92 Chapter 4 each sample type tested when comparing endometrial cancers with healthy controls. ADCYAP1 and CDH13 performed particularly well in urine samples, with AUC values of 0.83 and 0.90, respectively. In line with previous studies (17, 24, 32), CDO1 allowed endometrial cancer detection with excellent performance in all sample types tested (AUC 0.90-0.96). HAND2 performed moderately in all sample types (AUC 0.62-0.71), as opposed to previously described performances of 0.91 and 0.96 in vaginal swabs of stage IA and stage IB/II/III patients, respectively (23). On the other hand, the diagnostic performance of HAND2 found in our study is more similar to the AUC of 0.77 in cervical scrapes as reported by Liew et al. (24). GALR1 performed best in urine (AUC 0.79) but did not reach the AUC value of 0.93 as found in vaginal swabs by Doufekas et al. (21). The methylation markers included in our study were originally discovered on tissue material, rather than the presently tested samples types, which could explain differences in their performance. It has been demonstrated that endometrial cancer is detectable in cervical scrapes, vaginal tampons, vaginal brushes and urine by DNA methylation analysis (20, 23, 25, 33-37). The collection of paired samples in the present study allowed a comprehensive comparison of their diagnostic performance. Most methylation marker levels correlated moderately to strongly between the different sample types, except for CDH13, GALR1 and HAND2, which correlated poorly when comparing urine and self-samples with cervical scrapes. Among these markers, a clear difference in performance was observed for CDH13 in the different sample types, with an AUC value of 0.90 in urine as opposed to AUC values of 0.69 and 0.65 in self-samples and scrapes. These results indicate that methylation markers may not have equal performance in different sample types, which could be explained by the differences in background DNA of each sample type and the source of methylated DNA. While mostly shedded endometrial material is collected by self-samples and scrapes, the full void urine contains both shedded material and transrenally excreted cell-free DNA (25). The application of previously discovered methylation markers in endometrial carcinomas of non-endometrioid histologies has remained largely unexplored, as previous studies included mostly endometrioid carcinomas. Even though non-endometrioid carcinomas are rare, early detection of this aggressive subtype is critical as they have a higher risk to metastasize and a substantially worse prognosis (38). Our study revealed differences in DNA methylation changes between endometrioid and non-endometrioid cancers. While endometrioid carcinomas showed increased methylation of all methylation markers, the non-endometrioid carcinomas showed particularly increased CDO1 and GHSR methylation, followed by BHLHE22. Interestingly, CDO1 and GHSR are also known as pancancer markers, as they are described to be highly methylated in many human cancers
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