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Background Eye salvaging therapy of malignant melanomas of the uvea can preserve the eye in most cases, but still about half of patients die from metastatic disease. Previous analyses of cell‐free DNA from plasma had shown detectable levels of tumor‐specific GNAQ/GNA11 mutations in patients with the clinical diagnosis of progressive disease. However, data on the time span that elapses from the detection of ctDNA in plasma to the clinical detection of metastases (diagnostic lead time) are missing. Methods We examined 135 patients with uveal melanoma. Cell‐free DNA was isolated from a total of 807 blood samples which were taken over a period of up to 41 months and analyzed for the presence of GNAQ/GNA11 mutations by deep amplicon sequencing. Results Twenty‐one of the 135 patients developed metastases or recurrence. A ctDNA signal was identified in the plasma of 17 of the 21 patients. In 10 patients, this ctDNA signal preceded the clinical diagnosis of metastasis by 2–10 months. In 10 other patients, a ctDNA signal was only detected in samples obtained shortly before or after radiotherapy. The presence of a ctDNA signal in 16 of the remaining 125 patients was linked to clinical manifestation of metastases (n = 14) or tumor recurrence (n = 2) with a sensitivity and specificity of 80% and 96%, respectively. Conclusion Detection of ctDNA in plasma can provide a diagnostic lead time over the clinical diagnosis of metastases or tumor recurrence. Longer lead times are to be expected if intervals between sampling are shortened. In metastasized uveal melanoma (UM) patients, circulating tumor DNA (ctDNA) can be detected in blood. Here we explored if cfDNA is a suitable biomarker for the early detection of metastatic disease in UM patients. Our data show that this biomarker fulfills the expectation as, overall, about half of the patients who developed metastases showed a positive ctDNA signal prior to the clinical diagnosis of metastatic disease with a lead time ranging between 2 and 10 months. Moreover, it is reasonable that, with more frequent sampling time points, diagnostic lead times will be even longer.

Background Advanced gastric cancers are usually associated with incurable conditions for which systemic treatments are indicated. Recent studies suggest that circulating cell-free plasma DNA of tumour origin (tDNA) is a promising non-invasive biomarker that can be used to predict the prognosis and monitor the efficacy of systemic treatments in patients with certain types of cancer. We conducted a pilot study to analyse the potential role of tDNA as a biomarker in patients with advanced gastric cancer. Methods We included 30 patients with locally advanced unresectable or metastatic gastric cancer. We obtained samples (10 mL of total blood) from each patient every 3 months and performed concomitant CT until disease progression or death. Total cell-free circulating DNA (cfDNA) samples were measured using GeneQuant RNA/DNA Calculator-Amersham Pharmacia Biotech (Biochrom) Ltd. The cfDNA was used to evaluate the ALU DNA sequences 247 and 115. The level of tDNA was calculated from the ratio of the expression of ALU DNA sequences and the concentration of total cell-free DNA. We utilized the RECIST criteria 1.1 to evaluate the tumour response. Results Patients with advanced gastric cancer had significantly higher concentrations of cfDNA compared with normal controls ( p  = 0.00015), which allowed us to conclude that the cfDNA in the patients originated from the tumour. We did not find any significant correlation between the level of tDNA and OS or tumour response. However, after the first cycles of chemotherapy (at 3 months), we observed that patients with lower tDNA levels had significantly longer DFS compared with those with higher levels (Cox Regression p  = 0.0228). Conclusions At 3 months after the beginning of chemotherapy, the tDNA levels are correlated with DFS in patients with advanced gastric cancer who receive systemic chemotherapy. tDNA may be a specific, non-invasive and cost effective new biomarker for these patients.

Parkinson's disease (PD) is a progressive neurodegenerative disorder marked by the gradual and irreversible loss of neurons, especially within the substantia nigra region of the midbrain. Early and accurate diagnosis remains a significant challenge in both research and clinical practice. This difficulty is further compounded by the substantial clinical and molecular heterogeneity of PD, emphasizing the urgent need for reliable biomarkers to enhance diagnostic precision and guide therapeutic strategies. One promising candidate biomarker is cell-free DNA (cfDNA), comprising short DNA fragments composed of mitochondrial (cf-mtDNA) and nucleus-derived (cf-ntDNA) DNA. cfDNA is released into body fluids through physiological or pathological processes such as apoptosis, necrosis, NETosis, or active secretion. The presence of cfDNA in human biological fluids has been utilized for years in oncology and prenatal medicine and, more recently, it has gained attention as a non-invasive diagnostic tool in the context of neurodegenerative diseases such as PD. This review article aims to provide a comprehensive overview of the current knowledge on the origin of cfDNA, highlighting the roles of the mitochondria and cf-mtDNA in PD, mitochondria quality control, and neuroinflammation in cfDNA biogenesis. The review collates available research on cfDNA types in human serum, plasma, and CSF, sequence analysis, and its potential application as a biomarker in the diagnosis and monitoring of PD, contributing to the ongoing search for non-invasive biomarkers of neurodegenerative diseases.

