219 Summary and general discussion Technical considerations to enhance methylation test accuracy in urine • Alternative DNA methylation markers suitable for cancer detection in urine could be discovered by methylation sequencing of urine cfDNA. • The primer and probe design quality is crucial for obtaining reproducible methylation level measurements from urine. • The extraction of cfDNA from urine can potentially be advanced by increasing the urine input volume, pooling samples from different collection time points, collecting a particular urine portion, using an optimal urine extraction kit, and selectively enriching for methylated cfDNA. 8.4 Advancing urine-based molecular tests using alternative urine biomarkers Urine-based molecular tests could be enhanced by integrating multiple biomarker classes (44). In addition to DNA methylation signatures, tumor-derived cfDNA in the urine contains information about various other genetic and epigenetic events. Moreover, urine is a rich source of alternative biomarkers beyond DNA, including circulating RNA, proteins, metabolites, and exfoliated tumor cells (Figure 2). 8.4.1 Concurrent analysis of genetic and epigenetic events Recent advances in sequencing technologies and bioinformatics analyses unlock the potential of retrieving genetic and epigenetic changes from the same sample. The recently developed MethylSaferSeqS technology allows a concurrent analysis of copy number aberrations, mutations, and methylation signals from the same cfDNA molecules (79). This innovative approach separates the original DNA strands from the copied DNA strands during library preparation, which enables the analysis of epigenetic alterations in the original strand and the recognition of genetic alterations (including cytosine-to-thymine mutations) in the copied strand. The simultaneous analysis of genetic and epigenetic markers could add sensitivity to cancer detection methods (80, 81). Another advanced sequencing platform for assessing genetic and epigenetic information from the same strand is known as Five/Six-letter-seq (82). This approach is based on enzymatic conversion and also allows the recognition of both 5-methylcytosine and 5-hydroxymethylcytosine, besides the four regular nucleotides. DNA hydroxymethylation signatures in cfDNA appeared additive to DNA methylation signatures, as shown for the detection of colorectal and pancreatic cancer in plasma (83, 84). The development of novel profiling methods to accurately capture and analyze hydroxymethylation markers could further enhance its utility for cancer detection. 8
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