A Novel Approach for Glioblastoma Treatment by Combining Apoptosis Inducers (TMZ, MTX, and Cytarabine) with E.V.A. (Eltanexor, Venetoclax, and A1210477) Inhibiting XPO1, Bcl-2, and Mcl-1.

Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

Status epilepticus in patients with brain tumors and metastases: A multicenter cohort study of 208 patients and literature review.

OBJECTIVE: Brain tumors and metastases account for approximately 10% of all status epilepticus (SE) cases. This study described the clinical characteristics, treatment, and short- and long-term outcomes of this population. METHODS: This retrospective, multi-center cohort study analyzed all brain tumor patients treated for SE at the university hospitals of Frankfurt and Marburg between 2011 and 2017. RESULTS: The 208 patients (mean 61.5 ± 14.7 years of age; 51% male) presented with adult-type diffuse gliomas (55.8%), metastatic entities (25.5%), intracranial extradural tumors (14.4%), or other tumors (4.3%). The radiological criteria for tumor progression were evidenced in 128 (61.5%) patients, while 57 (27.4%) were newly diagnosed with tumor at admission and 113 (54.3%) had refractory SE. The mean hospital length of stay (LOS) was 14.8 days (median 12.0, range 1-57), 171 (82.2%) patients required intensive care (mean LOS 8.9 days, median 5, range 1-46), and 44 (21.2%) were administered mechanical ventilation. All patients exhibited significant functional status decline (modified Rankin Scale) post-SE at discharge (p < 0.001). Mortality at discharge was 17.3% (n = 36), with the greatest occurring in patients with metastatic disease (26.4%, p = 0.031) and those that met the radiological criteria for tumor progression (25%, p < 0.001). Long-term mortality at one year (65.9%) was highest in those diagnosed with adult-type diffuse gliomas (68.1%) and metastatic disease (79.2%). Refractory status epilepticus cases showed lower survival rates than non-refractory SE patients (log-rank p = 0.02) and those with signs of tumor progression (log-rank p = 0.001). CONCLUSIONS: SE occurrence contributed to a decline in functional status in all cases, regardless of tumor type, tumor progression status, and SE refractoriness, while long-term mortality was increased in those with malignant tumor entities, tumor progressions, and refractory SE. SE prevention may preserve functional status and improve survival in individuals with brain tumors.

Feasibility and comparability of different platelet function tests in acute stroke with or without prior antiplatelet therapy.

Background: The clinical course of ischemic and hemorrhagic strokes can be influenced by the coagulation status of individual patients. The prior use of antiplatelet therapy (APT) such as acetylsalicylic acid (ASA) or P2Y12-antagonists has been inconsistently described as possibly increasing the risk of hemorrhagic transformation or expansion. Since clinical studies describing prior use of antiplatelet medication are overwhelmingly lacking specific functional tests, we aimed to implement testing in routine stroke care. Methods: We used fluorescence-activated cell sorting (FACS) with antibodies against CD61 for thrombocyte identification and CD62p or platelet activation complex-1 (PAC-1) to determine platelet activation. Aggregometry and automated platelet functioning analyzer (PFA-200) were employed to test thrombocyte reactivity. FACS and aggregometry samples were stimulated in vitro with arachidonic acid (AA) and adenosine diphosphate (ADP) to measure increase in CD62p-/PAC-1-expression or aggregation, respectively. Results: Between February and July 2023, 20 blood samples (n = 11 ischemic strokes; n = 7 hemorrhagic strokes; n = 2 controls) were acquired and analyzed within 24 h of symptom onset. N = 11 patients had taken ASA, n = 8 patients no APT and n = 1 ASA+clopidogrel. ASA intake compared to no APT was associated with lower CD62p expression after stimulation with AA on FACS analysis (median 15.8% [interquartile range {IQR} 12.6-37.2%] vs. 40.1% [IQR 20.3-56.3%]; p = 0.020), lower platelet aggregation (9.0% [IQR 7.0-12.0%] vs. 88.5% [IQR 11.8-92.0%]; p = 0.015) and longer time to plug formation with PFA-200 (248.0 s [IQR 157.0-297] vs. 121.5 s [IQR 99.8-174.3]; p = 0.027). Significant correlations were noted between AA-induced CD62p expression and aggregometry analysis (n = 18; ρ = 0.714; p < 0.001) as well as a negative correlation between CD62p increase and PFA clot formation time (n = 18; ρ = -0.613; p = 0.007). Sensitivity for ASA intake was highest for PFA (81.8% for values ≥155.5 s). The combination of ASA + clopidogrel also affected ADP-induced CD62p and PAC-1 expression. Conclusion: In the clinical setting it is feasible to use differentiated platelet analytics to determine alterations caused by antiplatelet therapy. Among the tests under investigation, PFA-200 showed the highest sensitivity for the intake of ASA in stroke patients. FACS analysis on the other hand might be able to provide a more nuanced approach to altered platelet reactivity.

Mammalian target of rapamycin inhibition protects glioma cells from temozolomide-induced cell death.

Glioblastoma is an incurable brain tumor with a median survival below two years. Trials investigating targeted therapy with inhibitors of the kinase mTOR have produced ambiguous results. Especially combination of mTOR inhibition with standard temozolomide radiochemotherapy has resulted in reduced survival in a phase II clinical trial. To date, this phenomenon is only poorly understood. To recreate the therapeutic setting in vitro, we exposed glioblastoma cell lines to co-treatment with rapamycin and temozolomide and assessed cell viability, DNA damage and reactive oxygen species. Additionally, we employed a novel translatomic based mass spectrometry approach ("mePROD") to analyze acute changes in translated proteins. mTOR inhibition with rapamycin protected glioblastoma cells from temozolomide toxicity. Following co-treatment of temozolomide with rapamycin, an increased translation of reactive oxygen species (ROS)-detoxifying proteins was detected by mass spectrometry. This was accompanied by improved ROS-homeostasis and reduced DNA damage. Additionally, rapamycin induced the expression of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma cells with an unmethylated MGMT gene promotor. Inhibition of mTOR antagonized the cytotoxic effects of temozolomide in vitro. The induction of antioxidant defences and MGMT are two underlying candidate mechanisms. Further functional experiments in vitro and in vivo are warranted to characterize this effect that appears relevant for combinatorial therapeutic strategies.

The Acute Superficial Siderosis Syndrome - Clinical Entity, Imaging Findings, and Histopathology.

Superficial siderosis is a consequence of repetitive bleeding into the subarachnoid space, leading to toxic iron and hemosiderin deposits on the surface of the brain and spine. The clinical and radiological phenotypes of superficial siderosis are known to manifest over long time intervals. In contrast, this study defines the "acute superficial siderosis syndrome" and illustrates typical imaging and histopathological findings of this entity. We describe the case of a 61-year-old male patient who was diagnosed with a melanoma metastasis in the right frontal cortex in February 2019. Within a few weeks he developed a progressive syndrome characterized by cerebellar ataxia, gait disturbance, signs of myelopathy, and radiculopathy. MRI revealed ongoing hemorrhage from the metastasis into the lateral ventricle and the subarachnoid space. A semiquantitative assessment of three subsequent MRI within an 8-week period documented the rapid development of superficial siderosis along the surface of the cerebellum, the brain stem, and the lower parts of the supratentorial regions on T2*-weighted sequences. The diagnosis of a superficial siderosis was histopathologically confirmed by identifying iron and hemosiderin deposits on the cortex along with astrogliosis. The recognition of this "acute superficial siderosis syndrome" triggered surgical removal of the hemorrhagic metastasis. Based on a single case presentation, we define the "acute superficial siderosis syndrome" as a clinical entity and describe the radiological and histopathological characteristics of this entity. Early recognition of this syndrome may allow timely elimination of the bleeding source, in order to prevent further clinical deterioration.

Prevalence of acute neurological complications and pathological neuroimaging findings in critically ill COVID-19 patients with and without VV-ECMO treatment.