Cell loss and mitochondrial dysfunction are key pathological features of idiopathic Parkinson's disease (PD) and multiple system atrophy (MSA). It remains unclear whether disease-specific changes in plasma circulating cell-free nuclear DNA (cf-nDNA) and mitochondrial DNA (cf-mtDNA) occur in patients with PD and MSA. In this study, we investigated whether plasma cf-nDNA, cf-mtDNA levels, as well as cf-mtDNA integrity, are altered in patients with PD and MSA. TaqMan probe-based quantitative PCR was employed to measure plasma cf-nDNA levels, cf-mtDNA copy numbers, and cf-mtDNA deletion levels in 171 participants, including 76 normal controls (NC), 62 PD patients, and 33 MSA patients. A generalized linear model was constructed to analyze differences in circulating cell-free DNA (cfDNA) biomarkers across clinical groups, while a logistic regression model was applied to assess the predictive values of these biomarkers for developing PD or MSA. Spearman correlations were used to explore associations between the three cfDNA biomarkers, demographic data, and clinical scales. No significant differences in plasma cf-nDNA levels, cf-mtDNA copy numbers, or cf-mtDNA deletion levels were observed among the PD, MSA, and NC groups (all > 0.05). Additionally, these measures were not associated with the risk of developing PD or MSA. In PD patients, cf-nDNA levels were positively correlated with Hamilton Anxiety Rating Scale scores (Rho = 0.382, FDR adjusted = 0.027). In MSA patients, cf-nDNA levels were positively correlated with International Cooperative Ataxia Rating Scale scores (Rho = 0.588, FDR adjusted = 0.011) and negatively correlated with Montreal Cognitive Assessment scores (Rho = -0.484, FDR adjusted = 0.044). Subgroup analysis showed that PD patients with constipation had significantly lower plasma cf-mtDNA copy numbers than those without constipation ( = 0.049). MSA patients with cognitive impairment had significantly higher cf-nDNA levels compared to those without ( = 0.008). Plasma cf-nDNA level, cf-mtDNA copy number, and cf-mtDNA deletion level have limited roles as diagnostic biomarkers for PD and MSA. However, their correlations with clinical symptoms support the hypothesis that cell loss and mitochondrial dysfunction are involved in PD and MSA development.

The presence of DNA in EVs, including exosomes and microvesicles, remains contentious as this field of study is still evolving. This review explores what is known about the biogenesis of EV‐DNA and the possible biological roles of DNA packaging into EVs. Moreover, we also discuss the potential clinical applications of EV‐DNA and how those compare to cell‐free DNA. The study of extracellular vesicles (EVs), especially in the liquid biopsy field, has rapidly evolved in recent years. However, most EV studies have focused on RNA or protein content and DNA in EVs (EV‐DNA) has largely been unnoticed. In this review, we compile current evidence regarding EV‐DNA and provide an extensive discussion on EV‐DNA biology. We look into EV‐DNA biogenesis and mechanisms of DNA loading into EVs, as well as describe the particularly significant function of DNA‐carrying EVs in the maintenance of cellular homeostasis, intracellular communication, and immune response modulation. We also examine the current role of EV‐DNA in the clinical setting, specifically in cancer, infections, pregnancy, and prenatal diagnosis.

Background Most studies on cell‐free DNA (cfDNA) were only for single body fluids; however, the differences in cfDNA distribution between two body fluids are rarely reported. Hence, in this work, we compared the differences in cfDNA distribution between cerebrospinal fluid (CSF) and serum of patients with brain‐related diseases. Methods The fragment length of cfDNA was determined by using Agilent 2100 Bioanalyzer. The copy numbers of cell‐free mitochondrial DNA (cf‐mtDNA) and cell‐free nuclear DNA (cf‐nDNA) were determined by using real‐time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) with three pairs of mitochondrial ND1 and nuclear GAPDH primers, respectively. Results There were short (~60 bp), medium (~167 bp), and long (>250 bp) cfDNA fragment length distributions totally obtained from CSF and serum using Agilent 2100 Bioanalyzer. The results of both qPCR and ddPCR confirmed the existence of these three cfDNA fragment ranges in CSF and serum. According to qPCR, the copy numbers of long cf‐mtDNA, medium, and long cf‐nDNA in CSF were significantly higher than in paired serum. In CSF, only long cf‐mtDNA's copy numbers were higher than long cf‐nDNA. But in serum, the copy numbers of medium and long cf‐mtDNA were higher than the corresponding cf‐nDNA. Conclusion The cf‐nDNA and cf‐mtDNA with different fragment lengths differentially distributed in the CSF and serum of patients with brain disorders, which might serve as a biomarker of human brain diseases.

