Predictive MGMT status in a homogeneous cohort of IDH wildtype glioblastoma patients

Abstract

Methylation of the O(6)-Methylguanine-DNA methyltransferase (MGMT) promoter is predictive for treatment response in glioblastoma patients. However, precise predictive cutoff values to distinguish "MGMT methylated" from "MGMT unmethylated" patients remain highly debated in terms of pyrosequencing (PSQ) analysis. We retrospectively analyzed a clinically and molecularly very well-characterized cohort of 111 IDH wildtype glioblastoma patients, who underwent gross total tumor resection and received standard Stupp treatment. Detailed clinical parameters were obtained. Predictive cutoff values for MGMT promoter methylation were determined using ROC curve analysis and survival curve comparison using Log-rank (Mantel-Cox) test. MGMT status was analyzed using pyrosequencing (PSQ), semi-quantitative methylation specific PCR (sqMSP) and direct bisulfite sequencing (dBiSeq). Highly methylated (> 20%) MGMT correlated with significantly improved progression-free survival (PFS) and overall survival (OS) in our cohort. Median PFS was 7.2 months in the unmethylated group (UM, < 10% mean methylation), 10.4 months in the low methylated group (LM, 10-20% mean methylation) and 19.83 months in the highly methylated group (HM, > 20% mean methylation). Median OS was 13.4 months for UM, 17.9 months for LM and 29.93 months for HM. Within the LM group, correlation of PSQ and sqMSP or dBiSeq was only conclusive in 51.5% of our cases. ROC curve analysis revealed superior test precision for survival if additional sqMSP results were considered (AUC = 0.76) compared to PSQ (cutoff 10%) alone (AUC = 0.67). We therefore challenge the widely used, strict PSQ cutoff at 10% which might not fully reflect the clinical response to alkylating agents and suggest applying a second method for MGMT testing (e.g. MSP) to confirm PSQ results for patients with LM MGMT levels if therapeutically relevant.

Keywords: Glioblastoma; IDH (isocitrate dehydrogenase); Methylation specific PCR (MSP); O(6)-Methylguanine-DNA methyltransferase (MGMT); Pyrosequencing (PSQ); Temozolomide (TMZ).

Fig. 1

a, b: Kaplan-Meier curves for progression-free (PFS) and overall survival (OS) of subgroup analysis comparing the different methylation groups (mean MGMT promoter methylation): 0-9%, 10-20%, 21-30%, 31-40%, and > 40%. c, d: Kaplan-Meier curves for progression-free (PFS) and overall survival (OS) of subgroup analysis comparing the different methylation groups UM, LM, and HM according to mean MGMT methylation PSQ results

Fig. 2

a: Receiver operating characteristics (ROC) for 10% PSQ cutoff (AUC = 0.67) and 10% PSQ cutoff corrected for sqMSP results (AUC = 0.76) in terms of overall survival. b: Receiver operating characteristics (ROC) for determination of optimal cutoff values for PSQ results at 10% (AUC = 0.67), 12% (AUC = 0.72), 15% (AUC = 0.74), 17% (AUC = 0.77) and 20% (AUC = 0.75). c: Representative methylation-specific PCR (MSP) shows results obtained from 15 different, representative GBM samples and one negative control (NC). The DNA was extracted from formalin-fixed and paraffin-embedded tissue. Methylated samples demonstrated PCR products with primers detecting the methylated (M, 89bp) and unmethylated (U, 93bp) MGMT promoter sequence. Clearly unmethylated samples showed PCR products only for the unmethylated MGMT promoter sequence (U). The pyrogram (d) demonstrates the pyrosequencing result of patient #19 (PSQ mean = 14%). The yellow areas correspond to the internal control of conversion (arrows). The blue areas indicate the polymorphism (T/C) as result of the bisulfite treatment and show the level of the methylation (%) of each CpG. An exemplary section of direct Bisulfite Sanger sequencing trace (forward) of patient #26 demonstrates CpGs 13-18 of 27 (arrows) which are partly (T/C) or fully converted (e)