214 Chapter 8 acid composition in urine (48, 49). The low levels of tumor-derived cfDNA in the urine call for biomarkers that are unique to cancer cells. 8.2.3 Methylation changes in non-cancerous conditions Methylation levels can be elevated due to non-cancerous conditions, such as aging, smoking, and the presence of benign conditions (e.g. benign ovarian mass or lung nodule), potentially leading to false-positive results (50-52). The strong correlation between aging and changes in DNA methylation underlines the importance of including age-matched control groups (53). While the case-control studies described in this thesis all included age-matched healthy control groups, patients with a benign ovarian mass were only taken along in Chapter 5. An important finding of this chapter was that most methylation markers displayed similar methylation levels in the urine of patients with benign and malignant ovarian masses. Knowledge of changes in DNA methylation levels in the urine of individuals with benign conditions is essential to ensure a high specificity for cancer detection in a clinical setting. While other chapters on endometrial and lung cancer did not include patients with benign conditions, most methylation markers were selected from epigenome-wide discovery screens using tissue specimens of both cancerous and benign conditions (23, 27, 28, 54-56). It is expected that the selected methylation markers for endometrial and lung cancer detection also discriminate between cancerous and benign conditions in the urine, although this remains to be confirmed in future studies. The methylation markers tested in Chapters 6 and 7 were also analyzed for NSCLC detection in plasma and urine by others (29, 57) and further developed for our studies in collaboration with this research team. Liu and colleagues included a relatively small group of urine control samples from 27 age-matched controls with benign pulmonary nodules. They showed that the majority of controls showed no detectable methylation of CDO1, SOX17, and TAC1, and methylation levels were independent of age and smoking pack years (29). To confirm the diagnostic accuracy of DNA methylation testing in urine in future studies, it is essential to include suitable control groups. Challenges of cancer-specific DNA methylation marker testing in urine • Individual methylation markers may not be cancer-type-specific. • Urine may contain extremely low concentrations of tumor-derived nucleic acids in some cases which poses analytical challenges. • Changes in methylation may occur due to aging or the presence of benign conditions underlining the importance of including suitable control groups during test development.
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