L-RNA aptamer-based CXCL12 inhibition combined with radiotherapy in newly-diagnosed glioblastoma: dose escalation of the phase I/II GLORIA trial.

The chemokine CXCL12 promotes glioblastoma (GBM) recurrence after radiotherapy (RT) by facilitating vasculogenesis. Here we report outcomes of the dose-escalation part of GLORIA (NCT04121455), a phase I/II trial combining RT and the CXCL12-neutralizing aptamer olaptesed pegol (NOX-A12; 200/400/600 mg per week) in patients with incompletely resected, newly-diagnosed GBM lacking MGMT methylation. The primary endpoint was safety, secondary endpoints included maximum tolerable dose (MTD), recommended phase II dose (RP2D), NOX-A12 plasma levels, topography of recurrence, tumor vascularization, neurologic assessment in neuro-oncology (NANO), quality of life (QOL), median progression-free survival (PFS), 6-months PFS and overall survival (OS). Treatment was safe with no dose-limiting toxicities or treatment-related deaths. The MTD has not been reached and, thus, 600 mg per week of NOX-A12 was established as RP2D for the ongoing expansion part of the trial. With increasing NOX-A12 dose levels, a corresponding increase of NOX-A12 plasma levels was observed. Of ten patients enrolled, nine showed radiographic responses, four reached partial remission. All but one patient (90%) showed at best response reduced perfusion values in terms of relative cerebral blood volume (rCBV). The median PFS was 174 (range 58-260) days, 6-month PFS was 40.0% and the median OS 389 (144-562) days. In a post-hoc exploratory analysis of tumor tissue, higher frequency of CXCL12+ endothelial and glioma cells was significantly associated with longer PFS under NOX-A12. Our data imply safety of NOX-A12 and its efficacy signal warrants further investigation.

The proneural subtype is not associated with survival benefit from bevacizumab in newly diagnosed glioblastoma: a secondary analysis of the GLARIUS trial.

PURPOSE: The AVAglio trial reported a significant survival benefit for first line bevacizumab treatment in patients with IDH wildtype glioblastoma of the proneural gene expression subtype. We here aim to replicate these findings in an independent trial cohort. METHODS: We evaluate the treatment benefit of bevacizumab according to gene expression subtypes of pretreatment tumor samples (n = 123) in the GLARIUS trial (NCT00967330) for MGMT unmethylated glioblastoma patients with Kaplan-Meier analyses, log-rank tests and Cox regression models. RESULTS: Employing the Phillips classifier, bevacizumab conferred a significant PFS advantage in patients with proneural IDH wild-type tumors (10.4 vs. 6.0 months, p = 0.002), but no OS advantage (16.4 vs. 17.4 months, p = 0.6). Multivariable analysis adjusting for prognostic covariates confirmed the absence of a significant OS advantage from bevacizumab (hazard ratio, 1.05, 95% CI, 0.42 to 2.64; p = 0.14). Further, there was no interaction between the proneural subtype and treatment arm (p = 0.15). These results were confirmed in analyses of tumor subgroups according to the Verhaak classifier. CONCLUSION: In contrast to AVAglio, glioblastoma gene expression subgroups were not associated with a differential OS benefit from first-line bevacizumab in the GLARIUS trial.

Intraoperative or postoperative stereotactic radiotherapy for brain metastases: time to systemic treatment onset and other patient-relevant outcomes.

