Plasma Levels Of Cell-free DNA Research Articles

The value of plasma cell-free DNA levels as biomarker in patients with eating disorders: A preliminary study

ObjectiveCirculating cell-free DNA (cfDNA) holds promise as a rapid and convenient biomarker for identifying individuals with eating disorders. To investigate this hypothesis, we measured plasma cfDNA in patients with different eating disorders. MethodsIn this study, 110 participants (98 patients with eating disorders divided into 30 patients with bulimia nervosa, 33 patients with anorexia nervosa (AN) Restricting subtype, 35 patients with AN Binge-eating/purging subtype and 12 controls) were enrolled. We measured both cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) from plasma using two specific droplet digital PCR assays each, referring to two amplicon sizes. ResultsLevels of plasma cf-nDNA and cf-mtDNA showed no significant differences between control participants and those with eating disorders. However, we observed a higher proportion of long cf-nDNA fragments in patients with eating disorders, suggesting its potential as a biomarker for eating disorders. ConclusionThis is the first study of cfDNA in patients with eating disorders. Our findings highlight the potential for qualitative exploration of cfDNA, although not of quantitative interest. Full characterization of cfDNA may serve as a valuable biomarker for eating disorders and provide some insights into the hidden mechanisms underlying the chronic development of these conditions. Future studies are needed to confirm or refute this hypothesis.

PB0008 Increased Levels of Plasma Cell-Free DNA are Associated with Symptomatology in Patients with Carotid Stenosis and are Independent of Plasma DNaseI Activity

PB0008 Increased Levels of Plasma Cell-Free DNA are Associated with Symptomatology in Patients with Carotid Stenosis and are Independent of Plasma DNaseI Activity

Circulating Plasma Cell-free DNA (cfDNA) as a Predictive Biomarker for Radiotherapy: Results from a Prospective Trial in Head and Neck Cancer.

The plasma levels of cell-free DNA (cfDNA) in cancer patients increase due to rapid cancer cell proliferation and death. Therefore, cfDNA can be used to study specific tumor-DNA features. In addition, the non-specific cfDNA concentration may be an important biomarker of cancer prognosis. We prospectively examined the predictive role of cfDNA levels and the kinetics in the outcome of chemo-radiotherapy (CRT) in a cohort of 47 patients with locally advanced squamous cell head-neck cancer (SCHNC) treated with definitive chemo-radiotherapy. Increased cfDNA levels after therapy completion (after/before treatment ratio; A/B-ratio >1) were found in 26/47 patients (55.3%). Locally advanced T4-stage was significantly associated with higher cfDNA levels after CRT (3.3 ng/μl in T4-stage vs. 1.3 ng/μl in T1-3 stages, p=0.007). Patients who responded to CRT (partial/complete response) had significantly lower cfDNA levels before therapy (mean values 1.2 ng/μl vs. 2.7 ng/μl, p=0.03). A significantly worse locoregional progression-free survival in patients with an A/B-ratio >1 was documented (p=0.01; hazard ratio 3.5, 95%CI=1.2-9.7). This was also confirmed in multivariate analysis, where the A/B-ratio was an independent predictive variable of locoregional relapse (p=0.03, hazard ratio 3.9, 95%CI=1.2-13). High post-CRT cfDNA levels could be an early biomarker for the immediate recruitment of patients with SCHNC in consolidation chemo-immunotherapy protocols.

Plasma Cell-Free DNA and Caspase-3 Levels in Patients with Chronic Kidney Disease.

Cell-free plasma DNA (cfDNA) is circulating extracellular DNA arising from cell death mechanisms (apoptosis, necrosis, etc.). It is commonly existent in healthy individuals, but its ranks increase in diverse clinical circumstances, such as kidney disease, sepsis, myocardial infarction, trauma and cancer. In patients with advanced chronic kidney disease, cfDNA is connected to inflammation, and it has been associated with higher mortality. Caspase-3 plays a dominant role in apoptosis, a mechanism of programmed cell death involved in the pathogenesis and progression of chronic kidney disease (CKD). The aim of this pilot study was the evaluation of cfDNA levels and caspase-3 concentrations in patients with chronic kidney disease, in order to investigate the potential role of these molecules, deriving from inflammatory and apoptotic mechanisms, in the progression of renal damage. We compared cfDNA and caspase-3 levels in 25 CKD patients and in 10 healthy subjects, evaluating their levels based on CKD stage. We also explored correlations between cfDNA and caspase-3 levels in CKD patients and between cfDNA and caspase-3 levels and serum creatinine and urea in this population. We observed that cfDNA and caspase-3 levels were higher in patients with CKD compared to healthy subjects, in particular in patients with advanced renal disease (CKD stage 5). A positive correlation between cfDNA and caspase-3 levels and between cfDNA and caspase-3 and creatinine and urea were also noticed. Patients with chronic kidney disease show higher levels of cfDNA and caspase-3 levels compared to the control group. Based on these preliminary results, we speculated that the worsening of renal damage and the increase in uremic toxin concentration could be associated with higher levels of cfDNA and caspase-3 levels, thus reflecting the potential role of inflammation and apoptosis in the progression of CKD. Future studies should focus on the validation of these promising preliminary results.