Acute brain injuries such as intracerebral hemorrhage (ICH) and ischemic stroke have been reported in critically ill COVID-19 patients as well as in patients treated with veno-venous (VV)-ECMO independently of their COVID-19 status. The purpose of this study was to compare critically ill COVID-19 patients with and without VV-ECMO treatment with regard to acute neurological symptoms, pathological neuroimaging findings (PNIF) and long-term deficits. The single center study was conducted in critically ill COVID-19 patients between February 1, 2020 and June 30, 2021. Demographic, clinical and laboratory parameters were extracted from the hospital's databases. Retrospective imaging modalities included head computed tomography (CT) and magnetic resonance imaging (MRI). Follow-up MRI and neurological examinations were performed on survivors > 6 months after the primary occurrence. Of the 440 patients, 67 patients received VV-ECMO treatment (15%). Sixty-four patients (24 with VV-ECMO) developed acute neurological symptoms (pathological levels of arousal/brain stem function/motor responses) during their ICU stay and underwent neuroimaging with brain CT as the primary modality. Critically ill COVID-19 patients who received VV-ECMO treatment had a significantly lower survival during their hospital stay compared to those without (p < 0.001). Among patients treated with VV-ECMO, 10% showed acute PNIF in one of the imaging modalities during their ICU stay (vs. 4% of patients in the overall COVID-19 ICU cohort). Furthermore, 9% showed primary or secondary ICH of any severity (vs. 3% overall), 6% exhibited severe ICH (vs. 1% overall) and 1.5% were found to have non-hemorrhagic cerebral infarctions (vs. < 1% overall). There was a weak, positive correlation between patients treated with VV-ECMO and the development of acute neurological symptoms. However, the association between the VV-ECMO treatment and acute PNIF was negligible. Two survivors (one with VV-ECMO-treatment/one without) showed innumerable microhemorrhages, predominantly involving the juxtacortical white matter. None of the survivors exhibited diffuse leukoencephalopathy. Every seventh COVID-19 patient developed acute neurological symptoms during their ICU stay, but only every twenty-fifth patient had PNIF which were mostly ICH. VV-ECMO was found to be a weak risk factor for neurological complications (resulting in a higher imaging rate), but not for PNIF. Although logistically complex, repeated neuroimaging should, thus, be considered in all critically ill COVID-19 patients since ICH may have an impact on the treatment decisions and outcomes.

Molecular matched targeted therapies for primary brain tumors-a single center retrospective analysis.

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.

Pulmonary Resection after Radiosurgery and Neoadjuvant Immunochemotherapy for NSCLC Patients with Synchronous Brain Metastasis-A Case Series of Three Patients.

Brain metastases are a common finding upon initial diagnosis of otherwise locally limited non-small cell lung cancer. We present a retrospective case series describing three cases of patients with symptomatic, synchronous brain metastases and resectable lung tumors. The patients received local ablative treatment of the brain metastases followed by neoadjuvant immunochemotherapy with pemetrexed, cisplatin, and pembrolizumab. Afterwards, resection of the pulmonary lesion with curative intent was performed. One patient showed progressive disease 12 months after initial diagnosis, and passed away 31 months after initial diagnosis. Two of the patients are still alive and maintain a good quality of life with a progression-free survival and overall survival of 28 and 35 months, respectively, illustrating the potential of novel combinatorial treatment approaches.

Activating transcription factor 4 mediates adaptation of human glioblastoma cells to hypoxia and temozolomide.

The integrated stress response (ISR) is a central cellular adaptive program that is activated by diverse stressors including ER stress, hypoxia and nutrient deprivation to orchestrate responses via activating transcription factor 4 (ATF4). We hypothesized that ATF4 is essential for the adaptation of human glioblastoma (GB) cells to the conditions of the tumor microenvironment and is contributing to therapy resistance against chemotherapy. ATF4 induction in GB cells was modulated pharmacologically and genetically and investigated in the context of temozolomide treatment as well as glucose and oxygen deprivation. The relevance of the ISR was analyzed by cell death and metabolic measurements under conditions to approximate aspects of the GB microenvironment. ATF4 protein levels were induced by temozolomide treatment. In line, ATF4 gene suppressed GB cells (ATF4sh) displayed increased cell death and decreased survival after temozolomide treatment. Similar results were observed after treatment with the ISR inhibitor ISRIB. ATF4sh and ISRIB treated GB cells were sensitized to hypoxia-induced cell death. Our experimental study provides evidence for an important role of ATF4 for the adaptation of human GB cells to conditions of the tumor microenvironment characterized by low oxygen and nutrient availability and for the development of temozolomide resistance. Inhibiting the ISR in GB cells could therefore be a promising therapeutic approach.

