Transcriptome analysis reveals tumor microenvironment changes in glioblastoma.

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n = 322 test, n = 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells.

Glioma patient-reported outcomes: patients and clinicians.

INTRODUCTION: Routine assessment of patient-reported outcomes (PROs) in oncology has shown to improve the quality of the delivered care and to prolong survival. However, for successful implementation of routine assessment of PROs, more knowledge on their usability in clinical practice is needed. OBJECTIVE: This study aimed to cross-sectionally assess the perspective of patients and clinicians on the practicality of routinely measuring PROs in clinical practice for glioma patients. METHODS: Semistructured interviews were conducted evaluating the role of healthcare professionals (HCP) in discussing results of PRO measures (PROMs), and the preferred topics, methods and frequency of PRO assessment. Glioma patients, their proxies and HCPs involved in the treatment of glioma patients from eight centres in the Netherlands were included. RESULTS: Twenty-four patients, 16 proxies and 35 HCPs were interviewed. The majority of patients, proxies and HCPs (92%, 81% and 80%, respectively) were willing to discuss PRO results during consultations. Although HCPs prefer that results are discussed with the nurse specialist, only one-third of patients/proxies agreed. Functioning of daily life was considered important in all three groups. Most participants indicated that discussion of PROM results should take place during standard follow-up visits, and completed at home about 1 week in advance. On group level, there was no preference for administration of questionnaires on paper or digitally. Lastly, all centres had staff available to send questionnaires on paper. CONCLUSION: This study shows that routine assessment of PROs is desired by patients, proxies and HCP's in neuro-oncological care in Dutch hospitals.

The impact of different volumetric thresholds to determine progressive disease in patients with recurrent glioblastoma treated with bevacizumab.

Background: The optimal volumetric threshold for determining progressive disease (PD) in recurrent glioblastoma is yet to be determined. We investigated a range of thresholds in association with overall survival (OS). Methods: First recurrent glioblastoma patients treated with bevacizumab and/or lomustine were included from the phase II BELOB and phase III EORTC26101 trials. Enhancing and nonenhancing tumor volumes were measured at baseline, first (6 weeks), and second (12 weeks) follow-up. Hazard ratios (HRs) for the appearance of new lesions and several thresholds for tumor volume increase were calculated using cox regression analysis. Results were corrected in a multivariate analysis for well-established prognostic factors. Results: At first and second follow-up, 138 and 94 patients respectively, were deemed eligible for analysis of enhancing volumes, while 89 patients were included in the analysis of nonenhancing volumes at first follow-up. New lesions were associated with a significantly worse OS (3.2 versus 11.2 months, HR = 7.03, P < .001). At first follow-up a threshold of enhancing volume increase of ≥20% provided the highest HR (5.55, p = .001. At second follow-up, any increase in enhancing volume (≥0%) provided the highest HR (9.00, p < .001). When measuring nonenhancing volume at first follow-up, only 6 additional patients were scored as PD with the highest HR of ≥25% increase in volume (HR=3.25, p = .008). Conclusion: Early appearing new lesions were associated with poor OS. Lowering the volumetric threshold for PD at both first and second follow-up improved survival prediction. However, the additional number of patients categorized as PD by lowering the threshold was very low. The per-RANO added change in nonenhancing volumes to the analyses was of limited value.

The EGFRvIII transcriptome in glioblastoma: A meta-omics analysis.

BACKGROUND: EGFR is among the genes most frequently altered in glioblastoma, with exons 2-7 deletions (EGFRvIII) being among its most common genomic mutations. There are conflicting reports about its prognostic role and it remains unclear whether and how it differs in signaling compared with wildtype EGFR. METHODS: To better understand the oncogenic role of EGFRvIII, we leveraged 4 large datasets into 1 large glioblastoma transcriptome dataset (n = 741) alongside 81 whole-genome samples from 2 datasets. RESULTS: The EGFRvIII/EGFR expression ratios differ strongly between tumors and range from 1% to 95%. Interestingly, the slope of relative EGFRvIII expression is near-linear, which argues against a more positive selection pressure than EGFR wildtype. An absence of selection pressure is also suggested by the similar survival between EGFRvIII-positive and -negative glioblastoma patients. EGFRvIII levels are inversely correlated with pan-EGFR (all wildtype and mutant variants) expression, which indicates that EGFRvIII has a higher potency in downstream pathway activation. EGFRvIII-positive glioblastomas have a lower CDK4 or MDM2 amplification incidence than EGFRvIII-negative (P = .007), which may point toward crosstalk between these pathways. EGFRvIII-expressing tumors have an upregulation of "classical" subtype genes compared to those with EGFR-amplification only (P = 3.873e-6). Genomic breakpoints of the EGFRvIII deletions have a preference toward the 3'-end of the large intron-1. These preferred breakpoints preserve a cryptic exon resulting in a novel EGFRvIII variant and preserve an intronic enhancer. CONCLUSIONS: These data provide deeper insights into the complex EGFRvIII biology and provide new insights for targeting EGFRvIII mutated tumors.

