cfDNA Levels Research Articles

Preoperative Cell-Free DNA (cfDNA) in Muscle-Invasive Bladder Cancer Treatment Outcome.

Tumor heterogeneity and lack of personalized prognosis leads to bladder cancer (BlCa) patients' lifelong surveillance with invasive interventions, highlighting the need for modern minimally invasive tools for disease management. Herein, we have evaluated the clinical utility of preoperative serum cell-free DNA (cfDNA) in ameliorating patients' risk-stratification and prognosis. cfDNA was purified from 190 preoperative BlCa patients and 26 healthy individuals' serum samples and quantified by 2 assays: an in-house quantitative real-time PCR (qPCR) assay using LEP as reference control and a direct fluorometric assay using Qubit HS dsDNA. Capillary electrophoresis was performed in 31 samples for cfDNA fragment profiling. Tumor relapse/progression and metastasis/death were used as clinical endpoints for non-muscle-invasive bladder cancer and muscle-invasive bladder cancer (MIBC), respectively. cfDNA profiling by capillary electrophoresis highlighted that total and fragment-related cfDNA levels were significantly increased in BlCa and associated with advance disease stages. Evaluation of cfDNA levels by both Qubit/qPCR displayed highly consistent results (rs = 0.960; P < 0.001). Higher cfDNA was correlated with MIBC and stronger risk for early metastasis (Qubit:hazard ratio [HR] = 3.016, P = 0.009; qPCR:HR = 2.918, P = 0.004) and poor survival (Qubit:HR = 1.898, P = 0.042; qPCR:HR = 1.888, P = 0.026) of MIBC patients. Multivariate cfDNA-fitted models led to superior risk stratification and net benefit for MIBC prognosis compared to disease established markers. Elevated preoperative cfDNA levels are strongly associated with higher risk for short-term metastasis and poor outcome of MIBC, supporting modern noninvasive disease prognosis and management.

Kinetics of postoperative circulating cell-free DNA and impact on minimal residual disease detection rates in patients with resected stage I-III colorectal cancer.

5 Background: A growing body of evidence supports the utility of circulating tumor DNA (ctDNA) as a useful biomarker for detecting molecular residual disease (MRD) in colorectal cancer (CRC). Immediately after surgery or during adjuvant therapy, high levels of cell-free DNA (cfDNA) from normal tissue may limit the detection of tumor-derived ctDNA. The optimal timing of blood collection for reliable MRD detection after surgery or adjuvant therapy remains unclear. Methods: In this retrospective, U.S.-based, multi-institutional study, data from commercial ctDNA testing in 16,347 patients with stage I-III CRC were analyzed. Complete clinical data were available for 417 patients with 2,538 plasma samples collected between 6/2019 and 4/2022. The median follow-up for relapsed and non-relapsed patients was 730 and 615 days, respectively. A personalized, tumor-informed multiplex PCR-based next-generation sequencing assay (Signatera) was used to quantify ctDNA prior to surgery and postoperatively in a longitudinal manner. We analyzed the kinetics of total cfDNA and compared it with the ctDNA MRD positivity rates at various time points after surgery. Results: Among all patients, cfDNA levels were higher immediately after surgery (0-2 weeks) and gradually declined during the subsequent 2-8 weeks (p&lt;0.0001). Despite the higher cfDNA levels, among patients with immediate post-operative draws (0-2 weeks) 30.6% (113/369) of patients were ctDNA positive. Similar ctDNA detection rates were observed with conventional MRD windows after resection, whether the window was defined as 2-6 weeks (20.8%; 957/4605) or 2-8 weeks (20.9%; 1155/5534). In the clinically annotated cohort, ctDNA-positivity during the MRD time window of 2-8 weeks and during surveillance (&gt;6 months post-operatively and subsequent to any adjuvant therapy) was significantly associated with worse recurrence-free survival as compared to ctDNA negative patients (MRD: HR 14.1, 95% CI: 5.8-34; p&lt;0.0001; and surveillance HR 20.6, 95% CI: 10.6-37.6; p&lt;0.0001), respectively. On analyzing cfDNA dynamics during adjuvant therapy, cfDNA levels gradually increased between 8 weeks and 8 months after surgery (p=0.0001), suggesting a potential impact of treatment on cell death and cfDNA shed. Conclusions: This is one of the first, large-scale in-depth studies evaluating treatment-based fluctuations in post-operative cfDNA and its correlation with ctDNA-positivity. Our analyses demonstrated no significant impact of plasma cfDNA levels on ctDNA detection rates across different MRD windows using a tumor-informed approach. This may affect real-world application of personalized ctDNA testing by allowing earlier testing windows, as well as guide clinical trial designs using ctDNA as an integral biomarker.

Multiallelic Copy Number Variation in ORM1 is Associated with Plasma Cell-Free DNA Levels as an Intermediate Phenotype for Venous Thromboembolism.

