Rapid Assessment of the Heterogeneous Methylation Status of CEBPA in Patients with Acute Myeloid Leukemia by Using High-Resolution Melting Profile Tsung-Chin.

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Rapid Assessment of the Heterogeneous Methylation Status of CEBPA in Patients with Acute Myeloid Leukemia by Using High-Resolution Melting Profile Tsung-Chin Lin, Sin-Sien Jiang, Wen-Chien Chou, Hsin-An Hou, Yu-Min Lin, Chia-Ling Chang, Cherng-An Hsu, Hwei-Fang Tien, Liang- In Lin The Journal of Molecular Diagnostics Volume 13, Issue 5, Pages 514-519 (September 2011) DOI: 10.1016/j.jmoldx.2011.05.002 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 HRM assay for quantitative DNA methylation analysis. A: Illustration of Tm and width in HRM assay. Tm is the melting temperature at the point at which half of the amplicons are double-stranded and single-stranded, and width is the width at half the height of the melting peak. B: Melting profiles obtained from HL-60 and K562 cells. K562 genomic DNA (CEBPA methylation) was serially diluted into HL-60 genomic DNA (no CEBPA methylation) as 0%, 1%, 5%, 10%, 50%, and 100%, respectively, followed sequentially by bisulfite treatment, PCR, and HRM assay. Fluorescence intensity was differential to time, shown on the y axis. C: Simple linear regression analysis of quantitative DNA methylation analysis based on areas of melting peaks. Percentage of the area of methylated melting peak is shown on the x axis. The y axis indicates the exact dilution percentage. M, methylated fragment; U, unmethylated fragment. D: Melting peaks in three patients with AML with high CEBPA methylation and four healthy donors. The Tm and width for the three patients were 79.87, 81.02, and 80.77, and 3.02, 3.81, and 3.79, respectively, and for the donors ranged from 77.87 to 78.20 and 2.15 to 2.45, respectively. Fluorescence intensity was differential to time, shown on the y axis. BM, bone marrow. The Journal of Molecular Diagnostics 2011 13, 514-519DOI: (10.1016/j.jmoldx.2011.05.002) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Differentiation between high and low CEBPA methylation as determined using MassARRAY and HRM assay. A: Two-way hierarchic cluster analysis of CEBPA methylation in 181 patients with AML (columns) with complete linkage clustering algorithm. Two groups were established: high methylation and low methylation. DNA methylation values are presented on a continuous scale from 0 (green) to 1 (red). The threshold for high CEBPA methylation is 0.486. B: ROC curve of the methylation index in patients with high and low methylation. The open circle indicates the cutoff methylation index of 1.412, with the best sensitivity and specificity, 97.14% and 95.89%, respectively. The positive and negative case number determined using MassARRAY and methylation index is shown in the table to the right. C: Simple linear regression analysis between the methylation index from HRM and the methylation level from MassARRAY in 181 patients with AML. Dotted lines indicate cutoff (C-O) methylation level from MassARRAY and cutoff methylation index from HRM profile. Methylation index = (Widthpatient/Widthreference) + (Tmpatient – Tmreference). The Journal of Molecular Diagnostics 2011 13, 514-519DOI: (10.1016/j.jmoldx.2011.05.002) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Sequential analysis of methylation status in patients with AML before and after standard therapy. Dotted line indicates cutoff methylation index to differentiate high and low CEBPA methylation. CR1, first complete remission; CR2, second complete remission. Methylation index = (Widthpatient/Widthreference) + (Tmpatient – Tmreference). The Journal of Molecular Diagnostics 2011 13, 514-519DOI: (10.1016/j.jmoldx.2011.05.002) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions