cfDNA Levels Research Articles

Plasma cell-free DNA in canine lymphoma patients as a novel material for genotyping.

Canine lymphoma is a disease with high morbidity and poor long-term prognosis, despite a high response rate to chemotherapy. In this study, we focused on liquid biopsy, in which small amounts of substances from body fluids were analysed, to determine whether cell-free DNA (cfDNA) in the plasma can be used as a biomarker for lymphoma in dogs. We found that 23 patients with lymphoma had significantly higher cfDNA concentrations than the 12 healthy dogs (median 2360 ng/mL versus 299 ng/mL, p < .0001). Polymerase chain reaction for antigen receptor rearrangement (PARR) was also employed using cfDNA from the lymphoma group to investigate whether cfDNA could be used for the detection of genetic clonality of lymphomas, as well as the genomic DNA (gDNA) extracted from an original lesion in each case. The correlation of the PARR results between cfDNA and gDNA was observed in 100% of B-cell lymphomas (10/10), 77.8% of T-cell lymphomas (7/9), and 100% of other types of lymphomas (4/4), respectively. These results indicate that plasma cfDNA levels are increasing in canine lymphoma patients, that cfDNA concentration can be a novel diagnostic tool, and that it can be used as a diagnostic tool for PARR.

Towards effectiveness of cell free DNA based liquid biopsy in head and neck squamous cell carcinoma

Liquid biopsy is a minimally invasive procedure, that uses body fluids sampling to detect and characterize cancer fingerprints. It is of great potential in oncology, however there are challenges associated with the proper handling of liquid biopsy samples that need to be addressed to implement such analysis in patients’ care. Therefore, in this study we performed optimization of pre-analytical conditions and detailed characterization of cfDNA fraction (concentration, length, integrity score) in surgically treated HNSCC patients (n = 152) and healthy volunteers (n = 56). We observed significantly higher cfDNA concentration in patients compared to healthy controls (p < 0.0001) and a time dependent decrease of cfDNA concentration after tumor resection. Our results also revealed a significant increase of cfDNA concentration with age in both, healthy volunteers (p = 0.04) and HNSCC patients (p = 0.000002). Moreover, considering the multitude of HNSCC locations, we showed the lack of difference in cfDNA concentration depending on the anatomical location. Furthermore, we demonstrated a trend toward higher cfDNA length (range 35–10380 and 500–10380 bp) in the group of patients with recurrence during follow-up. In conclusion, our study provide a broad characterization of cfDNA fractions in HNSCC patients and healthy controls. These findings point to several aspects necessary to consider when implementing liquid biopsy in clinical practice including: (I) time required for epithelial regeneration to avoid falsely elevated levels of cfDNA not resulting from active cancer, (II) age-related accumulation of nucleic acids accompanied by less efficient elimination of cfDNA and (III) higher cfDNA length in patients with recurrence during follow-up, reflecting predominance of tumor necrosis.

Biomarkers of peripheral blood neutrophil extracellular traps in the diagnosis and progression of malignant tumors.

The mortality rate associated with malignant tumors remains high and there is a lack of effective diagnostic and tumor progression markers. Neutrophil extracellular traps (NETs) can promote tumor-associated thrombosis, invasive metastasis, and inflammatory responses, but there is a lack of research on the value of measuring NETs in the peripheral blood of patients with malignancies. We included 263 patients with malignancies (55 gliomas, 101 ovarian, 64 colorectal, and 43 lung cancers) and 75 healthy controls in this study. We compared the levels of citrullinated histone H3 (citH3), cell-free DNA (cfDNA), and systemic inflammation-related parameters, including neutrophils, lymphocytes, monocytes, platelets, neutrophil-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, platelet-to-lymphocyte ratio, systemic immune inflammation index, and systemic inflammation response index. We assessed the value of changes in NETs in peripheral blood to determine the diagnosis, venous thromboembolism, clinical staging, and systemic inflammatory response in patients with malignancy. The levels of citH3 and cfDNA in peripheral blood can distinguish between healthy controls and tumor patients. The levels of citH3 and cfDNA before clinical intervention did not predict the risk of combined venous thromboembolism in oncology patients in the short-term after clinical intervention. The levels of citH3, cfDNA, and systemic inflammation-related parameters in the peripheral blood of tumor patients increased with the clinical stage. There was a correlation between cfDNA levels in peripheral blood and systemic inflammation-related parameters in tumor patients, and this correlation was more significant in patients with advanced tumors. Changes in NETs in the peripheral blood differ between healthy controls and patients with malignant tumors. NETs may be involved in tumor-induced systemic inflammatory responses through interaction with circulating inflammatory cells, thus promoting tumor progression. NETs may be used as markers to assist in the diagnosis and progression of tumor malignancy.