Venous thromboembolism (VTE) is a common disease with high heritability. However, only a small portion of the genetic variance of VTE can be explained by known genetic risk factors. Neutrophil extracellular traps (NETs) have been associated with prothrombotic activity. Therefore, the genetic basis of NETs could reveal novel risk factors for VTE. A recent genome-wide association study of plasma cell-free DNA (cfDNA) levels in the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project showed a significant associated locus near ORM1. We aimed to further explore this candidate region by next-generation sequencing, copy number variation (CNV) quantification, and expression analysis using an extreme phenotype sampling design involving 80 individuals from the GAIT-2 Project. The RETROVE study with 400 VTE cases and 400 controls was used to replicate the results. A total of 105 genetic variants and a multiallelic CNV (mCNV) spanning ORM1 were identified in GAIT-2. Of these, 17 independent common variants, a region of 22 rare variants, and the mCNV were significantly associated with cfDNA levels. In addition, eight of these common variants and the mCNV influenced ORM1 expression. The association of the mCNV and cfDNA levels was replicated in RETROVE (p-value = 1.19 × 10-6). Additional associations between the mCNV and thrombin generation parameters were identified. Our results reveal that increased mCNV dosages in ORM1 decreased gene expression and upregulated cfDNA levels. Therefore, the mCNV in ORM1 appears to be a novel marker for cfDNA levels, which could contribute to VTE risk.

Comparison of phenomics and cfDNA in a large breast screening population: the Breast Screening and Monitoring Study (BSMS)

To assess their roles in breast cancer diagnostics, we aimed to compare plasma cell-free DNA (cfDNA) levels with the circulating metabolome in a large breast screening cohort of women recalled for mammography, including healthy women and women with mammographically detected breast diseases, ductal carcinoma in situ and invasive breast cancer: the Breast Screening and Monitoring Study (BSMS). In 999 women, plasma was analyzed by nuclear magnetic resonance (NMR) and Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and then processed to isolate and quantify total cfDNA. NMR and UPLC-MS results were compared with data for 186 healthy women derived from the AIRWAVE cohort. Results showed no significant differences between groups for all metabolites, whereas invasive cancers had significantly higher plasma cfDNA levels than all other groups. When stratified the supervised OPLS-DA analysis and total cfDNA concentration showed high discrimination accuracy between invasive cancers and the disease/medication-free subjects. Furthermore, comparison of OPLS-DA data for invasive breast cancers with the AIRWAVE cohort showed similar discrimination between breast cancers and healthy controls. This is the first report of agreement between metabolomics and plasma cfDNA levels for discriminating breast cancer from healthy subjects in a true screening population. It also emphasizes the importance of sample standardization. Follow on studies will involve analysis of candidate features in a larger validation series as well as comparing results with serial plasma samples taken at the next routine screening mammography appointment. The findings here help establish the role of plasma analysis in the diagnosis of breast cancer in a large real-world cohort.

Diagnostic and prognostic value of plasma cell-free DNA combined with VEGF-C in laryngeal squamous cell carcinoma

BackgroundCirculating cell-free DNA (cfDNA) and vascular endothelial growth factor-C (VEGF-C) can be utilized to detect cancer and predict its prognosis. However, their potential application in laryngeal squamous cell carcinoma (LSCC) is unclear. PurposeThis study aimed to identify the diagnostic and prognostic value of cfDNA and VEGF-C in LSCC patients. MethodsThe plasma cfDNA of 148 LSCC patients and 43 non-tumor patients were isolated. Quantitative real-time PCR (qRT-PCR) was performed to assess long and short DNA fragments in plasma by amplifying the ALU repeats. ALU-qPCR results (ALU247/ALU115) were used to calculate cfDNA integrity index. Vascular endothelial growth factor-C (VEGF-C) level was detected by ELISA assay. Correlation between cfDNA and clinical features was analyzed. For detecting the sensitivity and specificity of cfDNA and VEGF-C alone or in combination for diagnosing LSCC, receiver operator characteristic (ROC) was established. For evaluating the overall survival (OS) of LSCC, Kaplan-Meier curves were established. ResultsLSCC patients had significantly higher levels of plasma cfDNA (ALU115, ALU247, and cfDNA integrity index) and VEGF-C than those without cancer (p < 0.05), showing area under the curve (AUC) values of 0.79, 0.74, 0.62 and 0.80, when cutoff value was correspondingly defined at 2.14 ng/mL, 1.39 ng/mL, 0.73 and 412.90 pg/mL, respectively. The AUC for distinguishing LSCC patients from non-tumor patients by plasma cfDNA combined with VEGF-C was 0.89 (95% CI: 0.83–0.94). A significant correlation was found between plasma cfDNA levels and Ki-67, tumor size, pT stage, and smoking history (p < 0.05). Based on survival analysis, low VEGF-C concentration groups had longer OS than those with high VEGF-C concentration (p = 0.02). ConclusionIndicators such as plasma cfDNA and VEGF-C may be used to diagnose and monitor LSCC for its noninvasiveness and rapid accessibility.

The methylome and cell-free DNA: current applications in medicine and pediatric disease.

DNA methylation is an epigenetic mechanism that contributes to cell regulation and development, and different methylation patterns allow for the identification of cell and tissue type. Cell-free DNA (cfDNA) is composed of small circulating fragments of DNA found in plasma and urine. Total cfDNA levels correlate with the presence of inflammation and tissue injury in a variety of disease states. Unfortunately, the utility of cfDNA is limited by its lack of tissue or cell-type specificity. However, methylome analysis of cfDNA allows the identification of the tissue or cell type from which cfDNA originated. Thus, methylation patterns in cfDNA from tissues isolated from direct study may provide windows into health and disease states, thereby serving as a "liquid biopsy". This review will discuss methylation and its role in establishing cellular identity, cfDNA as a biomarker and its pathophysiologic role in the inflammatory process, and the ways cfDNA and methylomics can be jointly applied in medicine. IMPACT: Cell-free DNA (cfDNA) is increasingly being used as a noninvasive diagnostic and disease-monitoring tool in pediatric medicine. However, the lack of specificity of cfDNA limits its utility. Identification of cell type-specific methylation signatures can help overcome the limited specificity of cfDNA. As knowledge of the cfDNA methylome improves, cfDNA will be more broadly applied in medicine, such that clinicians will need to understand the methods and applications of its use.

Cell-Free DNA as a New Biomarker of IVF Success, Independent of Any Infertility Factor, Including Endometriosis.

Cell-free DNA fragments detected in blood and in other biological fluids are released from apoptotic/necrotic cells. In this study, we analyzed cfDNA levels in follicular fluid (FF) samples from patients with infertility. Samples were collected from 178 infertile women and cfDNA was extracted and quantified by qPCR, using ALU115 and ALU247 primers, and statistical correlations were performed. We found that cfDNA concentration was significantly higher in FF pools from women aged 35 and over than in women under 35 years of age (p = 0.017). We also found that q247 cfDNA levels were significantly higher in women with an associated female factor, such as endometriosis, PCOS and POF, compared with women with no specific cause of infertility (p = 0.033). The concentration of cfDNA did not vary significantly in each group of women with an associated female factor. The concentration of cfDNA was significantly higher in the FF of women that obtained embryos with a high fragmentation rate, compared to embryos with a low fragmentation rate (p = 0.007). Finally, we found that women who did not become pregnant during IVF treatments had higher q247 cfDNA levels (p = 0.043). The quantification of cfDNA could be an important biomarker of follicular micro-environment quality to predict embryo quality and the success of IVF, making them more specific and effective.

Abstract PR007: A liposomal priming agent increases the sensitivity of liquid biopsies

Abstract Liquid biopsy measurements, such as analysis of circulating tumor DNA (ctDNA) shed by cancer cells, have garnered significant attention for their potential to empower the field of precision oncology. Given that ctDNA tests could enable minimally invasive monitoring and molecular profiling of disease, they are being investigated for use in earlier detection via pan-cancer screening tests, for tracking tumor evolution to inform therapy selection, and for making treatment decisions during minimal residual disease surveillance. However, a typical blood draw carries ultra-low levels of ctDNA (as low as 1.7 copies of the tumor genome in 15mL of blood for a 1cm lung tumor), and this fundamentally limits the sensitivity and the clinical utility of ctDNA-testing in many settings. To push beyond current ctDNA detection limits, we present a first-in-class liquid biopsy priming agent that is given prior to a blood draw to increase the abundance of ctDNA in circulation. Our priming agent consists of liposomes that transiently block the uptake of cell free DNA (cfDNA) by macrophages in the liver, resulting in increased cfDNA available for diagnostic analysis in blood. Using an in vitro 2D assay, we first identified a DSPE-based liposomal formulation that inhibits the uptake of cfDNA by two independent murine macrophage cell lines. Next, we injected our liposomal agent into healthy or tumor-bearing mice and collected blood for further analysis. We found that in healthy mice priming increases the half-life and the recovery of cfDNA from a blood draw, assayed via qPCR. In tumor-bearing mice, the priming agent increases the recovery of ctDNA by up to 60-fold (P = 0.0103) and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. Importantly, cfDNA levels in mice return to baseline within five hours of agent administration and repeated dosing shows no evidence of toxicity. Our priming strategy should be of interest for precision oncology applications, including early detection, longitudinal monitoring of therapeutic response, and surveillance for minimal residual disease. This tumor-agnostic priming agent should also improve the performance of ctDNA analytical techniques other than mutational profiling and may even increase the recovery of cfDNA from other body fluids beyond plasma. Moreover, we believe that this work sets a precedent for the potential development of priming agents for liquid biopsy at large across other analytes. In summary, here we present a first-in-class liquid biopsy priming agent capable of improving the sensitivity and the robustness of ctDNA testing in tumor-bearing mice by modulating liver cfDNA clearance. We envision that further development of liquid biopsy priming agents will signify a major step forward for the successful deployment of precision oncology tools across all stages of cancer management. Citation Format: Carmen Martin-Alonso, Shervin Tabrizi, Kan Xiong, Ahmet Bekdemir Bekdemir, Sahil Patel, Zhenyi An, Timothy Blewett, Sainetra Sridhar, Douglas Shea, Ava Amini, Jesse D. Kirkpatrick, Jin Bae, Eugenia Roberts, Ruolin Liu, Justin Rhoades, Todd Golub, J. Christopher Love, Viktor A. Adalsteinsson, Sangeeta N. Bhatia. A liposomal priming agent increases the sensitivity of liquid biopsies. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR007.

