Total cfDNA Levels Research Articles

Circulating nucleases degrade high-molecular-weight cell-free DNA in coronary vessels during acute myocardial infarction

Abstract Introduction Extracellular cell-free DNA (cfDNA) originates from various biological processes, including cell turnover, apoptosis, necrosis, NETosis, and other forms of cell death. In healthy individuals, highly fragmented cfDNA is elevated in the bloodstream due to infection, cancer, inflammation and thrombosis. In thrombotic vascular diseases, neutrophils release elongated, high-molecular-weight DNA with bioactive and prothrombotic properties in a process termed NETosis. Circulating nucleases are thought to counteract pathogenic properties of NETs. However, attempts to connect total cfDNA concentration to nuclease activity have failed. Purpose We aimed to elucidate the in vivo connection between extracellular DNA and circulating nucleases in thrombotic vascular diseases by investigating DNA length as a potential novel biomarker in cardiovascular disease. Methods Citrated plasma from healthy volunteers (n=363) and from ST-elevation myocardial infarction (MI) coronary aspirates (n=59) and peripheral arterial sites (n=96) were analyzed for DNA concentration, length, and nuclease activity. Total DNA concentration was measured with a fluorescent DNA binding dye, and elongated DNA assessed using qPCR and Agilent bioanalyzer high-sensitivity DNA chip. Nuclease activity was measured by single radial enzyme-diffusion (SRED) assay. Cardiac MRI assessed infarct size acutely and six months after the event. Results Coronary aspirates exhibited an elevation in total DNA, with a significant rise in high-molecular weight cell-free DNA in addition to an increase in sub-nucleosome fragmentation (Figure 1). Peripheral and coronary plasma samples from individuals with myocardial infarction (MI) showed elevated levels of total and elongated cfDNA. When compared to healthy individuals, the integrity of cfDNA was lower in peripheral samples and higher in coronary aspirates. During myocardial infarction, circulating nuclease activity was elevated systemically but low at the coronary culprit lesion site. Elongated high-molecular-weight cfDNA accumulates primarily in coronary vessels but is also elevated systemically. In coronary aspirates, nuclease activity negatively correlated with elongated DNA and DNA integrity (rs=0.6255, p<0.0001). Conclusions Elongated DNA accumulates during MI at the culprit lesion site. Its inverse correlation with nuclease activity underscores the role of nucleases in cleaving elongated DNA to shorter, soluble fragments. This study establishes DNA length and integrity as novel biomarkers in thrombotic cardiovascular disease.Figure 1:Coronary cfDNA lengthFigure 2:Nuclease activity and cfDNA

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.

CELL‐FREE DNA KINETICS DECIPHER POTENTIAL MECHANISMS OF ACTION OF IBRUTINIB COMBINATION THERAPY IN MANTLE CELL LYMPHOMA (MCL)

