Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM

Abstract

O⁶-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR = 5.23, 95% CI [2.089-13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR = 3.076, 95% CI [1.301-7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.

Figure 1. MGMT methylation profiles of 70 GBM samples show four distinct groups.

Each row represents an individual patient. For each patient the qMSP^P positive result is shown as a black rectangle. The next column shows the results of the qMSP test (Labcorp). PFS is represented by a circle with a blackened circle representing greater than 1 year PFS. Positive MGMT protein expression (>15% tumor cells stained positive in IHC) is shown as black rounded rectangle and negative expression is shown as white rounded rectangle. MGMT mRNA expression is shown using color-coded rectangles. Absence of any of the shapes indicates results not available. All CpG sites belonging to the three regions, R1, R2 and R3 are shown within the blue outlined squares. The four distinct groups are outlined with red squares. The qMSP region is labeled with qMSP primers in R3. The transcription start site (TSS) of the MGMT gene is marked by an arrow.

Figure 2. Schematic of 3R classification.

All three regions of the MGMT promoter for each patient are classified as methylated (M) or unmethylated (UM). Based on the status of these three regions, patients are classified into four major groups: Group A) all regions unmethylated, Group B) one region methylated, Group C) two regions methylated, and Group D) all three regions methylated. For correlation analysis, Groups B and C were further subdivided as indicated.

Figure 3. 3R classification and MS-MLPA predict PFS in GBM.

Kaplan-Meier estimation of PFS according to MGMT promoter methylation status determined using: A) 3R classification method - The median PFS for the methylated group was 540 days vs. 210 days for the unmethylated group (HR  = 5.23, 95% CI [2.089–13.097], p<0.0001), B) qMSP ^P - The median PFS for the methylated group was 373 days vs. 224 days for the unmethylated group (HR  = 1.707, 95% CI [0.728–4.003], p = 0.213), and C) MS-MLPA - The median PFS for the methylated group was 540 days vs. 210 days for the unmethylated group (HR  = 3.076, 95% CI [1.301–7.27], p = 0.007).

Figure 4. MS-MLPA predicts PFS in 28 GBM patients.

Kaplan-Meier estimation of PFS according to MGMT promoter methylation status determined using: A. MS-MLPA (p-value  = 0.002) and B. qMSP (p-value  = 0.089).