Abstract
Background Extramammary Paget's disease (EMPD) is a rare skin tumor. Hypermethylation in the MSH2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of MSH2 may play an important role in the pathogenesis of EMPD. Objective This study aims to establish a rapid analysis strategy based on the methylation-sensitive high-resolution melting curve (MS-HRM) to detect the methylation level of the MSH2 promoter. Methods With the use of universal methylated human DNA products, we established the MS-HRM standard curve to quantitatively detect the methylation level of the MSH2 promoter. Then, all 57 EMPD tumor DNA samples were analyzed. Pyrosequencing assay was also carried out to test the accuracy and efficacy of MS-HRM. Besides, a total of 54 human normal and other cancerous tissues were included in this study to test the reliability and versatility of the MS-HRM standard curve. Results In this study, by using the established MS-HRM, we found that 96.5% (55/57) EMPD tumor samples had varying methylation levels in the MSH2 promoter ranging from 0% to 30%. Then, the methylation data were compared to the results obtained from pyrosequencing, which showed a high correlation between these two techniques by Pearson's correlation (r = 0.9425) and Bland–Altman plots (mean difference = −0.1069) indicating that the methylation levels analyzed by MS-HRM were consistent with DNA pyrosequencing. Furthermore, in 23 normal and 31 other cancerous tissue samples, there were two colorectal cancer (CRC) tissues that tested MSH2 methylation positive (1% and 5%) which confirmed that our established MS-HRM can be widely applied to various types of samples. Conclusion MS-HRM standard curve can be used for the detection of the methylation level of MSH2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze MSH2 promoter methylation in routine pathological procedure.
Highlights
Extramammary Paget’s disease (EMPD) is a rare intraepithelial adenocarcinoma which is most likely to appear in the skin containing apocrine sweat glands, such as anogenital and axillary regions [1]. e clinical presentation of this skin cancer is eczema-like, exhibiting pruritus, burning, or tenderness in affected lesions
23 normal controls and 31 samples of other cancer types were detected by methylation-sensitive high-resolution melting curve (MS-HRM) with the same standard curves in this study including 4 normal skin tissues, 19 nontumor adjacent tissues, 19 colorectal cancer (CRC) tissue samples, 5 breast cancer tissues, 4 bladder cancer tissues, 1 thyroid cancer tissue, 1 kidney cancer tissue, and 1 endometrial cancer tissue
mismatch repair (MMR) deficiency is strongly related to the development of multiple cancers, such as colorectal cancer (CRC), endometrial carcinoma (EC), and breast cancer, as well as glioma and lymphoma [18, 19]
Summary
Extramammary Paget’s disease (EMPD) is a rare intraepithelial adenocarcinoma which is most likely to appear in the skin containing apocrine sweat glands, such as anogenital and axillary regions [1]. e clinical presentation of this skin cancer is eczema-like, exhibiting pruritus, burning, or tenderness in affected lesions. Hypermethylation in the MSH2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of MSH2 may play an important role in the pathogenesis of EMPD. With the use of universal methylated human DNA products, we established the MS-HRM standard curve to quantitatively detect the methylation level of the MSH2 promoter. By using the established MS-HRM, we found that 96.5% (55/57) EMPD tumor samples had varying methylation levels in the MSH2 promoter ranging from 0% to 30%. MS-HRM standard curve can be used for the detection of the methylation level of MSH2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze MSH2 promoter methylation in routine pathological procedure
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