Molecular Evolution of IDH Wild-Type Glioblastomas Treated With Standard of Care Affects Survival and Design of Precision Medicine Trials: A Report From the EORTC 1542 Study.

PURPOSE: Precision medicine trials in glioblastoma (GBM) are often conducted at tumor recurrence. However, second surgeries for recurrent GBM are not routinely performed, and therefore, molecular data for trial inclusion are predominantly derived from the primary sample. This study aims to establish whether molecular targets change during tumor progression and, if so, whether this affects precision medicine trial design. MATERIALS AND METHODS: We collected 186 pairs of primary-recurrent GBM samples from patients receiving chemoradiotherapy with temozolomide and sequenced approximately 300 cancer genes. MGMT, TERT, and EGFRvIII status was individually determined. RESULTS: The molecular profile of our cohort was identical to that of other GBM cohorts (IDH wild-type [WT], 95%; EGFR amplified, approximately 50%), indicating that patients amenable to second surgery do not represent a specific molecular subtype. Molecular events in IDH WT GBMs were stable in approximately 80% of events, but changes in mutation status were observed for all examined genes (range, approximately 90% and 60% for TERT and EGFR mutations, respectively), and such changes strongly affected targeted trial size and design. A similar pattern of GBM driver instability was observed within MGMT promoter-methylated tumors. MGMT promoter methylation status remained prognostic at tumor recurrence. The observation that hypermutation at GBM recurrence was rare (8%) and not correlated with outcome was relevant for immunotherapy-based treatments. CONCLUSION: This large cohort of matched primary and recurrent IDH WT tumors establishes the frequency of GBM driver instability after chemoradiotherapy with temozolomide. This allows per gene or pathway calculation of trial size at tumor recurrence, using molecular data of the primary tumor only. We also identify genes for which repeat surgery is necessary because of low mutation retention rate.

A pilot randomized controlled trial of exercise to improve cognitive performance in patients with stable glioma: a proof of concept.

BACKGROUND: Patients with glioma often suffer from cognitive deficits. Physical exercise has been effective in ameliorating cognitive deficits in older adults and neurological patients. This pilot randomized controlled trial (RCT) explored the possible impact of an exercise intervention, designed to improve cognitive functioning in glioma patients, regarding cognitive test performance and patient-reported outcomes (PROs). METHODS: Thirty-four clinically stable patients with World Health Organization grades II/III glioma were randomized to a home-based remotely coached exercise group or an active control group. Patients exercised 3 times per week for 20-45 minutes, with moderate to vigorous intensity, during 6 months. At baseline and immediate follow-up, cognitive performance and PROs were assessed with neuropsychological tests and questionnaires, respectively. Linear regression analyses were used to estimate effect sizes of potential between-group differences in cognitive performance and PROs at 6 months. RESULTS: The exercise group (n = 21) had small- to medium-sized better follow-up scores than the control group (n = 11) on several measures of attention and information processing speed, verbal memory, and executive function, whereas the control group showed a slightly better score on a measure of sustained selective attention. The exercise group also demonstrated small- to medium-sized better outcomes on measures of self-reported cognitive symptoms, fatigue, sleep, mood, and mental health-related quality of life. CONCLUSIONS: This small exploratory RCT in glioma patients provides a proof of concept with respect to improvement of cognitive functioning and PROs after aerobic exercise, and warrants larger exercise trials in brain tumor patients.

Predicting disease progression in high-grade glioma with neuropsychological parameters: the value of personalized longitudinal assessment.

