13 General introduction Methylated DNA remains stable after long-term storage of clinical samples and can be analyzed efficiently using relatively inexpensive methods (27, 28). Bisulfite modification of the DNA is a fundamental first step of many methylation detection workflows. By treating the extracted DNA with sodium bisulfite, methylated cytosine can be distinguished from unmethylated cytosine by selective deamination of unmethylated cytosine to uracil. Methylation levels of a specific target region can be measured precisely using a quantitative methylation-specific PCR reaction (qMSP). Methylationspecific primers only amplify methylated cytosine-containing regions, which provides a highly sensitive quantification of methylated DNA in an excess of unmethylated DNA (29, 30). Methylation analysis does not require the presence of intact tumor cells for interpretation and can also be performed on fragmented DNA that has been shed by tumors (27). This offers opportunities for methylation testing in different types of patient material (e.g. tissue and liquid biopsies). 1.3 Cell-free DNA biology Both normal and tumor cells release nucleic acids, including cell-free DNA (cfDNA), into the circulation. Although the exact origin of cfDNA in the circulation is still under debate, proposed release mechanisms include apoptotic or necrotic cell death and active secretion (31). The presence of extracellular DNA was first described by Mandel and Métais in 1948 (32). This finding remained largely unnoticed by scientists until it was related to lupus disease in 1966 (33). Toward the end of the century, the discovery of fetal DNA in maternal blood (34), cancer-derived genetic mutations (35, 36), and aberrant promoter hypermethylation of tumor suppressor genes (37, 38) in serum and plasma followed. In the past decades, there has been a steep increase in research on the molecular profiling of cfDNA derived from tumor cells, referred to as tumor-derived cfDNA, for diagnostic purposes. Tumor-derived cfDNA accurately reflects molecular alterations in the tumor tissue (39, 40) and has high potential for non-invasive cancer detection (41). Molecular analysis of tumor-derived cfDNA allows the identification of a variety of biomarkers, including, but not limited to, copy number changes, differences in fragment lengths, fusion genes, methylation, and mutations (42, 43). The specific biological properties of cfDNA are related to its origin. The size of small cfDNA fragments in the circulation varies from 140 to 170 base pairs and peaks around 167 base pairs. This size is characteristic of the length needed to wrap DNA around a histone protein (147 base pairs), forming a mono-nucleosome, and the presence of a linker DNA (20 base pairs) which protects DNA from degradation (44-46). In healthy individuals, the majority of cfDNA is derived from leukocytes (47). Generally, the amount of cfDNA in circulation is extremely low (0-100 ng/mL) but elevates under normal physiological conditions, such as exercise, and pathological conditions, such 1
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