Abstract 81: Development of a methylation-sensitive high resolution melting assay (MS-HRMA) for Cystatin M (CST6)
Authors: Dimitrakopoulos Lambros , Vorkas Panayiotis , Sotiropoulou Georgia , Georgoulias Vasilis , Lianidou Evi S. 
Published: June 2012

Introduction: Cystatin M or E/M (encoded by the CST6 gene) is an endogenous inhibitor of lysosomal cysteine proteases that functions to protect cells against uncontrolled proteolysis. CST6 gene is featuring a CpG island within the promoter and first exon, and is frequently methylated in cancer. We have recently shown that detection of CST6 methylation is of prognostic significance in operable breast cancer patients (Kioulafa et al, Int J Cancer, 2009). Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) enables highly sensitive, labor- and cost-efficient single locus methylation studies on the basis of DNA high-resolution melting technology. The aim of our study was to develop a novel closed-tube MS-HRMA methodology for the investigation of CST6 methylation status that is based on the combination of PCR and high-resolution melting analysis.

Materials and methods: The study material consisted of a total of 117 breast formalin fixed paraffin-embedded tissues (FFPEs): 90 paraffin-embedded breast carcinomas from patients with operable breast cancer, 18 non cancerous breast tissues and 9 fibroadenomas, all obtained from the Department of Medical Oncology, University Hospital of Heraklion Crete. 1μg of extracted DNA was modified with sodium bisulfite, in order to convert all unmethylated, but not methylated-cytosines to uracil. In each sodium bisulfite conversion reaction, dH2O and MCF-7 were included as a negative and positive control, respectively. Our methodology was first optimized and evaluated by using certain mixtures of fully methylated to unmethylated bisulfite converted genomic DNA, as controls. We compared our results with Methylation-Specific PCR (MSP) for the same samples.

Results: The developed methodology can detect the presence of methylation within the studied region, and additionally provides a semi-quantitative estimation of CST6 methylation level in terms of percentage. By using MS-HRMA for CST6 methylation we observed 42/90 (47.7%) of breast cancer FFPEs samples analyzed to be methylated at various methylation levels. In non cancerous tissues CST6 methylation was detected at very low levels in 2/18 (11.1%), while in benign breast tumors (fibroadenomas) CST6 methylation was observed in 1/9 (11.1%). Our results for MS-HRMA when compared to MSP for the same samples show a strong correlation (P&lt;0.001).

Conclusions: The developed MS-HRMA assay for CST6 promoter methylation is highly sensitive, cost-effective, easy-to-perform, and can be used as a screening test prior to DNA sequencing for the exclusion of clearly unmethylated samples. MS-HRMA gives comparable results to MSP in less time, while its main advavantage is the ability to additionally provide an estimation of the level of methylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 81. doi:10.1158/1538-7445.AM2011-81