Circulating free DNA
Circulating free DNA (cfDNA) are degraded DNA fragments released to the blood plasma. cfDNA can be used to describe various forms of DNA freely circulating the bloodstream, including circulating tumor DNA (ctDNA) and cell-free fetal DNA (cffDNA). Elevated levels of cfDNA are observed in cancer, especially in advanced disease. There is evidence that cfDNA becomes increasingly frequent in circulation with the onset of age. cfDNA has been shown to be a useful biomarker for a multitude of ailments other than cancer and fetal medicine. This includes but is not limited to trauma, sepsis, aseptic inflammation, myocardial infarction, stroke, transplantation, diabetes, and sickle cell disease. cfDNA is mostly a double-stranded extracellular molecule of DNA, consisting of small fragments (70 to 200 bp) and larger fragments (21 kb). and has been recognized as an accurate marker for the diagnosis of prostate cancer and breast cancer.
Other publications confirm the origin of cfDNA from carcinomas and cfDNA occurs in patients with advanced cancer. Cell‐free DNA (cfDNA) is present in the circulating plasma, also in other body fluids.
The release of cfDNA into the bloodstream appears by different reasons, including the primary tumor, tumor cells that circulate in peripheral blood, metastatic deposits present at distant sites, and normal cell types, like hematopoietic and stromal cells. Tumor cells and cfDNA circulate in the bloodstream of patients with cancer. Its rapidly increased accumulation in blood during tumor development is caused by an excessive DNA release by apoptotic cells and necrotic cells. Active secretion within exosomes has been discussed, but it is still we don't know whether this is a relevant or rather minor source of cfDNA.
cfDNA circulates predominantly as nucleosomes, which are nuclear complexes of histones and DNA. They are frequently nonspecifically elevated in cancer but may be more specific for monitoring cytotoxic cancer therapy, mainly for the early estimation of therapy efficacy.
- 1 History
- 2 Based on intracellular origin, cfDNA and immune system
- 3 Methods
- 4 cfDNA and Illness
- 5 Future directions
- 6 References
Circulated cell-free DNA was 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. This increase in cfDNA in cancer patients has been shown to be due to circulating tumor cells (CTC) traveling in the peripheral blood. 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.
Based on intracellular origin, cfDNA and immune system
The intracellular origin of cfDNA, e.g., either from nucleus or mitochondria, can also influence the inflammatory potential of cfDNA. MtDNA, nuclear DNA, 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 RA .
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 ROS and relatively inefficient DNA repair mechanisms that can lead to the accumulation of DNA lesions.
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 NRLP3 inflammasome, leading to the production of pro-inflammatory cytokines, IL-1β, and IL-18. 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.
Collection and purification
cfDNA purification is prone to contamination 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 in Plasma
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). 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 detect down to a single targeted molecule present in a sample. 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
Massively parallel sequencing (MPS) has allowed the deep sequencing of cfDNA. this deep sequencing is required to detect ctDNA present in low concentrations in the plasma. Two main sequencing techniques are typically used for analysis of cfDNA; PCR amplicon sequencing and hybrid capture sequencing.
cfDNA and Illness
The majority of cfDNA research is focused on DNA originating from circulating tumor cells (ctDNA). In short, the DNA from circulating tumor cells gets released by means that are not fully understood.
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.
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.
Patients showing signs of myocardial infraction have been shown to have elevated cfDNA levels. 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 GcfDNA. GcfDNA values spike initially after a transplant procedure (>5%) and typically drop down (<0.5%) within one week. If the host body rejects the grafted organ the GcfDNA concentration in the blood will rise to a level greater than 5-fold higher than those without complications. This increase in GcfDNA can be detected prior to any other clinical or biochemical signs of complication.
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 or tumors. However, this method lacks uniformity on the type of sample (plasma/serum/synovial fluid), 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. Circulating nucleosomes, they are the primary repeating unit of DNA organization in chromatin, are quantified by enzyme-linked immunosorbent assays (ELISA).
- Shaw JA, Stebbing J (January 2014). "Circulating free DNA in the management of breast cancer". Annals of Translational Medicine. 2 (1): 3. doi:10.3978/j.issn.2305-5839.2013.06.06. PMC 4200656. PMID 25332979.
- Gravina S, Sedivy JM, Vijg J (June 2016). "The dark side of circulating nucleic acids". Aging Cell. 15 (3): 398–9. doi:10.1111/acel.12454. PMC 4854914. PMID 26910468.
- Butt AN, Swaminathan R (August 2008). "Overview of circulating nucleic acids in plasma/serum". Annals of the New York Academy of Sciences. 1137 (1): 236–42. Bibcode:2008NYASA1137..236B. doi:10.1196/annals.1448.002. PMID 18837954.
- Gall TM, Belete S, Khanderia E, Frampton AE, Jiao LR (January 2019). "Circulating Tumor Cells and Cell-Free DNA in Pancreatic Ductal Adenocarcinoma". The American Journal of Pathology. 189 (1): 71–81. doi:10.1016/j.ajpath.2018.03.020. PMID 30558725.
