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

Abstract

Purpose: The methylation status of the O⁶-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).

Results: For 4,041 patients valid MGMT results were obtained, whereof 1,725 were randomized. The unsupervised cutoff in the training dataset was 1.27 (log₂[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 ² = 0.94).

Conclusions: 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.

Figure 1.

Flow of patient samples through the study. all-P, All patients population; rand-P, randomized patient population; ACTB, β-actin gene.

Figure 2.

Reproducibility of qMSP MGMT assay. The original and retest dataset (corrected MGMT log₂ ratios, $\left[{\log }_2\left(\frac{MGMT+1}{ACT}\times 1,000\right)\right]$) from 218 paired samples are visualized in a scatter plot. The R² was 93%. Retests were performed using a second set of FFPE tumor sections in a different laboratory blinded to the original results.

Figure 3.

A, Unsupervised MGMT promoter methylation cutoff and OS. The unsupervised cutoff of 1.27 obtained in the all-P training cohort is indicated in green in the bimodal distribution of the corrected MGMT log₂ ratio values $\left[{\log }_2\left(\frac{MGMT+1}{ACTB}\times 1,000\right)\right]$. B, The Kaplan-Meier plot visualizes OS in the rand-P training cohort separated into patients with MGMT promoter-methylated and -unmethylated tumors (P < 0.0001, log-rank test). The shaded area represents the 95% CI.

Figure 4.

Optimal MGMT promoter methylation cutoff and OS. A, The position of the optimal cutoff point of −0.28 [corrected MGMT log₂ ratio value, log₂ $\left(\frac{MGMT+1}{ACTB}\times 1,000\right)$] is indicated in orange in the bimodal distribution of the entire all-P training cohort. It corresponds to a 96% chance to be unmethylated (4% chance to be methylated) as illustrated in the posterior probability plot (B) and defines the lower bound of the “gray zone” (−0.28, and ≤1.27). C, The Kaplan-Meier plot visualizes the outcome of patients in the randomized patient (rand-P) training cohort separated into MGMT promoter-methylated (<1.27), gray zone (—0.28, and <1.27), and truly unmethylated patients (<−0.28; P < 0.0001, log-rank test). The shaded area represents the 95% CI.