PURPOSE: Progressive disease in patients with high-grade glioma may be reflected in cognitive decline. However, the cognitive functions most sensitive to progression may differ between patients. We investigated whether decline on a personalized selection of tests predicted progressive disease according to RANO criteria in high-grade glioma patients. METHODS: Starting one day before surgery, patients underwent neuropsychological assessment every three months during standard treatment and clinical follow-up. We first made a personalized selection of three tests that showed the highest Reliable Change Index (RCI) values, i.e., most positive change, at the first post-surgical assessment for each patient. In subsequent follow up, a decline of RCI ≤ - 1 on at least two of the three tests in the selection was considered cognitive decline. We performed a discrete Cox proportional hazards model including a time-dependent coefficient cognitive decline (vs. stability) and covariate age to predict progressive disease. RESULTS: Twenty five patients were included. Cognitive decline on the personalized test selection preceded or had occurred by the time progression was established in 9/15 patients with RANO confirmed progressive disease (60%). Decline was absent in 8/10 patients (80%) with stable disease during participation. The independent hazard ratio for progression in case of cognitive decline was 5.05 (p < 0.01) compared to stable performance. CONCLUSIONS: Using only three patient-specific neuropsychological tests, we found a fivefold increased chance of disease progression in case of cognitive decline as compared to stable performance. Brief, patient-tailored cognitive assessment may be a noninvasive addition to disease monitoring without overburdening patients and clinical care.

Comparison of 2D (RANO) and volumetric methods for assessment of recurrent glioblastoma treated with bevacizumab-a report from the BELOB trial.

Background: The current method for assessing progressive disease (PD) in glioblastoma is according to the Response Assessment in Neuro-Oncology (RANO) criteria. Bevacizumab-treated patients may show pseudo-response on postcontrast T1-weighted (T1w) MRI, and a more infiltrative non-enhancing growth pattern on T2w/fluid attenuated inversion recovery (FLAIR) images. We investigated whether the RANO criteria remain the method of choice for assessing bevacizumab-treated recurrent glioblastoma when compared with various volumetric methods. Methods: Patients with assessable MRI data from the BELOB trial (n = 148) were included. Patients were treated with bevacizumab, lomustine, or both. At first and second radiological follow-up (6 and 12 wk), PD was determined using the 2D RANO criteria and various volumetric methods based on enhancing tumor only and enhancing plus non-enhancing tumor. Differences in overall survival (OS) between PD and non-PD patients were assessed with the log-rank test and a Cox model. Hazard ratios (HRs) and their 95% CIs were determined. Results: For all patients together, all methods (except subtraction of non-enhancing from enhancing volume at first follow-up) showed significant differences in OS between PD and non-PD patients (P < .001). The largest risk increase for death in case of PD at both first and second follow-up was found with the RANO criteria: HR = 2.81 (95% CI, 1.92-4.10) and HR = 2.80 (95% CI, 1.75-4.49), respectively. In the bevacizumab-treated patients, all methods assessed showed significant differences in OS between PD and non-PD patients. There were no significant differences between methods. Conclusions: In the first 12 weeks, volumetric methods did not provide significant improvement over the RANO criteria as a posttreatment prognostic marker.

Identification of Patients with Recurrent Glioblastoma Who May Benefit from Combined Bevacizumab and CCNU Therapy: A Report from the BELOB Trial.

The results from the randomized phase II BELOB trial provided evidence for a potential benefit of bevacizumab (beva), a humanized monoclonal antibody against circulating VEGF-A, when added to CCNU chemotherapy in patients with recurrent glioblastoma (GBM). In this study, we performed gene expression profiling (DASL and RNA-seq) of formalin-fixed, paraffin-embedded tumor material from participants of the BELOB trial to identify patients with recurrent GBM who benefitted most from beva+CCNU treatment. We demonstrate that tumors assigned to the IGS-18 or "classical" subtype and treated with beva+CCNU showed a significant benefit in progression-free survival and a trend toward benefit in overall survival, whereas other subtypes did not exhibit such benefit. In particular, expression of FMO4 and OSBPL3 was associated with treatment response. Importantly, the improved outcome in the beva+CCNU treatment arm was not explained by an uneven distribution of prognostically favorable subtypes as all molecular glioma subtypes were evenly distributed along the different study arms. The RNA-seq analysis also highlighted genetic alterations, including mutations, gene fusions, and copy number changes, within this well-defined cohort of tumors that may serve as useful predictive or prognostic biomarkers of patient outcome. Further validation of the identified molecular markers may enable the future stratification of recurrent GBM patients into appropriate treatment regimens.

The impact of bevacizumab on health-related quality of life in patients treated for recurrent glioblastoma: results of the randomised controlled phase 2 BELOB trial.