- Zoli, Wainer; Silvestrini, Rosella; Amadori, Dino; Carretta, Elisa; Gunelli, Roberta; Salvi, Samanta; Calistri, Daniele; Casadio, Valentina (2013). "Urine Cell-Free DNA Integrity as a Marker for Early Prostate Cancer Diagnosis: A Pilot Study". BioMed Research International. 2013: 270457. doi:10.1155/2013/270457. PMC 3586456. PMID 23509700.
- Teo YV, Capri M, Morsiani C, Pizza G, Faria AM, Franceschi C, Neretti N (February 2019). "Cell-free DNA as a biomarker of aging". Aging Cell. 18 (1): e12890. doi:10.1111/acel.12890. PMC 6351822. PMID 30575273.
- Thakur, Zhang H, BECKER A, MATEI I, HUANG Y, COSTA-SILVA B (2014). "Double-stranded DNA in exosomes: a novel biomarker in cancer detection". Cell Research.CS1 maint: Multiple names: authors list (link)
- Roth C, Pantel K, Müller V, Rack B, Kasimir-Bauer S, Janni W, Schwarzenbach H (January 2011). "Apoptosis-related deregulation of proteolytic activities and high serum levels of circulating nucleosomes and DNA in blood correlate with breast cancer progression". BMC Cancer. 11 (1): 4. doi:10.1186/1471-2407-11-4. PMC 3024991. PMID 21211028.
- Stoetzer OJ, Fersching DM, Salat C, Steinkohl O, Gabka CJ, Hamann U, Braun M, Feller AM, Heinemann V, Siegele B, Nagel D, Holdenrieder S (August 2013). "Prediction of response to neoadjuvant chemotherapy in breast cancer patients by circulating apoptotic biomarkers nucleosomes, DNAse, cytokeratin-18 fragments and survivin". Cancer Letters. 336 (1): 140–8. doi:10.1016/j.canlet.2013.04.013. PMID 23612068.
- Mandel P, Metais P (February 1948). "[Not Available]". Comptes Rendus des Seances de la Societe de Biologie et de Ses Filiales. 142 (3–4): 241–3. PMID 18875018.
- Tan EM, Schur PH, Carr RI, Kunkel HG (November 1966). "Deoxybonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus". The Journal of Clinical Investigation. 45 (11): 1732–40. doi:10.1172/jci105479. PMC 292857. PMID 4959277.
- Leon SA, Shapiro B, Sklaroff DM, Yaros MJ (March 1977). "Free DNA in the serum of cancer patients and the effect of therapy". Cancer Research. 37 (3): 646–50. PMID 837366.
- Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL (January 1994). "Soluble normal and mutated DNA sequences from single-copy genes in human blood". Cancer Epidemiology, Biomarkers & Prevention. 3 (1): 67–71. PMID 8118388.
- Arneth B (May 2018). "Update on the types and usage of liquid biopsies in the clinical setting: a systematic review". BMC Cancer. 18 (1): 527. doi:10.1186/s12885-018-4433-3. PMC 5935950. PMID 29728089.
- Babayan A, Pantel K (March 2018). "Advances in liquid biopsy approaches for early detection and monitoring of cancer". Genome Medicine. 10 (1): 21. doi:10.1186/s13073-018-0533-6. PMC 5861602. PMID 29558971.
- Lood C, Blanco LP, Purmalek MM, Carmona-Rivera C, De Ravin SS, Smith CK, Malech HL, Ledbetter JA, Elkon KB, Kaplan MJ (February 2016). "Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease". Nature Medicine. 22 (2): 146–53. doi:10.1038/nm.4027. PMC 4742415. PMID 26779811.
- Yang, D.; Oyaizu, Y.; Oyaizu, H.; Olsen, G. J.; Woese, C. R. (1985-07-01). "Mitochondrial origins". Proceedings of the National Academy of Sciences. 82 (13): 4443–4447. Bibcode:1985PNAS...82.4443Y. doi:10.1073/pnas.82.13.4443. ISSN 0027-8424.
- Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K, Hauser CJ (March 2010). "Circulating mitochondrial DAMPs cause inflammatory responses to injury". Nature. 464 (7285): 104–7. Bibcode:2010Natur.464..104Z. doi:10.1038/nature08780. PMC 2843437. PMID 20203610.
- Collins LV, Hajizadeh S, Holme E, Jonsson IM, Tarkowski A (June 2004). "Endogenously oxidized mitochondrial DNA induces in vivo and in vitro inflammatory responses". Journal of Leukocyte Biology. 75 (6): 995–1000. doi:10.1189/jlb.0703328. PMID 14982943.
- Duvvuri B, Lood C (2019-03-19). "Cell-Free DNA as a Biomarker in Autoimmune Rheumatic Diseases". Frontiers in Immunology. 10: 502. doi:10.3389/fimmu.2019.00502. PMC 6433826. PMID 30941136. Material was copied from this source, which is available under a Creative Commons Attribution 4.0 International License.