MRI characteristics in treatment for cerebral melanoma metastasis using stereotactic radiosurgery and concomitant checkpoint inhibitors or targeted therapeutics.

INTRODUCTION: Combination therapy for melanoma brain metastases (MM) using stereotactic radiosurgery (SRS) and immune checkpoint-inhibition (ICI) or targeted therapy (TT) is currently of high interest. In this collective, time evolution and incidence of imaging findings indicative of pseudoprogression is sparsely researched. We therefore investigated time-course of MRI characteristics in these patients. METHODS: Data were obtained retrospectively from 27 patients (12 female, 15 male; mean 61 years, total of 169 MMs). Single lesion volumes, total MM burden and edema volumes were analyzed at baseline and follow-up MRIs in 2 months intervals after SRS up to 24 months. The occurrence of intralesional hemorrhages was recorded. RESULTS: 17 patients (80 MM) received ICI, 8 (62 MM) TT and 2 (27 MM) ICI + TT concomitantly to SRS. MM-localization was frontal (n = 89), temporal (n = 23), parietal (n = 20), occipital (n = 10), basal ganglia/thalamus/insula (n = 10) and cerebellar (n = 10). A volumetric progression of MM 2-4 months after SRS was observed in combined treatment with ICI (p = 0.028) and ICI + TT (p = 0.043), whereas MMs treated with TT showed an early volumetric regression (p = 0.004). Edema volumes moderately correlated with total MM volumes (r = 0.57; p < 0.0001). Volumetric behavior did not differ significantly over time regarding lesions' initial sizes or localizations. No significant differences between groups were observed regarding rates of post-SRS intralesional hemorrhages. CONCLUSION: Reversible volumetric increases in terms of pseudoprogression are observed 2-4 months after SRS in patients with MM concomitantly treated with ICI and ICI + TT, rarely after TT. Edema volumes mirror total MM volumes. Medical treatment type does not significantly affect rates of intralesional hemorrhage.

Immune Checkpoint Inhibitor-Induced Cerebral Pseudoprogression: Patterns and Categorization.

Background: The inclusion of immune checkpoint inhibitors (ICIs) in therapeutic algorithms has led to significant survival benefits in patients with various metastatic cancers. Concurrently, an increasing number of neurological immune related adverse events (IRAE) has been observed. In this retrospective analysis, we examine the ICI-induced incidence of cerebral pseudoprogression and propose a classification system. Methods: We screened our hospital information system to identify patients with any in-house ICI treatment for any tumor disease during the years 2007-2019. All patients with cerebral MR imaging (cMRI) of sufficient diagnostic quality were included. cMRIs were retrospectively analyzed according to immunotherapy response assessment for neuro-oncology (iRANO) criteria. Results: We identified 12 cases of cerebral pseudoprogression in 123 patients treated with ICIs and sufficient MRI. These patients were receiving ICI therapy for lung cancer (n=5), malignant melanoma (n=4), glioblastoma (n=1), hepatocellular carcinoma (n=1) or lymphoma (n=1) when cerebral pseudoprogression was detected. Median time from the start of ICI treatment to pseudoprogression was 5 months. All but one patient developed neurological symptoms. Three different patterns of cerebral pseudoprogression could be distinguished: new or increasing contrast-enhancing lesions, new or increasing T2 predominant lesions and cerebral vasculitis type pattern. Conclusion: Cerebral pseudoprogression followed three distinct patterns and was detectable in 3.2% of all patients during ICI treatment and in 9.75% of the patients with sufficient brain imaging follow up. The fact that all but one of the affected patients developed neurological symptoms, which would be classified as progressive disease according to iRANO criteria, mandates vigilance in the diagnosis and treatment of ICI-induced cerebral lesions.

