221 Summary and general discussion conventional short-read sequencing, typically shorter fragments are found to be more abundant in the urine of cancer patients as compared to controls (88, 89). Nevertheless, this area of research continues to be actively explored as long-read sequencing revealed that a similar tumor fraction was found in larger fragments of over 300 base pairs (90). 8.4.3 Circulating histones and post-translational histone modifications Circulating histone proteins also carry valuable information for the detection of various cancer types (93-95). Changes in circulating histone levels and post-translational modifications on histone tails that modulate chromatin compaction (e.g. acetylation, methylation, phosphorylation) represent promising biomarker candidates as both are related to carcinogenesis and are measurable in liquid biopsies (95, 96). Circulating histones tend to be more stable as compared to cfDNA and can be detected using easy and established methods, such as ELISA (93). Yet, while histones are found in the urine, their use is currently unexplored and it is unclear whether these histones are translocated from the blood or locally shedded by cells of the genitourinary tract (85). 8.4.4 Circulating RNA Circulating RNA provides another promising biomarker class for cancer detection in liquid biopsies. Different types of circulating RNA include messenger RNAs, microRNAs, long non-coding RNAs, and circular RNAs. RNA molecules are typically stable due to their structural characteristics and/or the protection provided by enrichment in vesicles, such as exosomes (97, 98). The value of circulating microRNAs as cancer biomarkers in urine has been described for several cancer types, including endometrial (99, 100) and ovarian (100) cancer. Urine-derived expression profiles of long non-coding RNAs and circular RNAs are explored for the detection of urological cancers but remain limitedly investigated for non-urological cancer types (97, 101, 102). Currently, our lab is developing a long-read sequencing workflow applicable to urine samples to assess the use of circular RNAs as biomarkers for cervical cancer detection. Post-transcriptional RNA modifications, such as RNA methylation, present another avenue for detecting cancer but have remained unexplored in liquid biopsy (103). The integration of DNA and RNA analyses from a single urine sample could be facilitated by using the urine sediment for methylation measurements and the urine supernatant for RNA analysis, as shown previously for prostate cancer detection (104). Alternatively, cell-free RNA (cfRNA) and cfDNA could be extracted simultaneously from a single sample and subsequently be divided for independent cfRNA and cfDNA sequencing analyses. Direct comparisons have shown that cfRNA provides a more sensitive measurement of certain biomarkers, including mutations and fusion genes (105). 8
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