Introduction: The European MCL Network trial TRIANGLE (NCT02858258) evaluated the addition of ibrutinib to standard treatment with autologous stem cell transplantation (ASCT) (arm A+I) in comparison to ASCT alone (arm A) and ibrutinib plus chemoimmunotherapy without ASCT (arm I) demonstrating significant and clinically relevant superiority of additional ibrutinib on outcome. Cell-free (cf)DNA captures tumor genetics and can be used to assess disease kinetics and minimal residual disease (MRD). We used capture based targeted sequencing for genotyping and MRD monitoring in cfDNA in the TRIANGLE trial to gain insight into the mechanisms of action of Ibrutinib and to identify patients (pts) with high risk of relapse. Methods: Peripheral blood (PB) and plasma samples from 57 pts with a PB infiltration of ≥10% at baseline (BL) and samples at mid induction (MI, n = 57) and end of induction (EoI, n = 53) were sequenced using the EuroClonality (EC)-NDC assay (Univ8® Genomics UK) to detect immunoglobulin and T-cell receptor (IG/TR) rearrangements, structural- (SV) and single nucleotide variants (SNVs) in 72 genes. Bioinformatics by ARResT/Interrogate with adaptations for cfDNA. Pts were considered MRD+ by detecting ≥1 read of a lymphoma-specific IG, SV or SNV. MRD in PB was analyzed by ASO-qPCR. Results: cfDNA levels were high at BL (mean 93 ng/mL) and MI (124 mg/mL) and decreased at EoI (12 ng/mL). BL cfDNA levels correlated with MIPI scores and identified pts with poor outcome. Genotyping by EC-NDC showed fully concordant IG clonotypes and SVs among 57 paired PB gDNA and cfDNA samples. 126 SNVs were identified (VAF 2.7%–75%, median 30%) in genes described as drivers in MCL, ATM (44%), TP53 (19%), KMT2D (16%), SAMHD1 (8%), CCND1 (8%) and 18 others. On average, 6 MRD markers were identified per patient (range 3-10). Copy number variation analysis revealed deletions in ATM (10%), CDKN2A (16%) and TP53 (5%) pts. TP53 mutations/deletions are a poor prognostic indicator in MCL, but therapy in arms A+I/I (n = 6) overcomes this poor outcome in comparison to arm A (n = 5). At MI, total cfDNA and ctDNA levels were significantly increased in arms A+I/I as compared to arm A (p = 0.049). 20/57 (35%) pts were MRD+ in PB and 35/55 (64%) in cfDNA. ctDNA was detected in 17/21 pts (81%) in Arm A and 18/34 pts (53%) in Arms A+I/I, suggesting effective disease clearance in the lymph nodes by ibrutinib. ctDNA detection at MI predicted outcome only in arm A. At EoI 8/53 (15%) pts were MRD+ in PB and 20/40 (50%) pts were MRD+ in cfDNA. ctDNA was detected in 12/18 pts (66%) in Arm A and 8/22 pts (36%) in Arms A+I/I. Similar to MI, ctDNA at EoI predicted outcome only in arm A. Keywords: combination therapies, liquid biopsy, minimal residual disease Conflicts of interests pertinent to the abstract S. Ferrero Consultant or advisory role: EUSA Pharma, Jannsen Honoraria: Servier Research funding: Gilead, Morphosys, Jannsen V. H. van der Velden Other remuneration: EuroFlow: Patents and Royalties R. García Sanz Honoraria: Janssen, BeiGene, Gilead, Astellas, Amgen, Takeda Research funding: Janssen, Gilead, Astellas, Takeda Educational grants: Janssen M. Dreyling Consultant or advisory role: Novartis, Lilly/Loxo, AstraZeneca, Roche, Janssen, Gilead/Kite, BMS/Celgene, Beigene Honoraria: Novartis, Lilly/Loxo, Amgen, AstraZeneca, Roche, Janssen, Gilead/Kite Research funding: Roche, Janssen, Bayer, AbbVie A. W. Langerak Research funding: Genentech, Roche Other remuneration: Spearkers Bureau: Janssen, Gilead D. Gonzalez de Castro Employment or leadership position: Founder and director of Univ8 Genomics Ltd.

Circulating Cell-Free DNA Levels in Psychiatric Diseases: A Systematic Review and Meta-Analysis.

The cell-free DNA (cfDNA) levels are known to increase in biological fluids in various pathological conditions. However, the data on circulating cfDNA in severe psychiatric disorders, including schizophrenia, bipolar disorder (BD), and depressive disorders (DDs), is contradictory. This meta-analysis aimed to analyze the concentrations of different cfDNA types in schizophrenia, BD, and DDs compared with healthy donors. The mitochondrial (cf-mtDNA), genomic (cf-gDNA), and total cfDNA concentrations were analyzed separately. The effect size was estimated using the standardized mean difference (SMD). Eight reports for schizophrenia, four for BD, and five for DDs were included in the meta-analysis. However, there were only enough data to analyze the total cfDNA and cf-gDNA in schizophrenia and cf-mtDNA in BD and DDs. It has been shown that the levels of total cfDNA and cf-gDNA in patients with schizophrenia are significantly higher than in healthy donors (SMD values of 0.61 and 0.6, respectively; p < 0.00001). Conversely, the levels of cf-mtDNA in BD and DDs do not differ compared with healthy individuals. Nevertheless, further research is needed in the case of BD and DDs due to the small sample sizes in the BD studies and the significant data heterogeneity in the DD studies. Additionally, further studies are needed on cf-mtDNA in schizophrenia or cf-gDNA and total cfDNA in BD and DDs due to insufficient data. In conclusion, this meta-analysis provides the first evidence of increases in total cfDNA and cf-gDNA in schizophrenia but shows no changes in cf-mtDNA in BD and DDs. Increased circulating cfDNA in schizophrenia may be associated with chronic systemic inflammation, as cfDNA has been found to trigger inflammatory responses.

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.

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.

The clinical value of circulating free tumor DNA in testicular germ cell tumor patients.

Testicular germ cell tumors (TGCT) are unique malignancies of young adult men; their biology is, however, underexplored and there has not been much progress in their treatment for decades. Circulating free tumor DNA (cfDNA) analysis represents a promising way of discovering novel diagnostic and treatment options. The study evaluates the clinical value of cfDNA detection in TGCT patients. Total cfDNA concentration and ratio of its 2 main fragments (180 and 360 bp) were evaluated by spectrophotometry, capillary electrophoresis and qPCR in peripheral blood plasma of 96 TGCT patients (173 samples) and 31 normal controls. Non-parametric tests were used for statistical analyses. The total cfDNA concentration was significantly higher in TGCT than in controls (P < 0.0001), with the highest levels at disease progression, but with no clear threshold between malignant and normal samples. Patients with positive tumor markers had higher cfDNA concentrations than those with negative markers (P = 0.01). Longer 360 bp cfDNA fragments were found in 58% of TGCT patients including almost all samples from relapse or disease progression but no normal controls (P < 0.0001). Total cfDNA levels are significantly increased in TGCT patients but without a clear threshold separating normal and tumor samples, thus total cfDNA amount itself is not a sensitive enough marker to identify or monitor TGCT. Longer cfDNA fragments have been found exclusively in a proportion of tumors and predominantly at disease progression, representing a novel potential marker for TGCT monitoring that would deserve further exploration.

Abstract 533: A study of sequencing circulating cell free DNA from lung cancer patients

Abstract Precision oncology requires accurate cancer molecular information. While widely used to characterize tumors, tissue biopsy is limited in patient follow-up due to its invasive nature and the lack representation of the entire tumor bulk. To develop the method of circulating cell free DNA (cfDNA) for obtaining clinically valuable sequence information, we isolated cfDNA from the plasma samples of 10 advanced metastatic lung cancer patients and performed next generation sequencing (NGS) using a unique molecular marker (UMI) integrated 275-gene-target-panel at the average depth of 4,000 X coverage. We were able to identify common mutants of lung cancer driver genes, e.g. EGFR, KRAS, DNMT3 and TP53 with high confidence. In one example, the TKI resistant EGFR mutant T790M was found coexisting with EGFR 746-750del and TP53 splice variant in a baseline sample of a patient progressed from a first-generation EGFR inhibitor. And the dynamic changes of the allele frequency of EGFR and TP53 mutants were consistent with total cfDNA levels of the patients. Interestingly, an osimertinib resistance EGFR mutant C797S emerged at Day 355 post-treatment and followed by disease progression in 4 months. In addition to EGFR gene mutations, we further found increasing EGFR copy number associated with drug resistance and cancer progression of this patient. Our study demonstrated that it is feasible using cfDNA for patient follow-up to understand cancer resistance or disease progression through characterization of cancer mutations. Citation Format: Zhigang Kang, Liang Cao. A study of sequencing circulating cell free DNA from lung cancer patients [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 533.

Machine-Learning-Based Clinical Biomarker Using Cell-Free DNA for Hepatocellular Carcinoma (HCC).

Simple SummaryCirculating cell-free DNA (cfDNA) has attracted a great deal of scientific interest as a predictive biomarker for the diagnosis and prognosis of hepatocellular carcinoma (HCC). HCC result in high mortality due to the absence of blood biomarkers for early diagnosis and prognosis. We established cfDHCC as a new scoring system by applying a machine learning algorithm that integrates the expression profiles of cfDNA. Based on this, it was possible to accurately predict the clinico-pathological characteristics of patients with HCC as well as improve their survival. (1) Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Although various serum enzymes have been utilized for the diagnosis and prognosis of HCC, the currently available biomarkers lack the sensitivity needed to detect HCC at early stages and accurately predict treatment responses. (2) Methods: We utilized our highly sensitive cell-free DNA (cfDNA) detection system, in combination with a machine learning algorithm, to provide a platform for improved diagnosis and prognosis of HCC. (3) Results: cfDNA, specifically alpha-fetoprotein (AFP) expression in captured cfDNA, demonstrated the highest accuracy for diagnosing malignancies among the serum/plasma biomarkers used in this study, including AFP, aspartate aminotransferase, alanine aminotransferase, albumin, alkaline phosphatase, and bilirubin. The diagnostic/prognostic capability of cfDNA was further improved by establishing a cfDNA score (cfDHCC), which integrated the total plasma cfDNA levels and cfAFP-DNA expression into a single score using machine learning algorithms. (4) Conclusion: The cfDHCC score demonstrated significantly improved accuracy in determining the pathological features of HCC and predicting patients’ survival outcomes compared to the other biomarkers. The results presented herein reveal that our cfDNA capture/analysis platform is a promising approach to effectively utilize cfDNA as a biomarker for the diagnosis and prognosis of HCC.

Association Between Total Cell Free DNA and SARS-CoV-2 In Kidney Transplant Patients: A Preliminary Study

BackgroundKidney transplant (KT) recipients are at high risk for developing severe COVID-19. Lowering immunosuppression levels in KT recipients with COVID-19 encourages native immune responses but can raise the risk of rejection. Donor-derived cell-free DNA (dd-cfDNA), reported as a fraction of total cfDNA, is a proven biomarker for KT rejection. Total cfDNA levels are elevated in patients with COVID-19, which may depress dd-cfDNA fractions, potentially leading to missed rejections.MethodsA retrospective analysis of 29 KT recipients hospitalized with COVID-19 between April and November 2020 examined total and dd-cfDNA levels. Blood samples were collected after onset of COVID-19, with follow-up samples collected from a subset of patients, when infection had likely subsided.ResultsAfter COVID-19 diagnosis, the median total cfDNA level was elevated (7.9 multiples of median [MoM]). A significant decrease in total cfDNA levels was observed between the first and second time points (6.2 MoM, 1.0 MoM; P <001). A significant positive association was identified between total cfDNA levels and COVID-19 severity (P = .02; R2 = .19). Two patients with biopsy-proven acute cellular rejection had dd-cfDNA fractions below the 1% cutoff for rejection (0.20% and 0.78%), with elevated total cfDNA levels of 7.9 MoM and 41.8 MoM, respectively.ConclusionsIn this preliminary study, total cfDNA levels were elevated in KT patients with COVID-19, subsiding after resolution of infection. High total cfDNA levels may confound dd-cfDNA results, leading to failure to identify rejection. Considering total cfDNA levels is important in interpretation of dd-cfDNA tests for assessment of rejection in KT patients with COVID-19 or other infection.

Abstract 10765: Plasma Cell-Free DNA Predicts Survival and Maps Tissue-Specific Sources of Injury in Pulmonary Arterial Hypertension

Background: Cell-free DNA (cfDNA) is a non-invasive marker of tissue injury. Although being incrementally used as a clinical biomarker, its significance in pulmonary arterial hypertension (PAH) is unknown. Methods: Plasma cfDNA was isolated in two independent PAH cohorts (A, n=48; B, n=161) and healthy controls (n=48). Clinical variables and survival status were collected for Registry to Evaluate Early And Long-term PAH Disease Management (REVEAL 2.0) scores and outcome determinations. Total cfDNA levels were compared amongst controls and PAH patients and also used to predict transplant-free survival. A subset of samples from cohort B (n=96) and controls (n=16) were subjected to bisulfite sequencing and methylation analysis to quantify tissue-specific sources of cfDNA. Results: Median (IQR) cfDNA concentrations were higher in cohort A PAH patients (exploratory cohort) compared to controls and increased according to REVEAL 2.0 risk group: controls 13.7 (10.8-18.8) vs low risk 14.4 (10.0-19.9) vs medium risk 14.6 (11.3-28.2) vs high risk 31.5 (17.2-45.6) ng/ml (P&lt;0.01). cfDNA analysis in cohort B (validation cohort) demonstrated a similar cfDNA pattern: controls 13.7 (10.8-18.8) vs low risk 24.3 (16.0-57.9) vs medium risk 50.4 (29.4-70.9) vs high risk 51.5 (34.0-110.5) ng/ml (P&lt;0.0001). cfDNA tertiles (Log-Rank; P=0.0001) and REVEAL 2.0 risk group (Log-Rank; P&lt;0.0001) similarly predicted transplant-free survival, and the addition of cfDNA to REVEAL 2.0 improved model performance (AUC 0.64 to 0.70; P&lt;0.05). Tissue-specific methylation analysis revealed increased cfDNA originating from vascular endothelium, cardiac myocytes, monocytes, erythrocyte progenitors, neutrophils, natural killer cells, and adipocytes (all P&lt;0.005), but not colon, head/neck, liver, bladder, or kidney. With the exception of natural killer cells, cfDNA from enriched tissues also differentiated PAH patients of varying REVEAL 2.0 risk. Conclusions: cfDNA is elevated in PAH patients, correlates with disease severity and predicts worse survival. Methylation analysis of cfDNA in PAH patients is consistent with prevailing paradigms of disease pathogenesis. Thus, cfDNA may add prognostic power to PAH risk assessment and provide insight into disease progression.

Utility of total cell-free DNA levels for surgical damage evaluation in patients with urological surgeries

The evaluation of surgical damage is challenging because of the lack of specific biomarkers. Total cell-free DNA (cfDNA) levels have been reported to increase with external trauma and may be a biomarker for tissue damage. To investigate the utility of perioperative total cfDNA levels in evaluating surgical damage in urological surgeries. This multicenter, prospective, observational study included 196 patients scheduled for urological surgeries between September 2020 and July 2021. The primary outcome was the change in total cfDNA levels before and after urological surgery. The secondary outcome was the effect of surgical type on total cfDNA ratio before and after urological surgery. The postoperative median total cfDNA level of the 196 patients was significantly increased 2.5-fold compared to the preoperative level (185.2 ng/mL vs. 406.7 ng/mL, P < 0.001). The median total cfDNA before/after ratio was greater than four-fold for kidney transplantation, open cystectomy, and open adrenalectomy. The ratio was less than two-fold for laparoscopic adrenalectomy and robot-assisted radical prostatectomy. Major surgery showed a significant postoperative increase in total cfDNA levels, while minor surgery did not. Total cfDNA levels increased 2.5-fold after urological surgery and it can be used as an acute-phase biomarker for surgical damage.

Absolute or Relative Quantification of Donor-derived Cell-free DNA in Kidney Transplant Recipients: Case Series.

Background.Donor-derived cell-free DNA (dd-cfDNA) is increasingly recognized as a valuable biomarker for acute transplant injury, with possible indications in the detection of cellular or humoral rejection and the guidance of immunosuppressive therapy. There is an ongoing debate on whether relative or absolute quantification of dd-cfDNA is more reliable for the detection of acute transplant injury.Methods.We retrospectively reviewed all 22 kidney transplant recipients who underwent dd-cfDNA measurements (percentage and absolute) between April 2020 and April 2021 at our institution. Of these, 9 (41%) showed discrepancies between absolute (cutoff: 50 copies/mL) and relative (cutoff: 0.5%) quantification in at least 1 dd-cfDNA measurement.Results.We report on 9 of 22 cases with discrepancies in relative and absolute quantification of dd-cfDNA, which were predominantly late posttransplant patients. We found bacterial and viral infections, as well as low leukocyte count from chronic myeloid leukaemia treatment, to be reasons for variability in total cell-free DNA (cfDNA), leading to inter- and intraindividual variability in relative dd-cfDNA quantification. When correlating dd-cfDNA quantification and biopsy results, as well as clinical course, our data indicate that relying solely on relative dd-cfDNA can lead to false-negative and false-positive results.Conclusions.In summary, these cases argue that absolute quantification of dd-cfDNA is better suited in patients with underlying conditions affecting total cfDNA levels and suggest using both absolute and relative dd-cfDNA together for higher reliability and interindividual comparability in the clinical setting. Especially for patients with chronic active antibody–mediated rejection, further studies on the use of dd-cfDNA are desirable.

Aorta-specific DNA methylation patterns in cell-free DNA from patients with bicuspid aortic valve-associated aortopathy