Increased occurrence of status epilepticus in patients with brain metastases and checkpoint inhibition.

Integration of immune checkpoint inhibitors (ICIs) has improved the efficacy of treatment regimens for various cancers. The array of potential side effects keeps evolving and includes neurological complications. An increased risk of seizures and status epilepticus (SE) has been discussed and appears likely. In this report, we present clinical data from brain metastases patients undergoing ICI treatment revealing, for what we believe is the first time, SE as a serious adverse effect of ICI treatment. In our cohort of 3202 patients with brain metastases, we observed an increasing incidence of SE since the approval of ICIs in 2014 (16 patients in 2008-2013 vs. 36 patients in 2014-2019). Almost half of the patients treated in 2014-2019 received ICIs during the course of their disease, and in more than 80% of cases last dose of ICIs was given less than 30 days before SE. These findings suggest that ICIs may lead to an increased rate of SE in patients with brain metastases. Additional mechanistic research and prospective trials are necessary to elucidate the pathomechanism causing SE in patients treated with ICIs.

Non-Invasive Measurement of Drug and 2-HG Signals Using 19F and 1H MR Spectroscopy in Brain Tumors Treated with the Mutant IDH1 Inhibitor BAY1436032.

BACKGROUND: BAY1436032 is a fluorine-containing inhibitor of the R132X-mutant isocitrate dehydrogenase (mIDH1). It inhibits the mIDH1-mediated production of 2-hydroxyglutarate (2-HG) in glioma cells. We investigated brain penetration of BAY1436032 and its effects using 1H/19F-Magnetic Resonance Spectroscopy (MRS). METHODS: 19F-Nuclear Magnetic Resonance (NMR) Spectroscopy was conducted on serum samples from patients treated with BAY1436032 (NCT02746081 trial) in order to analyze 19F spectroscopic signal patterns and concentration-time dynamics of protein-bound inhibitor to facilitate their identification in vivo MRS experiments. Hereafter, 30 mice were implanted with three glioma cell lines (LNT-229, LNT-229 IDH1-R132H, GL261). Mice bearing the IDH-mutated glioma cells received 5 days of treatment with BAY1436032 between baseline and follow-up 1H/19F-MRS scan. All other animals underwent a single scan after BAY1436032 administration. Mouse brains were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: Evaluation of 1H-MRS data showed a decrease in 2-HG/total creatinine (tCr) ratios from the baseline to post-treatment scans in the mIDH1 murine model. Whole brain concentration of BAY1436032, as determined by 19F-MRS, was similar to total brain tissue concentration determined by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS), with a signal loss due to protein binding. Intratumoral drug concentration, as determined by LC-MS/MS, was not statistically different in models with or without R132X-mutant IDH1 expression. CONCLUSIONS: Non-invasive monitoring of mIDH1 inhibition by BAY1436032 in mIDH1 gliomas is feasible.

Cetuximab-Mediated Protection from Hypoxia- Induced Cell Death: Implications for Therapy Sequence in Colorectal Cancer.

Monoclonal antibodies like cetuximab, targeting the epidermal growth factor receptor (EGFR), and bevacizumab, targeting the vascular endothelial growth factor (VEGF), are an integral part of treatment regimens for metastasized colorectal cancer. However, inhibition of the EGFR has been shown to protect human glioma cells from cell death under hypoxic conditions. In colon carcinoma cells, the consequences of EGFR blockade in hypoxia (e.g., induced by bevacizumab) have not been evaluated yet. LIM1215 and SW948 colon carcinoma and LNT-229 glioblastoma cells were treated with cetuximab, PD153035, and erlotinib and analyzed for cell density and viability. The sequential administration of either cetuximab followed by bevacizumab (CET->BEV) or bevacizumab followed by cetuximab (BEV->CET) was investigated in a LIM1215 (KRAS wildtype) and SW948 (KRAS mutant) xenograft mouse model. In vitro, cetuximab protected from hypoxia. In the LIM1215 model, a survival benefit with cetuximab and bevacizumab monotherapy was observed, but only the sequence CET->BEV showed an additional benefit. This effect was confirmed in the SW948 model. Our observations support the hypothesis that bevacizumab modulates the tumor microenvironment (e.g., by inducing hypoxia) where cetuximab could trigger protective effects when administered later on. The sequence CET->BEV therefore seems to be superior as possible mutual adverse effects are bypassed.

