Circulating free DNA

Circulating nucleic acids were first discovered by Mandel and Metais in 1948. It was later discovered that the level of cfDNA is significantly increased in the plasma of diseased patients. This discovery was first made in lupus patients and later it was determined that the levels of cfDNA are elevated in over half of cancer patients. Molecular analysis of cfDNA resulted in an important discovery that blood plasma DNA from cancer patients contains tumor-associated mutations and it can be used for cancer diagnostics and follow up. The ability to extract circulating tumor DNA (ctDNA) from the human plasma has led to huge advancements in noninvasive cancer detection. Most notably, it has led to what is now known as liquid biopsy. In short, liquid biopsy is using biomarkers and cancer cells in the blood as a means of diagnosing cancer type and stage. This type of biopsy is noninvasive and allows for the routine clinical screening that is important in determining cancer relapse after initial treatment. == Different origins of cfDNA ==

The intracellular origin of cfDNA, e.g., either from nucleus or mitochondria, can also influence the inflammatory potential of cfDNA. mtDNA is a potent inflammatory trigger. mtDNA, due to its prokaryotic origin, holds many features that are similar to bacterial DNA, including the presence of a relatively high content of unmethylated CpG motifs, which are rarely observed in nuclear DNA. The unmethylated CpG motifs are of particular importance as TLR9, the only endolysosomal DNA-sensing receptor, has a unique specificity for unmethylated CpG DNA. mtDNA was shown to activate neutrophils through TLR9 engagement unless coupled to carrier proteins, mtDNA, but not nuclear DNA, can be recognized as a danger-associated molecular pattern inducing pro-inflammation through TLR9. Collins et al. reported that intra-articular injection of mtDNA induces arthritis in vivo, proposing a direct role of mtDNA extrusion in the disease pathogenesis of rheumatoid arthritis and autoimmune rheumatic diseases. They have shown that oxidative burst during NETosis can oxidize mtDNA and the released oxidized mtDNA by itself, or in complex with TFAM, can generate prominent induction of type I IFNs. MtDNA can also be recognized by cyclic GMP-AMP synthase (cGAS), a cytosolic dsDNA sensor to initiate a STING-IRF3-dependent pathway that in turn orchestrates the production of type I IFNs. == Methods ==

Collection and purification cfDNA purification is prone to contamination through genomic DNA due to ruptured blood cells during the purification process. Because of this, different purification methods can lead to significantly different cfDNA extraction yields. At the moment, typical purification methods involve collection of blood via venipuncture, centrifugation to pellet the cells, and extraction of cfDNA from the plasma. The specific method for extraction of cfDNA from the plasma depends on the protocol desired. Analysis of cfDNA Polymerase chain reaction In general, the detection of specific DNA sequences in cfDNA can be done by two means; sequence specific detection (using polymerase chain reaction, commonly called PCR) and general genomic analysis of all cfDNA present in the blood (DNA sequencing). The presence of cfDNA containing DNA from tumor cells was originally characterized using PCR amplification of mutated genes from extracted cfDNA. and hybrid capture sequencing. Other forms of genetic alterations can be analysed using ctDNA (e.g. somatic copy number alterations or genetic rearrangements). Here, methods based on untargeted sequencing, like WGS or low coverage WGS, are mainly used. Numerous epigenetic features of the tissue of origin may be extracted from cfDNA WGS, i.e. nucleosome spacing and DNA methylation state. == cfDNA and illness ==

Cancer The majority of cfDNA research is focused on DNA originating from cancer (ctDNA). In short, the DNA from cancer cells gets released by cell-death, secretion or other mechanisms still not known. The fraction of cfDNA released by tumor cells in circulation is influenced by the size of the tumor as well as the tumor stage and type. Early stage cancers and brain tumor are among the most difficult to detect with liquid biopsy. Trauma Elevated cfDNA has been detected with acute blunt trauma and burn victims. In both of these cases cfDNA concentration in the plasma were correlated to the severity of the injury, as well as outcome of the patient. Sepsis It has been shown that an increase cfDNA in the plasma of ICU patients is an indicator of the onset of sepsis. Due to the severity of sepsis in ICU patients, further testing in order to determine the scope of cfDNA efficacy as a biomarker for septic risk is likely. This elevation correlates to patient outcome in terms of additional cardiac issues and even mortality within two years. Transplant graft rejection Foreign cfDNA has been shown to be present in the plasma of solid organ transplant patients. This cfDNA is derived from the grafted organ and is termed dd-cfDNA (donor-derived cell-free DNA). Dd-cfDNA values spike initially after a transplant procedure (>5%) with values heavily depending on the transplanted organ and typically drop (<0.5%) within one week for most organs. If the host body rejects the grafted organ the ddcfDNA concentration in the blood (plasma) will rise to a level greater than 5-fold higher than those without complications. This increase in ddcfDNA can be detected prior to any other clinical or biochemical signs of complication. Attaching barcodes to the ligated adapters prior to NGS during library preparation make absolute ddcfDNA quantification possible without the need for prior donor genotyping. This has been shown to provide additional clinical benefits if the absolute number of cfDNA copies is considered combined with the fraction of ddcfDNA over cfDNA from the recipient to determine whether the allograft is being rejected or not. == Future directions ==

cfDNA allows a rapid, easy, non-invasive and repetitive method of sampling. A combination of these biological features and technical feasibility of sampling, position cfDNA as a potential biomarker of enormous utility for example for autoimmune rheumatic diseases and tumors. It offers also a potential biomarker with its own advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection as well as immunosuppression optimisation. However, this method lacks uniformity on the type of sample (plasma/serum/synovial fluid/urine), methods of sample collection/processing, free or cell-surface bound DNA, cfDNA extraction and cfDNA quantification, and also in the presentation and interpretation of quantitative cfDNA findings. cfDNA is quantified by fluorescence methods, such as PicoGreen staining and ultraviolet spectrometry, the more sensitive is quantitative polymerase chain reaction (PCR; SYBR Green or TaqMan) of repetitive elements or housekeeping genes, or deep sequencing methods. Circulating nucleosomes, the primary repeating unit of DNA organization in chromatin, are quantified by enzyme-linked immunosorbent assays (ELISA). == Databases ==

• NucPosDB: a database of nucleosome positioning in vivo and nucleosomics of cell-free DNA == References ==