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.
BACKGROUND
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.
Analysis of Circulating Tumor Cells, Lab of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece.
Sophia
S
Mastoraki
Analysis of Circulating Tumor Cells, Lab of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece.
Maria
M
Chimonidou
Analysis of Circulating Tumor Cells, Lab of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece.
Lampros
L
Dimitrakopoulos
Oncology Unit and Pathology Department, Helena Venizelou Hospital, Athens, Greece.
Sophia
S
Kounelis
Oncology Unit and Pathology Department, Helena Venizelou Hospital, Athens, Greece.
Nikos
N
Malamos
Laboratory of Tumor Cell Biology, Medical School, University of Crete, Heraklion, Greece.
Vassilis
V
Georgoulias
Analysis of Circulating Tumor Cells, Lab of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece. Electronic address: lianidou@chem.uoa.gr.
Evi
E
Lianidou
D001943:Breast Neoplasms / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D006801:Humans; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D055760:SOXF Transcription Factors / Q000235:genetics*; D044366:Transition Temperature
C109188:SOX17 protein, human; D055760:SOXF Transcription Factors
Breast cancer; DNA methylation; Methylation Sensitive High Resolution Melting Analysis; Methylation specific PCR; SOX17; Solid tumors