PURPOSE: Intraoperative radiotherapy (IORT) has become a viable treatment option for resectable brain metastases (BMs). As data on local control and radiation necrosis rates are maturing, we focus on meaningful secondary endpoints such as time to next treatment (TTNT), duration of postoperative corticosteroid treatment, and in-hospital time. METHODS: Patients prospectively recruited within an IORT study registry between November 2020 and June 2023 were compared with consecutive patients receiving adjuvant stereotactic radiotherapy (SRT) of the resection cavity within the same time frame. TTNT was defined as the number of days between BM resection and start of the next extracranial oncological therapy (systemic treatment, surgery, or radiotherapy) for each of the groups. RESULTS: Of 95 BM patients screened, IORT was feasible in 84 cases (88%) and ultimately performed in 64 (67%). The control collective consisted of 53 SRT patients. There were no relevant differences in clinical baseline features. Mean TTNT (range) was 36 (9 - 94) days for IORT patients versus 52 (11 - 126) days for SRT patients (p = 0.01). Mean duration of postoperative corticosteroid treatment was similar (8 days; p = 0.83), as was mean postoperative in-hospital time (11 versus 12 days; p = 0.97). Mean total in-hospital time for BM treatment (in- and out-patient days) was 11 days for IORT versus 19 days for SRT patients (p < 0.001). CONCLUSION: IORT for BMs results in faster completion of interdisciplinary treatment when compared to adjuvant SRT, without increasing corticosteroid intake or prolonging in-hospital times. A randomised phase III trial will determine the clinical effects of shorter TTNT.

Undetected pseudoprogressions in the CeTeG/NOA-09 trial: hints from postprogression survival and MRI analyses.

PURPOSE: In the randomized CeTeG/NOA-09 trial, lomustine/temozolomide (CCNU/TMZ) was superior to TMZ therapy regarding overall survival (OS) in MGMT promotor-methylated glioblastoma. Progression-free survival (PFS) and pseudoprogression rates (about 10%) were similar in both arms. Further evaluating this discrepancy, we analyzed patterns of postprogression survival (PPS) and MRI features at first progression according to modified RANO criteria (mRANO). METHODS: We classified the patients of the CeTeG/NOA-09 trial according to long vs. short PPS employing a cut-off of 18 months and compared baseline characteristics and survival times. In patients with available MRIs and confirmed progression, the increase in T1-enhancing, FLAIR hyperintense lesion volume and the change in ADC mean value of contrast-enhancing tumor upon progression were determined. RESULTS: Patients with long PPS in the CCNU/TMZ arm had a particularly short PFS (5.6 months). PFS in this subgroup was shorter than in the long PPS subgroup of the TMZ arm (11.1 months, p = 0.01). At mRANO-defined progression, patients of the CCNU/TMZ long PPS subgroup had a significantly higher increase of mean ADC values (p = 0.015) and a tendency to a stronger volumetric increase in T1-enhancement (p = 0.22) as compared to long PPS patients of the TMZ arm. CONCLUSION: The combination of survival and MRI analyses identified a subgroup of CCNU/TMZ-treated patients with features that sets them apart from other patients in the trial: short first PFS despite long PPS and significant increase in mean ADC values upon mRANO-defined progression. The observed pattern is compatible with the features commonly observed in pseudoprogression suggesting mRANO-undetected pseudoprogressions in the CCNU/TMZ arm of CeTeG/NOA-09.

Outcome assessment of intraoperative radiotherapy for brain metastases: results of a prospective observational study with comparative matched-pair analysis.

PURPOSE: Intraoperative radiation therapy (IORT) is an emerging alternative to adjuvant stereotactic external beam radiation therapy (EBRT) following resection of brain metastases (BM). Advantages of IORT include an instant prevention of tumor regrowth, optimized dose-sparing of adjacent healthy brain tissue and immediate completion of BM treatment, allowing an earlier admission to subsequent systemic treatments. However, prospective outcome data are limited. We sought to assess long-term outcome of IORT in comparison to EBRT. METHODS: A total of 35 consecutive patients, prospectively recruited within a study registry, who received IORT following BM resection at a single neuro-oncological center were evaluated for radiation necrosis (RN) incidence rates, local control rates (LCR), distant brain progression (DBP) and overall survival (OS) as long-term outcome parameters. The 1 year-estimated OS and survival rates were compared in a balanced comparative matched-pair analysis to those of our institutional database, encompassing 388 consecutive patients who underwent adjuvant EBRT after BM resection. RESULTS: The median IORT dose was 30 Gy prescribed to the applicator surface. A 2.9% RN rate was observed. The estimated 1 year-LCR was 97.1% and the 1 year-DBP-free survival 73.5%. Median time to DBP was 6.4 (range 1.7-24) months in the subgroup of patients experiencing intracerebral progression. The median OS was 17.5 (0.5-not reached) months with a 1 year-survival rate of 61.3%, which did not not significantly differ from the comparative cohort (p = 0.55 and p = 0.82, respectively). CONCLUSION: IORT is a safe and effective fast-track approach following BM resection, with comparable long-term outcomes as adjuvant EBRT.

