BACKGROUND: Multiple studies have reported the prognostic impact of DNA methylation changes in acute myeloid leukemia (AML). However, these epigenetic markers have not been thoroughly validated and therefore are still not considered in clinical practice. Hence, we aimed to independently verify results of selected studies describing the relationship between DNA methylation of specific genes and their prognostic potential in predicting overall survival (OS) and event-free survival (EFS). RESULTS: Fourteen studies (published 2011-2019) comprising of 27 genes were subjected to validation by a custom NGS-based sequencing panel in 178 newly diagnosed non-M3 AML patients treated by 3 + 7 induction regimen. The results were considered as successfully validated, if both the log-rank test and multivariate Cox regression analysis had a p-value ≤ 0.05. The predictive role of DNA methylation was confirmed for three studies comprising of four genes: CEBPA (OS: p = 0.02; EFS: p = 0.03), PBX3 (EFS: p = 0.01), LZTS2 (OS: p = 0.05; EFS: p = 0.0003), and NR6A1 (OS: p = 0.004; EFS: p = 0.0003). For all of these genes, higher methylation was an indicator of longer survival. Concurrent higher methylation of both LZTS2 and NR6A1 was highly significant for survival in cytogenetically normal (CN) AML group (OS: p < 0.0001; EFS: p < 0.0001) as well as for the whole AML cohort (OS: p = 0.01; EFS < 0.0001). In contrast, for two studies reporting the poor prognostic effect of higher GPX3 and DLX4 methylation, we found the exact opposite, again linking higher GPX3 (OS: p = 0.006; EFS: p < 0.0001) and DLX4 (OS: p = 0.03; EFS = 0.03) methylation to a favorable treatment outcome. Individual gene significance levels refer to the outcomes of multivariate Cox regression analysis. CONCLUSIONS: Out of twenty-seven genes subjected to DNA methylation validation, a prognostic role was observed for six genes. Therefore, independent validation studies are necessary to reveal truly prognostic DNA methylation changes and to enable the introduction of these promising epigenetic markers into clinical practice.

• Keywords
• AML, DNA methylation, Prognosis, Validation,
• MeSH
• Leukemia, Myeloid, Acute diagnosis   genetics   MeSH
• Adult MeSH
• Immunochemistry methods   statistics & numerical data   MeSH
• Middle Aged MeSH
• Humans MeSH
• DNA Methylation genetics   physiology   MeSH
• Biomarkers, Tumor analysis   genetics   MeSH
• Prognosis MeSH
• Sequence Analysis, DNA methods   statistics & numerical data   MeSH
• Transcription Factors genetics   MeSH
• Validation Studies as Topic MeSH
• Treatment Outcome MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers, Tumor MeSH
• Transcription Factors MeSH

Here, we present a practical overview of four commonly used validation methods for DNA methylation assessment: methylation specific restriction endonucleases (MSRE) analysis, pyrosequencing, methylation specific high-resolution DNA melting (MS-HRM) and quantitative methylation specific polymerase chain reaction (qMSP). Using these methods, we measured DNA methylation levels of three loci in human genome among which one was highly methylated, one intermediately methylated and one unmethylated. We compared the methods in terms of primer design demands, methods' feasibility, accuracy, time and money consumption, and usability for clinical diagnostics. Pyrosequencing and MS-HRM proved to be the most convenient methods. Using pyrosequencing, it is possible to analyze every CpG in a chosen region. The price of the instrument may represent the main limitation of this methodology. MS-HRM is a simple PCR-based method. The measurement was quick, cheap and very accurate. MSRE analysis is based on a methylation specific digestion of DNA. It does not require a bisulfite conversion of DNA as the other methods. MSRE analysis was very easy to perform, however, it was not suitable for intermediately methylated regions and it was also quite expensive. qMSP is a qPCR-based method that uses primers designed specifically for methylated and unmethylated alleles of a chosen region. This was the least accurate method and also the primer design and optimization of PCR conditions were highly demanding.

• Keywords
• DNA methylation, MS-HRM, MSRE, Pyrosequencing, Validation methods, qMSP,
• Publication type
• Journal Article MeSH
• Review MeSH