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

The benefit of the addition of temozolomide to radiotherapy in people with newly diagnosed glioblastoma was first demonstrated in 2005 in the pivotal European Organization for Research and Treatment of Cancer (EORTC) 26981/22981-NCIC CE3 randomized clinical trial (1). The efficacy of temozolomide in combination with radiotherapy was confirmed in a study on elderly patients with glioblastoma (2). In both studies, the clinical benefit of temozolomide treatment was largely confined to patients with glioblastomas with a methylated O⁶-methylguanine DNA methyltransferase (MGMT) promoter (2, 3). In other clinical trials, a survival benefit of single-agent treatment with temozolomide was demonstrated in patients with MGMT promoter methylated high-grade gliomas (4–6). In the CATNON trial, the efficacy of the addition of temozolomide during and after radiotherapy was investigated in patients with grade 3 astrocytoma. In the recently published second interim analysis of the study, the benefit of temozolomide was found to be restricted to patients with astrocytoma grade 3 with isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations (mt), and only for adjuvant temozolomide treatment (7). There was no clinical benefit of temozolomide in patients with IDH1/2 wildtype (wt) gliomas regardless of MGMT promoter status (7). However, IDH1/2wt gliomas are not a single entity, and molecular subtyping of grade 2 and 3 IDH1/2wt gliomas has identified prognostically significant patient subgroups (8–10). In particular, a major subgroup of grade 2 and 3 IDH1/2wt glioma has emerged with molecular features of glioblastoma. These are characterized by either (i) a mutation of the telomerase reverse transcriptase promoter (pTERT), and/or (ii) paired chromosome 7 trisomy and loss of heterozygosity of chromosome 10 (7+/10− signature), and/or (iii) amplification of EGFR (EGFRamp; refs. 11–13). With outcomes resembling those of glioblastoma, the 2021 World Health Organization (WHO) classification of central nervous system (CNS) tumors now labels these tumors as glioblastoma, isocitrate dehydrogenase–wildtype (IDH-wt) and consequently most guidelines recommend to treat them with radiotherapy and both concurrent and adjuvant temozolomide (14). However, the benefit of adding temozolomide to radiotherapy has not been proven in patients with tumors meeting the molecular criteria of glioblastoma, IDH-wt but not the histologic criteria.

The randomized CATNON trial (NCT00626990) with a control arm of radiotherapy alone allows the retrospective analyses of the effect of adjuvant and concurrent temozolomide in patients with histologically grade 3 astrocytomas with molecular features of glioblastoma (in the WHO 2021 classified as glioblastoma, IDH-wt), also in relation to the MGMT promoter methylation status.

Patients with tumors meeting the molecular criteria for glioblastoma, IDH-wt were identified in the EORTC, nonblinded, multicenter, randomized CATNON trial. This trial examined the effect of concurrent and adjuvant temozolomide given in addition to radiotherapy in adult patients with a diagnosis of primary 1p/19q non-codeleted anaplastic glioma (n = 751) according to the 2007 WHO classification of CNS tumors (7). Patient randomization (1:1:1:1) was based on a 2×2 factorial design; after primary surgery patients were treated with radiotherapy (59.4 Gy in 33 fractions of 1.8 Gy) without any temozolomide, or radiotherapy with concurrent temozolomide (75 mg/m² daily, max 7 weeks), or radiotherapy with adjuvant temozolomide (12 four-week cycles: 150–200 mg/m² on day 1–5), or radiotherapy with concurrent and adjuvant temozolomide. Patients were stratified on the basis of MGMT promoter status as determined by quantitative methylation-specific PCR. The collection of formalin-fixed paraffin-embedded (FFPE) tumor material was part of the study design. All institutions obtained ethics approval from their Institutional Review Boards or ethics review committees before enrollment started. The study was conducted in accordance with the Declaration of Helsinki. All patients gave written informed consent according to local, national, and international guidelines.

