Molecular differences in IDH wildtype glioblastoma according to MGMT promoter methylation

Abstract

Background: O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma. We investigated whether this marker furthermore defines a molecularly distinct tumor subtype with clinically different outcome.

Methods: We analyzed copy number variation (CNV) and methylation profiles of 1095 primary and 92 progressive isocitrate dehydrogenase wildtype glioblastomas, including paired samples from 49 patients. DNA mutation data from 182 glioblastoma samples of The Cancer Genome Atlas (TCGA) and RNA expression from 107 TCGA and 55 Chinese Glioma Genome Atlas samples were analyzed.

Results: Among untreated glioblastomas, MGMT promoter methylated (mMGMT) and unmethylated (uMGMT) tumors did not show different CNV or specific gene mutations, but a higher mutation count in mMGMT tumors. We identified 3 methylation clusters. Cluster 1 showed the highest average methylation and was enriched for mMGMT tumors. Seventeen genes including gastrulation brain homeobox 2 (GBX2) were found to be hypermethylated and downregulated on the mRNA level in mMGMT tumors. In progressive glioblastomas, platelet derived growth factor receptor alpha (PDGFRA) and GLI2 amplifications were enriched in mMGMT tumors. Methylated MGMT tumors gain PDGFRA amplification of PDGFRA, whereas uMGMT tumors with amplified PDGFRA frequently lose this amplification upon progression. Glioblastoma patients surviving <6 months and with mMGMT harbored less frequent epidermal growth factor receptor (EGFR) amplifications, more frequent TP53 mutations, and a higher tumor necrosis factor-nuclear factor-kappaB (TNF-NFκB) pathway activation compared with patients surviving >12 months.

Conclusions: MGMT promoter methylation status does not define a molecularly distinct glioblastoma subpopulation among untreated tumors. Progressive mMGMT glioblastomas and mMGMT tumors of patients with short survival tend to have more unfavorable molecular profiles.

Fig. 1

Copy number variations in primary glioblastoma according to MGMT promoter methylation. (A) Left panel: overall survival of 143 glioblastoma patients with IDH1 wildtype tumors according to MGMT promoter methylation. Right panel: survival analysis of patients with tumors from the dataset from TCGA regardless of the treatment. (B) Left panel: survival analysis of tumors of the TCGA glioblastoma database not receiving chemotherapy according to MGMT promoter methylation. Right panel: survival analysis of the dataset from TCGA treated with chemotherapy. (C) Graphical illustration of the percentage of CNV in the mMGMT and uMGMT groups. (D) Relative fractions of DNA copy number variations detected in the database from TCGA according to MGMT promoter methylation. Shown are the means ± standard deviation. (E) Average fraction of mutations detected in the dataset from TCGA according to MGMT promoter methylation. Shown are the means ± standard deviation.

Fig. 2

Differential methylation in primary glioblastoma according to MGMT promoter methylation. From outside to inside of the circle: First circle: hypermethylated genes in mMGMT tumors according to their chromosome position. Genes that were additionally downregulated in the RNAseq TCGA dataset are labeled in black, others in light red. Second circle: all DMPs within a promoter region of a gene. Third circle: area of each DMR between mMGMT and uMGMT glioblastomas. Fourth circle: all DMPs, including MGMT related on chromosome 10. Center: GBX2 mRNA expression in the dataset from TCGA in mMGMT and uMGMT glioblastomas.

Fig. 3

IDH wildtype glioblastoma cluster into 3 different clusters. (A) Heatmaps of 2024 positions that are differentially methylated between mMGMT and uMGMT tumors in the dataset of primary tumors, excluding MGMT related and intergenomic positions. (B) Consensus clustering using k-means of all samples of the primary tumor dataset. Shown is the result for k = 3. (C) Plot showing the relative change in area under the cumulative distribution function (CDF) curve in the k-means unsupervised clustering of primary glioblastoma. (D) CDF plot with examples from k = 2 to k = 8. Three clusters were chosen as the optimal number. (E) Distribution of mMGMT and uMGMT primary tumors into the clusters 1, 2, and 3. (F) Comparison of the cluster assignments with the clusters from Ceccarelli et al and Sturm et al for 65 glioblastoma samples of the database from TCGA. (G) Mean methylation beta-values of the 2024 differentially methylated CpGs, (H) relative TERT expression, and (I) TNF-NFκB score according to methylation clusters. The lower and the upper hinges correspond to the first and third quartiles. The upper and lower ends of the whiskers correspond to the 1.5× interquartile range from the hinge.

Fig. 4

CNV changes in pairs of primary and progressive glioblastoma. (A) CNV of the genes CDK4, CDKN2A, MDM2, and PDGFRA in a set of 49 paired glioblastoma samples at primary diagnosis and progression. MGMT promoter methylation is indicated in the first row. (B) Shift in gene dosages of CDK4, CDKN2A, MDM2, and PDGFRA in paired samples of primary and progressive glioblastoma according to MGMT promoter methylation. (C) Examples of copy number profiles of 3 paired primary and progressive glioblastoma samples. Color code: PDGFRA = red, CDKN2A = yellow, CDK4 = blue, MDM2 = green.

Fig. 5

Molecular differences between uMGMT and mMGMT progressive glioblastomas. (A) CNV of CDK4, MDM2, PDGFRA, and CDKN2A in the cohort of the progressive tumors, grouped according to MGMT promoter methylation. (B) Comparison of CDK4, MDM2, and PDGFRA copy number variations between mMGMT and uMGMT tumors in the progressive situation. (C) Heatmap of CNVs of a set of 19 frequently altered genes in the progressive tumor collective. High frequencies of alterations are shown in red, low frequencies in blue. MGMT promoter methylation is indicated in the first row. (D) Copy number variation profiles in mMGMT and uMGMT tumors in the progressive situation.

Fig. 6

Comparison between glioblastomas of patients with different survival times (<6 mo vs >12 mo) according to MGMT promoter methylation status. (A) Copy number variations of CDK4, EGFR, and PDGFRA according to MGMT promoter methylation status and survival groups (<6 mo vs >12 mo) of glioblastoma patients in the cohort of TCGA. (B) Mutations of EGFR and TP53 according to MGMT promoter methylation status and survival groups of TCGA glioblastoma patients. (C) GSEA (left) and IPA (right) comparing results obtained for mMGMT tumors of patients with survival times <6 months vs >12 months. Prediction for upregulation is indicated in orange. (D) IPA of patients with survival times <6 months vs >12 months. Enriched differentially regulated functions are visualized and the topics cellular movement, cell survival, signaling, and proliferation as well as infectious disease are highlighted in red. (E) GSEA (left) and IPA (right) comparing results obtained for uMGMT tumors of patients with survival times <6 months vs >12 months. Prediction for upregulation is indicated in orange, for downregulation in blue.