to benefit from starvation-inducing therapies.

Purpose Classification and treatment of WHO grade II/III gliomas have dramatically changed. Implementing molecular markers into the WHO classification raised discussions about the significance of grading and clinical trials showed overall survival (OS) benefits for combined radiochemotherapy. As molecularly stratified treatment data outside clinical trials are scarce, we conducted this retrospective study. Methods We identified 343 patients (1995–2015) with newly diagnosed WHO grade II/III gliomas and analyzed molecular markers, patient characteristics, symptoms, histology, treatment, time to treatment failure (TTF) and OS. Results IDH-status was available for all patients (259 mutant, 84 IDH1-R132H-non-mutant). Molecular subclassification was possible in 173 tumors, resulting in diagnosis of 80 astrocytomas and 93 oligodendrogliomas. WHO grading remained significant for OS in astrocytomas/IDH1-R132H-non-mutant gliomas (p < 0.01) but not for oligodendroglioma (p = 0.27). Chemotherapy (and temozolomide in particular) showed inferior OS compared to radiotherapy in astrocytomas (median 6.1/12.1 years; p = 0.03) and oligodendrogliomas (median 13.2/not reached (n.r.) years; p = 0.03). While radiochemotherapy improved TTF in oligodendroglioma (median radiochemotherapy n.r./chemotherapy 3.8/radiotherapy 7.3 years; p < 0.001/ = 0.06; OS data immature) the effect, mainly in combination with temozolomide, was weaker in astrocytomas (median radiochemotherapy 6.7/chemotherapy 2.3/radiotherapy 2.0 years; p < 0.001/ = 0.11) and did not translate to improved OS (median 8.4 years). Conclusion This is one of the largest retrospective, real-life datasets reporting treatment and outcome in low-grade gliomas incorporating molecular markers. Current histologic grading features remain prognostic in astrocytomas while being insignificant in oligodendroglioma with interfering treatment effects. Chemotherapy (temozolomide) was less effective than radiotherapy in both astrocytomas and oligodendrogliomas while radiochemotherapy showed the highest TTF in oligodendrogliomas.