DNA was isolated from FFPE tumor material (15). For samples with ≥60 ng DNA available, the DNA methylation and sequencing data were produced and reported in a previous study (16). In short, IDH1/2 and H3F3A mutation status were determined by a standard glioma-tailored next-generation sequencing (NGS) panel (17). Mutation status of pTERT was determined with a SNaPshot assay of the two hotspot mutations in gliomas (C228T and C250T; ref. 18). DNA methylation profiles were acquired with the Infinium MethylationEPIC BeadChip (Illumina) according to the manufacturer's instructions after using the Infinium FFPE DNA Restoration Kit. Copy-number data (presence of EGFRamp, and the 7+/10− signature) were derived and interpreted from the DNA methylation data as described previously (16). MGMT promoter status was assessed from the DNA methylation data with the MGMT-STP27 algorithm (19). For samples with <60 ng DNA available, DNA methylation profiling and the standard NGS panel could not both be performed. Instead, IDH1/2, H3F3A, and pTERT mutation status, and copy-number data were determined by an in-house developed NGS panel requiring less DNA for successful analysis (20, 21). Two dedicated neuropathologists centrally reviewed all tumor samples (J.M. Kros: European and Australian samples, K. Aldape: North-American samples). Clinical data such as survival data, sex, age at enrollment, use of corticosteroids at enrollment, type of surgery, mini-mental state examination (MMSE) score at enrollment, and treatment regimen were collected from the study entry forms.

For the analyses, the temozolomide treatment arms were combined into several larger cohorts. The “temozolomide and radiotherapy” cohort is comprised of the concurrent arm, the adjuvant arm, and the concurrent/adjuvant arm. The “adjuvant temozolomide” cohort consists of the adjuvant arm and the concurrent/adjuvant arm. The “no adjuvant temozolomide” cohort consists of the concurrent arm and the radiotherapy only arm. The “concurrent temozolomide” cohort consists of the concurrent arm and the concurrent/adjuvant arm. The “no concurrent temozolomide” cohort consists of the adjuvant arm and the radiotherapy only arm. The primary endpoint of overall survival and the secondary endpoint of progression-free survival were measured from the date of randomization until the date of event (death or death/progression, respectively) or censored at the date of last follow-up. Survival curves were created using the Kaplan–Meier technique and compared with the log-rank test. The Cox regression model was used for univariable and multivariable analysis to determine HRs with 95% confidence intervals (CI). Significance was set at P values below 0.05 unless otherwise specified. Statistical analysis was performed using R version 3.6.3 and packages minfi, and survival.

Data sharing requests for all interested researchers are welcome and can be made through the official EORTC data sharing policy committee via https://www.eortc.be/services/forms/erp/request.aspx.

We identified 202 patients with IDH1/2wt and H3F3Awt astrocytomas grade 3 from the 751 patients with astrocytomas grade 3 enrolled in the CATNON study (standard NGS panel: n = 194, in-house NGS panel: n = 8). The tumors of 159 patients fulfilled the molecular characteristics of glioblastoma, IDH-wt, that is, presence of EGFRamp, and/or pTERTmt, and/or the 7+/10− signature (EGFRamp: n = 83, pTERTmt: n = 144, 7+/10− signature: n = 105; Supplementary Table S1). Of this patient cohort, 47 (29.6%) patients received radiotherapy alone and 112 (70.4%) patients received radiotherapy with adjuvant and/or concurrent temozolomide. These data are summarized in Fig. 1.

Table 1 illustrates the baseline characteristics of the temozolomide and radiotherapy cohort, and the patient cohort treated with radiotherapy only. No significant differences were found between the two cohorts based on age, sex, type of surgery, corticosteroid use, WHO performance score, MMSE score, and presence of necrosis and/or microvascular proliferation. There was a trend towards more tumors with unmethylated MGMT promoter in the temozolomide and radiotherapy cohort (P = 0.066).

