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Background   CST6  promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of  CST6  promoter methylation.    Methods  We designed primers that amplify both methylated and unmethylated  CST6  sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.    Results  The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated  CST6  sequence and provides additionally a semi-quantitative estimation of  CST6  promoter methylation.  CST6  promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.    Conclusions  The developed MS-HRMA assay for  CST6  promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.

bmc cancer

A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples