Validation of Real-Time Methylation-Specific PCR to Determine O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma Ilse Vlassenbroeck,

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Validation of Real-Time Methylation-Specific PCR to Determine O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma Ilse Vlassenbroeck, Stéphane Califice, Annie-Claire Diserens, Eugenia Migliavacca, Josef Straub, Ivano Di Stefano, Fabrice Moreau, Marie-France Hamou, Isabelle Renard, Mauro Delorenzi, Bruno Flamion, James DiGuiseppi, Katja Bierau, Monika E. Hegi The Journal of Molecular Diagnostics Volume 10, Issue 4, Pages 332-337 (July 2008) DOI: 10.2353/jmoldx.2008.070169 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Decision tree for real-time MSP results. The Journal of Molecular Diagnostics 2008 10, 332-337DOI: (10.2353/jmoldx.2008.070169) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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. The Journal of Molecular Diagnostics 2008 10, 332-337DOI: (10.2353/jmoldx.2008.070169) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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). The Journal of Molecular Diagnostics 2008 10, 332-337DOI: (10.2353/jmoldx.2008.070169) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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). The Journal of Molecular Diagnostics 2008 10, 332-337DOI: (10.2353/jmoldx.2008.070169) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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). The Journal of Molecular Diagnostics 2008 10, 332-337DOI: (10.2353/jmoldx.2008.070169) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions