213 Summary and general discussion as it can be collected non-invasively in large volumes, which allows for serial sampling from home at high patient acceptance (9). However, despite clear advantages, DNA methylation marker testing in urine faces several challenges, including 1) the identification of cancertype-specific methylation markers, 2) the low quantity of tumor-derived nucleic acids in urine, and 3) methylation changes in non-cancerous conditions. 8.2.1 The identification of cancer-type-specific methylation markers Biomarkers that allow the detection of multiple cancer types are known as pan-cancer markers. For example, in this thesis, the methylation marker CDO1 appeared valuable for the detection of endometrial (Chapter 4), ovarian (Chapter 5), and lung (Chapters 6 and 7) cancer. CDO1 gene methylation has also been described by others for a wide variety of cancer types, including bladder (17), brain (18), breast (17, 19), bile duct (20, 21), colorectal (22), endometrial (23, 24), gallbladder (25), gastric (17, 26), lung (17, 27-29), oesophageal (17, 30), ovarian (31), pancreatic (32), prostate (33), renal cell (34), and small bowel cancer (35). From an implementation perspective, pan-cancer markers might be most applicable in a targeted setting to rule out malignancies in a certain high-risk group. When implemented into a population-wide screening setting, cancer localization after detection forms a critical test element. To this end, pan-cancer methylation markers could potentially be combined with tissue-specific methylation markers to determine the primary tumor site (36-38). Previous work has shown that methylation patterns derived from urine cell-free (cfDNA) can serve as signatures to determine their tissue of origin (39). For plasma cfDNA, it has been shown that tumor type, molecular subtype, and histological classification can be derived from changes in DNA methylation (40-43). Identifying the primary tumor site and other tumor characteristics from urinary cfDNA would extend the use of urine for screening and diagnostics. 8.2.2 Low quantity of tumor-derived nucleic acids in urine The low abundance of tumor-derived cfDNA in urine poses analytical challenges. Generally, the concentrations of tumor-derived nucleic acids in liquid biopsies can be extremely low in some cases, depending on disease stage, cancer type, and treatment (44). Total tumor-derived cfDNA levels can be particularly low in patients with earlystage disease or patients diagnosed with poorly shedding cancer types, such as renal cancer or glioma (45, 46). Treatment with senescence-inducing cytotoxic therapies may also reduce the release of tumor DNA (47). The tumor-derived cfDNA fraction in urine is often further diluted by the large volumes of voided urine and the presence of nontumor-derived nucleic-acids. Nucleic acids in the urine predominantly originate from non-malignant cells, considering the presence of intact urinary tract and inflammatory cells. Moreover, the continuous release of cfDNA from virtually any tissue type, coupled with its translocation into the urine, further contributes to the heterogeneous nucleic 8
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