Five-Fraction Stereotactic Radiotherapy for Brain Metastases-A Retrospective Analysis.

PURPOSE: To determine the safety and outcome profile of five-fraction stereotactic radiotherapy (FSRT) for brain metastases (BM), either as a definitive or adjuvant treatment. METHODS: We assessed clinical data of patients receiving five fractions of 7 Gy each (cumulative physical dose of 35 Gy) to BM or surgical cavities. The primary endpoints were toxicity and radiation necrosis (RN) rates. Secondary endpoints were 1-year cumulative local control rate (LCR) and estimated overall survival (OS). RESULTS: A total of 36 eligible patients receiving FSRT to a total of 49 targets were identified and included. The median follow up was 9 (1.1-56.2) months. The median age was 64.5 (34-92) years, the median ECOG score was 1, and the median Diagnostic-Specific Graded Prognostic Assessment (DS-GPA) score was 2. Treatment was well tolerated and there were no grade 3 adverse events or higher. The overall RN rate was 14.3% and the median time to RN was 12.9 (1.8-23.8) months. RN occurrence was associated with immunotherapy, young age (≤45 years), and large PTV. The cumulative 1-year local control rate was 83.1% and the estimated median local progression free-survival was 18.8 months. The estimated median overall survival was 11 (1.1-56.2) months and significantly superior in those patients presenting with RN. CONCLUSIONS: FSRT with 5 × 7 Gy represents a feasible, safe, and efficient fast track approach of intensified FSRT with acceptable LC and comparable RN rates for both the adjuvant and definitive RT settings.

Impact of the SARS-CoV-2 pandemic on the survival of patients with high-grade glioma and best practice recommendations.

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has changed the clinical day-to-day practice. The aim of this study was to evaluate the impact of the pandemic on patients with high-grade glioma (HGG) as well as to derive best practice recommendations. We compared a multi-institutional cohort with HGG (n = 251) from 03/2020 to 05/2020 (n = 119) to a historical cohort from 03/2019 to 05/2019 (n = 132). The endpoints were outcome (progression-free survival (PFS) and overall survival (OS)) as well as patterns of care and time intervals between treatment steps. The median OS for WHO grade 4 gliomas was 12 months in 2019 (95% Confidence Interval 9.7-14.3 months), and not reached in 2020 (p = .026). There were no other significant differences in the Kaplan-Meier estimates for OS and PFS between cohorts of 2019 and 2020, neither did stratification by WHO grade reveal any significant differences for OS, PFS or for patterns of care. The time interval between cranial magnetic resonance imaging (cMRI) and biopsy was significantly longer in 2020 cohort (11 versus 21 days, p = .031). Median follow-up was 10 months (range 0-30 months). Despite necessary disease containment policies, it is crucial to ensure that patients with HGG are treated in line with the recent guidelines and standard of care (SOC) algorithms. Therefore, we strongly suggest pursuing no changes to SOC treatment, a timely diagnosis and treatment with short time intervals between first symptoms, initial diagnosis, and treatment, as well as a guideline-based cMRI follow-up.

Prognostic impact of obesity in newly-diagnosed glioblastoma: a secondary analysis of CeTeG/NOA-09 and GLARIUS.