Depiction of the experimental design. While tissue biopsy has for the longest time been the gold-standard in biomedicine, precision/personalized medicine is making the shift toward liquid biopsies. Cell-free DNA (cfDNA) based genetic and epigenetic biomarkers reflect the molecular status of its tissue-of-origin allowing for early and non-invasive diagnostics of different pathologies. However, selection of preanalytical procedures (including cfDNA isolation) as well as analytical methods are known to impact the downstream results. Calls for greater standardization are made continuously, yet comprehensive assessments of the impact on diagnostic parameters are lacking. This study aims to evaluate the preanalytic and analytic factors that influence cfDNA diagnostic parameters in blood and semen. Text mining analysis has been performed to assess cfDNA research trends, and identify studies on isolation methods, preanalytical and analytical impact. Seminal and blood plasma were tested as liquid biopsy sources. Traditional methods of cfDNA isolation, commercial kits (CKs), and an in-house developed protocol were tested, as well as the impact of dithiothreitol (DTT) on cfDNA isolation performance. Fluorimetry, qPCR, digital droplet PCR (ddPCR), and bioanalyzer were compared as cfDNA quantification methods. Fragment analysis was performed by qPCR and bioanalyzer while the downstream application (cfDNA methylation) was analyzed by pyrosequencing. In contrast to blood, semen as a liquid biopsy source has only recently begun to be reported as a liquid biopsy source, with almost half of all publications on it being review articles. Experimental data revealed that cfDNA isolation protocols give a wide range of cfDNA yields, both from blood and seminal plasma. The addition of DTT to CKs has improved yields in seminal plasma and had a neutral/negative impact in blood plasma. Capillary electrophoresis and fluorometry reported much higher yields than PCR methods. While cfDNA yield and integrity were highly impacted, cfDNA methylation was not affected by isolation methodology or DTT. In conclusion, NucleoSnap was recognized as the kit with the best overall performance. DTT improved CK yields in seminal plasma. The in-house developed protocol has shown near-kit isolation performance. ddPCR LINE-1 assay for absolute detection of minute amounts of cfDNA was established and allowed for quantification of samples inhibited in qPCR. cfDNA methylation was recognized as a stable biomarker unimpacted by cfDNA isolation method. Finally, semen was found to be an abundant source of cfDNA offering potential research opportunities and benefits for cfDNA based biomarkers development related to male reproductive health.

Glioma‐derived cell‐free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma‐derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient’s tumor, we detected tumor‐derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10 −3 , 3.1 × 10 −5 , and 4.7 × 10 −5 , respectively. We identified a shift in the size distribution of tumor‐derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole‐genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non‐malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non‐malignant brain disorders ( P  = 1.7 × 10 −2 ) and healthy individuals ( P  = 5.2 × 10 −9 ). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80–0.91) suggesting possibilities for truly non‐invasive cancer detection. SYNOPSIS Gliomas are challenging to detect using tumor derived cell‐free DNA (cfDNA) in body fluids. In this study, two novel analysis methods (tumor‐guided sequencing and sWGS) were developed to explore the potential of using plasma and urine cfDNA as a liquid biopsy for this malignancy. Multiple tumor regions were sequenced to recover a high number of mutations for designing tumor‐guided sequencing panels. Using tumor‐guided sequencing and the INVAR analysis approach, mutations were detected in 7/8 CSF, 10/12 plasma and 10/16 urine gliomas samples. Using low coverage whole genome sequencing, cfDNA fragmentation patterns were analysed in urine samples from 35 glioma patients, 27 individuals with non‐malignant brain disorders, and 26 healthy individuals. Fragment lengths differed significantly between these groups; Machine learning models (LR, SVM, RF, GLMEN) integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80–0.91). Graphical Abstract Gliomas are challenging to detect using tumor derived cell‐free DNA (cfDNA) in body fluids. In this study, two novel analysis methods (tumor‐guided sequencing and sWGS) were developed to explore the potential of using plasma and urine cfDNA as a liquid biopsy for this malignancy.

