63 DNA methylation markers for endometrial cancer appeared valuable for the detection of EC in minimally invasive samples (24, 25, 27-29). Yet, it would be interesting to investigate whether a discovery screen using minimally invasive sample types would yield even more accurate markers. This approach has been proven valuable for the discovery of novel diagnostic DNA methylation markers for the early detection of cervical (pre)cancer in self-collected lavage samples (33). The methylation status of the extracted DNA can be measured by several techniques (34) which may also contribute to differences in marker performance (35). Studies selected in this systematic review used three different methods to assess methylation status, including MethyLight PCR, pyrosequencing and qMSP. Although it has been shown that the use of diverse assays could lead to differences in clinical decision making (35), this has not fully been elucidated yet for our marker selection. Draht et al. (36) emphasized the importance of precise optimization of the chosen assay to measure DNA methylation levels. They showed that the prognostic value of a DNA methylation marker was not affected by using different techniques if the chosen assay was adequately optimized. Reporting information on optimization of the used assay to measure DNA methylation is therefore of high importance. Yet, none of the selected articles in our review discussed the optimization of the used method in sufficient detail. Different sources of DNA were often assessed for DNA methylation using the same conditions and cut-off values, possibly leading to skewed sensitivity and specificity of the assay. The lack of a standardized cut-off for the assessment of methylation levels makes DNA methylation research challenging. Marketed DNA methylation assays use standardized cut-off values and often rely on a biomarker panel instead of a single gene (37). Similarly, the included studies in this review indicated increased sensitivity of markers when combined in a marker panel. For example, the sensitivity of the individual DNA methylation markers HOXA9, HTR1B, and RASSF1 described by Sangtani et al. (26) ranged from 37.8 to 40% but increased to 60% when combined in a marker panel without any impact on specificity. Combining DNA methylation markers with genetic alterations, such as DNA mutations or copy number alterations, seems another attractive approach to strengthen minimally invasive cancer detection (13, 26, 38, 39). Genetic alterations provide a binary readout and may complement DNA methylation markers. A strategy for completely non-invasive EC detection could be the use of urine as an alternative DNA source, which has already been proven feasible for cervical cancer detection (40, 41). Considering the anatomical proximity of the uterus and cervix, urine could also be a valuable specimen for EC detection. Notably, the presence of EC-specific microRNAs in urine has been demonstrated previously (42, 43). 3
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