MGMT Promoter Methylation Cutoff with Safety Margin for Selecting Glioblastoma Patients into Trials Omitting Temozolomide: A Pooled Analysis of Four Clinical Trials.

The methylation status of the O <sup>6</sup> -methylguanine DNA methyltransferase (MGMT) gene promoter is predictive for benefit from temozolomide in glioblastoma (GBM). A clinically optimized cutoff was sought allowing patient selection for therapy without temozolomide, while avoiding to withhold it from patients who may potentially benefit.Experimental Design: Quantitative MGMT methylation-specific PCR data were obtained for newly diagnosed patients with GBM screened or treated with standard radiotherapy and temozolomide in four randomized trials. The pooled dataset was randomly split into a training and test dataset. The unsupervised cutoff was obtained at a 50% probability to be (un)methylated. ROC analysis identified an optimal cutoff supervised by overall survival (OS). For 4,041 patients valid MGMT results were obtained, whereof 1,725 were randomized. The unsupervised cutoff in the training dataset was 1.27 (log <sub>2</sub> [1,000 × (MGMT+1)/ACTB]), separating unmethylated and methylated patients. The optimal supervised cutoff for unmethylated patients was -0.28 (AUC = 0.61), classifying "truly unmethylated" (≤-0.28) and "gray zone" patients (>-0.28, ≤1.27), the latter comprising approximately 10% of cases. In contrast, for patients with MGMT methylation (>1.27) more methylation was not related to better outcome. Both methylated and gray zone patients performed significantly better for OS than truly unmethylated patients [HR = 0.35, 95% confidence interval (CI), 0.27-0.45, P < 0.0001; HR = 0.58, 95% CI, 0.43-0.78, P < 0.001], validated in the test dataset. The MGMT assay was highly reproducible upon retesting of 218 paired samples (R <sup>2</sup> = 0.94). Low MGMT methylation (gray zone) may confer some sensitivity to temozolomide treatment, hence the lower safety margin should be considered for selecting patients with unmethylated GBM into trials omitting temozolomide

Bevacizumab for the Treatment of Recurrent Glioblastoma

Despite advances in upfront therapy, the prognosis in the great majority of patients with glioblastoma (GBM) is poor as almost all recur and result in disease-related death. Glioblastoma are highly vascularized cancers with elevated expression levels of vascular endothelial growth factor (VEGF), the dominant mediator of angiogenesis. A compelling biologic rationale, a need for improved therapy, and positive results from studies of bevacizumab in other cancers led to the evaluation of bevacizumab in the treatment of recurrent GBM. Bevacizumab, a humanized monoclonal antibody that targets VEGF, has been shown to improve patient outcomes in combination with chemotherapy (most commonly irinotecan) in recurrent GBM, and on the basis of positive results in two prospective phase 2 studies, bevacizumab was granted accelerated approval by the US Food and Drug Administration (FDA) as a single agent in recurrent GBM. Bevacizumab therapy is associated with manageable, class-specific toxicity as severe treatment-related adverse events are observed in only a minority of patients. With the goal of addressing questions and controversies regarding the optimal use of bevacizumab, the objective of this review is to provide a summary of the clinical efficacy and safety data of bevacizumab in patients with recurrent GBM, the practical issues surrounding the administration of bevacizumab, and ongoing investigations of bevacizumab in managing GBM

Prognostic significance of DNA methylation profiles at MRI enhancing tumor recurrence: a report from the EORTC 26091 TAVAREC Trial

Purpose:Despite recent advances in the molecular characterization of gliomas, it remains unclear which patients benefit most from which second-line treatments. The TAVAREC trial was a randomized, open-label phase II trial assessing the benefit of the addition of the angiogenesis inhibitor bevacizumab to treatment with temozolomide in patients with a first enhancing recurrence of World Health Organization grade 2 or 3 glioma without 1p/19q codeletion. We evaluated the prognostic significance of genome-wide DNA methylation profiles and copy-number variations on the TAVAREC trial samples.Experimental Design:Isocitrate dehydrogenase (IDH) mutation status was determined via Sanger sequencing and IHC. DNA methylation analysis was performed using the MethylationEPIC BeadChip (Illumina) from which 1p/19q codeletion, MGMT promoter methylation (MGMT-STP27), and homozygous deletion of CDKN2A/B were determined. DNA methylation classes were determined according to classifiers developed in Heidelberg and The Cancer Genome Atlas (TCGA; “Heidelberg” and “TCGA” classifier respectively).Results:DNA methylation profiles of 122 samples were successfully determined. As expected, most samples were IDH-mutant (89/122) and MGMT promotor methylated (89/122). Methylation classes were prognostic for time to progression. However, Heidelberg methylation classes determined at time of diagnosis were no longer prognostic following enhancing recurrence of the tumor. In contrast, TCGA methylation classes of primary samples remained prognostic also following enhancing recurrence. Homozygous deletions in CDKN2A/B were found in 10 of 87 IDH-mutated samples and were prognostically unfavorable at recurrence.Conclusions:DNA methylome Heidelberg classification at time of diagnosis is no longer of prognostic value at the time of enhancing recurrence. CDKN2A/B deletion status was predictive of survival from progression of IDH-mutated tumors.Neurolog

Non-IDH1-R132H IDH1/2 mutations are associated with increased DNA methylation and improved survival in astrocytomas, compared to IDH1-R132H mutations

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1(R132H) mutations. Patients harbouring IDH1(R132H) mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations ("non-R132H IDH1/2 mutations"). This reduced methylation is seen in multiple tumour types and thus appears independent of the site of origin. For 1p/19q non-codeleted glioma (astrocytoma) patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring tumours with IDH mutations other than IDH1(R132H) have a better outcome (hazard ratio 0.41, 95% CI [0.24, 0.71], p = 0.0013). Such non-R132H IDH1/2-mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p < 0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H IDH1/2-mutated astrocytomas have a more favourable prognosis than their IDH1(R132H) mutated counterpart indicates that not all IDH-mutations are identical. This difference is clinically relevant and should be taken into account for patient prognostication.MTG6Molecular tumour pathology - and tumour genetic

Associations of anticoagulant use with outcome in newly diagnosed glioblastoma.

To test the hypothesis that despite bleeding risk, anticoagulants improve the outcome in glioblastoma because of reduced incidence of venous thromboembolic events and modulation of angiogenesis, infiltration and invasion. We assessed survival associations of anticoagulant use from baseline up to the start of temozolomide chemoradiotherapy (TMZ/RT) (period I) and from there to the start of maintenance TMZ chemotherapy (period II) by pooling data of three randomised clinical trials in newly diagnosed glioblastoma including 1273 patients. Progression-free survival (PFS) and overall survival (OS) were compared between patients with anticoagulant use versus no use; therapeutic versus prophylactic versus no use; different durations of anticoagulant use versus no use; anticoagulant use versus use of anti-platelet agents versus neither anticoagulant nor anti-platelet agent use. Cox regression models were stratified by trial and adjusted for baseline prognostic factors. Anticoagulant use was documented in 75 patients (5.9%) in period I and in 104 patients (10.2%) in period II. Anticoagulant use during period II, but not period I, was associated with inferior OS than no use on multivariate analysis (p = 0.001, hazard ratio [HR] = 1.52, 95% confidence interval [CI]: 1.18-1.95). No decrease in OS became apparent when only patients with prophylactic anticoagulant use were considered. No survival association was established for anti-platelet agent use. Anticoagulant use was not associated with improved OS. Anticoagulants may not exert relevant anti-tumour properties in glioblastoma

Health-Related Quality of Life in a Randomized Phase III Study of Bevacizumab, Temozolomide, and Radiotherapy in Newly Diagnosed Glioblastoma