Circulating Cell-Free DNA as a Biomarker for Prognosis and Response to Systemic Therapy in Patients with Unresectable Hepatocellular Carcinoma.

Systemic therapy provides clinical benefits to a subset of patients with advanced unresectable hepatocellular carcinoma (HCC). However, few biomarkers are available for predicting prognosis and treatment response in patients with advanced HCC undergoing treatment with systemic therapies. This study aimed to examine whether circulating cell-free DNA (cfDNA) containing circulating tumor DNA can act as a therapeutic response and prognostic biomarker in patients with advanced HCC. We analyzed longitudinally collected plasma cfDNA of patients with advanced HCC who were naïve to systemic therapy, and assessed their prognostic and predictive values to determine treatment responses. cfDNA concentration positively correlated with entire tumor volume on computed tomography before (p = 0.0231) and at the end (p &lt; 0.0001) of the first-line systemic therapy. The overall survival rate was higher in patients with cfDNA concentrations lower than the median cfDNA level at baseline compared to patients with higher cfDNA concentrations (hazard ratio, 0.2765; 95% confidence interval, 0.08-0.81; p = 0.0197). The ratio of cfDNA at 4 weeks to that at baseline was predictive of radiographic disease response. In patients with progressive disease, cfDNA concentration at 4 weeks increased significantly (p = 0.0245), whereas the concentration remained unchanged in patients with other disease courses (p = 0.9375). The baseline plasma cfDNA concentration can be used as a prognostic biomarker in patients with advanced HCC. cfDNA kinetics may also predict the tumor response to therapy and disease progression.

Clinical Potential of Circulating Cell-Free DNA (cfDNA) for Longitudinally Monitoring Clinical Outcomes in the First-Line Setting of Non-Small-Cell Lung Cancer (NSCLC): A Real-World Prospective Study

Despite the increasing implementation of targeted and immunotherapy-based treatments, the prognosis of patients with advanced NSCLC remains dismal. We prospectively evaluated longitudinal plasma cfDNA kinetics as an early marker of therapeutic efficacy in patients with advanced NSCLC undergoing standard first-line treatments. From February 2020 to May 2022, treatment-naïve patients with advanced NSCLC were consecutively enrolled at the Medical Oncology Unit of the Paolo Giaccone University Hospital, Palermo (Italy). We quantified cfDNA in terms of ng/μL using a QubitTM dsDNA HS Assay Kit. The agreement between the cfDNA and radiologic response was evaluated from baseline (T0) to the radiologic evaluation (T1). A total of 315 liquid biopsy samples were collected from 63 patients at baseline, with a total of 235 paired plasma samples from 47 patients at disease re-evaluation. A fair concordance was observed between early and durable radiographic and cfDNA response (Cohen's kappa coefficient = 0.001); 11 and 18 patients receiving TKI (Pearson's chi-squared test = 4.278; Cohen's kappa coefficient = 0.039) and IO treatments (Pearson's chi-squared test = 7.481; Cohen's kappa coefficient = 0.006) showed a significant and durable association between cfDNA dynamics and the first radiologic evaluation, whereas among the 18 patients undergoing CT, no significant correlation was observed (Pearson's chi-squared test = 0.720; Cohen's kappa coefficient = 0.396). The ECOG-PS 2 patients presented with the mean baseline cfDNA levels 2.6-fold higher than those with ECOG-PS 0-1 (1.71 vs. 0.65 ng/µL; p = 0.105). Our real-world study demonstrates that quantitative changes in cfDNA values correlated with responses to therapy and relapse of disease in treatment-naïve patients with advanced NSCLC undergoing TKI- and IO-based treatments.

The Correlation between Periodontal Parameters and Cell-Free DNA in the Gingival Crevicular Fluid, Saliva, and Plasma in Chinese Patients: A Cross-Sectional Study.

