Existence nukleových kyselin volně cirkulujících v plazmě byla sice odhalena již kolem poloviny minulého století, s nástupem nových citlivých technologií – především kvantifikace specifických sekvencí pomocí Real-Time PCR se jejich analýza dostala do popředí zájmu molekulárních genetiků a klinických pracovníků hledajících nové efektivní markery pro využití v klinické medicíně. Kvalitativní i kvantitativní analýza volně cirkulujících nukleových kyselin zaznamenala obrovský nárůst počtu studií především ve vztahu ke studiu karcinogeneze, detekce nádoru, monitorování jejich rozvoje či úspěšnosti terapie. Objev volné DNA fetálního původu v mateřské plazmě byl příčinou nástupu efektních diagnostických strategií umožňujících neinvazivní prenatální diagnostiku. Současnost je charakterizována snahou o využití kvantitativní analýzy celkového množství volně cirkulující DNA (cfDNA) v oběhu jako prognostického markeru u pacientů s polytraumaty, mozkovou příhodou či akutním infarktem myokardu. Recentní studie prokázaly korelaci zvýšených hodnot celkové cfDNA s přežíváním pacientů jednotek intenzivní péče. Z výše vedených důvodů považujeme za důležité rozvíjení znalostí o chování cfDNA v cirkulaci za patologických podmínek, k nimž nepochybně narušení funkce ledvin patří. Této oblasti byla zatím ve světové literatuře věnována poměrně malá pozornost. Pouze dvě studie se dosud zabývaly změnami koncentrací cfDNA u hemodialyzovaných pacientů, obě prokázaly významný nárůst celkové koncentrace cfDNA v plazmě pacientů bezprostředně po proceduře. Vzhledem k výše uvedeným potenciálním klinickým aplikacím stanovení cfDNA v plazmě pacientů se jedná o důležité zjištění mající dopad na vývoj diagnostické strategie u pacientů, jejichž klinický stav vyžaduje hemodialýzu. V našem příspěvku diskutujeme současné znalosti v této oblasti a další úkoly a perspektivy, které studium cfDNA přináší nejen nefrologum.
Though circulating free nucleic acids in plasma were first described in 1950s, it was the advent of new sophisticated technologies – namely the quantification of specific sequences using real-time PCR – that made it a highly attractive target for molecular geneticists and clinicians looking for new effective markers applicable in clinical medicine. There has been an enormous increase in the number of studies analyzing qualitatively and quantitatively the circulating free nucleic acids, namely with regard to carcinogenesis, tumor detection and its progression or treatment monitoring. The discovery of free DNA of fetal origin in maternal plasma has enabled efficient strategies of non-invasive prenatal diagnostics. Quantitative analysis of total circulating free DNA (cfDNA) has recently been utilized and evaluated as a prognostic marker in the patients with polytrauma, stroke, or acute myocardial infarction. Recent studies have proven a correlation between the increased levels of total cfDNA and patient survival in the intensive care units. We therefore consider further studies on cfDNA level changes in various diseases, including renal failure, as important. In nephrology, however, these questions have only rarely been addressed. Only two studies have reported cfDNA plasma level changes in hemodialyzed patients; in both a significant increase immediately after the procedure was found. In accord with the above-mentioned possible clinical applications of cfDNA measurement, this is an important finding that might affect our diagnostic strategies in hemodialyzed patients. In this article we discuss the current knowledge in the field, as well as further tasks and perspectives regarding cfDNA measurement not only in nephrology.
- MeSH
- Renal Dialysis methods trends utilization MeSH
- DNA isolation & purification blood MeSH
- Genetic Markers MeSH
- Humans MeSH
- Evidence-Based Medicine trends MeSH
- Kidney Neoplasms diagnosis genetics MeSH
- Nephrology methods trends MeSH
- Polymerase Chain Reaction methods utilization MeSH
- Prognosis MeSH
- Check Tag
- Humans MeSH
Výskyt krátkých fragmentů volných DNA (cell-free DNA – cfDNA) jaderného či mitochondriálního původu v tělesných tekutinách je fyziologický. Jejich koncentrace násobně narůstá následkem zranění, zánětu či intenzivní fyzické námahy. Zvýšené koncentrace se rychle vracejí na původní hladiny díky masivní degradaci restrikčními endonukleázami a extenzivnímu katabolizmu v játrech, slezině a částečně i díky vylučování ledvinami. Vyšší koncentrace cfDNA byly popsány v souvislosti s přítomností většiny rizikových faktorů aterosklerózy. Existuje silná korelace mezi cfDNA a koncentracemi kreatinkinázy a troponinu. cfDNA může být použita jako marker cévní mozkové příhody či k rozlišení STEMI a nonSTEMI infarktu myokardu. Mitochondriální cfDNA pak stimuluje zánětlivé procesy. Přestože řada nálezů potřebuje ověřit na rozsáhlejších souborech, cfDNA se mohou v budoucnu stát novými slibnými markery onemocnění a zdravotních prognóz i v kardiologii.
The presence of short fragments of cell-free DNA (cfDNA), of nuclear or mitochondrial origin, in body fluids is physiological. Their concentration increases manifold as a result of injury, inflammation or intensive physical exercise. Increased concentrations quickly return to their baseline levels due to intense degradation by restriction endonucleases and extensive catabolism in the liver, spleen and partly excretion by the kidneys. Higher cfDNA concentrations have been described in association with the presence of majority atherosclerosis risk factors. There is a strong correlation between cfDNA and creatine kinase and troponin concentrations. CfDNA can be used as a marker of stroke or to differentiate between STEMI and non-STEMI myocardial infarction. Mitochondrial cfDNA then stimulates inflammatory processes. Although many of the findings need validation on larger datasets, cfDNAs may become new promising markers of disease and health prognosis in the future also in cardiology.
- MeSH
- Biomarkers MeSH
- Cardiovascular Diseases * diagnosis blood MeSH
- Plasma physiology MeSH
- Humans MeSH
- Cell-Free Nucleic Acids * physiology blood metabolism therapeutic use MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Úvod: Nemocní s pokročilými stadii zhoubných novotvarů mívají často zvýšené hladiny cirkulující volné DNA (cell-free DNA, cfDNA) v periferní krvi. Část cfDNA pocházející z tumoru představuje zdroj materiálu pro molekulární profilaci tumoru především za účelem predikce léčebné odpovědi biologické cílené léčby. Ve skupině nemocných s pokročilým NSCLC jsme s využitím detekce nádorově-specifických mutací sledovali výskyt nádorové složky cfDNA a v ní následně prováděli metylační vyšetření zaměřená na panel 30ti genů. Metodika: V souboru 107 NSCLC pacientů jsme v tumorózní tkáni vyšetřovali přítomnost somatické mutace EGFR nebo KRAS. U pacientů s nalezenou mutací byla vyšetřena přítomnost dané mutace v cfDNA získané z plazmy. U pozitivních vzorků bylo provedeno vyšetření DNA metylace v cfDNA. Výsledky: Z celkového počtu 107 pacientů ve studii byla některá z mutací EGFR nebo KRAS nalezena u 26 pacientů (14 x EGFR, 12 x KRAS). Z tohoto počtu byla přítomnost tumorové cfDNA detekována v plazmě 8 pacientů, u kterých bylo následně provedeno metylační vyšetření. U 7 cfDNA-pozitivních pacientů (7/8, 87 %) byla potvrzena shoda metylace v tumoru i v cfDNA u minimálně 3 ze sledované sady genů. Závěr: Přítomnosti cfDNA v plazmě pacientů s pokročilým NSCLC může být využita pro profilování primárního tumoru, a to jak pro neinvazivní vyšetřování genových mutací, tak i pro vyšetřování metylačních nádorových aberací majících význam pro biologické chování nemalobuněčného karcinomu.
Introduction: Patients in advanced stages of cancer often display elevated levels of circulating cell-free DNA (cfDNA) in peripheral blood. The part of cfDNA originating from a tumor represents a viable source of material for molecular profiling of the tumor, mainly in order to predict the biological response of targeted therapy. In a group of patients with advanced NSCLC, the frequency of tumor cfDNA was assessed by detecting tumor-specific gene mutations. The tumor cfDNA was subsequently subjected to methylation analysis using a panel of 30 genes. Methods: In a group of 107 NSCLC patients, primary tumor tissue was evaluated for the presence of somatic mutations in EGFR and KRAS genes. In mutation-positive patients, the presence of a particular mutation in cfDNA obtsdned from plasma was studied. The positive samples were subjected to analysis of DNA methylation in cfDNA. Results: Either of the EGFR or KRAS mutations was found in 26 out of the 107 patients (EGFR 14 x, KRAS 12 x). Of those, eight were found to have tumor cfDNA present in plasma. Subsequently, they underwent methylation tests. In 7 of these cfDNA-positive patients (87 %), methylation was found in both tumor and cfDNA in at least 3 of the observed set of genes. Conclusion: The presence cfDNA in plasma of patients with advanced NSCLC may be used for primary tumor protiling, for both non-invasive investigation of gene mutations and investigation of tumor methylation aberrations of relevance for the biological behaviour of non-small cell lung carcinoma.
- Keywords
- mutace EGFR, mutace KRAS, cirkulující volná DNA, nemalobuněčný karcinom,
- MeSH
- Biological Therapy methods utilization MeSH
- Financing, Organized MeSH
- Genes, erbB-1 genetics MeSH
- Genes, ras genetics MeSH
- Humans MeSH
- DNA Methylation genetics MeSH
- Mutation genetics MeSH
- Neoplastic Cells, Circulating MeSH
- Carcinoma, Non-Small-Cell Lung genetics blood MeSH
- Statistics as Topic MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
Obesity is a disease that affects about 13 % of the world population (2016) (Who 2018). This condition generates a process of systemic inflammation that may contribute to the release of cell-free DNA (cfDNA) into the bloodstream. cfDNA has been considered a potential biomarker to monitor several physiological and pathological conditions, such as tumors, exercise intensity and obesity. Therefore, the objective of this study was to evaluate the association of cfDNA levels with the amount of weight and fat mass lost six months after bariatric surgery. Thirty-eight subjects classified as obese (BMI, 43.5+/-6.2; BFP, 46.6+/-4.8) were evaluated anthropometrically and underwent bariatric surgery. Weight, BMI, body fat percentage (BFP), waist circumference, C-Reactive Protein (CRP) and cfDNA levels were evaluated before and six months after surgery; furthermore, a correlation was performed between cfDNA levels and BFP and CRP. Decrease in total body weight and CRP were observed after bariatric surgery; however, the cfDNA levels remained unchanged. There was a weak correlation between cfDNA levels and BFP before the bariatric surgery, and a moderate correlation between cfDNA and CRP. Obese subjects who underwent bariatric surgery, the decrease in body fat percentage did not result in changes in cfDNA levels six months after surgery.
- MeSH
- Anthropometry methods MeSH
- Bariatric Surgery methods MeSH
- Biomarkers blood MeSH
- C-Reactive Protein analysis MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Obesity blood diagnosis genetics surgery MeSH
- Cell-Free Nucleic Acids blood MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
The cell-free DNA (cfDNA) is always present in plasma, and it is biomarker of growing interest in prenatal diagnostics as well as in oncology and transplantology for therapy efficiency monitoring. But does this cfDNA have a physiological role? Here we show that cfDNA presence and clearance in plasma of healthy individuals plays an indispensable role in immune system regulation. We exposed THP1 cells to healthy individuals' plasma with (NP) and without (TP) cfDNA. In cells treated with NP, we found elevated expression of genes whose products maintain immune system homeostasis. Exposure of cells to TP triggered an innate immune response (IIR), documented particularly by elevated expression of pro-inflammatory interleukin 8. The results of mass spectrometry showed a higher abundance of proteins associated with IIR activation due to the regulation of complement cascade in cells cultivated with TP. These expression profiles provide evidence that the presence of cfDNA and its clearance in plasma of healthy individuals regulate fundamental mechanisms of the inflammation process and tissue homeostasis. The detailed understanding how neutrophil extracellular traps and their naturally occurring degradation products affect the performance of immune system is of crucial interest for future medical applications.
