94 Chapter 4 abnormalities, benign endometrial conditions and cancer) or patient populations with an increased risk of developing endometrial cancer. During finalization of the current article, the clinical utility of simple methylation-based tests in self-collected samples for endometrial cancer detection was also evaluated by Herzog et al. (35). Methylation levels of two regions of the GYPC gene and the ZCAIN12 gene allowed endometrial cancer detection in cervical scrapes, vaginal swabs and self-collected cervicovaginal samples with high accuracy in a cohort of women presenting with postmenopausal blood loss. Of note, the specificity in clinician-collected cervical scrapes was substantially lower (76%) as compared to present findings (92%). Nevertheless, their results are complementary to ours and independently exemplify the potential of epigenomic testing in self-collected samples. Our study is limited by the fact that the distribution of histopathological subtypes included in our study does not reflect the natural prevalence. The inclusion of patients diagnosed with endometrial cancer mainly occurred in tertiary care cancer centers treating high-grade cancers and rare histopathological subtypes. It is, however, worth noting that early detection of high-grade non-endometrioid cancers is of utmost importance given their worse prognosis (38). Samples were not collected at first clinical presentation but after endometrial cancer diagnosis was made based on a pipelle or hysteroscopic biopsy. This order would be different when home-based sampling would be applied in clinical practice. Sample collection after endometrial biopsy might have facilitated the release of tumor DNA into the urine or vaginal fluid. Yet, the influence of biopsy procedures on the presence of tumor DNA in self-collected specimen is most likely limited as the median time between biopsy and self-sampling was 37 days. In some excluded cases the complete carcinoma was biopsied with no or minimal residual cancer being found during histopathological evaluation of the uterus. It is conceivable that DNA methylation testing will be even more accurate when used at first clinical presentation. Finally, this comparative study had no access to paired samples from controls and did not include controls with postmenopausal bleeding symptoms or benign endometrial conditions. Even though others have shown that most of the markers tested in our study enable discrimination between benign endometrial pathology and cancer (17, 18, 20, 21, 23, 24, 32), this was not validated in the current study. Strengths of the current study are that nine DNA methylation markers, originated from different discovery screens, were tested on a large series of 626 samples. Over a 100 patients diagnosed with endometrial cancer were included, encompassing the full and heterogeneous range of endometrial cancer histotypes, grades and FIGO (2009) stages. Methylation marker assays were multiplexed to measure the methylation levels of three genes and a reference gene within the same reaction, without loss of PCR efficiency, to
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