Purpose: To investigate the correlation between periodontal parameters and cell-free DNA (cfDNA) concentrations in gingival crevicular fluid (GCF), saliva, and plasma. Methods: Full mouth periodontal parameters, including probing depth (PD), bleeding on probing (BOP), and plaque index (PI) were recorded from 25 healthy volunteers, 31 patients with untreated gingivitis, and 25 patients with untreated periodontitis. GCF, saliva, and plasma samples were collected from all subjects. Extraction and quantification assays were undertaken to determine cfDNA concentrations of each sample. Results: GCF and salivary cfDNA levels were increased with aggravation of periodontal inflammation (GCF p < 0.0001; saliva p < 0.001). Plasma cfDNA concentrations in patients with periodontitis were significantly higher than those in healthy volunteers and patients with gingivitis. GCF and salivary cfDNA were positively correlated with mean PD, max PD, BOP, and mean PI (p < 0.0001), whereas plasma cfDNA was not correlated with BOP (p = 0.099). Conclusion: GCF, saliva, and plasma concentrations of cfDNA were significantly elevated in patients with periodontal disease. There were also positive correlations between cfDNA levels in GCF and saliva and periodontal parameters.

501 Serum cell-free DNA as a new biomarker in cutaneous T-cell lymphoma

In recent years, circulating cell-free DNA (cfDNA) has received a great attention as a biomarker for various cancers. Many reports have shown that serum cfDNA levels are elevated in cancer patients and their levels correlate with prognosis and disease activity. The aim of this study was to measure serum cfDNA levels in cutaneous T-cell lymphomas (CTCL) patients and to evaluate their correlations with hematological and clinical findings. Serum cfDNA levels in CTCL patients were significantly higher than those in healthy controls, and their levels gradually increased with the progression of the disease stage.

Neutrophil Extracellular Traps (NETs) Thrombogenicity and Endothelial Cell Toxicity Are Ameliorated By Platelet Factor 4 (PF4): Implications to the Treatment of Sepsis

Sepsis, the dysregulated response to infection, is a leading cause of mortality worldwide for which there are no targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens. However, when NETs are degraded by circulating nucleases, they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger thrombosis, induce complement activation and cause oxidative vascular damage. We propose a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4 (CXCL4), which enhances NET resistance to DNase degradation, promoting NDP sequestration and bacterial capture, improving sepsis outcomes. As NETs are traditionally considered to be prothrombotic, we investigated whether PF4-stabilization alters the thrombogenicity of NETs. We examined the effect of PF4 on both NET- and DNA-mediated thrombus formation and endothelial cell injury using NET and DNA fragments of different lengths. Human neutrophils were stimulated to release NETs and treated with mild DNase I (4U/mL) to liberate high-molecular-weight (hmw) NETs (>50kb), and then digested with restriction enzymes to generate low-molecular-weight (lmw) fragments of ~4kb and ~250bp. We also studied single-stranded (ss) DNA, generated by heating double stranded (ds) DNA fragments to 100°C. We assessed the effects of PF4 on NETs/dsDNA/ssDNA-induced thrombin and fibrin generation in normal and in factor (F) XII- and FXI-depleted plasma. PF4 significantly reduced plasma thrombin and fibrin generation induced by digested NET and DNA fragments and by ssDNA without impairing thrombolysis. PF4 did not exert any effects on DNA-induced fibrin generation in FXII- or FXI-depleted plasma, while supplementation with purified FXII or FXI restored the anticoagulant effects of PF4, suggesting that PF4 binding reduces the ability of cfDNA and NETs to activate the contact pathway of coagulation. We next studied how PF4 alters the ability of DNA to induce a prothrombotic phenotype in human umbilical vein endothelial cells (HUVECs) incubated with dsDNA or ssDNA fragments by measuring von Willebrand factor (VWF) release with immunofluorescence and by quantifying tissue factor (TF) expression with a chromogenic substrate assay. To study the mechanism of endothelial activation, HUVECs were treated with inhibitors of toll-like receptor (TLR) 9, a pattern recognition receptor that binds to dsDNA. Unlike intact NETs or hmwDNA, both digested dsDNA and ssDNA upregulated VWF release and TF expression by endothelial cells via a TLR9-dependent mechanism, which was prevented by the addition of PF4. To study these observations in vivo, hmwDNA was administered intravenously to wildtype (WT) and Cxcl4-/- mice and plasma thrombin-anti-thrombin (TAT) levels were measured 4h post infusion. Cxcl4-/- mice exhibited higher TAT levels as compared to WT mice. We also studied two distinct sepsis-like states: (1) WT mice exposed to lipopolysaccharide (LPS, IP; 35mg/kg) ± human (h)PF4 (IV; 40mg/kg), where plasma cfDNA and TAT levels were measured at 6h, and (2) the cecal slurry model of polymicrobial infection in endothelial-specific TLR9-/- mice, where plasma TAT levels were measured at 4h. In the LPS endotoxemia model (1), TAT and cfDNA levels were elevated at 6h post LPS challenge, which were prevented with the administration of hPF4. In the cecal slurry model (2), endothelial TLR9-/- mice exhibited lower TAT levels at 4h post challenge as compared to WT controls. In summary, while intact NETs help to contain infection, NET degradation liberates lmwDNA and ssDNA that activate the contact pathway and induce a prothrombotic phenotype in endothelial cells via activation of TLR9. PF4-stabilization of NETs decreases the risk of NET-mediated toxicity by (1) inhibiting DNase cleavage of intact NETs and (2) neutralizing released lmwDNA and ssDNA. NET stabilization by PF4 tips the balance, limiting NET- and NDP-induced thrombosis and vascular toxicity, while enhancing NET's ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis. Strategies that augment the beneficial effects of PF4 merit further investigation.