- MeSH
- Biomarkers blood MeSH
- Chromatography, Liquid MeSH
- Adult MeSH
- Extracellular Traps immunology MeSH
- Mass Spectrometry MeSH
- Plasma MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Monocytes immunology MeSH
- Immunity, Innate * MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Tandem Mass Spectrometry MeSH
- THP-1 Cells MeSH
- Cell-Free Nucleic Acids blood MeSH
- Inflammation MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Volná cirkulující DNA (cf‑DNA) je charakterizována jako extracelulární DNA, která může být přítomna v nízkých koncentracích v krvi zdravých jedinců. Cf‑DNA je do krevního řečiště uvolňována apoptózou, ale i nekrózou. Zvýšené hladiny se vyskytují u patologických stavů, jako je zánět, stres či autoimunitní onemocnění. Výrazně zvýšené hodnoty cf‑DNA jsou patrné zejména u pacientů s malignitami, a to především v pokročilých stadiích nemoci. V takovémto případě je nádorově specifická cf‑DNA uvolňována nekrózou z buněk primárního nádoru a metastáz. V poslední době se hodně studií zabývá tzv. liquid biopsy (tekutou biopsií), která umožňuje poměrně snadnou detekci cirkulujících nádorových buněk i cirkulujících molekul nukleových kyselin z periferní krve k diagnostice nádorových onemocnění. Kvantitativní stanovení a detekce genetických a epigenetických změn v cf‑DNA u pacientů s různými malignitami má potenciální využití v molekulární diagnostice, prognóze, monitorování průběhu nemoci a odpovědi na léčbu. Tento článek je zaměřen na potenciální využití cf‑DNA jako krevního biomarkeru u vybraných solidních nádorů a hematologických malignit.
Circulating cell‑free DNA (cf‑DNA) is characterized as extracellular DNA that may be present in the blood of healthy individuals in low concentrations. Cf‑DNA is released by apoptosis or necrosis into the bloodstream. Increased levels are found in pathological conditions, such as inflammation, autoimmune diseases, or stress. Significant increase of cf‑DNA is particularly evident in patients with malignancies, especially in the advanced stages of the disease. In this case, the tumor specific cf‑DNA is released by necrosis from the cells of primary tumor and metastases. Recently, many studies concentrate on the so‑called ‘liquid biopsies’ that allow detection of circulating tumor cells and circulating nucleic acids from peripheral blood for tumor diagnostics. Quantitative methods and detection of genetic and epigenetic alternations of cf‑DNA in patients with different malignancies have potential applications in molecular diagnosis, prognosis, monitoring of disease progression and response to treatment. This review focuses on potential utility of cf‑DNA as a blood biomarker in selected solid tumors and hematologic malignancies. Key words: circulating cell‑free DNA – tumor marker – solid tumors – hematological malignancies This study was supported by grant of Internal Grant Agency of the Czech Ministry of Health NT14575. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 13. 3. 2015 Accepted: 18. 5. 2015
- MeSH
- Biopsy methods MeSH
- DNA * diagnostic use genetics blood MeSH
- Hematologic Neoplasms genetics blood MeSH
- Colorectal Neoplasms blood MeSH
- Humans MeSH
- DNA Methylation MeSH
- Mutation MeSH
- Biomarkers, Tumor MeSH
- Lung Neoplasms genetics blood MeSH
- Breast Neoplasms genetics blood MeSH
- Neoplasms * genetics blood MeSH
- Sensitivity and Specificity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
As the number of cancer patients globally increases, a need for reliable biomarkers including circulating tumour DNA from liquid biopsy for diagnosis, prognosis and monitoring of the disease is rising. Currently, mainly tissue samples from biopsy are used, but there are certain limitations: firstly, it is an invasive technique, and secondly, in some cases it is almost impossible to obtain an acceptable tissue sample. This could be changed by using circulating cell-free DNA from liquid biopsy, which also gives the possibility of repeated examination. Here, we focus on the options of isolating circulating cell-free DNA from plasma samples using two isolation techniques: precision manual QIAamp Circulating Nucleic Acid Kit and automatic MagNA Pure Compact (MPC) using Nucleic Acid Isolation Kit I. Manual extraction gave significantly better yields of circulating tumour DNA (P < 0.05). This DNA also had less contaminants (organic compounds or proteins). DNA obtained by both tested methods of isolation is suitable for subsequent molecular genetic methods.
- MeSH
- Circulating Tumor DNA * MeSH
- Humans MeSH
- Neoplasms * MeSH
- Liquid Biopsy MeSH
- Cell-Free Nucleic Acids * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Although tumor cells are the most reliable source of tumor DNA, biopsy of the tumor is an invasive procedure that should be avoided in some cases. The main limitation of any biopsy is sampling of one tumor site, which may not represent all malignant clones due to the heterogeneity of the tumor. These clones respond to treatment differently and thus directly influence survival of the patient. Circulating cell-free DNA (cfDNA) is released from multiple tumor sites, reflects overall heterogeneity of the tumor, and correlates with its progression. Detection of tumor-specific genetic and epigenetic aberrations in cfDNA could have a direct impact on molecular diagnosis, prognosis, follow-up of disease, monitoring of minimal residual disease, and response to treatment. While most cfDNA data are still experimental, they are very promising. This review focuses on cfDNA in hematological malignancies.
PURPOSE: Cell-free plasma DNA (cfDNA) levels originating predominantly from apoptotic leukocytes were found to rise during hemodialysis (HD) session, and as such are considered a marker of HD membrane biocompatibility. The purpose of our study was to determine the changes of cfDNA during two consecutive high-flux polysulphone HD sessions after a long (HD-L) and short (HD-S) interdialytic interval. METHODS: A total of 17 HD patients were examined. Prior to HD and at 30 and 240 min, cfDNA (using real-time PCR) and leukocyte count were determined. RESULTS: No significant difference was found when comparing pre-HD-S with pre-HD-L cfDNA [4893 (1090-28804) vs. 4589 (691-73796) genomic equivalent/mL]. A significant increase in cfDNA at 240 min was seen in HD-L (p < 0.05) but not in HD-S. Leukocyte count correlated with cfDNA levels before HD-S (r = 0.8; p < 0.01); however, no other correlation was seen between routinely measured biochemical markers and pre-HD cfDNA levels or cfDNA changes during HD. The increase in plasma cfDNA during HD did not correlate with dialysis duration, its efficacy, or ultrafiltration. An association between magnitude of diuresis and cfDNA levels in HD-S was found (r = 0.58; p < 0.05). CONCLUSIONS: The behavior of cfDNA during HD after long and short interdialytic interval is inconsistent and cannot be explained by changes in laboratory and clinical parameters. The observed relationship of plasma cfDNA levels with diuresis deserves further investigation.
- MeSH
- Cell-Free System MeSH
- Biomarkers blood MeSH
- C-Reactive Protein analysis MeSH
- Time Factors MeSH
- Kidney Failure, Chronic blood mortality therapy MeSH
- Renal Dialysis methods mortality MeSH
- DNA blood MeSH
- Adult MeSH
- Risk Assessment MeSH
- Cohort Studies MeSH
- Middle Aged MeSH
- Humans MeSH
- Membranes, Artificial MeSH
- Survival Rate MeSH
- Plasma Cells MeSH
- Leukocyte Count MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Probability MeSH
- Predictive Value of Tests MeSH
- Prognosis MeSH
- Reference Values MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Alleles MeSH
- Biomarkers blood MeSH
- Tissue Donors MeSH
- Major Histocompatibility Complex genetics MeSH
- Humans MeSH
- Arm transplantation MeSH
- Transplant Recipients MeSH
- Prospective Studies MeSH
- Graft Rejection blood diagnosis MeSH
- Facial Transplantation adverse effects MeSH
- Cell-Free Nucleic Acids blood MeSH
- Check Tag
- Humans MeSH
- Publication type
- Letter MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