At the time of database lock (May 7, 2019), 143 of 159 patients (89.9%) with tumors meeting the criteria of glioblastoma, IDH-wt, were deceased, and in 154 patients (96.9%) progression of disease was reported. The median overall survival of this patient cohort was 1.4 years (95% CI, 1.3–1.8 years; Supplementary Fig. S1A) and the median progression-free survival was 0.5 years (95% CI, 0.5–0.7 years; Supplementary Fig. S1B). In this cohort of patients, there was no added effect of temozolomide in relation to radiotherapy alone on either overall survival (HR, 1.19; 95% CI, 0.82–1.71; Fig. 2A), or progression-free survival (HR, 0.87; 95% CI, 0.61–1.24; Fig. 2B). This lack of effect in overall and progression-free survival was not associated with the timing of the temozolomide treatment, that is, concurrent, adjuvant, or both concurrent and adjuvant temozolomide. Neither the adjuvant temozolomide nor the concurrent temozolomide was associated with clinical benefit in this cohort of patients. Moreover, no significant differences in overall survival or progression-free survival were found between the radiotherapy alone treatment arm and the radiotherapy with both concurrent and adjuvant temozolomide treatment arm (Supplementary Fig. S2).

For 152 of the 159 patients in this cohort, tumor DNA methylation data were available allowing the determination of the MGMT promoter methylation status by the MGMT-STP27 algorithm. Of these, 53 (34.9%) tumors were MGMT methylated, and the remaining 99 tumors (65.1%) were MGMT unmethylated. Patients with MGMT-methylated tumors had superior overall survival (HR, 0.65; 95% CI, 0.45–0.92; Fig. 3A); the median overall survival for the cohort with MGMT-methylated tumors was 1.8 years (95% CI, 1.4–2.2 years), and for the cohort with MGMT-unmethylated tumors was 1.4 years (95% CI, 1.2–1.6 years). MGMT promoter methylation was not prognostic for progression-free survival (HR, 0.95; 95% CI, 0.68–1.34; Supplementary Fig. S3A) with a median progression-free survival of 0.5 years for both the cohort with MGMT-methylated tumors (95% CI, 0.4–0.8 years), and the cohort with MGMT-unmethylated tumors (95% CI, 0.5–0.7 years). No survival benefit was detected for temozolomide in addition to radiotherapy on overall survival in either the cohort with MGMT-methylated tumors (HR, 1.36; 95% CI, 0.75–2.48; Fig. 3B), or the cohort with MGMT-unmethylated tumors (HR, 0.88; 95% CI, 0.54–1.42; Fig. 3C). Similarly, no predictive effect for temozolomide efficacy was identified on progression-free survival in patients with MGMT-methylated and MGMT-unmethylated tumors (Supplementary Fig. S3B and C). The lack of predictive effect of MGMT promoter methylation extended to overall survival and progression-free survival and to each temozolomide cohort, that is, in none of the three temozolomide arms a clinical benefit was observed (Supplementary Fig. S4).

To correct for possible confounding factors, we selected all available factors from univariable analyses with likelihood ratio test P values ≤ 0.10. For overall survival, these factors included age group (<50 vs. ≥50 years), MMSE score (≤26 vs. 27–30), type of surgery (biopsy vs. resection), use of corticosteroids at randomization (yes vs. no), and MGMT promoter status (methylated vs. unmethylated; Supplementary Table S2). For progression-free survival, the significant factors included age group, type of surgery, and use of corticosteroids at randomization (Supplementary Table S3). Lack of clinical benefit of temozolomide was still apparent after correction for these factors in this cohort of patients on overall survival (temozolomide and radiotherapy vs. radiotherapy only: HR, 0.89; 95% CI, 0.60–1.33; Table 2), and in progression-free survival (HR, 0.79; 95% CI, 0.55–1.13; Supplementary Table S4).