BackgroundThe dilation of the aorta that occurs as a consequence of a congenitally bicuspid aortic valve (BAV) is associated with a risk of dissection, aneurysm or rupture. With progressive aortopathy, surgery is often recommended, but current patient selection strategies have limitations. A blood-based assay to identify those who would most benefit from prophylactic surgery would be an important medical advance. In a proof-of-concept study, we sought to identify aorta-specific differentially methylated regions (DMRs) detectable in plasma cell-free DNA (cfDNA) obtained from patients undergoing surgery for BAV-associated aortopathy.MethodsWe used bioinformatics and publicly available human methylomes to identify aorta-specific DMRs. We used data from 4D-flow cardiac magnetic resonance imaging to identify regions of elevated aortic wall shear stress (WSS) in patients with BAV-associated aortopathy undergoing surgery and correlated WSS regions with aortic tissue cell death assessed using TUNEL staining. Cell-free DNA was isolated from patient plasma, and levels of candidate DMRs were correlated with aortic diameter and aortic wall cell death.ResultsAortic wall cell death was not associated with maximal aortic diameter but was significantly associated with elevated WSS. We identified 24 candidate aorta-specific DMRs and selected 4 for further study. A DMR on chromosome 11 was specific for the aorta and correlated significantly with aortic wall cell death. Plasma levels of total and aorta-specific cfDNA did not correlate with aortic diameter.ConclusionsIn a cohort of patients undergoing surgery for BAV-associated aortopathy, elevated WSS created by abnormal flow hemodynamics was associated with increased aortic wall cell death which supports the use of aorta-specific cfDNA as a potential tool to identify aortopathy and stratify patient risk.

Determinants of Circulating Tumor DNA Levels across Lymphoma Histologic Subtypes

Background: Cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) analysis has an emerging diagnostic role in multiple malignancies including lymphomas. As levels of these biomarkers have been correlated with clinical prognosis, knowledge of baseline values could have utility given wide clinical variation among lymphoma subtypes. We examined cfDNA and ctDNA levels for a pooled international cohort representing both classical Hodgkin (cHL) and non-Hodgkin (NHL) lymphomas. In addition, we analyzed corresponding clinical laboratory data for confounding factors.Methods: A total of 196 patients from six centers were analyzed - Stanford University (65), University of Eastern Piedmont (36), the National Cancer Institute (33), Hospital Le Bocage, Dijon (25), MD Anderson Cancer Center (21), and University Hospital Essen (16). Pre-treatment cfDNA concentration was obtained by fluorimetry after DNA extraction. All samples underwent deep sequencing with error correction by CAPP-Seq (Newman et al Nat Biotech 2016). After genotyping, ctDNA levels were calculated as the product of the cfDNA concentration and the mean allelic fraction of somatic single nucleotide mutations. Where available, clinical laboratory data from the time of ctDNA measurement were correlated.Results: Across lymphoma subtypes, total cfDNA levels were observed to distinguish between aggressive and indolent histologies (Fig 1A). The highest cfDNA levels were seen in a statistically distinct subset of aggressive thoracic tumors including mediastinal grey zone lymphoma (MGZL) and primary mediastinal tumors (PMBCL). These did not differ significantly from each other (p>0.999), but were higher than other aggressive lymphomas including diffuse large B cell lymphoma (DLBCL), cHL, and transformed low grade lymphomas (p<0.013 for all comparisons). Follicular lymphoma (FL) and cHL had the lowest median cfDNA levels.Circulating tumor DNA was detected in 97.4% of patients (191/196) with no difference in detection rate by subtype. Median ctDNA level followed a similar distribution to cfDNA, with highest levels in aggressive thoracic MGZL and PMBCL and lowest in FL (Fig 1B). Variation in ctDNA levels was notably broader than cfDNA, with DLBCL varying across a 10-million fold concentration range.To further characterize our DLBCL cohort we separated cases on cell of origin (COO) determined by Hans classification, and by histological transformation status. No difference in cfDNA or ctDNA levels was seen for GCB versus non-GCB histology (Fig 1C), or for de novo versus transformed DLBCL. However, transformed FL had significantly higher levels of both cfDNA and ctDNA than primary FL (Fig 1D, p<0.0001 for cfDNA, p=0.0032 for ctDNA).We correlated our findings with clinical laboratory data for potential confounders. Although cfDNA is renally cleared, we found no significant correlation between serum creatinine and cfDNA level (p=0.77). While the majority of cfDNA originates in the hematologic compartment, we found no significant correlation with WBC (p=0.10) and a negative correlation with hematocrit (p=0.0016) and serum albumin levels (p=0.0003). Confirming prior observations, LDH was positively correlated with cfDNA level (r=0.51, p=0.0004). All described laboratory correlations remained significant after Bonferroni correction.Conclusions: Cell free and ctDNA levels vary significantly across lymphoma histologies, suggesting that both total levels and genotype may play a role in future assays. The highest levels were observed in a subset of aggressive thoracic tumors which may reflect higher disease volumes, proliferative rates, and/or proximity to venous circulation. Minimal variation with renal and hepatic functions reinforces the reliability of cfDNA as a biomarker, while correlations with anemia, hypoalbuminemia, and LDH elevation are likely reflective of inflammatory disease burden.Figure 1: Variation by lymphoma subtype of cfDNA and ctDNA levels.A) A scatter plot demonstrates variation in cell-free DNA (cfDNA) levels across lymphoma subtypes.B) Circulating tumor DNA (ctDNA) levels vary across lymphoma subtypes.C) Neither cfDNA nor ctDNA levels differ significantly between DLBCL cell of origin subtypes.D) Primary and transformed FL display significant variation in cfDNA and ctDNA levels.(hGE/mL: Haploid genome equivalents / mL) [Display omitted] DisclosuresHüttmann:Bristol-Myers Squibb, Takeda, Celgene, Roche: Honoraria; Gilead, Amgen: Other: Travel cost. Casasnovas:BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Westin:Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Apotex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees. Gaidano:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Advani:Juno Therapeutics: Consultancy; Bayer Healthcare Pharmaceuticals: Research Funding; Infinity: Research Funding; Gilead: Consultancy; Celgene: Research Funding; Spectrum: Consultancy; Millennium: Research Funding; FortySeven: Research Funding; Kura: Research Funding; Genentech: Research Funding; Cell Medica: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Nanostring: Consultancy; Regeneron: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Pharmacyclics: Consultancy; Merck: Research Funding; Agensys: Research Funding; Sutro: Consultancy; Pharmacyclics: Research Funding. Diehn:Roche: Consultancy; Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Varian Medical Systems: Research Funding. Alizadeh:CiberMed: Consultancy; Genentech: Consultancy; Celgene: Consultancy; Roche: Consultancy; Gilead: Consultancy.

Extremely High Cell-free DNA Levels Observed in Renal Allograft Patient With SARS-CoV-2 Infection.

Beyond its widely recognized morbidity and mortality, coronavirus disease 2019 poses an additional health risk to renal allograft recipients. Detection and measurement of donor-derived cell-free DNA (dd-cfDNA), expressed as a fraction of the total cell-free DNA (cfDNA), has emerged as a noninvasive biomarker for allograft rejection. Here, we present a case report of a patient who was infected with severe acute respiratory syndrome coronavirus 2, 11 mo post–kidney transplant. The patient was serially monitored using an analytically and clinically validated massively multiplex PCR-based dd-cfDNA assay to assess allograft injury and risk for rejection. Over the course of infection, low dd-cfDNA fractions were observed (below the 1% cutoff) and were accompanied by unusually highly elevated levels of total cfDNA, which gradually declined as the infection resolved. The case study highlights the variability in total cfDNA levels during and after viral infection, and the need to consider both total and dd-cfDNA levels when clinically interpreting the results for allograft rejection. Furthermore, the study highlights the importance of serial testing, wherein an interplay between total cfDNA and dd-cfDNA can inform the optimization of a patient’s immunosuppressive treatment regimen in response to infection.

Autoantibody-mediated impairment of DNASE1L3 activity in sporadic systemic lupus erythematosus.

Antibodies to double-stranded DNA (dsDNA) are prevalent in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis, yet the nature and regulation of antigenic cell-free DNA (cfDNA) are poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity. We report that >50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation, which was associated with neutralizing autoantibodies to DNASE1L3. These patients had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens included DNA-associated proteins such as HMGB1. Our results reveal autoantibody-mediated impairment of DNASE1L3 activity as a common nongenetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE.

Prognostic significance of total plasma cell-free DNA level and androgen receptor amplification in castration-resistant prostate cancer.