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 that undergo hematopoietic stem cell transplantation (HCT). Current diagnosis of chronic GVHD (cGVHD) in bone marrow transplant patients is based on inaccurate, operator-dependent clinical markers, and less often on biopsies. These methods are time consuming, costly, invasive and yield late-stage diagnoses that negatively affect morbidity and mortality. Here we demonstrate the use of circulating cell-free DNA (cfDNA) for detection of chronic GVHD and damage to 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 203 samples from 82 HCT patients(Fig 1). Patients with active cGVHD show elevated concentration of cfDNA, as well as tissue-specific and immune cell driven methylation markers that agree with the 2014 National Institute of Health (NIH) clinical scores. Interestingly transplanted patients with no clinical symptoms show abnormally high levels of cfDNA and tissue-specific markers, suggesting hidden tissue turnover even in the absence of evident pathology. We used machine learning to create a model, which can aid predicting the likelihood that a patient has cGVHD. We compared XG boost and random forest classifiers on our data set. Algorithms were tested on data that included pre-selected specific measures (cfDNA of skin, lung, liver, GI, neutrophils, monocyte, eosinophil, T lymphocytes, CD8+ cells, Treg cells and B lymphocytes, as well as white blood cell (WBC) counts, BIL, AST, ALT and ALP liver enzyme levels). The data was randomly divided to a training set consisting of 67% of the samples and a testing set consisting of the remaining 33%. The model was constructed based on the training set alone that was later verified on the testing set. As the XG boost model had both higher accuracy as well as more robust results by cross-validation, we applied XG boost for further analyses. A total of 179 samples were divided between the training and test set. One hundred nineteen samples were randomly chosen for training the machine learning classifier, while 60 samples were used solely for testing the model. Each sample was intended either for the training or test sets. The integrative model was able to correctly identify GVHD with a sensitivity of 91.49% and precision of 89%% (AUC of 0.9, Fig 2). Our study shows that tissue-specific DNA methylation patterns can serve as plasma biomarkers for detection of tissue damage in cGVHD. To our knowledge, this is the first report of potential tissue-specific cfDNA utility in the context of chronic GVHD. Larger cohorts will be needed to assess the prognostic potential of methylation-based biomarkers. Tissue-specific cfDNA markers can open a window into uncovering underlying tissue dynamics in patients who underwent allogeneic stem cell transplantation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Haplotype-Assisted Noninvasive Prenatal Diagnosis of Genetic Diseases by Massively Parallel Sequencing of Maternal Plasma Cell-Free DNA.

Early prenatal diagnosis of genetic diseases allows for timely intervention or prevention of thediseases in newborns. Conventional prenatal diagnosis of most genetic diseases relies on testing fetal DNA obtained by invasive procedures such as amniocentesis or chorionic villus sampling, which are associated with small risks of fetal loss. Maternal circulating blood contains cell-free DNA (cfDNA) from the fetal genome and can thus be used to noninvasively detect fetal genetic diseases such as chromosomal abnormalities, copy number variants, and single gene diseases. However, due to the presence of a high level of maternal cfDNA in the maternal blood stream, a relative haplotype dosage (RHDO) analysis is required to detect the mutant loci in the fetal genome when performing noninvasive prenatal diagnosis (NIPD) by massively parallel sequencing (MPS) of cfDNA. In this chapter, we describe a protocol utilizing the RHDO strategy for NIPD of any gene of interest associating with single gene diseases.

Longitudinal Evaluation of Donor-Derived Cellfree DNA in Pediatric Kidney Transplantation.

Donor-derived cellfree DNA (cfDNA) is a less-invasive marker of allograft injury compared with kidney biopsy. However, donor-derived cfDNA has not yet been extensively tested in children, where the test may have different characteristics. We assayed donor-derived cfDNA (AlloSure; CareDx) from 290 stored plasma samples from a prospective biobank at our center, collected from 57 children monthly in the first year postkidney transplant between January 2013 and December 2019. We assessed the kinetic changes in donor-derived cfDNA levels within the first year post-transplant. We analyzed donor-derived cfDNA levels for associations with biopsy-proven acute rejection using area under the receiver operating characteristic curve to longitudinal plasma and urine BK viral loads using linear mixed models. We analyzed the prognostic effect of an elevated donor-derived cfDNA level on the eGFR 30 days after the assay via Kolmogorov-Smirnov two-sample tests or on measured GFR or interstitial fibrosis at 12 months post-transplant. The donor-derived cfDNA levels in children remained persistently elevated for at least 4 months post-transplant, more so if there is greater disparity in size between the donor and the recipient, before reaching a steady low level. A donor-derived cfDNA level of >1% discriminated between biopsy-proven acute rejection with a receiver operating characteristic area under the curve of 0.82 (95% confidence interval, 0.71 to 0.93). During BK viruria or viremia, patients had a significantly higher median donor-derived cfDNA than before or after and a significant rise within the same patient. A donor-derived cfDNA of >0.5% predicted a wider spread in the eGFR over the next 30 days but not the 12-month outcomes. In children, donor-derived cfDNA is a valuable, less invasive biomarker for assessment of allograft rejection and injury. This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2022_10_27_CJN03840322.mp3.