Neurolog

Temozolomide and Radiotherapy versus Radiotherapy Alone in Patients with Glioblastoma, IDH-wildtype: Post Hoc Analysis of the EORTC Randomized Phase III CATNON Trial

Purpose: In a post hoc analysis of the CATNON trial (NCT00626990), we explored whether adding temozolomide to radiotherapy improves outcome in patients with IDH1/2 wildtype (wt) anaplastic astrocytomas with molecular features of glioblas-toma [redesignated as glioblastoma, isocitrate dehydrogenase- wildtype (IDH-wt) in the 2021 World Health Organization (WHO) classification of central nervous system tumors].Patients and Methods: From the randomized phase III CATNON study examining the addition of adjuvant and con-current temozolomide to radiotherapy in anaplastic astrocyto-mas, we selected a subgroup of IDH1/2wt and H3F3Awt tumors with presence of TERT promoter mutations and/or EGFR amplifications and/or combined gain of chromosome 7 and loss of chromosome 10. Molecular abnormalities including MGMT promoter methylation status were determined by next-generation sequencing, DNA methylation profiling, and SNaPshot analysis.Results: Of the 751 patients entered in the CATNON study, 670 had fully molecularly characterized tumors. A total of 159 of these tumors met the WHO 2021 molecular criteria for glioblastoma, IDH-wt. Of these patients, 47 received radiotherapy only and 112 received a combination of radiotherapy and temozolomide. There was no added effect of temozolomide on either overall survival [HR, 1.19; 95% confidence interval (CI), 0.82-1.71] or progression-free survival (HR, 0.87; 95% CI, 0.61-1.24). MGMT promoter methyl-ation was prognostic for overall survival, but was not predictive for outcome to temozolomide treatment either with respect to overall survival or progression-free survival.Conclusions: In this cohort of patients with glioblastoma, IDH-wt temozolomide treatment did not add benefit beyond that observed from radiotherapy, regardless of MGMT promoter status. These findings require a new well-powered prospective clinical study to explore the efficacy of temozolomide treatment in this patient population

Prognostic significance of genome-wide DNA methylation profiles within the randomized, phase 3, EORTC CATNON trial on non-1p/19q deleted anaplastic glioma

Background. Survival in patients with IDH1/2-mutant (mt) anaplastic astrocytomas is highly variable. We have used the prospective phase 3 CATNON trial to identify molecular factors related to outcome in IDH1/2mt anaplastic astrocytoma patients.Methods. The CATNON trial randomized 751 adult patients with newly diagnosed 1p/19q non-codeleted anaplastic glioma to 59.4 Gy radiotherapy +/- concurrent and/or adjuvant temozolomide. The presence of necrosis and/or microvascular proliferation was scored at central pathology review. Infinium MethylationEPIC BeadChip arrays were used for genome-wide DNA methylation analysis and the determination of copy number variations (CNV). Two DNA methylation-based tumor classifiers were used for risk stratification. Next-generation sequencing (NGS) was performed using 1 of the 2 glioma-tailored NGS panels. The primary endpoint was overall survival measured from the date of randomization.Results. Full analysis (genome-wide DNA methylation and NGS) was successfully performed on 654 tumors. Of these, 432 tumors were IDH1/2mt anaplastic astrocytomas. Both epigenetic classifiers identified poor prognosis patients that partially overlapped. A predictive prognostic Cox proportional hazard model identified that independent prognostic factors for IDH1/2mt anaplastic astrocytoma patients included; age, mini-mental state examination score, treatment with concurrent and/or adjuvant temozolomide, the epigenetic classifiers, PDGFRA amplification, CDKN2A/B homozygous deletion, PI3K mutations, and total CNV load. Independent recursive partitioning analysis highlights the importance of these factors for patient prognostication.Conclusion. Both clinical and molecular factors identify IDH1/2mt anaplastic astrocytoma patients with worse outcome. These results will further refine the current WHO criteria for glioma classification