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

Abstract

The methylation status of the O⁶-methylguanine DNA methyltransferase (<i>MGMT</i>) 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.<b>Experimental Design:</b> Quantitative <i>MGMT</i> 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). For 4,041 patients valid <i>MGMT</i> results were obtained, whereof 1,725 were randomized. The unsupervised cutoff in the training dataset was 1.27 (log₂[1,000 × (<i>MGMT</i>+1)/<i>ACTB</i>]), 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 <i>MGMT</i> 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, <i>P</i> < 0.0001; HR = 0.58, 95% CI, 0.43-0.78, <i>P</i> < 0.001], validated in the test dataset. The MGMT assay was highly reproducible upon retesting of 218 paired samples (<i>R</i> ² = 0.94). Low <i>MGMT</i> 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.