14 Chapter 1 as inflammation, tissue trauma, and cancer (48). The abundance of tumor-derived DNA in the blood of cancer patients is described to be highly variable (0.01-90% of total cfDNA) and differs per tumor type and stage, depending on tumor localization and vascularization (49, 50). The presence of cfDNA is also largely determined by degradation and clearance rates (51). The described half-life of cfDNA ranges from 16 minutes to 2.5 hours (52). During the last decades, several routes of cfDNA clearance have been proposed. Yet, the exact underlying mechanisms remain elusive as this complex process involves multiple filtering organs. It is thought that the majority (71-85%) of nucleosomes are removed from circulation by the liver (53), followed by transrenal glomerular excretion and absorption in the spleen (54, 55). Enzymes that degrade DNA, known as deoxyribonucleases, play a role in generating cfDNA during cell death and clearing cfDNA in the bloodstream (56). The cfDNA release and clearance mechanisms are summarized in Figure 2. Apoptosis Necrosis Active secretion Cell-free DNA release Cell-free DNA clearance Liver Kidneys Extracellular vesicle White blood cell Red blood cell Platelet Tumor cell Normal cell-free DNA Tumor cell-free DNA Nucleosome Deoxyribonucleases Spleen Figure 2: Cell-free DNA release and clearance mechanisms. Normal and tumor-derived cell-free DNA is released into the bloodstream by cell death (apoptosis or necrosis) and active secretion. Cell-free DNA is cleared from the circulation by absorption in the liver and spleen, transrenal excretion through the kidneys, and nuclease digestion by deoxyribonucleases. Created with BioRender.com. Transrenal excretion of cfDNA was first described by Botezatu and colleagues in 2000 (57). In this pioneer study, human DNA and radioactively labeled DNA were injected in mice and both were detected in the urine. They also described the presence of Y-chromosomal DNA in the urine of women carrying a male fetus and women who were transfused with blood from a male donor. Furthermore, KRAS gene mutations were detected in the urine of patients diagnosed with colorectal cancer whose tissue
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