PURPOSE: The role of obesity in glioblastoma remains unclear, as previous analyses have reported contradicting results. Here, we evaluate the prognostic impact of obesity in two trial populations; CeTeG/NOA-09 (n = 129) for MGMT methylated glioblastoma patients comparing temozolomide (TMZ) to lomustine/TMZ, and GLARIUS (n = 170) for MGMT unmethylated glioblastoma patients comparing TMZ to bevacizumab/irinotecan, both in addition to surgery and radiotherapy. METHODS: The impact of obesity (BMI ≥ 30 kg/m2) on overall survival (OS) and progression-free survival (PFS) was investigated with Kaplan-Meier analysis and log-rank tests. A multivariable Cox regression analysis was performed including known prognostic factors as covariables. RESULTS: Overall, 22.6% of patients (67 of 297) were obese. Obesity was associated with shorter survival in patients with MGMT methylated glioblastoma (median OS 22.9 (95% CI 17.7-30.8) vs. 43.2 (32.5-54.4) months for obese and non-obese patients respectively, p = 0.001), but not in MGMT unmethylated glioblastoma (median OS 17.1 (15.8-18.9) vs 17.6 (14.7-20.8) months, p = 0.26). The prognostic impact of obesity in MGMT methylated glioblastoma was confirmed in a multivariable Cox regression (adjusted odds ratio: 2.57 (95% CI 1.53-4.31), p < 0.001) adjusted for age, sex, extent of resection, baseline steroids, Karnofsky performance score, and treatment arm. CONCLUSION: Obesity was associated with shorter survival in MGMT methylated, but not in MGMT unmethylated glioblastoma patients.

Phase I/II trial of meclofenamate in progressive MGMT-methylated glioblastoma under temozolomide second-line therapy-the MecMeth/NOA-24 trial.

BACKGROUND: Glioblastoma is the most frequent and malignant primary brain tumor. Even in the subgroup with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and favorable response to first-line therapy, survival after relapse is short (12 months). Standard therapy for recurrent MGMT-methylated glioblastoma is not standardized and may consist of re-resection, re-irradiation, and chemotherapy with temozolomide (TMZ), lomustine (CCNU), or a combination thereof. Preclinical results show that meclofenamate (MFA), originally developed as a nonsteroidal anti-inflammatory drug (NSAID) and registered in the USA, sensitizes glioblastoma cells to temozolomide-induced toxicity via inhibition of gap junction-mediated intercellular cytosolic traffic and demolishment of tumor microtube (TM)-based network morphology. METHODS: In this study, combined MFA/TMZ therapy will be administered (orally) in patients with first relapse of MGMT-methylated glioblastoma. A phase I component (6-12 patients, 2 dose levels of MFA + standard dose TMZ) evaluates safety and feasibility and determines the dose for the randomized phase II component (2 × 30 patients) with progression-free survival as the primary endpoint. DISCUSSION: This study is set up to assess toxicity and first indications of efficacy of MFA repurposed in the setting of a very difficult-to-treat recurrent tumor. The trial is a logical next step after the identification of the role of resistance-providing TMs in glioblastoma, and results will be crucial for further trials targeting TMs. In case of favorable results, MFA may constitute the first clinically feasible TM-targeted drug and therefore might bridge the idea of a TM-targeted therapeutic approach from basic insights into clinical reality. TRIAL REGISTRATION: EudraCT 2021-000708-39 . Registered on 08 February 2021.

Erythropoietin Abrogates Post-Ischemic Activation of the NLRP3, NLRC4, and AIM2 Inflammasomes in Microglia/Macrophages in a TAK1-Dependent Manner.

