Validation of real-time methylation-specific PCR to determine O6-methylguanine-DNA methyltransferase gene promoter methylation in glioma

Abstract

Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.

Figure 1

Decision tree for real-time MSP results.

Figure 2

Reproducibility of duplicate measurements. Dotted line represents identity line (x = y), and dashed lines represent the cutoff between classification as methylated or non-methylated according to the mixture Gaussian model (see Figures 3 and 4). Pearson correlation 0.996, Spearman correlation 0.93, N = 94. Black dots represent samples with m_MGMT copies >20; gray stars represent samples with m_MGMT copies <20 and Ct <40.

Figure 3

Density plot of normalized methylated MGMT copy number in glioma. Histogram of average results from 94 samples with duplicate measurements; the lines represent results from each replicate. Only samples with Ct values <40 for m_MGMT are included. The minimum of the density of the Gaussian mixture between the two local maxima is at the ratio of log₂(1000 * m_MGMT/ACTB) = 3 (ratio value of 8).

Figure 4

Definition of natural cut-off for methylated MGMT. Density (A), Classification (B), Uncertainty of classification (C), and posterior Probability (D) of class 2 (promoter methylation) obtained by fitting a mixture model to the average log₂(1000 * m_MGMT/ACTB). In the classification plot (B), all of the data are displayed at the bottom; assignment to the class with highest posterior probability at two different levels is above (M, class 2, methylated; U, class 1, non-methylated). Uncertainty (C) was defined as the smaller of the two posterior probabilities. A gray dashed line represents the optimal cutoff according to the selected model (log₂ ratio = 3; ratio value = 8). The region between the gray dotted lines (D) defines a classification “gray zone”; the thresholds for 95% posterior probability of class 1 and class 2, respectively, are 2 (ratio value = 4) and 4 (ratio value = 16).

Figure 5

Comparison between direct, real-time MSP and nested, gel-based MSP results. Box-plots and strip-charts compare the log₂(1000 * m_MGMT/ACTB) (y axis) values determined for 91 samples by direct, real-time MSP assay to the classification obtained with the nested, gel-based assay into non-methylated (green) and methylated (red) samples (x axis). A dashed line represents the cutoff defined with the Gaussian mixture model (Figures 3 and 4).