Activation of Epidermal Growth Factor Receptor Sensitizes Glioblastoma Cells to Hypoxia-Induced Cell Death.

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.

A Randomized Phase II Trial (TAMIGA) Evaluating the Efficacy and Safety of Continuous Bevacizumab Through Multiple Lines of Treatment for Recurrent Glioblastoma.

BACKGROUND: We assessed the efficacy and safety of bevacizumab (BEV) through multiple lines in patients with recurrent glioblastoma who had progressed after first-line treatment with radiotherapy, temozolomide, and BEV. PATIENTS AND METHODS: TAMIGA (NCT01860638) was a phase II, randomized, double-blind, placebo-controlled, multicenter trial in adult patients with glioblastoma. Following surgery, patients with newly diagnosed glioblastoma received first-line treatment consisting of radiotherapy plus temozolomide and BEV, followed by six cycles of temozolomide and BEV, then BEV monotherapy until disease progression (PD1). Randomization occurred at PD1 (second line), and patients received lomustine (CCNU) plus BEV (CCNU + BEV) or CCNU plus placebo (CCNU + placebo) until further disease progression (PD2). At PD2 (third line), patients continued BEV or placebo with chemotherapy (investigator's choice). The primary endpoint was survival from randomization. Secondary endpoints were progression-free survival in the second and third lines (PFS2 and PFS3) and safety. RESULTS: Of the 296 patients enrolled, 123 were randomized at PD1 (CCNU + BEV, n = 61; CCNU + placebo, n = 62). The study was terminated prematurely because of the high drop-out rate during first-line treatment, implying underpowered inferential testing. The proportion of patients receiving corticosteroids at randomization was similar (BEV 33%, placebo 31%). For the CCNU + BEV and CCNU + placebo groups, respectively, median survival from randomization was 6.4 versus 5.5 months (stratified hazard ratio [HR], 1.04; 95% confidence interval [CI], 0.69-1.59), median PFS2 was 2.3 versus 1.8 months (stratified HR, 0.70; 95% CI, 0.48-1.00), median PFS3 was 2.0 versus 2.2 months (stratified HR, 0.70; 95% CI, 0.37-1.33), and median time from randomization to a deterioration in health-related quality of life was 1.4 versus 1.3 months (stratified HR, 0.76; 95% CI, 0.52-1.12). The incidence of treatment-related grade 3 to 4 adverse events was 19% (CCNU + BEV) versus 15% (CCNU + placebo). CONCLUSION: There was no survival benefit and no detriment observed with continuing BEV through multiple lines in patients with recurrent glioblastoma. IMPLICATIONS FOR PRACTICE: Previous research suggested that there may be value in continuing bevacizumab (BEV) beyond progression through multiple lines of therapy. No survival benefit was observed with the use of BEV through multiple lines in patients with glioblastoma who had progressed after first-line treatment (radiotherapy + temozolomide + BEV). No new safety concerns arose from the use of BEV through multiple lines of therapy.

Akt and mTORC1 signaling as predictive biomarkers for the EGFR antibody nimotuzumab in glioblastoma.

Glioblastoma (GB) is the most frequent primary brain tumor in adults with a dismal prognosis despite aggressive treatment including surgical resection, radiotherapy and chemotherapy with the alkylating agent temozolomide. Thus far, the successful implementation of the concept of targeted therapy where a drug targets a selective alteration in cancer cells was mainly limited to model diseases with identified genetic drivers. One of the most commonly altered oncogenic drivers of GB and therefore plausible therapeutic target is the epidermal growth factor receptor (EGFR). Trials targeting this signaling cascade, however, have been negative, including the phase III OSAG 101-BSA-05 trial. This highlights the need for further patient selection to identify subgroups of GB with true EGFR-dependency. In this retrospective analysis of treatment-naïve samples of the OSAG 101-BSA-05 trial cohort, we identify the EGFR signaling activity markers phosphorylated PRAS40 and phosphorylated ribosomal protein S6 as predictive markers for treatment efficacy of the EGFR-blocking antibody nimotuzumab in MGMT promoter unmethylated GBs. Considering the total trial population irrespective of MGMT status, a clear trend towards a survival benefit from nimotuzumab was already detectable when tumors had above median levels of phosphorylated ribosomal protein S6. These results could constitute a basis for further investigations of nimotuzumab or other EGFR- and downstream signaling inhibitors in selected patient cohorts using the reported criteria as candidate predictive biomarkers.