BACKGROUND: The BELOB study, a randomised controlled phase 2 trial comparing lomustine, bevacizumab and combined lomustine and bevacizumab in patients with recurrent glioblastoma, showed that the 9-month overall survival rate was most promising in the combination arm. Here we report the health-related quality of life (HRQoL) results, a secondary trial end-point. METHODS: HRQoL was measured at baseline and every 6weeks until progression using the European Organisation for Research and Treatment of Cancer (EORTC) core questionnaire (QLQ-C30) and brain module (QLQ-BN20). HRQoL was assessed over time for five preselected scales (global health (GH), physical (PF) and social functioning (SF), motor dysfunction (MD) and communication deficit (CD)). Moreover, mean changes in HRQoL from baseline until progression were determined. RESULTS: 138/148 patients with at least a baseline HRQoL assessment were analysed. Over time, HRQoL remained relatively stable in all treatment arms for all five scales, at least during the first three treatment cycles. More than half (54-61%) of the patients showed stable (<10 point change) or improved (⩾10 point change) HRQoL during their progression-free time, except for SF (43%), irrespective of treatment arm. Deterioration of mean HRQoL was most profound at disease progression for all scales except SF, which deteriorated earlier in disease course. Compared to baseline, 40% of patients had clinically relevant (⩾10 points) worse GH, PF and SF, while 44% and 31% had increased MD and CD at disease progression, irrespective of treatment arm. CONCLUSIONS: Bevacizumab, whether or not in combination with lomustine, did not negatively affect HRQoL in patients treated for recurrent glioblastoma in this randomised study.

Single-agent bevacizumab or lomustine versus a combination of bevacizumab plus lomustine in patients with recurrent glioblastoma (BELOB trial): a randomised controlled phase 2 trial.

BACKGROUND: Treatment options for recurrent glioblastoma are scarce, with second-line chemotherapy showing only modest activity against the tumour. Despite the absence of well controlled trials, bevacizumab is widely used in the treatment of recurrent glioblastoma. Nonetheless, whether the high response rates reported after treatment with this drug translate into an overall survival benefit remains unclear. We report the results of the first randomised controlled phase 2 trial of bevacizumab in recurrent glioblastoma. METHODS: The BELOB trial was an open-label, three-group, multicentre phase 2 study undertaken in 14 hospitals in the Netherlands. Adult patients (≥18 years of age) with a first recurrence of a glioblastoma after temozolomide chemoradiotherapy were randomly allocated by a web-based program to treatment with oral lomustine 110 mg/m(2) once every 6 weeks, intravenous bevacizumab 10 mg/kg once every 2 weeks, or combination treatment with lomustine 110 mg/m(2) every 6 weeks and bevacizumab 10 mg/kg every 2 weeks. Randomisation of patients was stratified with a minimisation procedure, in which the stratification factors were centre, Eastern Cooperative Oncology Group performance status, and age. The primary outcome was overall survival at 9 months, analysed by intention to treat. A safety analysis was planned after the first ten patients completed two cycles of 6 weeks in the combination treatment group. This trial is registered with the Nederlands Trial Register (www.trialregister.nl, number NTR1929). FINDINGS: Between Dec 11, 2009, and Nov 10, 2011, 153 patients were enrolled. The preplanned safety analysis was done after eight patients had been treated, because of haematological adverse events (three patients had grade 3 thrombocytopenia and two had grade 4 thrombocytopenia) which reduced bevacizumab dose intensity; the lomustine dose in the combination treatment group was thereafter reduced to 90 mg/m(2). Thus, in addition to the eight patients who were randomly assigned to receive bevacizumab plus lomustine 110 mg/m(2), 51 patients were assigned to receive bevacizumab alone, 47 to receive lomustine alone, and 47 to receive bevacizumab plus lomustine 90 mg/m(2). Of these patients, 50 in the bevacizumab alone group, 46 in the lomustine alone group, and 44 in the bevacizumab and lomustine 90 mg/m(2) group were eligible for analyses. 9-month overall survival was 43% (95% CI 29-57) in the lomustine group, 38% (25-51) in the bevacizumab group, 59% (43-72) in the bevacizumab and lomustine 90 mg/m(2) group, 87% (39-98) in the bevacizumab and lomustine 110 mg/m(2) group, and 63% (49-75) for the combined bevacizumab and lomustine groups. After the reduction in lomustine dose in the combination group, the combined treatment was well tolerated. The most frequent grade 3 or worse toxicities were hypertension (13 [26%] of 50 patients in the bevacizumab group, three [7%] of 46 in the lomustine group, and 11 [25%] of 44 in the bevacizumab and lomustine 90 mg/m(2) group), fatigue (two [4%], four [9%], and eight [18%]), and infections (three [6%], two [4%], and five [11%]). At the time of this analysis, 144/148 (97%) of patients had died and three (2%) were still on treatment. INTERPRETATION: The combination of bevacizumab and lomustine met prespecified criteria for assessment of this treatment in further phase 3 studies. However, the results in the bevacizumab alone group do not justify further studies of this treatment. FUNDING: Roche Nederland and KWF Kankerbestrijding.