To investigate the prognostic significance of total cell-free DNA (cfDNA) level and androgen receptor amplification (AR-amp) in patients with castration-resistant prostate cancer (CRPC). We retrospectively compared the total cfDNA level and AR-amp in 42 individuals without prostate cancer, 57 patients with localized prostate cancer without androgen-deprivation therapy (ADT), 97 patients with castration-sensitive prostate cancer (CSPC) with ADT, and 97 patients with CRPC. The association of these cfDNA biomarkers on disease status and overall survival was evaluated using Kaplan-Meier analysis and multivariable Cox regression analysis. Finally, a simple risk model was developed including total cfDNA and AR-amp to predict poor prognosis. The median total cfDNA level and AR-amp in patients with CRPC was 387pg/μL and 1.07 copies, respectively. The total cfDNA levels and AR-amp were significantly higher in the patients with CRPC than in individuals without prostate cancer, patients with localized prostate cancer without ADT, and patients with CSPC with ADT. Total cfDNA-high (> 600pg/μL) and AR-amp-high (> 1.26 copies) were significantly associated with poor overall survival. Multivariable Cox regression analysis showed cfDNA-high and AR-amp-high were significantly associated with poor overall survival in patients with CRPC. We developed a risk model using cfDNA-high (score 1) and AR-amp-high (score 1). The risk score 1-2 was significantly associated with worse overall survival than score 0. Total cfDNA level and AR-amp are potential biomarkers for poor prognosis in patients with CRPC.

Circulating Total Cell-Free DNA Levels Are Increased in Hypertensive Disorders of Pregnancy and Associated with Prohypertensive Factors and Adverse Clinical Outcomes.

Previous studies have described increased circulating cell-free DNA (cfDNA) in hypertensive disorders of pregnancy (HDP). Here, we aimed first to confirm this information using a simple, but sensible fluorescent assay, and second to investigate whether total cfDNA is associated with circulating factors known to be linked to the pathophysiology of HDP as well as with poor maternal-fetal outcomes. We studied 98 women with healthy pregnancies (HP), 88 with gestational hypertension (GH), and 91 with preeclampsia (PE). Total DNA was extracted from plasma using the QIAamp DNA blood mini kit and quantified using Quant-iT™ PicoGreen® dsDNA fluorescent detection kit. We found higher total cfDNA levels in GH and PE (197.0 and 174.2 ng/mL, respectively) than in HP (140.5 ng/mL; both p < 0.0001). Interestingly, total cfDNA levels were elevated in both male and female-bearing pregnancies diagnosed with either HDP, and in more severe versus less severe HDP cases, as classified according to responsiveness to antihypertensive therapy. In addition, total cfDNA was independently associated with HDP, and a cutoff concentration of 160 ng/mL provided appropriate sensitivity and specificity values for diagnosing GH and PE compared to HP (70–85%, both p < 0.0001). Moreover, high total cfDNA was associated with adverse clinical outcomes (high blood pressure, low platelet count, preterm delivery, fetal growth restriction) and high prohypertensive factors (sFLT-1, sEndoglin, MMP-2). These findings represent a step towards to the establishment of cfDNA as a diagnostic tool and the need to understand its role in HDP.

Abstract 13079: An Increase in Circulating Cardiomyocyte-specific Cell-free DNA in Heart Failure

Introduction: Cardiomyocyte cell death is closely linked with the pathophysiology of heart failure. Cell-free DNA (cfDNA) is found in circulating blood as fragmented double-strand DNA, which is released from dying cells. However, the clinical significance of cfDNA in heart failure has not been elucidated. Methods and Results: The present study enrolled 32 heart failure patients and 28 control subjects. Circulating cfDNA was isolated from plasma samples in peripheral blood. The levels of total cfDNA assessed by quantitative PCR using LINE1-specific primers were not different between the two groups (P=0.343). Next, cfDNA was subjected to bisulfite conversion to identify cardiomyocyte-derived cfDNA, based on that FAM101A locus is unmethylated in a cardiac-specific manner. Digital PCR targeting the unmethylated FAM101A locus demonstrated that the copy numbers of cardiomyocyte-specific cfDNA were significantly elevated in heart failure patients compared to controls (0.99 [0.77 - 1.98] vs 0 [0 - 0.91] copies/mL, P=0.003, Figure A ). The receiver operating characteristic curve revealed that cardiomyocyte-specific cfDNA significantly discriminated heart failure patients from control subjects (AUC, 0.716, P=0.003). Correlation analysis showed that the levels of cardiomyocyte-specific cfDNA were significantly correlated with serum levels of high-sensitivity cardiac troponin I (r = 0.438, P = 0.012, Figure B ), but not with BNP (r = 0.275, P = 0.058), suggesting that the elevated cardiomyocyte-specific cfDNA might be associated with enhanced cardiomyocyte death but not with high ventricular filling pressure. Conclusions: A newly developed bisulfite-digital PCR system identified an increase in cardiomyocyte-specific cfDNA in heart failure patients. cfDNA may be a novel biomarker to measure the actual numbers of cardiomyocyte death in heart failure.