A rapid and accurate closed-tube Methylation-Sensitive High Resolution Melting Analysis assay for the semi-quantitative determination of SOX17 promoter methylation in clinical samples

• Published in: Clinica chimica acta Vol. 444; pp. 303 - 309
• Main Authors: Mastoraki, Sophia, Chimonidou, Maria, Dimitrakopoulos, Lampros, Kounelis, Sophia, Malamos, Nikos, Georgoulias, Vassilis, Lianidou, Evi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.04.2015
• Subjects: Breast cancer; Breast Neoplasms - genetics; DNA methylation; DNA Methylation - genetics; Female; Humans; Methylation Sensitive High Resolution Melting Analysis; Methylation specific PCR; Nucleic Acid Denaturation; Promoter Regions, Genetic - genetics; Solid tumors; SOX17; SOXF Transcription Factors - genetics; Transition Temperature; Methylation Sensitive High Resolution Melting Analysis; Methylation specific PCR; FFPEs; Breast cancer; MSP; SB; SOX17; cfDNA; DNA methylation; MS-HRMA; Solid tumors; SOX; CTCs
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2015.02.035

SOX17 promoter methylation can provide important prognostic information in cancer. We developed a novel semi-quantitative MS-HRMA assay for SOX17 promoter methylation. The assay was optimized by using synthetic control samples and validated by analyzing 165 clinical samples: a) 107 formalin fixed paraffin embedded (FFPEs) samples of patients with early breast cancer, b) 27 FFPE samples of patients with metastatic breast cancer, c) 15 reduction mammoplasty specimens obtained from healthy women and d) 16 genomic DNA samples isolated from healthy blood donors. Comparison with real time MSP was also performed. The assay is highly specific and sensitive and provides a semi-quantitative estimation of SOX17 promoter methylation. SOX17 promoter was found methylated in 96/134 (71.6%) breast cancer samples, while none of the 31 non-cancerous samples tested was positive (0%). SOX17 promoter methylation levels varied significantly among samples. When 165 clinical samples were analyzed both by MS-HRMA and real time MSP results were significantly comparable (concordance: 146/165, 88.5%). This novel MS-HRMA assay for SOX17 promoter methylation is closed-tube, highly sensitive, specific, cost-effective, rapid and easy-to-perform. It gives comparable results to Real-Time MSP in less time, while it offers the advantage of additionally providing an estimation of SOX17 promoter methylation levels. •SOX17 plays a tumor suppressor role through suppression of Wnt/β-catenin pathway.•SOX17 promoter methylation activates the Wnt signaling pathway.•SOX17 promoter is frequently methylated in many types of cancer.•SOX17 methylation is evaluated so far qualitatively.•We present a semi-quantitative MS-HRMA assay for SOX17 promoter methylation.•SOX17 promoter methylation levels differ between breast cancer samples.