Chronic graft-versus-host disease detected by tissue-specific cell-free DNA methylation biomarkers.

Accurate detection of graft-versus-host disease (GVHD) is a major challenge in the management of patients undergoing hematopoietic stem cell transplantation (HCT). Here, we demonstrated the use of circulating cell-free DNA (cfDNA) for detection of tissue turnover and chronic GVHD (cGVHD) in specific organs. We established a cocktail of tissue-specific DNA methylation markers and used it to determine the concentration of cfDNA molecules derived from the liver, skin, lungs, colon, and specific immune cells in 101 patients undergoing HCT. Patients with active cGVHD showed elevated concentrations of cfDNA, as well as tissue-specific methylation markers that agreed with clinical scores. Strikingly, transplanted patients with no clinical symptoms had abnormally high levels of tissue-specific markers, suggesting hidden tissue turnover even in the absence of evident clinical pathology. An integrative model taking into account total cfDNA concentration, monocyte/macrophage cfDNA levels and alanine transaminase was able to correctly identify GVHD with a specificity of 86% and precision of 89% (AUC of 0.8). cfDNA markers can be used for the detection of cGVHD, opening a window into underlying tissue dynamics in patients that receive allogeneic stem cell transplants. This work was supported by grants from the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation and the Helmsley Charitable Trust (to YD).

Cell-Free DNA as a Biomarker at Diagnosis and Follow-Up in 256 B and T-Cell Lymphomas.

Cell-free DNA (cfDNA) analysis has become a promising tool for the diagnosis, prognosis, and monitoring of lymphoma cases. Until now, research in this area has mainly focused on aggressive lymphomas, with scanty information from other lymphoma subtypes. We selected 256 patients diagnosed with lymphomas, including a large variety of B-cell and T-cell non-Hodgkin and Hodgkin lymphomas, and quantified cfDNA from plasma at the time of diagnosis. We further selected 49 large B-cell lymphomas (LBCL) and analyzed cfDNA levels at diagnosis (pre-therapy) and after therapy. In addition, we performed NGS on cfDNA and tissue in this cohort of LBCL. Lymphoma patients showed a statistically significant higher cfDNA concentration than healthy controls (mean 53.0 ng/mL vs. 5.6 ng/mL, p < 0.001). The cfDNA concentration was correlated with lymphoma subtype, lactate dehydrogenase, the International Prognostic Index (IPI) score, Ann Arbor (AA), and B-symptoms. In 49 LBCL cases, the cfDNA concentration decreased after therapy in cases who achieved complete response (CR) and increased in non-responders. The median cfDNA at diagnosis of patients who achieved CR and later relapsed was higher (81.5 ng/mL) compared with levels of those who did not (38.6 ng/mL). A concordance of 84% was observed between NGS results in tumor and cfDNA samples. Higher VAF in cfDNA is correlated with advanced stage and bulky disease. cfDNA analysis can be easily performed in almost all lymphoma cases. The cfDNA concentration correlated with the characteristics of the aggressiveness of the lymphomas and, in LBCL, with the response achieved after therapy. These results support the utility of cfDNA analysis as a complementary tool in the management of lymphoma patients.

Circulating cell-free DNA correlate to disease activity and treatment response of patients with radiographic axial spondyloarthritis.