A prospective cohort study of dynamic cell-free DNA elevation during cardiac surgery with cardiopulmonary bypass.

Cardiac surgery and cardiopulmonary bypass (CPB) are associated with a systemic inflammatory reaction that occasionally induces a life-threatening organ dysfunction caused by the dysregulated host response to the damage-associated molecular patterns (DAMPs). In severe inflammation, cell-free DNA (cfDNA) and histones are released by inflammatory cells and damaged tissue and act as DAMPs. We sought to characterize the changes in circulating cell-free DNA (cfDNA) levels during CPB. Primary outcomes were renal failure, ventilation time (>18 hr), length of stay (LOS) in the intensive care unit (ICU) (>48hr), hospital LOS (>15 days), and death. We looked for associations with blood tests and comparison to standard scores. In a prospective cohort study, we enrolled 71 patients undergoing non-emergent coronary artery bypass grafting. Blood was drawn at baseline, 20 and 40 minutes on CPB, after cross-clamp removal, and 30 minutes after chest closure. cfDNA was measured by our fast fluorescent method. Baseline cfDNA levels [796 (656-1063) ng/ml] increased during surgery, peaked after cross-clamp removal [2403 (1981-3357) ng/ml] and returned to baseline at recovery. The difference in cfDNA from 20 to 40 minutes on CPB (ΔcfDNA 40-20) inversely correlated with peripheral vascular disease (PVD), longer ventilation time, and longer ICU and hospital length of stay (LOS). Receiver operating characteristic (ROC) curve of ΔcfDNA 40-20 for long ICU-LOS (>48hr) was with an area under the curve (AUC) of 0.738 (p = 0.022). ROC AUC of ΔcfDNA 40-20 to long Hospital LOS (>15 days) was 0.787 (p = 0.006). Correction for time on CPB in a multivariate logistic regression model improved ROC-AUC to 0.854 (p = 0.003) and suggests that ΔcfDNA 40-20 is an independent risk factor. To conclude, of measured parameters, including STS and Euroscore, the predictive power of ΔcfDNA 40-20 was the highest. Thus, measurement of ΔcfDNA 40-20 may enable early monitoring of patients at higher risk. Further studies on the mechanism behind the negative association of ΔcfDNA 40-20 with PVD and outcomes are warranted.

A genome-wide cell-free DNA methylation analysis identifies an episignature associated with metastatic luminal B breast cancer.

Breast cancers of the luminal B subtype are frequent tumors with high proliferation and poor prognosis. Epigenetic alterations have been found in breast tumors and in biological fluids. We aimed to profile the cell-free DNA (cfDNA) methylome of metastatic luminal B breast cancer (LBBC) patients using an epigenomic approach to discover potential noninvasive biomarkers. Plasma cfDNA was analyzed using the Infinium MethylationEpic array in a cohort of 14 women, including metastatic LBBC patients and nontumor controls. The methylation levels of cfDNA and tissue samples were validated with droplet digital PCR. The methylation and gene expression data of 582 primary luminal breast tumors and 79 nontumor tissues were obtained from The Cancer Genome Atlas (TCGA). We found an episignature of 1,467 differentially methylated CpGs that clearly identified patients with LBBC. Among the genes identified, the promoter hypermethylation of WNT1 was validated in cfDNA, showing an area under the ROC curve (AUC) of 0.86 for the noninvasive detection of metastatic LBBC. Both paired cfDNA and primary/metastatic breast tumor samples showed hypermethylation of WNT1. TCGA analysis revealed significant WNT1 hypermethylation in the primary tumors of luminal breast cancer patients, with a negative association between WNT1 methylation and gene expression. In this proof-of-principle study, we discovered an episignature associated with metastatic LBBC using a genome-wide cfDNA methylation approach. We also identified the promoter hypermethylation of WNT1 in cfDNA as a potential noninvasive biomarker for luminal breast cancer. Our results support the use of EPIC arrays to identify new epigenetic noninvasive biomarkers in breast cancer.

Clinical Efficacy of Pyrotinib Combined with Capecitabine in the Second-Line or Above Treatment for HER-2 Positive Advanced Breast Cancer and Its Association with Cell-Free DNA.