Twenty-nine of the 159 patients with tumors meeting the criteria of glioblastoma, IDH-wt (18.2%) had a mutation of pTERT, and were negative for EGFRamp and the 7+/10− signature (pTERTmt only). Although this cohort is limited in size, we examined this subgroup in more detail. Patients with pTERTmt only tumors did not differ from other patients with tumors meeting the criteria of glioblastoma, IDH-wt with respect to overall survival (median overall survival pTERTmt only 1.4 years vs. other glioblastoma, IDH-wt 1.4 years, HR, 0.78; 95% CI, 0.50–1.21; Supplementary Fig. S5A), or in progression-free survival (median progression-free survival pTERTmt only 0.6 years vs. other glioblastoma, IDH-wt 0.5 years, HR, 0.79; 95% CI, 0.52–1.19; Supplementary Fig. S5B). We also failed to identify a beneficial effect of temozolomide in the patient cohort with pTERTmt only tumors on either overall survival or progression-free survival, though this analysis is based on few patients (radiotherapy only: n = 9, temozolomide and radiotherapy: n = 20; Supplementary Fig. S5C and D).

As per study protocol, all samples included in the CATNON trial were diagnosed as an astrocytoma grade 3 by at least one central dedicated neuropathologist according to the 2007 WHO classification of CNS tumors. Therefore, all glioblastoma, IDH-wt analyzed in the current study were also diagnosed as such. Despite the strict histologic criteria, subtle signs of necrosis and/or microvascular proliferation were reported at central histology review in 28 tumor samples (necrosis only: n = 4, microvascular proliferation only: n = 14, both necrosis and microvascular proliferation: n = 10) which are considered histologic features of glioblastoma but did not lead to exclusion from the study. Presence of these histologic features of glioblastoma in the patients with molecularly defined glioblastoma, IDH-wt was associated with shorter overall survival (HR, 1.62; 95% CI, 1.06–2.48; Supplementary Fig. S6A), but no significant difference was found in progression-free survival (HR, 1.46; 95% CI, 0.96–2.22; Supplementary Fig. S6B). After adjustment for significant factors from univariable analysis including histologic factors for glioblastoma, futility of temozolomide treatment remained for this cohort of patients with glioblastoma, IDH-wt in overall survival (HR, 0.90; 95% CI, 0.60–1.35; Supplementary Table S5), and progression-free survival (HR, 0.79; 95% CI, 0.55–1.14; Supplementary Table S6).

This is the first dataset investigating temozolomide treatment efficacy in patients with IDH1/2wt astrocytomas grade 3 with molecular features of glioblastoma treated in a randomized clinical trial with a control arm of radiotherapy only. Of 202 IDH1/2wt and H3F3Awt tumors present in the CATNON trial, 159 tumors fulfilled the WHO 2021 molecular criteria of glioblastoma, IDH-wt (14). We did not observe benefit of the addition of temozolomide to radiotherapy in this cohort of patients, neither for overall nor progression-free survival. Similarly, no benefit of temozolomide was observed in the subgroup of patients with tumors harboring a methylated MGMT promoter. Moreover, the timing of the temozolomide treatment (concurrent, adjuvant, or both) was not related to survival outcome.

Our data conflict with the results from earlier randomized clinical trials examining the efficacy of temozolomide in combination with radiotherapy in glioblastoma (1, 2). The study by Stupp and colleagues showed efficacy of concurrent temozolomide followed by six cycles of adjuvant temozolomide (1). However, during enrollment and primary analysis of this trial the role of IDH1/2 in glioma had not yet been described. The exact percentage of patients with IDH1/2mt tumors in that study is unspecified, although in a later subgroup analysis of 160 tumor samples only 8% had an IDH1 mutation (22). In the study by Perry and colleagues on elderly patients with glioblastoma, the percentage of patients with IDH1/2mt tumors is described, comprising less than 1% of patients (2). Intriguingly, there is a possibility that age-based selection could have had an effect on outcome in this study; patients younger than 70 years had no survival benefit of additional temozolomide in combination with radiotherapy, whereas patients older than 70 years did show a prolonged survival due to temozolomide treatment (2). Thus, the results we find from the CATNON trial might not be as easily comparable to these previous studies due to a younger patient population in our study and a well-documented molecular subtyping of the tumors of the investigated patients.

With the possible exception of 28 samples with subtle signs of necrosis and/or microvascular proliferation, we prognosticated patients solely on molecular data. For patients with an IDH1/2wt lower-grade glioma, both EGFRamp and the 7+/10− signature are now indicators of a grade 4 diagnosis on their own (9, 23, 24). Conversely, cohorts of patients with pTERTmt only tumors have been described with variable patient outcome (13, 23, 25). In our cohort, we did not observe a difference in overall survival or progression-free survival between patients with pTERTmt only tumors and patients with the typical glioblastoma, IDH-wt. However, the number of patients with IDH-wt, pTERTmt only tumors was limited, and we emphasize the importance of excluding other pTERTmt tumor diagnoses (e.g., 1p/19q codeleted tumors, pleomorphic xanthoastrocytomas) when performing similar analyses, or when setting up prospective trials (12). It is also important to note, that the CATNON trial only included patients with a grade 3 tumor, and it remains to be determined whether patients with pTERTmt only tumors, grade 2 have a comparable prognosis (25).

The major limitations of this study are the modest sample size, and the post hoc design. The CATNON trial was not specifically powered to answer the clinical questions of the current study; the lack of predictive effect in this post hoc study of a subset of tumors now meeting the criteria for glioblastoma, IDH-wt and the lack of impact of MGMT promoter methylation on overall survival could be due to the limited small sample size. However, a recent randomized study in 37 patients with grade 2 or 3 IDH-wt glioma showing TERT promoter mutations did observe a survival benefit of the addition of temozolomide to radiotherapy (26). Despite the mentioned limitations of our study, we extracted a 159-patient cohort by clearly defined and now official diagnostic criteria from the entire CATNON dataset without an indication of temozolomide efficacy. At the minimum, our study therefore questions whether the addition of temozolomide treatment to radiotherapy is beneficial for patients with molecularly defined glioblastoma, IDH-wt.

In short, we found no effect of adjuvant and concurrent temozolomide treatment in patients with anaplastic astrocytomas now meeting the molecular criteria for glioblastoma, IDH-wt, regardless of MGMT promoter status. At present, these findings are insufficient to warrant a change in the management of these patients; that is, given the outcome of other studies we believe these patients should be offered radiotherapy in combination with temozolomide chemotherapy. However, these findings do warrant a well-powered prospective study on the effectiveness of temozolomide when added to radiotherapy in tumors meeting the contemporary WHO 2021 molecular criteria for glioblastoma, IDH-wt. The choice for a trial design will depend on whether the trial should demonstrate that adding temozolomide will improve patient outcome as compared with radiotherapy alone, or whether it should demonstrate that temozolomide can safely be left out.

M. Sanson reports grants and other support from AstraZeneca, as well as personal fees from Genenta, Orion, and Mundi Pharma outside the submitted work. W. Wick reports non-financial support from Apogenix, Pfizer, and Roche; personal fees from Enterome; and other support from AstraZeneca, Mundipharma, and Bayer outside the submitted work. P.M. Clement reports personal fees and non-financial support from MSD during the conduct of the study. P.M. Clement also reports personal fees and non-financial support from BMS and Bayer; personal fees from Merck, Takeda, Rakuten, and Leo; non-financial support from Teva; and grants and non-financial support from AstraZeneca outside the submitted work. M.A. Vogelbaum reports grants from Oncosynergy, Celgene, and Denovo; grants and other support from Infuseon Therapeutics; and personal fees from Chimerix outside the submitted work. A.K. Nowak reports grants and personal fees from AstraZeneca and Douglas Pharmaceuticals, as well as personal fees from Seagen, Trizell, Bristol Myers Squibb, and PharmAbcine outside the submitted work. M. Weller reports grants and personal fees from Apogenix; grants from MSD, Merck (EMD), and Quercis; and personal fees from Philogen, BMS, Medac, Nerviano, Novartis, Orbus, and Y-mAbs outside the submitted work. H.J. Dubbink reports personal fees from AbbVie, Bayer, Janssen, Lilly, and Pfizer; grants and personal fees from AstraZeneca and Merck; and non-financial support from Illumina outside the submitted work. B.G. Baumert reports personal fees and non-financial support from SAMO (Swiss Academy of Multi-disciplinary Oncology) and Roche outside the submitted work. P.J. French reports personal fees from Aurikamed outside the submitted work. M.J. van den Bent reports grants from Dutch Cancer Society, UK Brain Tumor Society, Strijd van Salland, and Schering Plough/MSD during the conduct of the study, as well as personal fees from AGIOS, Boehringer, AstraZeneca, Carthera, Karyopharm, and Genenta outside the submitted work. No disclosures were reported by the other authors.

