Circulating free DNA

Circulating nucleic acids were first discovered by Mandel and Metais in 1948.{{cite journal | vauthors = Mandel P, Metais P | title = Les Acides Nucléiques Du Plasma Sanguin Chez l'Homme | journal = Comptes Rendus des Séances de la Société de Biologie et de ses Filiales | volume = 142 | issue = 3–4 | pages = 241–3 | date = February 1948 | pmid = 18875018 }} 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{{cite journal | vauthors = Tan EM, Schur PH, Carr RI, Kunkel HG | title = Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus | journal = The Journal of Clinical Investigation | volume = 45 | issue = 11 | pages = 1732–40 | date = November 1966 | pmid = 4959277 | pmc = 292857 | doi = 10.1172/jci105479 }} and later it was determined that the levels of cfDNA are elevated in over half of cancer patients.{{cite journal | vauthors = Leon SA, Shapiro B, Sklaroff DM, Yaros MJ | title = Free DNA in the serum of cancer patients and the effect of therapy | journal = Cancer Research | volume = 37 | issue = 3 | pages = 646–50 | date = March 1977 | pmid = 837366 }} 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.{{cite journal |vauthors= Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun M |title= Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia |journal= British Journal of Haematology |volume= 86 |issue= 4 |pages= 774–779 |date= April 1994 |pmid= 7918071 |doi= 10.1111/j.1365-2141.1994.tb04828.x |s2cid= 26365875 }}{{cite journal | vauthors = Vasioukhin V, Stroun M, Maurice P, Lyautey J, Lederrey C, Anker P | title = K-ras point mutations in the blood plasma DNA of patients with colorectal tumors | journal = Challenges of Modern Medicine: Biotechnology Today| volume = 5 |pages = 141–150 | date = May 1994 }}

The ability to extract circulating tumor DNA (ctDNA) from the human plasma has led to huge advancements in noninvasive cancer detection.{{cite journal | vauthors = Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL | title = Soluble normal and mutated DNA sequences from single-copy genes in human blood | journal = Cancer Epidemiology, Biomarkers & Prevention | volume = 3 | issue = 1 | pages = 67–71 | date = January 1994 | pmid = 8118388 }} 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.{{cite journal | vauthors = Arneth B | title = Update on the types and usage of liquid biopsies in the clinical setting: a systematic review | journal = BMC Cancer | volume = 18 | issue = 1 | pages = 527 | date = May 2018 | pmid = 29728089 | pmc = 5935950 | doi = 10.1186/s12885-018-4433-3 | doi-access = free }} This type of biopsy is noninvasive and allows for the routine clinical screening that is important in determining cancer relapse after initial treatment.{{cite journal | vauthors = Babayan A, Pantel K | title = Advances in liquid biopsy approaches for early detection and monitoring of cancer | journal = Genome Medicine | volume = 10 | issue = 1 | pages = 21 | date = March 2018 | pmid = 29558971 | pmc = 5861602 | doi = 10.1186/s13073-018-0533-6 | doi-access = free }}

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.{{cite journal | vauthors = Lood C, Blanco LP, Purmalek MM, Carmona-Rivera C, De Ravin SS, Smith CK, Malech HL, Ledbetter JA, Elkon KB, Kaplan MJ | title = Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease | journal = Nature Medicine | volume = 22 | issue = 2 | pages = 146–53 | date = February 2016 | pmid = 26779811 | pmc = 4742415 | doi = 10.1038/nm.4027 }} 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.{{cite journal | vauthors = Yang D, Oyaizu Y, Oyaizu H, Olsen GJ, Woese CR | title = Mitochondrial origins | journal = Proc Natl Acad Sci U S A | volume = 82 | issue = 13 | pages = 4443–7 | date = July 1985 | pmid = 3892535 | pmc = 391117 | doi = 10.1073/pnas.82.13.4443 | bibcode = 1985PNAS...82.4443Y | doi-access = free}} 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 {{cite journal | vauthors = Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K, Hauser CJ | title = Circulating mitochondrial DAMPs cause inflammatory responses to injury | journal = Nature | volume = 464 | issue = 7285 | pages = 104–7 | date = March 2010 | pmid = 20203610 | pmc = 2843437 | doi = 10.1038/nature08780 | bibcode = 2010Natur.464..104Z }} unless coupled to carrier proteins, mtDNA, but not nuclear DNA, can be recognized as a danger-associated molecular pattern inducing pro-inflammation through TLR9.{{cite journal | vauthors = Collins LV, Hajizadeh S, Holme E, Jonsson IM, Tarkowski A | title = Endogenously oxidized mitochondrial DNA induces in vivo and in vitro inflammatory responses | journal = Journal of Leukocyte Biology | volume = 75 | issue = 6 | pages = 995–1000 | date = June 2004 | pmid = 14982943 | doi = 10.1189/jlb.0703328 | s2cid = 6180899 }} 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.

MtDNA, in contrast to nuclear DNA, is characterized by elevated basal levels of 8-OHdG, a marker of oxidative damage. The high content of oxidative damage in mtDNA is attributed to the close proximity of mtDNA to Reactive oxygen species (ROS) and relatively inefficient DNA repair mechanisms that can lead to the accumulation of DNA lesions.{{cite book | vauthors = Clayton DA, Doda JN, Friedberg EC | title = Molecular Mechanisms for Repair of DNA | chapter = Absence of a Pyrimidine Dimer Repair Mechanism for Mitochondrial DNA in Mouse and Human Cells | series = Basic Life Sciences | volume = 5B | pages = 589–91 | date = 1975 | pmid = 1238079 | doi = 10.1007/978-1-4684-2898-8_26 | doi-broken-date = 1 November 2024 | isbn = 978-1-4684-2900-8 }}

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. Oxidized mtDNA generated during programmed cell death is not limited to activate TLR9, but was shown to also engage the NLRP3 inflammasome, leading to the production of pro-inflammatory cytokines, IL-1β, and IL-18.{{cite journal | vauthors = Shimada K, Crother TR, Karlin J, Dagvadorj J, Chiba N, Chen S, Ramanujan VK, Wolf AJ, Vergnes L, Ojcius DM, Rentsendorj A, Vargas M, Guerrero C, Wang Y, Fitzgerald KA, Underhill DM, Town T, Arditi M | title = Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis | journal = Immunity | volume = 36 | issue = 3 | pages = 401–14 | date = March 2012 | pmid = 22342844 | pmc = 3312986 | doi = 10.1016/j.immuni.2012.01.009 }} 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.{{cite journal | vauthors = West AP, Khoury-Hanold W, Staron M, Tal MC, Pineda CM, Lang SM, Bestwick M, Duguay BA, Raimundo N, MacDuff DA, Kaech SM, Smiley JR, Means RE, Iwasaki A, Shadel GS | title = Mitochondrial DNA stress primes the antiviral innate immune response | journal = Nature | volume = 520 | issue = 7548 | pages = 553–7 | date = April 2015 | pmid = 25642965 | pmc = 4409480 | doi = 10.1038/nature14156 | bibcode = 2015Natur.520..553W }}

cfDNA purification is prone to contamination through genomic DNA due to ruptured blood cells during the purification process.{{cite journal | vauthors = Lui YY, Chik KW, Chiu RW, Ho CY, Lam CW, Lo YM | title = Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation | journal = Clinical Chemistry | volume = 48 | issue = 3 | pages = 421–7 | date = March 2002 | doi = 10.1093/clinchem/48.3.421 | pmid = 11861434 | doi-access = free }} Because of this, different purification methods can lead to significantly different cfDNA extraction yields.{{cite journal | vauthors = Page K, Guttery DS, Zahra N, Primrose L, Elshaw SR, Pringle JH, Blighe K, Marchese SD, Hills A, Woodley L, Stebbing J, Coombes RC, Shaw JA | title = Influence of plasma processing on recovery and analysis of circulating nucleic acids | journal = PLOS ONE | volume = 8 | issue = 10 | pages = e77963 | date = 2013-10-18 | pmid = 24205045 | pmc = 3799744 | doi = 10.1371/journal.pone.0077963 | bibcode = 2013PLoSO...877963P | doi-access = free }}{{cite journal | vauthors = Barták BK, Kalmár A, Galamb O, Wichmann B, Nagy ZB, Tulassay Z, Dank M, Igaz P, Molnár B | title = Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection | journal = Pathology & Oncology Research | volume = 25 | issue = 3 | pages = 915–923 | date = January 2018 | pmid = 29374860 | doi = 10.1007/s12253-018-0382-z | s2cid = 24629831 }} 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.{{cite journal | vauthors = Pérez-Barrios C, Nieto-Alcolado I, Torrente M, Jiménez-Sánchez C, Calvo V, Gutierrez-Sanz L, Palka M, Donoso-Navarro E, Provencio M, Romero A | title = Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing | journal = Translational Lung Cancer Research | volume = 5 | issue = 6 | pages = 665–672 | date = December 2016 | pmid = 28149760 | pmc = 5233878 | doi = 10.21037/tlcr.2016.12.03 | doi-access = free }}

In general, the detection of specific DNA sequences in cfDNA can be done by two means; sequence specific detection (PCR based) and general genomic analysis of all cfDNA present in the blood (DNA sequencing).{{cite journal | vauthors = Volik S, Alcaide M, Morin RD, Collins C | title = Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies | journal = Molecular Cancer Research | volume = 14 | issue = 10 | pages = 898–908 | date = October 2016 | pmid = 27422709 | doi = 10.1158/1541-7786.MCR-16-0044 | url = http://mcr.aacrjournals.org/content/14/10/898 | doi-access = free | url-access = subscription }} The presence of cfDNA containing DNA from tumor cells was originally characterized using PCR amplification of mutated genes from extracted cfDNA. PCR based analysis of cfDNA typically rely on the analytical nature of qPCR and digital PCR. Both of these techniques can be sensitive and cost-effective for detecting limited number of hotspots mutations. For this reason the PCR based method of detection is still very prominent tool in cfDNA detection. This method has the limitation of not being able to detect larger structural variant present in ctDNA and for this reason massively parallel next generation sequencing is also used to determine ctDNA content in cfDNA

Massively parallel sequencing (MPS) has allowed the deep sequencing of cfDNA. This deep sequencing is required to detect mutant circulating tumor DNA (ctDNA) present in low concentrations in the plasma. Two main sequencing techniques are typically used for targeted analysis of mutant cfDNA; PCR amplicon sequencing{{cite journal | vauthors = Forshew T, Murtaza M, Parkinson C, Gale D, Tsui DW, Kaper F, Dawson SJ, Piskorz AM, Jimenez-Linan M, Bentley D, Hadfield J, May AP, Caldas C, Brenton JD, Rosenfeld N | title = Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA | journal = Science Translational Medicine | volume = 4 | issue = 136 | pages = 136ra68 | date = May 2012 | pmid = 22649089 | doi = 10.1126/scitranslmed.3003726 | s2cid = 34723244 }} and hybrid capture sequencing.{{cite journal | vauthors = Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, Liu CL, Neal JW, Wakelee HA, Merritt RE, Shrager JB, Loo BW, Alizadeh AA, Diehn M | title = An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage | journal = Nature Medicine | volume = 20 | issue = 5 | pages = 548–54 | date = May 2014 | pmid = 24705333 | pmc = 4016134 | doi = 10.1038/nm.3519 }}

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.

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.{{cite journal | vauthors = Schwarzenbach H, Hoon DS, Pantel K | title = Cell-free nucleic acids as biomarkers in cancer patients | journal = Nature Reviews. Cancer | volume = 11 | issue = 6 | pages = 426–37 | date = June 2011 | pmid = 21562580 | doi = 10.1038/nrc3066 | s2cid = 6061607 }} 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.{{cite journal | pmid=31614115 | doi=10.1016/j.ccell.2019.09.003 | volume=36 | issue=4 | pages=350–368 | title=Toward the early detection of cancer by decoding the epigenetic and environmental fingerprints of cell-free DNA. | year=2019 | author=van der Pol Y, Mouliere F | journal=Cancer Cell| doi-access=free }}{{cite journal | vauthors = Mouliere F, Smith CG, Heider K, Su J, van der Pol Y, Thompson M, Morris J, Wan JM, Chandrananda D, Hadfield J, Grzelak M, Hudecova I, Couturier DL, Cooper W, Zhao H, Gale D, Eldridge M, Watts C, Brindle K, Rosenfeld N, Mair R | title = Fragmentation patterns and personalized sequencing of cell-free DNA in urine and plasma of glioma patients | journal = EMBO Mol Med | volume = 13 | issue = 8 | pages = e12881 | date = August 2021 | pmid = 34291583 | pmc = 8350897 | doi = 10.15252/emmm.202012881 |doi-access = free}}{{cite journal | vauthors = Eibl RH, Schneemann M | title = Cell-free DNA as a biomarker in cancer | journal = Extracell Vesicles Circ Nucleic Acid | volume = 3 | pages = 178–98| date = August 2022| issue = 3 | doi = 10.20517/evcna.2022.20 |doi-access = free| pmc = 11648514 }}

Elevated cfDNA has been detected with acute blunt trauma{{cite journal | vauthors = Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA | title = Plasma DNA as a prognostic marker in trauma patients | journal = Clinical Chemistry | volume = 46 | issue = 3 | pages = 319–23 | date = March 2000 | doi = 10.1093/clinchem/46.3.319 | pmid = 10702517 | doi-access = free }} and burn victims.{{cite journal | vauthors = Chiu TW, Young R, Chan LY, Burd A, Lo DY | title = Plasma cell-free DNA as an indicator of severity of injury in burn patients | journal = Clinical Chemistry and Laboratory Medicine | volume = 44 | issue = 1 | pages = 13–7 | date = 2006 | pmid = 16375578 | doi = 10.1515/CCLM.2006.003 | s2cid = 37876738 }} 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.

It has been shown that an increase cfDNA in the plasma of ICU patients is an indicator of the onset of sepsis.{{cite journal | vauthors = Rhodes A, Wort SJ, Thomas H, Collinson P, Bennett ED | title = Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients | journal = Critical Care | volume = 10 | issue = 2 | pages = R60 | date = 2006 | pmid = 16613611 | pmc = 1550922 | doi = 10.1186/cc4894 | doi-access = free }}{{cite journal | vauthors = Martins GA, Kawamura MT, Carvalho M | title = Detection of DNA in the plasma of septic patients | journal = Annals of the New York Academy of Sciences | volume = 906 | issue = 1 | pages = 134–40 | date = April 2000 | pmid = 10818609 | doi = 10.1111/j.1749-6632.2000.tb06603.x | bibcode = 2000NYASA.906..134M | s2cid = 36198236 }} 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.

Patients showing signs of myocardial infarction have been shown to have elevated cfDNA levels.{{cite journal | vauthors = Chang CP, Chia RH, Wu TL, Tsao KC, Sun CF, Wu JT | title = Elevated cell-free serum DNA detected in patients with myocardial infarction | journal = Clinica Chimica Acta; International Journal of Clinical Chemistry | volume = 327 | issue = 1–2 | pages = 95–101 | date = January 2003 | pmid = 12482623 | doi = 10.1016/S0009-8981(02)00337-6 }} This elevation correlates to patient outcome in terms of additional cardiac issues and even mortality within two years.{{cite journal | vauthors = Rainer TH, Lam NY, Man CY, Chiu RW, Woo KS, Lo YM | title = Plasma beta-globin DNA as a prognostic marker in chest pain patients | journal = Clinica Chimica Acta; International Journal of Clinical Chemistry | volume = 368 | issue = 1–2 | pages = 110–3 | date = June 2006 | pmid = 16480967 | doi = 10.1016/j.cca.2005.12.021 }}

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.{{cite journal | vauthors = Beck J, Oellerich M, Schulz U, Schauerte V, Reinhard L, Fuchs U, Knabbe C, Zittermann A, Olbricht C, Gummert JF, Shipkova M, Birschmann I, Wieland E, Schütz E | title = Donor-Derived Cell-Free DNA Is a Novel Universal Biomarker for Allograft Rejection in Solid Organ Transplantation | journal = Transplantation Proceedings | volume = 47 | issue = 8 | pages = 2400–3 | date = October 2015 | pmid = 26518940 | doi = 10.1016/j.transproceed.2015.08.035 }} 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.

Besides dd-cfDNA in plasma, some research also focused on the excretion of ddcfDNA through urine. This is of special interest in kidney allografts transplantation.

When dd-cfDNA is measured using targeted next-generation sequencing, assays were used with a population specific genome wide SNP panel.{{cite journal | vauthors = Grskovic M | title = Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients | journal = The Journal of Molecular Diagnostics | volume = 18 | issue = 6 | pages = 890–902 | date = November 2016 | pmid = 27727019 | doi = 10.1016/j.jmoldx.2016.07.003 | doi-access = free }} Attaching barcodes to the ligated adapters prior to NGS during library preparation make absolute ddcfDNA quantification possible without the need for prior donor genotyping.{{cite journal | vauthors = Kueng N, Arcioni S, Sandberg F, Kuhn C, Banz V, Largiadèr CR, Sidler D, Amstutz U | display-authors = 6 | title = Comparison of methods for donor-derived cell-free DNA quantification in plasma and urine from solid organ transplant recipients | journal = Frontiers in Genetics | volume = 14 | pages = 1089830 | date = 2023 | pmid = 36777723 | pmc = 9916053 | doi = 10.3389/fgene.2023.1089830 | doi-access = free }} This has been shown to provide additional clinical benefits if the absolute number of cfDNA copies is considered combined together with the fraction of ddcfDNA over cfDNA from the recipient to determine whether the allograft is being rejected or not.

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.{{cite journal | vauthors = Duvvuri B, Lood C | title = Cell-Free DNA as a Biomarker in Autoimmune Rheumatic Diseases | journal = Frontiers in Immunology | volume = 10 | pages = 502 | date = 2019-03-19 | pmid = 30941136 | pmc = 6433826 | doi = 10.3389/fimmu.2019.00502 | doi-access = free }} 50px Material was copied from this source, which is available under a [https://creativecommons.org/licenses/by/4.0/ Creative Commons Attribution 4.0 International License].

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).{{cite journal | vauthors = Pinzani P, Salvianti F, Pazzagli M, Orlando C | title = Circulating nucleic acids in cancer and pregnancy | journal = Methods | volume = 50 | issue = 4 | pages = 302–7 | date = April 2010 | pmid = 20146940 | doi = 10.1016/j.ymeth.2010.02.004 }}

• [https://generegulation.org/nucposdb/ NucPosDB: a database of nucleosome positioning in vivo and nucleosomics of cell-free DNA]

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Category:DNA