215 Summary and general discussion 8.3 Technical considerations to enhance methylation test accuracy in urine Several technical optimizations could be considered to enhance methylation test accuracy in urine. The careful selection of the targeted genomic location and design of PCR-based assays are crucial to obtain clinically meaningful results. Moreover, maximizing the cfDNA yield from urine and the selective enrichment of methylated DNA could improve methylation detection. 8.3.1 Selecting a clinically relevant differentially methylated region The genomic location of the targeted PCR assay is crucial for the development of clinically relevant methylation assays. Genomic locations of the methylation markers tested in this thesis were derived from tissue methylation data, of which the majority also appeared valuable for cancer detection in patient-friendly samples. Yet, it would be interesting to investigate whether alternative DNA methylation markers suitable for cancer detection in urine could be discovered by methylation sequencing of urine cfDNA. Previous work has shown that the genome-wide methylation profiling of lavage-based self-samples enabled the discovery of a new methylation classifier for early cervical (pre)cancer detection, applicable to self-collected brush and lavage samples (58). Bisulfite sequencing of urine samples can be technically challenging due to low amounts of fragmented cfDNA and DNA degradation during sodium bisulfite treatment (59, 60). The use of bisulfite-free sequencing approaches using immunoprecipitation-based enrichment of methylated cfDNA, such as methylated cfDNA sequencing (MeD-Seq) (61), or cytosine conversion based on enzymatic processes, such as enzymatic methyl sequencing (EM-Seq) (62), could overcome this issue. In Chapter 5, the EM-Seq method was successfully applied to urine supernatant samples of ovarian cancer patients with a low quantity of tumor-derived cfDNA. Alternatively, Oxford Nanopore Technologies (ONT) long-read sequencing could be utilized for DNA methylation analysis at a singlebase, single-molecule resolution, by exploiting the differences in electric current of methylated and unmethylated cytosines (63). A technical challenge for ONT sequencing of urine samples is, however, the high cfDNA input needed, which might be particularly challenging for early-stage cancers with a low quantity of tumor-derived cfDNA. 8.3.2 Revisiting PCR-based methylation assay design Instead of looking for alternative methylation markers, test accuracy of PCR-based methylation assays could be improved by revisiting its design. The primer and probe design quality is crucial for obtaining reproducible methylation level measurements and often underlies the differences observed across studies investigating the same methylation marker (64, 65). Methylation assays described in this thesis were carefully developed in preparation for Chapters 4 to 7 to facilitate reliable cancer detection in 8
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