Suppression of oxidative phosphorylation confers resistance against bevacizumab in experimental glioma.

Although bevacizumab initially shows high response rates in gliomas and other tumours, therapy resistance usually develops later. Because anti-angiogenic agents are supposed to induce hypoxia, we asked whether rendering glioma cells independent of oxidative phosphorylation modulates their sensitivity against hypoxia and bevacizumab. LNT-229 glioma cells without functional mitochondria (rho0 ) and control (rho+ ) cells were generated. LNT-229 rho0 -cells displayed reduced expression of oxidative phosphorylation-related genes and diminished oxygen consumption. Conversely, glycolysis was up-regulated in these cells, as shown by increased lactate production and stronger expression of glucose transporter-1 and lactate dehydrogenase-A. However, hypoxia-induced cell death in vitro was nearly completely abolished in the LNT-229 rho0 -cells, these cells were more sensitive towards glucose restriction and the treatment with the glycolysis inhibitor 2-deoxy-D-glucose. In an orthotopic mouse xenograft experiment, bevacizumab induced hypoxia as reflected by elevated Hypoxia-inducible factor 1-alpha staining in both, rho+ - and rho0 -tumours. However, it prolonged survival only in the mice bearing rho+ -tumours (74 days vs. 105 days, p = 0.024 log-rank test) and had no effect on survival in mice carrying LNT-229 rho0 -tumours (75 days vs. 70 days, p = 0.52 log-rank test). Interestingly, inhibition of glycolysis in vivo with 2-deoxy-D-glucose re-established sensitivity of rho0 -tumours against bevacizumab (98 days vs. 80 days, p = 0.0001). In summary, ablation of oxidative phosphorylation in glioma cells leads to a more glycolytic and hypoxia-resistant phenotype and is sufficient to induce bevacizumab-refractory tumours. These results add to increasing evidence that a switch towards glycolysis is one mechanism how tumour cells may evade anti-angiogenic treatments and suggest anti-glycolytic strategies as promising approaches to overcome bevacizumab resistance.

Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

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 -ß). 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.

Best practice for analysis of shared clinical trial data.

BACKGROUND: Greater transparency, including sharing of patient-level data for further research, is an increasingly important topic for organisations who sponsor, fund and conduct clinical trials. This is a major paradigm shift with the aim of maximising the value of patient-level data from clinical trials for the benefit of future patients and society. We consider the analysis of shared clinical trial data in three broad categories: (1) reanalysis - further investigation of the efficacy and safety of the randomized intervention, (2) meta-analysis, and (3) supplemental analysis for a research question that is not directly assessing the randomized intervention. DISCUSSION: In order to support appropriate interpretation and limit the risk of misleading findings, analysis of shared clinical trial data should have a pre-specified analysis plan. However, it is not generally possible to limit bias and control multiplicity to the extent that is possible in the original trial design, conduct and analysis, and this should be acknowledged and taken into account when interpreting results. We highlight a number of areas where specific considerations arise in planning, conducting, interpreting and reporting analyses of shared clinical trial data. A key issue is that that these analyses essentially share many of the limitations of any post hoc analyses beyond the original specified analyses. The use of individual patient data in meta-analysis can provide increased precision and reduce bias. Supplemental analyses are subject to many of the same issues that arise in broader epidemiological analyses. Specific discussion topics are addressed within each of these areas. Increased provision of patient-level data from industry and academic-led clinical trials for secondary research can benefit future patients and society. Responsible data sharing, including transparency of the research objectives, analysis plans and of the results will support appropriate interpretation and help to address the risk of misleading results and avoid unfounded health scares.