- Clayton, David A.; Doda, Jackie N.; Friedberg, Errol C. (1975), Hanawalt, Philip C.; Setlow, Richard B. (eds.), "Absence of a Pyrimidine Dimer Repair Mechanism for Mitochondrial DNA in Mouse and Human Cells", Molecular Mechanisms for Repair of DNA, Springer US, pp. 589–591, doi:10.1007/978-1-4684-2898-8_26, ISBN 9781468429008
- 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 (March 2012). "Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis". Immunity. 36 (3): 401–14. doi:10.1016/j.immuni.2012.01.009. PMC 3312986. PMID 22342844.
- 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 (April 2015). "Mitochondrial DNA stress primes the antiviral innate immune response". Nature. 520 (7548): 553–7. Bibcode:2015Natur.520..553W. doi:10.1038/nature14156. PMC 4409480. PMID 25642965.
- Lui YY, Chik KW, Chiu RW, Ho CY, Lam CW, Lo YM (March 2002). "Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation". Clinical Chemistry. 48 (3): 421–7. PMID 11861434.
- 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 (2013-10-18). "Influence of plasma processing on recovery and analysis of circulating nucleic acids". PLOS ONE. 8 (10): e77963. Bibcode:2013PLoSO...877963P. doi:10.1371/journal.pone.0077963. PMC 3799744. PMID 24205045.
- Barták BK, Kalmár A, Galamb O, Wichmann B, Nagy ZB, Tulassay Z, Dank M, Igaz P, Molnár B (January 2018). "Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection". Pathology Oncology Research. doi:10.1007/s12253-018-0382-z. PMID 29374860.
- 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 (December 2016). "Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing". Translational Lung Cancer Research. 5 (6): 665–672. doi:10.21037/tlcr.2016.12.03. PMC 5233878. PMID 28149760.
- Volik S, Alcaide M, Morin RD, Collins C (October 2016). "Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies". Molecular Cancer Research. 14 (10): 898–908. doi:10.1158/1541-7786.MCR-16-0044. PMID 27422709.
- Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun M (April 1994). "Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia". British Journal of Haematology. 86 (4): 774–9. doi:10.1111/j.1365-2141.1994.tb04828.x. PMID 7918071.
- 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 (May 2012). "Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA". Science Translational Medicine. 4 (136): 136ra68. doi:10.1126/scitranslmed.3003726. PMID 22649089.
- 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 (May 2014). "An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage". Nature Medicine. 20 (5): 548–54. doi:10.1038/nm.3519. PMC 4016134. PMID 24705333.
- Schwarzenbach H, Hoon DS, Pantel K (June 2011). "Cell-free nucleic acids as biomarkers in cancer patients". Nature Reviews. Cancer. 11 (6): 426–37. doi:10.1038/nrc3066. PMID 21562580.
- Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA (March 2000). "Plasma DNA as a prognostic marker in trauma patients". Clinical Chemistry. 46 (3): 319–23. PMID 10702517.
- Chiu TW, Young R, Chan LY, Burd A, Lo DY (2006). "Plasma cell-free DNA as an indicator of severity of injury in burn patients". Clinical Chemistry and Laboratory Medicine. 44 (1): 13–7. doi:10.1515/CCLM.2006.003. PMID 16375578.
- Rhodes A, Wort SJ, Thomas H, Collinson P, Bennett ED (2006). "Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients". Critical Care. 10 (2): R60. doi:10.1186/cc4894. PMC 1550922. PMID 16613611.
- Martins GA, Kawamura MT, Carvalho M (April 2000). "Detection of DNA in the plasma of septic patients". Annals of the New York Academy of Sciences. 906 (1): 134–40. Bibcode:2000NYASA.906..134M. doi:10.1111/j.1749-6632.2000.tb06603.x. PMID 10818609.
- Chang CP, Chia RH, Wu TL, Tsao KC, Sun CF, Wu JT (January 2003). "Elevated cell-free serum DNA detected in patients with myocardial infarction". Clinica Chimica Acta; International Journal of Clinical Chemistry. 327 (1–2): 95–101. doi:10.1016/S0009-8981(02)00337-6. PMID 12482623.
- Rainer TH, Lam NY, Man CY, Chiu RW, Woo KS, Lo YM (June 2006). "Plasma beta-globin DNA as a prognostic marker in chest pain patients". Clinica Chimica Acta; International Journal of Clinical Chemistry. 368 (1–2): 110–3. doi:10.1016/j.cca.2005.12.021. PMID 16480967.
- 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 (October 2015). "Donor-Derived Cell-Free DNA Is a Novel Universal Biomarker for Allograft Rejection in Solid Organ Transplantation". Transplantation Proceedings. 47 (8): 2400–3. doi:10.1016/j.transproceed.2015.08.035. PMID 26518940.
- Pinzani P, Salvianti F, Pazzagli M, Orlando C (April 2010). "Circulating nucleic acids in cancer and pregnancy". Methods. 50 (4): 302–7. doi:10.1016/j.ymeth.2010.02.004. PMID 20146940.