33 Non-invasive detection of endometrial cancer by DNA methylation analysis in urine BACKGROUND Endometrial cancer (EC) is the most common gynecological cancer in developed countries and the sixth most common cancer worldwide (1). Its incidence is rising globally (2) with over 380,000 new cases and 89,929 deaths reported in 2018 (3). The increasing incidence of EC is partly attributable to the rise in the prevalence of risk factors associated with EC development, like obesity (4, 5). Despite the rising incidence of EC and proven value of early diagnosis, no screening program for EC exists (6, 7). In addition, if EC is suspected, invasive biopsy procedures remain necessary in routine clinical practice to detect EC in symptomatic women. Besides, the opportunity to detect EC in asymptomatic women by cytological evaluation of cervical scrapes during cervical cancer screening programs will be missed by the transition toward a primary high-risk human papillomavirus screening approach in many countries. Hence, there is a need to detect EC using less invasive sampling methods, combined with the analysis of cancer-specific markers (6). One of the emerging biomarkers for early cancer detection is DNA methylation, which involves the addition of a methyl group to a cytosine-guanine dinucleotide (CpG). Altered DNA methylation is a common epigenetic event that occurs during the early stages of carcinogenesis of many cancer types, including EC, and has been linked to gene silencing of tumor suppressor genes. Testing for elevated DNA methylation levels of specific genes is promising in early cancer detection (8). Previous studies have shown that aberrant EC-specific DNA methylation signatures can be measured in various minimally-invasive sample types, including cervical scrapes (912), endometrial brushes (13), vaginal swabs (14, 15) and vaginal tampons (16, 17). The ability to detect EC in cervicovaginal samples implicates shedding of endometrial cells and cell fragments into the lower genital tract, and, potentially, also into the urine. Apart from cellular tumor DNA, tumor-derived DNA can be released into the bloodstream as cell free DNA (cfDNA) and pass to the urine by filtration through transrenal excretion (18, 19). The suitability of EC detection in urine has been supported by the presence of EC-specific micro-RNAs in urine (20, 21). The measurement of DNA methylation markers in urine, has been proven useful for the detection of cervical cancer (22, 23), as well as other cancers, including bladder (24-27), lung (28), and prostate cancer (29-32). However, to the best of our knowledge, no such approach has been investigated for the detection of EC. 2
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