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(A) Normalized melt curves in duplicates showing the melt behavior of methylation standards (red=0 %, pink=25 %, blue=50 %, green=75 %, orange=100 %) and an unknown sample (black). (B) Regression model used for MGMT promoter methylation quantification. Area under the curve (AUC) from the normalized melt curves are used and regressed to the known methylation level of the standards. The linear regression model was chosen for quantification (R² > 0.98). (C) Pyrogram of the MGMT promoter of a patient tumor sample with a mean methylation of 31.4 %. (D) Typical pyrogram obtained from a grade III patient tumor sample indicating a heterozygous G-to-A point mutation of the IDH1 gene resulting in a mutation at codon 132 (R132H). (E) Relationship between MGMT protein activity and promoter methylation (r3) in 14 GBM cell lines. Figure S2. (A) Pyrogram of the whole HRM (R4) assay region from a patient sample. (B) Dot-plot of the methylation values obtained in a subsample by HRM and pyrosequencing showing a high correlation of this two methods. The dotted line indicates the 95 % CI. Figure S3. Overlay of Kaplan-Meier estimates of PFS and OS according to MGMT promoter methylation status. Kaplan-Meier estimates for (A) PFS and (B) OS of 65 high-grade glioma patients determined by HRM (red lines), MSP (blue) and PSQ (green). The solid and the dashed lines indicate the group as being categorized unmethylated (UM) or methylated (ME), respectively. Table S1. Primers used for HRM and pyrosequencing. Table S2. MGMT promoter methylation status determined by HRM, MSP, and PSQ in dependence of the IDH1 status. Table S3. ROC curves were plotted for 15 methylation cut-off scores (1-15 %) for predicting PFS ≥ 12 months and OS ≥ 18 months. (PPTX 312 kb)