Microdamage and its related inflammation contribute to the development of radiographic axial spondyloarthritis (r-axSpA). Inflammation and cell death in damaged tissues are associated with cell-free DNA (cfDNA) release. Here we investigated whether circulating cfDNA could be a potential biomarker for evaluating disease activity and treatment response in r-axSpA. Circulating cfDNA was detected in the discovery and validation cohort with 79 and 60 newly diagnosed r-axSpA patients respectively and 42 healthy controls using the Quant-iT PicoGreen dsDNA reagent and kit. As a result, cfDNA levels were significantly higher in r-axSpA patients compared with healthy controls in the discovery and validation cohort. Moreover, cfDNA levels were positively correlated with CRP, ASDAS-CRP and neutrophil counts. Additionally, non-steroid anti-inflammatory drugs (NSAIDs) combined with disease-modifying anti-rheumatic drugs or tumor necrosis factor inhibitors but not NSAIDs alone could reduce cfDNA levels. Moreover, a decrease of cfDNA levels after treatment was associated with an effective therapeutic response. Intriguingly, patients with higher levels of cfDNA at diagnosis responded better to combination therapy rather than NSAIDs. However, patients with lower levels of cfDNA displayed similar responses to combination or mono-NSAID treatment. In conclusion, circulating cfDNA levels showed a significant correlation with disease activity as well as treatment efficacy in patients with r-axSpA. Moreover, cfDNA at diagnosis might predict the response to different therapy. Consequently, cfDNA may serve as a useful biomarker of inflammation in r-axSpA.

Role of cell-free DNA levels in the diagnosis and prognosis of sepsis and bacteremia: A systematic review and meta-analysis.

Sepsis remains a major cause of mortality in intensive care units (ICUs). Prompt diagnosis and effective management are imperative for better outcomes. In this systematic review and meta-analysis, we explore the potential of circulating cell-free DNA (cfDNA), as a promising tool for early sepsis detection and prognosis assessment, aiming to address limitations associated with traditional diagnostic methods. Following PRISMA guidelines, we collected relevant literature from thirteen databases. Studies were included if they analyzed quantitative diagnostic or prognostic cfDNA levels in humans in case of sepsis. We collected data on basic study characteristics, baseline patient demographics (e.g. age and sex), and cfDNA levels across different stages of sepsis. Pooled SMD with 95%-CI was calculated, and Comprehensive Meta-Analysis (CMA) software facilitated meta-analysis. Receiver operating characteristic (ROC) curves were generated to assess cfDNA's combined sensitivity and specificity in diagnostics and prognostics. We included a final of 44 studies, of which, only 32 with 2950 participants were included in the meta-analysis. cfDNA levels were higher in septic patients compared to healthy controls (SMD = 3.303; 95%-CI [2.461-4.145], p<0.01). Furthermore, cfDNA levels were higher in non-survivors than survivors (SMD = 1.554; 95%-CI [0.905-2.202], p<0.01). Prognostic studies demonstrated a pooled sensitivity and specificity of 0.78, while diagnostic studies showed a sensitivity of 0.81 and a specificity of 0.87. These findings show that cfDNA levels are significantly higher in sepsis patients compared to control groups and non-survivors in comparison to survivors among both adult and pediatric populations.

Plasma cell-free DNA as predictor of disease status in patients with differentiated thyroid cancer - a prospective study from a tertiary care institution.

Plasma cell-free DNA (cfDNA) estimation offers a non-invasive method to potentially diagnose, monitor, and prognosticate patients with malignancy. This prospective study aimed to assess plasma cfDNA levels in patients with differentiated thyroid cancer (DTC) to determine its role in predicting disease status in the post-operative setting. This was a single-center prospective observational study conducted at a public medical research university and hospital in New Delhi, India. 254 patients with DTC in the post-operative setting were included: 95 in Group 1 (active structural disease) and 159 in Group 2 (disease-free). Blood samples were collected for plasma separation and cfDNA extraction. The cfDNA concentrations were quantified and compared across various disease states. Median values of plasma cfDNA (ng/µL) in groups 1 and 2 were found to be 0.272 (IQR: 0.137-0.442) and 0.222 (IQR: 0.123-0.398), respectively with no significant difference (p=0.122). cfDNA levels were significantly higher in patients in the age group ≥55 years (p=0.016). However, the cfDNA levels were not significantly associated with any of the other known prognostic markers of DTC. Based on the results of this study, plasma cfDNA levels did not significantly predict disease status in patients with DTC in the post-operative setting.

Cell-Free DNA As Peripheral Biomarker of Alzheimer's Disease.

Alzheimer's disease (AD) and Alzheimer's disease-related disorders (ADRD) are progressive neurodegenerative diseases without cure. Alzheimer's disease occurs in 2 forms, early-onset familial AD and late-onset sporadic AD. Early-onset AD is a rare (~1%), autosomal dominant, caused by mutations in presenilin-1, presenilin-2, and amyloid precursor protein genes and the other is a late-onset, prevalent and is evolved due to age-associated complex interactions between environmental and genetic factors, in addition to apolipoprotein E4 polymorphism. Cellular senescence, promoting the impairment of physical and mental functions is constituted to be the main cause of aging, the primary risk factor for AD, which results in progressive loss of cognitive function, memory, and visual-spatial skills for an individual to live or act independently. Despite significant progress in the understanding of the biology and pathophysiology of AD, we continue to lack definitive early detectable biomarkers and/or drug targets that can be used to delay the development of AD and ADRD in elderly populations. However, recent developments in the studies of DNA double-strand breaks result in the release of fragmented DNA into the bloodstream and contribute to higher levels of cell-free DNA (cf-DNA). This fragmented cf-DNA can be released into the bloodstream from various cell types, including normal cells and cells undergoing apoptosis or necrosis and elevated levels of cf-DNA in the blood have the potential to serve as blood blood-based biomarker for early detection of AD and ADRD. The overall goal of our study is to discuss the latest developments in circulating cell-free DNA into the blood in the progression of AD and ADRD. Our article summarized the status of research on double-strand breaks and circulating cell-free DNA in both healthy and disease states and how these recent developments can be used to develop early detectable biomarkers for AD and ADRD. Our article also discussed the impact of lifestyle and epigenetic factors that are involved in DNA double-strand breaks and circulating cell-free DNA in AD and ADRD.

The potential of liquid biopsy for detection of the KIAA1549-BRAF fusion in circulating tumor DNA from children with pilocytic astrocytoma.

Low-grade gliomas (LGGs) represent children's most prevalent central nervous system tumor, necessitating molecular profiling to diagnose and determine the most suitable treatment. Developing highly sensitive screening techniques for liquid biopsy samples is particularly beneficial, as it enables the early detection and molecular characterization of tumors with minimally invasive samples. We examined CSF and plasma samples from patients with pilocytic astrocytoma (PA) using custom multiplexed droplet digital polymerase chain reaction (ddPCR) assays based on whole genome sequencing data. These assays included a screening test to analyze BRAF duplication and a targeted assay for the detection of patient-specific KIAA1549::BRAF fusion junction sequences or single nucleotide variants. Our findings revealed that 5 out of 13 individual cerebrospinal fluid (CSF) samples tested positive for circulating tumor DNA (ctDNA). Among these cases, 3 exhibited the KIAA1549::BRAF fusion, which was detected through copy number variation (CNV) analysis (n = 1) or a fusion-specific probe (n = 2), while 1 case each displayed the BRAF V600E mutation and the FGFR1 N577K mutation. Additionally, a quantitative analysis of cell-free DNA (cfDNA) concentrations in PA CSF samples showed that most cases had low cfDNA levels, below the limit of detection of our assay (<1.9ng). While CNV analysis of CSF samples from LGGs still has some limitations, it has the potential to serve as a valuable complementary tool. Furthermore, it can also be multiplexed with other aberrations, for example, to the BRAF V600 test, to provide important insights into the molecular characteristics of LGGs.

Neutrophil extracellular trap biomarkers in aneurysmal subarachnoid hemorrhage: early decline of DNase 1 activity associated with delayed cerebral ischemia.

High-mobility group box 1 (HMGB1) protein is a critical mediator of neutrophil extracellular trap (NET) formation (NETosis). Myeloperoxidase (MPO)-DNA complexes, a biomarker of NETs, and HMGB1 have been associated with delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH). Additional mechanistic NET-related biomarkers and their role in the neuroinflammatory cascade surrounding DCI remain to be explored. A post-hoc analysis of a prospective, blinded, single-center biomarker observational study was performed. De novo measurements of serum citrullinated histone H3-DNA complexes (H3Cit-DNA), peptidylarginine deiminase 4 (PAD4), cell-free DNA (cf-DNA), and DNase 1 activity were conducted on admission (D0) and day 4 (D4). Delayed cerebral infarction (DCI) was defined as new cerebral infarction on CT head not present on the post-treatment scan. H3Cit-DNA, PAD4, cf-DNA, and DNase 1 activity were within quantifiable ranges in all serum samples analyzed at D0 and D4. Admission biomarker levels were not associated with DCI development. From D0 to D4, in both the DCI and the non-DCI groups, H3Cit-DNA levels significantly decreased, cf-DNA levels significantly increased, and PAD4 levels remained stable. In contrast, DNase 1 activity significantly decreased from D0 to D4 in the DCI group (p < 0.001) but not in the non-DCI group. This exploratory analysis demonstrated NET-related biomarkers such as H3Cit-DNA, PAD4, cf-DNA, and DNase 1 activity in all aSAH patients. A decline of systemic DNase 1 activity in the early phase might increase the risk of delayed cerebral ischemia.

Exercise-Induced Cell-Free DNA Correlates with Energy Expenditure in Multiple Exercise Protocols.

Exercise-induced cell-free DNA (ei-cfDNA) has been studied in response to various types of exercise. Its correlation with exercise intensity and duration has been observed consistently. However, comprehensive measurements and exploration of the tissue of origin are lacking. The aim of this study is to establish precise connections between exercise variables and the distribution of tissue of origin, aiming to provide further evidence supporting its use as a biomarker for exercise. Twelve self-identified active adults (six men and six women) performed a crossover study starting with either endurance testing or resistance testing under different intensities and protocols. We obtained blood before and after each exercise session and measured the levels of cfDNA and determined its tissue of origin utilizing cell type-specific DNA methylation patterns in plasma. We found that when duration and intensity are fixed, ei-cfDNA fold change correlates with energy expenditure ( P = 0.001) in endurance testing and years trained ( P = 0.001) in resistance testing. Most of the ei-cfDNA comes from increases in white blood cells (~95%) where neutrophils make up the majority (~74%) and the distribution is different between exercise modalities and protocols. This study highlights the potential of exercise-induced cfDNA as a biomarker for exercise, showing correlations with energy expenditure and a consistent pattern of tissue origin. Additional research is needed to investigate potential sex differences in the response of cfDNA to exercise, further exploring its clinical implications.

Levels of Cell-Free DNA in Kidney Failure Patients before and after Renal Transplantation.

Circulating cell-free DNA (cfDNA) has diverse applications in oncological, prenatal, toxicological, cardiovascular, and autoimmune diseases, diagnostics, and organ transplantation. In particular, mitochondrial cfDNA (mt-cfDNA) is associated with inflammation and linked to early vascular ageing (EVA) in end-stage kidney failure (ESKF), which could be a noninvasive marker for graft rejection and organ damage. Plasma samples from 44 ESKF patients, of whom half (n = 22) underwent either conservative therapy (non-HD) or hemodialysis (HD) before kidney transplantation (KT). These samples were analyzed at baseline and two years after KT. cfDNA was extracted from plasma and quantified using the fluorometric method. qPCR was used to quantify and differentiate the fractions of mt-cfDNA and nuclear cfDNA (nc-cfDNA). mt-cfDNA levels in KT patients decreased significantly from baseline to two years post-KT (p < 0.0268), while levels of total cfDNA and nc-cfDNA did not differ. Depending on therapy modality (HD vs. non-HD) before KT, total cfDNA levels were higher in HD patients at both baseline (p = 0.0133) and two years post-KT (p = 0.0421), while nc-cfDNA levels were higher in HD only at baseline (p = 0.0079). Males showed a nonsignificant trend of higher cfDNA levels. Patients with assessed vascular fibrosis (p = 0.0068), either alone or in combination with calcification plus fibrosis, showed reduced mt-cfDNA post-KT (p = 0.0195). Changes in mt-cfDNA levels suggests the impact of KT on the inflammatory state of ESKF, as evidenced via its correlation with high sensitivity C-reactive protein after KT. Further studies are warranted to assess if cfDNA could serve as a noninvasive method for monitoring the response to organ transplantation and even for amelioration of EVA status per se.

A comparative study on ctDNA and tumor DNA mutations in lung cancer and benign cases with a high number of CTCs and CTECs

BackgroundLiquid biopsy provides a non-invasive approach that enables detecting circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) using blood specimens and theoretically benefits early finding primary tumor or monitoring treatment response as well as tumor recurrence. Despite many studies on these novel biomarkers, their clinical relevance remains controversial. This study aims to investigate the correlation between ctDNA, CTCs, and circulating tumor-derived endothelial cells (CTECs) while also evaluating whether mutation profiling in ctDNA is consistent with that in tumor tissue from lung cancer patients. These findings will help the evaluation and utilization of these approaches in clinical practice.Methods104 participants (49 with lung cancer and 31 with benign lesions) underwent CTCs and CTECs detection using integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) strategy. The circulating cell-free DNA (cfDNA) concentration was measured and the mutational profiles of ctDNA were examined by Roche AVENIO ctDNA Expanded Kit (targeted total of 77 genes) by next generation sequencing (NGS) in 28 patients (20 with lung cancer and 8 with benign lesions) with highest numbers of CTCs and CTECs. Mutation validation in matched tumor tissue DNA was then performed in 9 patients with ctDNA mutations using a customized xGen pan-solid tumor kit (targeted total of 474 genes) by NGS.ResultsThe sensitivity and specificity of total number of CTCs and CTECs for the diagnosis of NSCLC were 67.3% and 77.6% [AUC (95%CI): 0.815 (0.722–0.907)], 83.9% and 77.4% [AUC (95%CI): 0.739 (0.618–0.860)]. The concentration of cfDNA in plasma was statistically correlated with the size of the primary tumor (r = 0.430, P = 0.022) and CYFRA 21–1 (r = 0.411, P = 0.041), but not with the numbers of CTCs and CTECs. In this study, mutations were found to be poorly consistent between ctDNA and tumor DNA (tDNA) in patients, even when numerous CTCs and CTECs were present.ConclusionDetection of CTCs and CTECs could be the potential adjunct tool for the early finding of lung cancer. The cfDNA levels are associated with the tumor burden, rather than the CTCs or CTECs counts. Moreover, the poorly consistent mutations between ctDNA and tDNA require further exploration.

Examining the Relationship and Prognostic Significance of Cell-Free DNA Levels and the PSMA-Positive Tumor Volume in Men with Prostate Cancer: A Retrospective-Prospective [68Ga]Ga-PSMA-11 PET/CT Study.

Functional imaging with prostate-specific membrane antigen (PSMA) ligands has emerged as the standard imaging method for prostate cancer (PCA). In parallel, the analysis of blood-derived, cell-free DNA (cfDNA) has been shown to be a promising quantitative biomarker of PCA aggressiveness and patient outcome. This study aimed to evaluate the relationship and prognostic value of cfDNA concentrations and the PSMA-positive tumor volume (PSMA-TV) in men with PCA undergoing [68Ga]Ga-PSMA-11 PET/CT imaging. Methods: We recruited 148 men with histologically proven PCA (mean age, 70.7 ± 7.7 y) who underwent [68Ga]Ga-PSMA-11 PET/CT (184.9 ± 18.9 MBq) and blood sampling between March 2019 and August 2021. Among these, 74 (50.0%) had hormone-sensitive PCA and 74 (50.0%) had castration-resistant PCA (CRPC). All patients provided written informed consent before blood sample collection and imaging. The cfDNA was extracted and quantified, and PSMA-expressing tumor lesions were delineated to extract the PSMA-TVs. The Spearman coefficient assessed correlations between PSMA-TV and cfDNA concentrations and cfDNA's relation with clinical parameters. The Kruskal-Wallis test examined the mean cfDNA concentration differences based on PSMA-TV quartiles for significantly correlated patient groups. Log-rank and multivariate Cox regression analyses evaluated the prognostic significance of high and low cfDNA and PSMA-TV levels for overall survival. Results: Weak positive correlations were found between cfDNA concentration and PSMA-TV in the overall group (r = 0.16, P = 0.049) and the CRPC group (r = 0.31, P = 0.007) but not in hormone-sensitive PCA patients (r = -0.024, P = 0.837). In the CRPC cohort, cfDNA concentrations significantly differed between PSMA-TV quartiles 4 and 1 (P = 0.002) and between quartiles 4 and 2 (P = 0.016). Survival outcomes were associated with PSMA-TV (P < 0.0001, P = 0.004) but not cfDNA (P = 0.174, P = 0.12), as per the log-rank and Cox regression analysis. Conclusion: These findings suggest that cfDNA might serve as a biomarker of advanced, aggressive CRPC but does not reliably reflect total tumor burden or prognosis. In comparison, [68Ga]Ga-PSMA-11 PET/CT provides a highly granular and prognostic assessment of tumor burden across the spectrum of PCA disease progression.

A Reliable Method for Quantifying Plasma Cell-Free DNA Using an Internal Standard Strategy: Evaluation in a Cohort of Non-Pregnant and Pregnant Women.

The use of plasma cell-free DNA (cfDNA) as a useful biomarker in obstetric clinical practice has been delayed due to the lack of reliable quantification protocols. We developed a protocol to quantify plasma cfDNA using an internal standard strategy to overcome difficulties posed by low levels and high fragmentation of cfDNA. cfDNA was isolated from plasma samples of non-pregnant (NP, n = 26) and pregnant (P, n = 26) women using a commercial kit and several elution volumes were evaluated. qPCR parameters were optimized for cfDNA quantification, and several quantities of a recombinant standard were evaluated as internal standard. Absolute quantification was performed using a standard curve and the quality of the complete method was evaluated. cfDNA was eluted in a 50-μl volume, actin-β (ACTB) was selected as the target gene, and qPCR parameters were optimized. The ACTB standard was constructed and 1000 copies were selected as internal standard. The standard curve showed R2 = 0.993 and E = 109.7%, and the linear dynamic range was defined between 102 and 106 ACTB copies/tube. Repeatability and reproducibility in terms of CV were 19% and up to 49.5% for ACTB copies per milliliter of plasma, respectively. The range of cfDNA levels was 428-18,851 copies/mL in NP women and 4031-2,019,363 copies/mL in P women, showing significant differences between the groups. We recommend the application of internal standard strategy for a reliable plasma cfDNA quantification. This methodology holds great potential for a future application in the obstetric field.

Three-week sprint interval training (SIT) reduces cell-free DNA and low-frequency fatigue but does not induce VO2max improvement in older men.

This study aimed to investigate the impact of sprint interval training (SIT) on both the acute and 3-week modulations of cell-free DNA (cfDNA), as well as its association with neuromuscular fatigue and physical performance in healthy young and old men. Ten young (20-25year old) and nine elderly (63-72year old) healthy men performed nine SIT sessions consisting of 4-to-6-all-out cycling repetitions of 30s interspaced with 4-min rest intervals. We compared the maximal voluntary contractions torque, central activation ratio, low-frequency fatigue (LFF), and cfDNA concentrations between the groups before, immediately after, 1h after, and 24h after the first and ninth SIT sessions. The plasma cfDNA levels were increased post-exercise (from 1.4 ± 0.258 to 1.91 ± 0.278ng/ml (P < 0.01) on a log10 scale), without significant differences between the groups. However, older individuals showed a slight decrease in the baseline cfDNA values, from 1.39 ± 0.176 to 1.29 ± 0.085ng/ml on a log10 scale, after 3weeks (P = 0.043). Importantly, the elevation of the post-exercise cfDNA values was correlated with an increase in LFF in both groups. Three weeks of SIT induced an improvement in the recovery of LFF (main session effect, P = 0.0029); however, only the young group showed an increase in aerobic capacity (VO2max) (from 40.8 ± 6.74 to 43.0 ± 5.80ml/kg/min, P = 0.0039). Three weeks of SIT diminished the baseline cfDNA values in the old group, together with an improvement in the recovery of LFF. However, VO2max was increased only in the young group.

High cell-free DNA is associated with disease progression, inflammasome activation and elevated levels of inflammasome-related cytokine IL-18 in patients with myelofibrosis.

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1β and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.

Cell-free DNA as diagnostic and prognostic biomarkers for adult sepsis: a systematic review and meta-analysis

Although cell-free DNA (cfDNA) is an emerging sepsis biomarker, the use of cfDNA, especially as diagnostic and prognostic indicators, has surprisingly not been systemically analyzed. Data of adult patients with sepsis that conducted cfDNA measurement within 24 h of the admission was collected from PubMed, ScienceDirect, Scopus, and Cochrane Library until October 2022. The Quality in Prognosis Studies (QUIPS) and Quality Assessment in Diagnostic Studies-2 (QUADAS-2) tools were used to reduce the risk of biased assessment. The mean difference (MD) of cfDNA concentration and the standardized mean difference (SMD) between populations was calculated using Review Manager (RevMan) version 5.4.1 package software. Pooled analysis from 18 included studies demonstrated increased serum cfDNA levels in sepsis when compared with healthy control (SMD = 1.02; 95% confidence interval (CI) 0.46–1.57) or non-sepsis patients in the intensive care unit (ICU) (SMD = 1.03; 95% CI 0.65–1.40), respectively. Meanwhile, a slight decrease in the statistical value was observed when compared with non-sepsis ICU patients with SIRS (SMD = 0.74; 95% 0.41–1.06). The lower cfDNA levels were also observed in sepsis survivors compared to the non-survivors (SMD at 1.43; 95%CI 0.69–2.17) with the pooled area under the receiver operating characteristic curve (AUC) of 0.76 (95% CI 0.64–0.87) for the mortality prediction. Levels of cfDNA showed a pooled sensitivity of 0.81 (95% CI 0.75–0.86) and specificity of 0.72 (95% CI 0.65–0.78) with pooled diagnostic odd ratio (DOR) at 25.03 (95% CI 5.48–114.43) for the identification of sepsis in critically ill conditions. The cfDNA levels were significantly higher in patients with sepsis and being a helpful indicator for the critically ill conditions of sepsis. Nevertheless, results of the test must be interpreted carefully with the context of all clinical situations.

CTNI-55. A MULTICENTER STUDY OF LOW-INTENSITY FOCUSED ULTRASOUND WITH SYSTEMIC MICROBUBBLE OSCILLATORS FOR BLOOD-BRAIN BARRIER DISRUPTION FOR LIQUID BIOPSY IN GLIOBLASTOMA (LIBERATE)

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by a lack of requisite circulating-free DNA (cfDNA) blood levels in due to the blood-brain barrier (BBB). Real-time image-guided low-intensity focused ultrasound (LIFU) with intravenously administered microbubble oscillators non-invasively causes BBB disruption (BBBD). This clinical trial aimed to evaluate the utility of LIFU for increasing cfDNA in blood for liquid biopsy in GBM. METHODS LIBERATE is an ongoing, prospective, multi-center, self-controlled, ongoing, pivotal trial evaluating the safety and technical efficacy of LIFU for BBBD to increase cfDNA in blood for GBM. Patients aged 18-80 years with suspected GBM planned for tumor biopsy or resection at ten centers in the US are being included. Patients are administered intravenous oscillating microbubbles for enhancing sonication, after which MR-guided BBBD using 220 kHz LIFU device is performed, with real-time acoustic feedback for effective sonication. Pre- and post-procedure, phlebotomies, and MRI brain are performed. The primary study endpoint is defined, per subject, as the ratio between cfDNA levels in blood 1-hour post-LIFU procedure compared to cfDNA level in blood pre-procedure. The primary study hypothesis is that BBBD with LIFU leads to ≥2-fold increase in cfDNA in blood. The secondary hypothesis is that there exists ≥75% concordance between biomarker patterns in the cfDNA sample from 1-hour post-LIFU sample and tumor tissue obtained later. Exploratory endpoints include (1) sensitivity of detection of known somatic mutations in cfDNA from blood samples collected before and after LIFU, (2) estimation of cfDNA levels in samples collected at 30 minutes, 1 hour, 2 hours, and 3 hours post-LIFU to determine the time of greatest yield, (3) correlation of MRI parameters related to BBBD grading and biomarkers in cfDNA from post-LIFU samples. Patient enrollment commenced in 2022, and 13 patients have been recruited by 06/09/2023. Clinical trial information: NCT05383872.