Background: The roles of plasma cell-free (pcf) mitochondrial DNA (mtDNApcf) and nuclear DNA (nDNApcf) in the pathogenesis of systemic lupus erythematosus (SLE) remain unclear. We analyzed the relative copies of mtDNApcf and nDNApcf and investigated their association with the levels of plasma 8-hydroxy-2’-deoxyguanosine (8-OHdG), plasma malondialdehyde (MDA) and mRNA of leukocyte C-type lectin domain family 5 member A (CLEC5A) in SLE patients. Methods: A total of 80 SLE patients and 43 healthy controls (HCs) were enrolled. Their plasma samples were subjected to the measurements of mtDNApcf copies, nDNApcf copies, 8-OHdG and MDA, respectively. Their leukocytes were analyzed for CLEC5A mRNA expression. Results: SLE patients had higher nDNApcf copies (2.84 ± 1.99 vs. 2.00 ± 0.88, p = 0.002), lower mtDNApcf copies (4.81 ± 6.33 vs. 9.83 ± 14.20, p = 0.032), higher plasma 8-OHdG (0.227 ± 0.085 vs. 0.199 ± 0.041 ng/mL, p = 0.016), lower plasma MDA (3.02 ± 2.20 vs. 4.37 ± 2.16 μM, p = 0.001) and similar leukocyte CLEC5A mRNA expression levels (1.21 ± 1.17 vs. 1.26 ± 1.05, p = 0.870), as compared with those of HCs. Among the HCs, SLE patients with SLE Disease Activity Index (SLEDAI) ≤8, and SLE patients with SLEDAI >8, their respective mtDNApcf copies decreased stepwisely (9.83 ± 14.20 vs. 6.28 ± 7.91 vs. 3.19 ± 3.35, p = 0.054). The nDNApcf copies of HCs, SLE patients without nephritis, and SLE patients with nephritis were increased stepwisely (2.00 ± 0.88 vs. 2.63 ± 1.74 vs. 3.16 ± 2.34, p = 0.043). Among SLE patients, higher nDNApcf copies were associated with higher levels of plasma 8-OHdG (p < 0.001) but lower plasma MDA (p = 0.019). Among HCs but not SLE patients, higher nDNApcf copies (p = 0.013) or lower mtDNApcf copies (p < 0.001) were related to higher levels of leukocyte CLEC5A mRNA expression. Conclusions: Higher nDNApcf, lower mtDNApcf, increased ROS-elicited oxidative DNA damage and dysregulated leukocyte CLEC5A expression might be implicated in the pathogenesis of SLE.

Increased plasma cell‐free mitochondrial DNA (cf‐mDNA), a damage‐associated molecular pattern (DAMP) produced by cellular injury, contributes to neutrophil activation/inflammation in trauma patients and arises in cancer and autoimmunity. To further understand relationships between cf‐mDNA released by tissue injury, inflammation, and health benefits of exercise, we examined cf‐mDNA response to prolonged moderate aerobic exercise. Seven healthy moderately trained young men (age = 22.4 ± 1.2) completed a treadmill exercise trial for 90 min at 60% VO2 max and a resting control trial. Blood was sampled immediately prior to exercise (0 min = baseline), during (+18, +54 min), immediately after (+90 min), and after recovery (R40). Plasma was analyzed for cf‐mDNA, IL‐6, and lactate. A significant difference in cf‐mDNA response was observed between exercise and control trials, with cf‐mDNA levels reduced during exercise at +54 and +90 (with or without plasma volume shift correction). Declines in cf‐mDNA were accompanied by increased lactate and followed by an increase in IL‐6, suggesting a temporal association with muscle stress and inflammatory processes. Our novel finding of cf‐mDNA decline with prolonged moderate treadmill exercise provides evidence for increased clearance from or reduced release of cf‐mDNA into the blood with prolonged exercise. These studies contrast with previous investigations involving exhaustive short‐term treadmill exercise, in which no change in cf‐mDNA levels were reported, and contribute to our understanding of differences between exercise‐ and trauma‐induced inflammation. We propose that transient declines in cf‐mDNA may induce health benefits, by reducing systemic inflammation. Increased plasma cell‐free mitochondrial DNA (cf‐mDNA), a damage‐associated molecular pattern (DAMP) produced by cellular injury, contributes to neutrophil activation/inflammation in trauma patients, and arises in cancer and autoimmunity. To further understand relationships between cf‐mDNA released by tissue injury, inflammation, and health benefits of exercise, we examined cf‐mDNA response to prolonged moderate aerobic exercise. We find a decline in plasma cf‐mDNA with prolonged treadmill exercise followed by increased plasma IL‐6, and we propose that transient cf‐mDNA clearance events may contribute to exercise suppression of systemic inflammation.