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Background  Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described  RET  promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series.    Methods  In the current study, we analyzed the  RET  promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of  RET  methylation with overall survival for three and five years of follow-up.    Results  Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed  RET  promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined  RET  methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected  RET  promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While  RET  methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97–3.15; HR 1.85, 95 % CI 0.93–3.86; HR 1.83, 95 % CI 0.92–3.65, respectively).    Conclusions  Our results show that upon optimizing and aligning four  RET  methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker’s prognostic outcome is minimal.    Electronic supplementary material  The online version of this article (doi:10.1186/s13148-016-0211-8) contains supplementary material, which is available to authorized users.

clinical epigenetics

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome