Release Of Cell-free DNA Research Articles

Assessment of cfDNA levels in saliva samples of stressed young adults: Preliminary study

Purpose: The aim of this pilot study was to examine the levels of cell-free DNA (cfDNA) in the saliva samples of second-year medical students before and after a stress-inducing event, exams. Materials and Methods: Saliva samples were collected from 20 students, whose stress profiles were assessed using the DASS-21 scale, both before and after the exams. Cell-free DNA were isolated from these samples, and measured using spectrophotometry and automated electrophoresis system. Results: Spectrophotometric analysis revealed a significant decrease in cfDNA levels in the saliva samples collected after the exams (p ≤ 0.05). This difference was confirmed by the automated electrophoresis system, particularly showing a reduction in cfDNA amounts in the 40-200 bp range with the reduction of stress (p ≤ 0.05). Conclusion: Psychosocial stress affects the release of cell-free DNA. This study reports that the amount of cfDNA found in saliva samples significantly changes in relation to stress. This preliminary study suggests that cfDNA in saliva samples could potentially serve as a biological marker for stress in healthy individuals.

Deficiency of hasB accelerated the clearance of Streptococcus equi subsp. Zooepidemicus through gasdermin d-dependent neutrophil extracellular traps

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus, SEZ) is an essential zoonotic bacterial pathogen that can cause various inflammation, such as meningitis, endocarditis, and pneumonia. UDP-glucose dehydrogenase (hasB) is indispensable in synthesizing SEZ virulence factor hyaluronan capsules. Our study investigated the infection of hasB on mice response to SEZ by employing a constructed capsule-deficient mutant strain designated as the ΔhasB strain. This deficiency was associated with a reduced SEZ bacterial load in the mice’s blood and peritoneal lavage fluid (PLF) post-infection. Besides, the ΔhasB SEZ strain exhibited a higher propensity for neutrophil infiltration and release of cell-free DNA (cfDNA) in vivo compared to the wild-type (WT) SEZ strain. In vitro experiments further revealed that ΔhasB SEZ more effectively induced the formation of neutrophil extracellular traps (NETs) containing histone 3 (H3), neutrophil elastase (NE), and DNA, than its WT counterpart. Moreover, the release of NETs was determined to be gasdermin D (GSDMD)-dependent during the infection process. Taken together, these findings underscore that the deficiency of the hasB gene in SEZ leads to enhanced GSDMD-dependent NET release from neutrophils, thereby reducing SEZ’s capacity to resist NETs-mediated eradication during infection. Our finding paves the way for the development of innovative therapeutic strategies against SEZ.

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

AbtractClinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.

Distinct Molecular Processes Mediate Donor-derived Cell-free DNA Release From Kidney Transplants in Different Disease States: Erratum.

Distinct Molecular Processes Mediate Donor-derived Cell-free DNA Release From Kidney Transplants in Different Disease States: Erratum.

Circadian rhythm and circulating cell-free DNA release on healthy subjects

In the last decade, clinical studies have investigated the clinical relevance of circulating cell-free-DNA (ccfDNA) as a diagnostic and prognosis tool in various diseases including cancers. However, limited knowledge on ccfDNA biology restrains its full development in the clinical practice. To improve our understanding, we evaluated the impact of the circadian rhythm on ccfDNA release in healthy subjects over a 24-h period. 10 healthy female subjects underwent blood sampling at 8am and 20 healthy male subjects underwent serial blood sampling (8:00 AM, 9:00 AM, 12:00 PM, 4:00 PM, 8:00 PM, 12:00 AM, 4 AM (+ 1 Day) and 8 AM (+ 1 Day)). We performed digital droplet-based PCR (ddPCR) assays to target 2 DNA fragments (69 & 243 bp) located in the KRAS gene to determine the ccfDNA concentration and fragmentation profile. As control, half of the samples were re-analyzed by capillary miniaturized electrophoresis (BIAbooster system). Overall, we did not detect any influence of the circadian rhythm on ccfDNA release. Instead, we observed a decrease in the ccfDNA concentration after meal ingestion, suggesting either a post-prandial effect or a technical detection bias due to a higher plasma load in lipids and triglycerides. We also noticed a potential effect of gender, weight and creatinine levels on ccfDNA concentration.

The origin and possible mechanism of embryonic cell-free DNA release in spent embryo culture media: a review.

The presence of cell-free DNA in spent embryo culture media (SECM) has unveiled its possible utilization for embryonic ploidy determination, opening new frontiers for the development of a non-invasive pre-implantation genetic screening technique. While a growing number of studies have shown a high concordance between genetic screening using cell-free DNA (cfDNA) and trophectoderm (TE), the mechanism pertaining to the release of cfDNA in SECM is largely unknown. This review aims to evaluate research evidence on the origin and possible mechanisms for the liberations of embryonic DNA in SECM, including findings on the self-correction abilities of embryos which might contribute to the presence of cfDNA. Several databases including EMBASE, PUBMED, and SCOPUS were used to retrieve original articles, reviews, and opinion papers. The keywords used for the search were related to the origins and release mechanism of cfDNA. cfDNA in SECM originates from embryonic cells and, at some levels, non-embryonic cells such as maternal DNA and exogenous foreign DNA. The apoptotic pathway has been demonstrated to eliminate aneuploid cells in developing mosaic embryos which might culminate to the release of cfDNA in SECM. Nonetheless, there is a recognized need for exploring other pathways such as cross-talk molecules called extracellular vesicles (EVs) made of small, round bi-layer membranes. During in vitro development, embryos physiologically and actively expel EVs containing not only protein and microRNA but also embryonic DNA, hence, potentially releasing cfDNA of embryonic origin into SECM through EVs.

Abstract WP60: Greater Enrichment Of Neuronal Function Can Be Reflected In Blood Plasma Samples Following The Stroke.

Acute ischemic stroke induces widespread cellular necrosis and apoptosis and activation of inflammation. These mediate release of cell-free DNA (cfDNA) into the circulation. Although increased cfDNA concentrations have been associated with clinical outcome in stroke, data concerning neuronal cfDNA is rare. In this study, we assessed the association of cfDNA with neuron biomarkers in patients with acute ischemic stroke. Methods: Thirteen patients without stroke and four patients with middle cerebral artery occlusion were recruited at UT Memorial Hermann Hospital. Blood plasma samples were collected and cfDNA samples were prepared for whole-genome bisulfite sequencing (WGBS) in order to generate quantitative DNA methylation datasets. The resulting WGBS data were analyzed for epigenetic biomarker identification using bioinformatic statistical approaches. CelFiE was used for cell deconvolution, designed to accurately estimate the relative abundances of cell types and tissues present in plasma cfDNA from methylation sequencing, a technique frequently referred to as cell deconvolution. Results: Differential methylation statistical analysis comparing the stroke against the non-stroke group of samples found 3493 DNA methylation differences. Pathway Analysis discovered greater enrichment for neuronal function with the top hit being “Neuronal System” followed by activity surrounding synapses, demonstrating that a clear enrichment of methylation change in neuronal pathways can be reflected from blood plasma samples following the stroke. Cell deconvolution showed trends of increased megakaryocyte and neutrophils which are involved in the post-stroke immune response. It may be that the secreted neutrophil nuclear DNA in blood plasma drives the increase in the observed proportion of neutrophil cell type in the stroke group samples. Principal Component Analysis demonstrated good separation between the stroke and normal groups. Conclusion: Blood sample can be used for circulating cfDNA analysis and neuron biomarker discovery for acute ischemic stroke prediction/recovery.

EGFR inhibition in EGFR-mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) elicit potent cell cycle arrest in EGFR-mutant non-small-cell lung cancer (NSCLC) cells. However, little is known about the mechanisms through which these drugs alter the tumor phenotype that contributes to the immune escape of EGFR-mutant cells. Using EGFR-mutant NSCLC cell lines and tissue samples from patients, we investigated the changes in immune checkpoints expressed in tumor cells following EGFR inhibition. Subsequently, we also analyzed the role of soluble factors from the dying tumor cells in the activation of immune signaling pathways involved in therapy resistance. Upon EGFR-TKI treatment, we found that EGFR-mutant cells upregulated the expression of innate immune checkpoint CD24 in vitro. We then analyzed biopsy samples from six patients who developed resistance to a first-generation EGFR-TKI without the acquired T790M mutation. Immunohistochemistry revealed that levels of tumor CD24 expression were increased upon treatment compared with those from pre-treatment samples. Monocyte-derived macrophages facilitated antibody-dependent cellular phagocytosis when EGFR-TKI-treated EGFR-mutant cells were incubated with anti-CD24 antibodies in vitro, suggesting that CD24 may be a therapeutical target for EGFR-mutant lung cancer. Moreover, EGFR inhibition accelerated the release of cell-free DNA (cfDNA) from dying tumor cells, which activated the type I interferon signaling pathways in human THP-1 monocytes in a stimulator of interferon genes-dependent manner. Our study indicates that EGFR inhibition in EGFR-mutant NSCLC cells fosters a tumor microenvironment associated with immune escape. Thus, CD24 targeted therapy and cfDNA monitoring may contribute to improved treatment outcomes in patients with EGFR-mutant NSCLC.

Abstract 13: Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro

Abstract Despite the well-documented benefits of circulating tumor DNA (ctDNA) based liquid biopsy, including screening, prognostication, and treatment monitoring, there remain several concerns with its application. Chief among these concerns are imperfect sensitivity and specificity, particularly pre-diagnosis and after tumor resection, when ctDNA burden is low. Cell death, either through apoptosis or necrosis, has been the most widely accepted mechanism of DNA release. Recent studies have also suggested active release through extracellular vesicles as a paradigm for DNA release. However, this pathway remains highly debated in terms of its contribution, mechanism, and even credibility. To address the deficiencies in ctDNA detection in the clinic and the lack of basic mechanistic knowledge of cf/ctDNA release, we performed an in vitro CRISPR screen on MCF10A cells to identify regulators of DNA release. This normal breast epithelial cell line was chosen because MCF10A cells release DNA into cell culture media with a similar fragmentation pattern to that observed in human blood. After lentiviral infection with the genome-wide Brunello CRISPR-Cas9 knockout library, the population of knockouts identified in the genomic DNA of cells attached to the plates was compared to the population of knockouts found in the DNA released into the cell-depleted culture media. Genes with skewed representation were considered potential regulators of DNA release. Our analysis identified several genes in the TRAIL apoptotic pathway including FADD, as well as several genes encoding RNA-binding proteins including Sam68. Knockout of Sam68 and FADD in MCF10As, MDA-MB-468 breast cancer cells, and NCI-H841 lung cancer cells lead to decreases in DNA release. Previous studies have hypothesized that the fragment size of released DNA is dependent on release mechanism. However, the decreases observed in our knockout cell lines were not limited to a specific fragment size of DNA. Application of the small molecule apoptosis inhibitor Z-VAD-FMK also recapitulated the DNA release phenotype seen in our knockout cell lines. From these results, we conclude that apoptosis is the major source of DNA release from cancer cells, and that fragment size is not the indicator of release mechanism it has been presumed to be. Mechanistic studies of DNA release such as this could lead to the ability to modify the amount of ctDNA in circulation, which would significantly improve accurate detection in the clinic. Citation Format: Brad A. Davidson, Adam X. Miranda, Sarah Croessmann, Ben Ho Park. Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 13.

Cell-free DNA Release in the Plasma of Patients with Cardiac Disease is Associated with Cell Death Processes.

Cell-free DNA (cfDNA) is released into the plasma of patients with cardiac disease. Here, the source and mechanism of plasma cfDNA release in patients with myocardial infarction (MI) and other cardiac diseases (n = 59) were investigated. Plasma levels of various markers including M30 (apoptosis), M65 (apoptosis and necrosis), cyclophilin A (CyPA) (necrosis), and myeloperoxidase (MPO) (neutrophil activation) were assayed. The plasma cfDNA concentrations in MI and other cardiac diseases were significantly higher than that in the healthy control subjects. Significant differences were not observed among the cardiac disease patients (MI and other cardiac diseases) and healthy control subjects in M30, M65, and CyPA levels. In contrast,the MPO levels were significantly elevated in cardiac disease patients when compared to control groups, and MPO levels in MI patients were significantly higher than other cardiac diseases patients. These results suggest that cfDNA is mainly released by neutrophils via NETosis in addition to apoptosis except for epithelial apoptosis in patients with cardiac disease and the degree is greater in MI patients. The results from this study provide basic information for diagnosis marker of MI.

Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy

Physical activity impacts immune homeostasis and leads to rapid and marked increase in cell-free DNA (cfDNA). However, the origin of cfDNA during exercise remains elusive and it is unknown if physical activity could improve or interfere with methylation based liquid biopsy. We analyzed the methylation levels of four validated CpGs representing cfDNA from granulocytes, lymphocytes, monocytes, and non-hematopoietic cells, in healthy individuals in response to exercise, and in patients with hematological malignancies under resting conditions. The analysis revealed that physical activity almost exclusively triggered DNA release from granulocytes, highlighting the relevance as a pre-analytical variable which could compromise diagnostic accuracy.Graphical

Anticancer effects of mifepristone on human uveal melanoma cells

BackgroundUveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds.MethodsThe effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR.ResultsMF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor.ConclusionThis study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

Combined Genotype Analysis of Tumor and Cell-Free DNA By Ultra-Deep Targeted Sequencing: Correlation with PET-Scan Imaging in a Prospective Cohort of Diffuse Large B-Cell Lymphoma Patients

The emerging concept of liquid biopsy in diffuse large B-cell lymphoma (DLBCL) is based on the release of Cell-free DNA (cfDNA) into the blood by tumor cells undergoing apoptosis or necrosis that can be used for the identification of mutations (Bohers et al., Haematologica 2015 ; Rossi et al., Blood 2017). cfDNA theoretically can give therefore information regarding potentially actionable mutations, clonal evolution, and genetic mechanisms of resistance. To determine the clinical relevance of cfDNA, we assessed prospectively by Ultra-deep targeted next generation sequencing (NGS) the basal DLBCL genetic pattern, its modification upon treatment using plasma cfDNA analysis and its correlation with PET scan imaging (clinical.gov number: NCT02339805).MethodsThirty consecutive untreated DLBCL or primary mediastinal B-cell lymphoma (PMBL) were enrolled and followed after rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) or R-CHOP-like chemotherapies. Mutational status of a 34-genes lymphopanel (CIITA, B2M, TNFRSF14, CD58, NOTCH1, NOTCH2, MFHAS1, XPO1, MYC, CDKN2A/B, FOXO1, TP53, GNA13, BCL2, TNFAIP3, MYD88, PIM1, CARD11, IRF4, PRDM1, EZH2, KMT2D, EP300, MEF2B, CREBBP, BRAF, CD79A/B, ITPKB, TCF3, ID3, SOCS1 and STAT6) was assessed in both tumor (genomic DNA, gDNA) and cfDNA at the time of diagnosis using AmpliSeq®/PGM® technologies (Dubois et al., Clinical Cancer Research 2016) . Longitudinal analysis of plasma samples collected at mid-treatment (C4) and at the end of treatment or at relapse/progression was performed and correlated with PET scan characteristics obtained at baseline (Metabolic tumor volume, MTV), mid and end-treatment (Delta SUV and Deauville score). To increase the NGS sensitivity and for cost-effectiveness reasons, only mutations detected at baseline were monitored during the follow-up. Variant analysis was performed using an in-house generated bioinformatics pipeline.ResultsPatient clinical features were as follow: IPI: 0-1, n =8; IPI: 2-3, n=14; IPI: 4-5, n =8; median age was 64 y; bone marrow involvement: 0/25 patients; elevated LDH: 12/30; Fifteen patients were sub-classified as GCB, 9 as ABC/non GCB and 3 as PMBL. cfDNA was available for the 30 patients at the time of diagnosis. Mean plasma concentration was 74 ng/mL (14.6 - 433) and sequencing was carried out with a mean depth of 3750x. cfDNA mutations were identified in 20 patients (67%) and were consistent with the pattern usually observed in the different subtypes of DLBCL. gDNA mutations from tissue biopsies (11 frozen/14 FFPE) were found in 22/25 (88%) cases. In 3/22 cases for whom gDNA analysis failed, an informative mutational pattern was finally successfully obtained in matched cfDNA. Variant allelic frequency (VAF) was lower in cfDNA (mean 15%) compared to tumor DNA (mean 33%). Finally, among the 22 tumor/plasma matched samples, 8 patients had no detectable tumoral cfDNA, whereas 4 patients displayed additional mutations in plasma compared to tumor. At the time of diagnosis, a high [cfDNA] correlated with the presence of tumoral circulating DNA and cfDNA mutations were observed in all patients with elevated LDH. MTV correlated with the highest concentrations of cfDNA and VAF rate. Moreover, 3 / 4 patients presenting additional mutations in cfDNA, as compared to gDNA, had a high MTV (> 2000cm3), suggesting that cfDNA mutations reflect more accurately tumor heterogeneity than gDNA analysis. After R-CHOP/ R-CHOP-like regimens, a rapid clearance of cfDNA mutations was observed in 13/17 cases. Conversely, among the 4/17 patients whose basal DLBCL mutations did not disappear at mid-treatment, 3 were in partial response according to Deauville score, including 2 with a delta SUVmax < 70%. One patient in partial remission had progressive disease 6 months after the end of the treatment and still harboured tumoral cfDNA, as shown by a persistent detectable TP53 mutation.ConclusionOur study demonstrates that cfDNA genotyping of DLBCL is an accurate genotyping tool and represents a real-time and non-invasive approach to tracking treatment-resistant clones. It highlights the major interest of the liquid biopsy in the context of bulky tumors where cfDNA is more representative than gDNA by capturing the entire tumoral mutation profile. cfDNA analysis represents therefore a complementary approach to TEP scan imaging at baseline and during follow-up for the management of DLBCL. DisclosuresStamatoullas:Takeda: Consultancy; Celgene Corporation: Honoraria. Tilly:Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Immunogen: Honoraria; Gilead: Honoraria; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria. Jardin:Roche: Honoraria; celgen: Honoraria; Janssen: Honoraria.

Targeted Liquid Biopsy Using Irradiation to Facilitate the Release of Cell-Free DNA from a Spatially Aimed Tumor Tissue.

PurposeWe investigated the feasibility of using an anatomically localized, target-enriched liquid biopsy (TLB) in mouse models of lung cancer.Materials and MethodsAfter irradiating xenograft mouse with human lung cancer cell lines, H1299 (NRAS proto-oncogene, GTPase [NRAS] Q61K) and HCC827 (epidermal growth factor receptor [EGFR] E746–750del), circulating (cell-free) tumor DNA (ctDNA) levels were monitored with quantitative polymerase chain reaction on human long interspersed nuclear element-1 and cell line-specific mutations. We checked dose-dependency at 6, 12, or 18 Gy to each tumor-bearing mouse leg using 6-MV photon beams. We also analyzed ctDNA of lung cancer patients by LiquidSCAN, a targeted deep sequencing to validated the clinical performances of TLB method.ResultsIrradiation could enhance the detection sensitivity of NRAS Q61K in the plasma sample of H1299-xenograft mouse to 4.5-fold. While cell-free DNA (cfDNA) level was not changed at 6 Gy, ctDNA level was increased upon irradiation. Using double-xenograft mouse with H1299 and HCC827, ctDNA polymerase chain reaction analysis with local irradiation in each region could specify mutation type matched to transplanted cell types, proposing an anatomically localized, TLB. Furthermore, when we performed targeted deep sequencing of cfDNA to monitor ctDNA level in 11 patients with lung cancer who underwent radiotherapy, the average ctDNA level was increased within a week after the start of radiotherapy.ConclusionTLB using irradiation could temporarily amplify ctDNA release in xenograft mouse and lung cancer patients, which enables us to develop theragnostic method for cancer patients with accurate ctDNA detection.

Mitoxantrone Inhibits FMLP-Induced Degenerative Changes in Human Neutrophils

Neutrophils fight with invading pathogens through various mechanisms including degranulation, phagocytosis, and the release of neutrophil extracellular traps (NETs). This study aimed to determine the impact of a synthetic formyl-peptide (FMLP) on human neutrophils in vitro, and to determine the role of mitoxantrone (MTX), a pharmacological blocker of mitochondrial Ca^(2+) Uniporter (MCU), on FMLP-induced alterations. Isolated neutrophils and a whole-blood preparation of neutrophils were pre-treated with MTX and then stimulated with FMLP. Field's-stained smears and brightfield microscopy were employed for morphological characterization and quantification of neutrophils. The release of cell-free DNA (cfDNA) was also measured for determining neutrophil damage. Our data demonstrated degenerative changes in neutrophils and a greater cfDNA release upon stimulation with FMLP which was negatively associated with the presence of resting platelets in whole blood preparation. Interestingly, MTX pre-treatment significantly reduced FMLP-triggered neutrophil damage and cfDNA release. Metformin, a known inhibitor of NETs formation, also decreased the FMLP-induced changes in neutrophils. In addition to confirming the degenerative potential of FMLP, this study reveals a novel contribution of MCU in regulating FMLP-induced morphological alterations in human neutrophils.

Cell-free DNA as an obesity biomarker.

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.

Senescence, Necrosis, and Apoptosis Govern Circulating Cell-free DNA Release Kinetics

The kinetics of circulating cell-free DNA (cfDNA) release may provide a real-time assessment of induced cell death. However, there is a limited understanding of the underlying biological rationale for cfDNA release following distinct treatments and cell death mechanisms. Here, we uncover a complex interplay between apoptosis, necrosis, and senescence in determining cfDNA release kinetics. Utilizing multiple invitro and invivo preclinical models, we show how cfDNA release is modulated through a combination of apoptotic and senescent triggers and inhibitors. Interestingly, we identify treatment-induced senescence as a previously unrecognized determinant of cfDNA kinetics that can counteract its release. Necrosis is the predominant cell death mechanism that consistently contributes to cfDNA release in response to ionizing radiation, and, surprisingly, apoptosis plays a comparatively minor role in some tumors. Based on our results, we propose a model to explain cfDNA release from cells over time, with important implications for future studies.

P1. Abstract Title: Markers of Coagulation as Potential Indicators for the Diagnosis of Sepsis

<h3>Introduction</h3> The emergency department (ED) is an important venue for early diagnosis of sepsis; however, it remains a clinical challenge due to its heterogenous presentation and lack of a definitive biomarker. Neutrophil extracellular trap formation, with the release of cell free DNA (cfDNA) and activation of the coagulation cascade, and inhibition of the anticoagulant Protein C (PC) through extracellular histone, have been shown to be important in sepsis prognosis. It is unknown whether these or other molecules important in the interface between infection and coagulation, might also be diagnostic. <h3>Objectives</h3> 1) To investigate if coagulation factors known to be involved in sepsis prognosis such as PC, cfDNA can aid in sepsis diagnosis. 2) To explore other potential coagulation markers such as the ADAMTS13. 3) To determine if a panel of biomarkers, in correlation with previously known laboratory tests and clinical scores of abnormal vital signs and organ dysfunction improves sepsis diagnosis in the emergency department. <h3>Materials and Methods</h3> This is a prospective cohort study of patients with suspicion of sepsis presenting to two tertiary hospital EDs in Hamilton, Ontario. Plasma samples were obtained on initial presentation, usually before antibiotic administration, (T1) and 4 hrs later (T2). The demographic and clinical data of the patients admitted to the ED was collected from electronic medical records, including vital signs to calculate an early warning score and evidence of organ dysfunction. Plasma cfDNA levels were determined by silica-membrane-based DNA purification and UV spectrophotometry. PC, PCT and ADAMTS13 antigen were measured by enzyme-linked immunosorbent assay. <h3>Results</h3> We analysed 144 patients included in the study, mean age 68.2 years (SD 17.8), 50% males and a mean highest Hamilton Early Warning Score of 5.05 (SD 3.4). Procalcitonin, a biomarker known to increase in bacterial infections was significantly higher in the cohort at both time points compared to our healthy adult volunteers (p<0.005), while cfDNA levels did not show any change. In contrast, ADAMTS13 levels (T1and T2, p<0.0001) and Protein C levels (T2, p=0.02) were lower among the cohort compared to the healthy controls. Out of the 144 patients, 108 patients with positive body fluid cultures and antibiotic administration, had significantly higher respiratory rate and body temperature compared to culture negative patients. Notably, culture positive patients also showed a notable decline in the levels of ADAMTS13 antigen 4 hours after the initial presentation (p=0.007). <h3>Conclusions</h3> These findings suggest that when ED physicians suspect sepsis there is already evidence of coagulation abnormalities. Evaluating biomarkers associated with increased risk of thrombosis could aid in the diagnosis of sepsis.

The Effect of Maternal Obesity on Placental Cell-Free DNA Release in a Mouse Model.

The fetal fraction of cell-free DNA (cfDNA) in maternal plasma is decreased in obese women. The underlying mechanism is not well understood. The amount of cfDNA released from the placenta has not been directly examined in maternal obesity. We sought to quantify release of cfDNA from the placenta and fetal membranes in maternal diet-induced obesity using explant cultures in an established mouse model. C57BL6/J females were fed either 60% high-fat diet or 10% fat-matched control diet for 14 weeks prepregnancy and throughout gestation. Placentas and fetal membranes were collected on e18 and randomly allocated to time 0-, 1-, or 6-hour culture times. The CfDNA was isolated from culture media, quantified, and normalized to tissue weight. Placentas from obese dams released significantly less cfDNA compared to those of lean dams at time 0 (45.8 ± 4.3 ng/mg vs 65.6 ± 7.9 ng/mg, P = .02). Absolute cfDNA levels increased with longer placental culture, with no significant differences between obese and lean dams at 1 and 6 hours. Membranes released significantly less cfDNA than did placentas at every time point. Maternal obesity is associated with decreased release of cfDNA from the placenta compared to lean controls immediately after tissue harvest. This may provide an alternative explanation for the lower fetal fraction of cfDNA noted in maternal obesity.

Biological Rationale for Circulating Cell-Free DNA Release

The kinetics of circulating cell-free (cf)DNA release may provide a real-time assessment of induced cell death. However, there is limited understanding of the underlying biological rationale for cfDNA release following distinct treatments and cell death mechanisms. Here, we uncover a complex interplay between apoptosis, necrosis, and senescence in determining cfDNA release kinetics. We utilized multiple in vitro and in vivo preclinical models to show how cfDNA release is modulated through a combination of apoptotic and senescent triggers and inhibitors. Using cfDNA from head and neck squamous cell carcinoma (HNSCC) cell lines and fibroblasts grown in culture, or as mouse xenografts, we identified treatment-induced senescence as a previously unrecognized determinant of cfDNA kinetics that counteracts its release. Necrosis was the predominant cell death mechanism that consistently contributed to cfDNA release from HNSCC in response to ionizing radiation, while, surprisingly, apoptosis played a comparatively minor role in some tumours. Based on these results, we propose a generalizable model to explain cfDNA release from cells over time. These findings will have important implications for future studies evaluating the utility of measuring cfDNA kinetics in clinical settings.