Purpose To evaluate the clinical efficacy of pyrotinib combined with capecitabine in the treatment of HER-2 positive breast cancer in real world and its correlation with cfDNA. Methods From September 2020 to June 2021, 181 cases of HER-2 positive advanced breast cancer patients who were treated in Jiangsu Cancer Hospital and Nantong Cancer Hospital were analyzed. Patients were given pyrotinib combined with capecitabine or trastuzumab combined with capecitabine. Eighty-one patients who received pyrotinib+capecitabine second-line or above treatment were included in the pyrotinib group, and 100 patients who received trastuzumab+capecitabine second-line or above treatment were included in the trastuzumab group. The objective response rate (ORR) and clinical benefit rate (CBR) of the two groups were compared. The follow-up results of the patients were analyzed, and the progression-free survival (PFS) and adverse reactions were compared between the two groups. Plasma cfDNA was detected by real-time fluorescence quantitative PCR. The cfDNA levels of patients before and after treatment were compared, and the change of cfDNA levels in patients with different curative effects over time was recorded. The patients were further divided into high cfDNA expression and low cfDNA expression groups, and the PFS of patients with different cfDNA levels was analyzed. COX univariate and multivariate analysis of factors influencing posttreatment survival in patients with HER-2-positive breast cancer were performed. Results The ORR of the pyrotinib group (58.02%) was significantly higher than that of the trastuzumab group (42.00%, P = 0.0369). Similarly, the CBR of the pyrotinib group (65.43%) was significantly higher than that of the trastuzumab group (49.00%, P = 0.0347). The incidence of adverse reactions between the two groups was not statistically significant (P > 0.05). The results of survival analysis showed that the PFS of the pyrotinib group was 8.02 ± 3.05 months, the PFS of the trastuzumab group was 7.11 ± 3.06 months, and the PFS of the pyrotinib group was significantly longer than that of the trastuzumab group (P = 0.035). The comparison of cfDNA levels between the two groups showed that on the 28th and 56th day of treatment, the cfDNA levels in the pyrotinib group were significantly lower than those in the trastuzumab group (P < 0.05). Long-term follow-up results showed that compared with patients with high cfDNA expression, the PFS of patients with low cfDNA expression was significantly prolonged (P < 0.05). The level of cfDNA is an independent risk factor affecting the prognosis of patients with HER-2-positive breast cancer. Conclusion The combined use of pyrotinib and capecitabine has good clinical efficacy and high safety in patients with HER-2 positive breast cancer. The combined use of pyrotinib and capecitabine prolongs the PFS of patients and reduces the level of plasma cfDNA. Changes in cfDNA levels can reflect the therapeutic efficacy of patients with HER-2-positive breast cancer to a certain extent and can be used as a potential indicator for evaluating the prognosis of patients with HER-2-positive breast cancer.

Dynamics of cell-free DNA levels in the in vivo LPS-induced inflammation model

An increased concentration of extracellular cell free DNA (cfDNA) is a distinctive characteristic of pathologies that mainly occur in acute inflammation (myocardial infarction, sepsis, stroke, trauma). The increase of cfDNA in chronic inflammatory processes, oncological, autoimmune diseases is less significant and is mainly due to aberrant cell death processes. One of such diseases is systemic lupus erythematosus (SLE). It has recently been shown that, in addition to increased cfDNA concentration, the degree of inflammation can reflect the N/L index (neutrophil to lymphocyte ratio), being a simple and informative marker of disease activity in patients with SLE. The aim of the study was to study the dynamics of the level of cfDNA and the N/L index in the model of LPS-induced inflammatory response as observed in intact mice, and their relation to the phenotypic heterogeneity of model SLE. We used female hybrid mice (C57Bl/6xDBA/2) F1 and female DBA/2 mice at the age of 6-8 weeks. LPS of E. coli strain 111: B4 (Sigma) was injected intraperitoneally once at doses of 10 ng, 1 g and 100 g per mouse in PBS. The control group was injected with the appropriate volume of buffer. The TNF-binding domain of the variola virus CRMB protein was used as an inhibitor of TNF, which was administered 30 min before the introduction of LPS. The dynamics of the response to LPS was assessed after 4, 8, 11, 24 hours by the N/L index and the level of cfDNA; at the zero point, the parameters were determined before the introduction of LPS. A day after a single injection of LPS at a dose of 1 g/mouse, a SLE model was induced on the same hybrid mice (double intravenous administration with an interval of 6 days of spleen cells of the DBA/2 line, 60-70 106 cells each). Three months later, with proteinuria of 3 mg/ mL or more, mice were assigned to the SLEnephritis+ group, with a protein of less than 3 mg/mL, to the SLEnephritis- group. Statistical processing of the results was carried out by nonparametric statistics using the MannWhitney test. Differences were considered statistically significant at p 0.05. It was found that the change in the N/L index, as well as the change in the level of cfDNA, depends on the dose of LPS administered. It was shown that the level of cfDNA reaches its maximum after 8 and 11 hours after the introduction of LPS is reliably reduced when using the inhibitor TNF. A retrospective analysis indicates that there is a definite relationship between the response of intact mice to LPS before induction of cGVHD, and their subsequent division into variants of SLEnephritis + and SLEnephritis - in the course of disease development.