Sophia Mastoraki1, Maria Chimonidou1, Lampros Dimitrakopoulos1, Sophia Kounelis2, Nikos Malamos2, Vassilis Georgoulias3, Evi Lianidou4. 1. Analysis of Circulating Tumor Cells, Lab of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece. 2. Oncology Unit and Pathology Department, Helena Venizelou Hospital, Athens, Greece. 3. Laboratory of Tumor Cell Biology, Medical School, University of Crete, Heraklion, Greece. 4. Analysis of Circulating Tumor Cells, Lab of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece. Electronic address: lianidou@chem.uoa.gr.
Abstract
INTRODUCTION: SOX17 promoter methylation can provide important prognostic information in cancer. We developed a novel semi-quantitative MS-HRMA assay for SOX17 promoter methylation. METHODS: 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. RESULTS: 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%). CONCLUSIONS: 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.
INTRODUCTION: SOX17 promoter methylation can provide important prognostic information in cancer. We developed a novel semi-quantitative MS-HRMA assay for SOX17 promoter methylation. METHODS: 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. RESULTS: 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%). CONCLUSIONS: 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.