Inflammasomes are known to contribute to brain damage after acute ischemic stroke (AIS). TAK1 is predominantly expressed in microglial cells and can regulate the NLRP3 inflammasome, but its impact on other inflammasomes including NLRC4 and AIM2 after AIS remains elusive. EPO has been shown to reduce NLRP3 protein levels in different disease models. Whether EPO-mediated neuroprotection after AIS is conveyed via an EPO/TAK1/inflammasome axis in microglia remains to be clarified. Subjecting mice deficient for TAK1 in microglia/macrophages (Mi/MΦ) to AIS revealed a significant reduction in infarct sizes and neurological impairments compared to the corresponding controls. Post-ischemic increased activation of TAK1, NLRP3, NLRC4, and AIM2 inflammasomes including their associated downstream cascades were markedly reduced upon deletion of Mi/MΦ TAK1. EPO administration improved clinical outcomes and dampened stroke-induced activation of TAK1 and inflammasome cascades, which was not evident after the deletion of Mi/MΦ TAK1. Pharmacological inhibition of NLRP3 in microglial BV-2 cells did not influence post-OGD IL-1ß levels, but increased NLRC4 and AIM2 protein levels, suggesting compensatory activities among inflammasomes. Overall, we provide evidence that Mi/MΦ TAK1 regulates the expression and activation of the NLRP3, NLRC4, AIM2 inflammasomes. Furthermore, EPO mitigated stroke-induced activation of TAK1 and inflammasomes, indicating that EPO conveyed neuroprotection might be mediated via an EPO/TAK1/inflammasome axis.

Microglial-specific depletion of TAK1 is neuroprotective in the acute phase after ischemic stroke.

Transforming growth factor-ß-activated kinase 1 (TAK1) is upregulated after cerebral ischemia and contributes to an aggravation of brain injury. TAK1 acts as a key regulator of NF-ΚB and the MAP kinases JNK and p38 and modulates post-ischemic neuroinflammation and apoptosis. Microglia are the main TAK1-expressing immunocompetent cells of the brain. However, little is known about the function and regulation of microglial TAK1 after cerebral ischemia. Tamoxifen-dependent conditional depletion of TAK1 in microglial cells was induced in Cx3cr1creER-Tak1fl/fl mice. The creER-negative Tak1fl/fl mice and vehicle-treated (corn oil) mice served as control groups. A transient intraluminal middle cerebral artery occlusion of 30 min followed by 6 h and 72 h of reperfusion was performed in male mice. Oxygen-glucose-deprivation (OGD) was performed with primary cortical glial cell cultures to examine the effect of microglial-specific and general (5Z-7-Oxozeaenol) TAK1 inhibition after different reperfusion times (1 h, 6 h, and 72 h). Cx3cr1creER-Tak1fl/fl mice showed reduced infarct sizes and improved neurological outcomes compared to the control group. The mRNA and protein levels of pro-inflammatory Il1b/IL-1ß and Tnf/TNF-α in the peri-infarct zones of microglial-specific TAK1-depleted mice were significantly reduced. Furthermore, TAK1 depletion in vitro led to reduced cell death rates after OGD. Moreover, hypoxia-mediated activation of TAK1 and its downstream signalling proteins, JNK and p38, were dampened by microglial TAK1 depletion. In contrast, 5Z-7-Oxozeaenol-induced pharmacological inhibition of TAK1 completely diminished MAPK-signalling including the kinases JNK and p38 in all cells. Microglial TAK1 depletion abrogates post-ischemic neuroinflammation and apoptosis in the acute phase, hence might be considered as a potential target in the treatment of cerebral hypoxia. KEY MESSAGES: TAK1 is activated after cerebral ischemia and induces MAP kinases p38 and JNK. Activated TAK1 increases apoptosis rate and the level pro-inflammatory cytokines IL-1ß and TNF-α. Microglial cells seem to be the main source of TAK1-mediated post-ischemic neuroinflammation. Microglial-specific TAK1-depletion mediates sustainable neuroprotective effects, which might be superior to global TAK1 inhibition.

EPO regulates neuroprotective Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members GRINA and FAIM2 after cerebral ischemia-reperfusion injury.

BACKGROUND AND PURPOSE: Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members exert inhibitory activities in apoptosis and necroptosis. FAIM2 (TMBIM-2) is neuroprotective against murine focal ischemia and is regulated by erythropoietin (EPO). Similar to FAIM2, GRINA (TMBIM-3) is predominantly expressed in the brain. The role of GRINA in transient brain ischemia, its potential synergistic effects with FAIM2 and its regulation by EPO treatment were assessed. METHODS: We performed transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 72 h of reperfusion in GRINA-deficient (GRINA-/-), FAIM2-deficient (FAIM2-/-), double-deficient (GRINA-/-FAIM2-/-) and wildtype littermates (WT) mice. We administered EPO or saline 0, 24 and 48 h after tMCAo. We subjected primary murine cortical neurons (pMCN) of all mouse strains to oxygen-glucose deprivation (OGD) after GRINA and/or FAIM2 gene transfection. RESULTS: Compared to wildtype controls GRINA deficiency led to a similar increase in infarct volumes as FAIM2 deficiency (p < .01). We observed the highest neurological deficits and largest infarct sizes in double-deficient mice. EPO administration upregulated GRINA and FAIM2 mRNA levels in wildtype littermates. EPO decreased infarct sizes and abrogated neurological impairments in wildtype controls. GRINA and/or FAIM2 deficient mice showed increased expression levels of cleaved-caspase 3 and of pro-apoptotic BAX mRNA. Further, caspase 8 was upregulated in FAIM2-/- and caspase 9 in GRINA-/- mice. Overexpression of GRINA and FAIM2 in wildtype and in double deficient pMCN decreased cell death rate after OGD. CONCLUSIONS: GRINA and FAIM2 are highly expressed in the brain and convey EPO-mediated neuroprotection after ischemic stroke involving different caspases.

Fingolimod (FTY720) is not protective in the subacute MPTP mouse model of Parkinson's disease and does not lead to a sustainable increase of brain-derived neurotrophic factor.

Parkinson's disease (PD) is characterized by the loss of midbrain dopaminergic neurons and aggregates of α-synuclein termed Lewy bodies. Fingolimod (FTY720) is an agonist of sphingosine-1 phosphate receptors and an approved oral treatment for multiple sclerosis. Fingolimod elevates brain-derived neurotrophic factor (BDNF), an important neurotrophic factor for dopaminergic neurons. BDNF and fingolimod are beneficial in several animal models of PD. In order to validate the therapeutic potential of fingolimod for the treatment of PD, we tested its effect in the subacute MPTP mouse model of PD. MPTP or vehicle was applied i.p. in doses of 30 mg/kg MPTP on five consecutive days. In order to recapitulate the combination of dopamine loss and α-synuclein aggregates found in PD, MPTP was first administered in Thy1-A30P-α-synuclein transgenic mice. Fingolimod was administered i.p. at a dose of 0.1 mg/kg every second day. Nigrostriatal degeneration was assayed by stereologically counting the number of dopaminergic neurons in the substantia nigra pars compacta, by analysing the concentration of catecholamines and the density of dopaminergic fibres in the striatum. MPTP administration produced a robust nigrostriatal degeneration, comparable to previous studies. Unexpectedly, we found no difference between mice with and without fingolimod treatment, neither at baseline, nor at 14 or 90 days after MPTP. Also, we found no effect of fingolimod in the subacute MPTP mouse model when we used wildtype mice instead of α-synuclein transgenic mice, and no effect with an increased dose of 1 mg/kg fingolimod administered every day. In order to explain these findings, we analysed BDNF regulation by fingolimod. We did find an increase of BDNF protein after a single injection of fingolimod 0.1 or 1.0 mg/kg, but not after multiple injections, indicating that the BDNF response to fingolimod is unsustainable over time. Taken together we did not observe a neuroprotective effect of fingolimod in the subacute MPTP mouse model of PD. We discuss possible explanations for this discrepancy with previous findings and conclude fingolimod might be beneficial for the nonmotor symptoms of PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* and *Open Data* because it provided all relevant information to reproduce the study in the manuscript and because it made the data publicly available. The data can be accessed at https://osf.io/6xgfn/. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.