C.M.S. Tesileanu: Conceptualization, data curation, formal analysis, investigation, visualization, methodology, writing–original draft, writing–review and editing, data collection. M. Sanson: Writing–review and editing, data collection. W. Wick: Writing–review and editing, data collection. A.A. Brandes: Writing–review and editing, data collection. P.M. Clement: Writing–review and editing, data collection. S.C. Erridge: Writing–review and editing, data collection. M.A. Vogelbaum: Writing–review and editing, data collection. A.K. Nowak: Writing–review and editing, data collection. J.-F. Baurain: Writing–review and editing, data collection. W.P. Mason: Writing–review and editing, data collection. H. Wheeler: Writing–review and editing, data collection. O.L. Chinot: Writing–review and editing, data collection. S. Gill: Writing–review and editing, data collection. M. Griffin: Writing–review and editing, data collection. L. Rogers: Writing–review and editing, data collection. W. Taal: Writing–review and editing, data collection. R. Rudà: Writing–review and editing, data collection. M. Weller: Writing–review and editing, data collection. C. McBain: Writing–review and editing, data collection. M.E. van Linde: Writing–review and editing, data collection. K. Aldape: Writing–review and editing, data collection. R.B. Jenkins: Writing–review and editing, data collection. J.M. Kros: Writing–review and editing, data collection. P. Wesseling: Writing–review and editing, data collection. A. von Deimling: Writing–review and editing, data collection. Y. Hoogstrate: Data curation, formal analysis, investigation, visualization, methodology, writing–review and editing, data collection. I. de Heer: Data curation, formal analysis, investigation, visualization, methodology, writing–review and editing, data collection. P.N. Atmodimedjo: Writing–review and editing, data collection. H.J. Dubbink: Writing–review and editing, data collection. R.W.W. Brouwer: Writing–review and editing, data collection. W.F.J. van IJcken: Writing–review and editing, data collection. K.J. Cheung: Writing–review and editing, data collection. V. Golfinopoulos: Writing–review and editing, data collection. B.G. Baumert: Conceptualization, writing–review and editing, data collection. T. Gorlia: Data curation, formal analysis, visualization, methodology, writing–review and editing, data collection. P.J. French: Conceptualization, data curation, formal analysis, supervision, investigation, visualization, methodology, writing–original draft, writing–review and editing, data collection. M.J. van den Bent: Conceptualization, data curation, formal analysis, supervision, investigation, visualization, methodology, writing–original draft, writing–review and editing, data collection.

This study was funded by Merck, Sharp & Dohme (MSD) formerly Schering-Plough by an educational grant, and by the provision of temozolomide. The clinical study was also supported by the NRG (grants U10CA180868 and U10CA180822), Cancer Research UK grant CRUK/07/028, and Cancer Australia (project grants 1026842 and 1078655). The molecular study was funded by grant GN-000577 from The Brain Tumour Charity, grant 10685 from the Dutch Cancer Society, and financial support from the Vereniging Heino “Strijd van Salland.” We thank our patients and their relatives for their willingness to participate to this study. We also thank all sites and their staff for contributing to this study. We further acknowledge the support of this study by the staff at the EORTC Headquarters, Brussels, Belgium; the NRG Oncology (formerly the Radiation Therapy Oncology Group) staff at the American College of Radiology; the staff at the Australian National Health and Medical Research Council (NHMRC) Clinical Trials Centre (COGNO Coordinating Centre); and the staff at MRC Clinical Trials Unit, London, United Kingdom.

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