Calibration of cell-free DNA measurements by next-generation sequencing.

Accurate monitoring of disease burden depends on accurate disease marker quantification. Although next-generation sequencing (NGS) is a promising technology for noninvasive monitoring, plasma cell-free DNA levels are often reported in misleading units that are confounded by non-disease-related factors. We proposed a novel strategy for calibrating NGS assays using spiked normalizers to improve precision and to promote standardization and harmonization of analyte concentrations. In this study, we refined our NGS protocol to calculate absolute analyte concentrations to (1) adjust for assay efficiency, as judged by recovery of spiked synthetic normalizer DNAs, and (2) calibrate NGS values against droplet digital polymerase chain reaction (ddPCR). As a model target, we chose the Epstein-Barr virus (EBV) genome. In patient (n = 12) and mock (n = 12) plasmas, NGS and 2 EBV ddPCR assays were used to report EBV load in copies per mL of plasma. Next-generation sequencing was equally sensitive to ddPCR, with improved linearity when NGS values were normalized for spiked DNA read counts (R2 = 0.95 for normalized vs 0.91 for raw read concentrations). Linearity permitted NGS calibration to each ddPCR assay, achieving equivalent concentrations (copies/mL). Our novel strategy for calibrating NGS assays suggests potential for a universal reference material to overcome biological and preanalytical variables hindering traditional NGS strategies for quantifying disease burden.

Development of a High-Throughput Urosepsis Mouse Model.

Murine sepsis models are typically polymicrobial, and are associated with high mortality. We aimed to develop a high-throughput murine model that mimics a slow-paced, monomicrobial sepsis originating from the urinary tract. A total of 23 male C57Bl/6 mice underwent percutaneous insertion of a 4 mm catheter into the bladder using an ultrasound-guided method, previously developed by our group. The following day, Proteus mirabilis (PM) was introduced percutaneously in the bladder in three groups: g1-50 µL 1 × 108 CFU/mL solution (n = 10); g2-50 µL 1 × 107 CFU/mL solution (n = 10); and g3 (sham mice)-50 µL sterile saline (n = 3). On day 4, mice were sacrificed. The number of planktonic bacteria in urine, adherent to catheters, and adherent to/invaded into the bladder and spleen was assessed. Cell-free DNA, D-dimer, thrombin-antithrombin complex (TAT), and 32 pro-/anti-inflammatory cytokines/chemokines were quantified in the blood. All mice survived the 4 day postinterventional period. Mean weight loss was 11% in g1, 9% in g2, and 3% in the control mice. Mean urine CFU counts were highest in group 1. All catheters showed high catheter-adhered bacterial counts. Of the infected mice, 17/20 had CFU counts in the splenic tissue, indicating septicemia. Plasma levels of cell-free DNA, D-dimer, and the proinflammatory cytokines IFN-γ, IL-6, IP-10, MIG, and G-CSF were significantly elevated in infected mice versus controls. We present a reproducible, monomicrobial murine model of urosepsis that does not lead to rapid deterioration and death, and is useful for studying prolonged urosepsis.

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.

Netosis and the Formation of Neutrophil Extracellular Traps (NETs) in Patients with Severe and Critical COVID-19 May Contribute to Disease Progression and Thrombosis

Introduction. NETosis and the formation of neutrophil extracellular traps (NETs) are known to be associated with the pathogenesis of various infectious diseases and thrombosis. However, the extent and clinical significance of NETosis and its released plasma markers in patients with a SARS-CoV2 infection resulting in severe and critical coronavirus disease 2019 (COVID-19) are not fully understood. Patients and Methods. COVID-19 patients on admission were prospectively enrolled for the study. Patients were classified according to WHO guidelines into asymptomatic (control), mild-moderate (not requiring oxygenation), severe (requiring oxygenation), and critical (multiple organ failure and/or requiring ventilator). Whole blood anticoagulated with EDTA from each patient was collected. Cells were fixed and stained for flow cytometry analysis following incubation with anti-CitrH3 and anti-MPO, and plasmas were used for assessing the soluble NETs. Results. Seventy-six hospitalized patients with PCR positive SARS-CoV2 were categorized into asymptomatic control (n=11), mild-moderate (n=28), severe (n=22), and critical (n=15) groups. Flow cytometry detected only few CitH3+/MPO+ positive neutrophils (undergoing NETosis) in blood of asymptomatic controls on admission. However, the number of CitH3+/MPO+ neutrophil dramatically increased following a 15-minute stimulation with a bacterial Shigatoxin-2 at 100 ng/mL or polyinosinic-polycytidylic acid [Poly (I:C)], a TLR3 agonist at 5 ug/mL for 3 hours (Fig. 1). The number of the CitH3+/MPO+ positive neutrophils in the samples from severe COVID-19 patients was significantly higher than that in asymptomatic controls (p=0.021). Interestingly, the number of the CitH3+/MPO+ positive neutrophils in critical patients was in fact lower than that in severe patients (p=0.013). Moreover, upon stimulation with Poly (I:C), the number of the CitH3+/MPO+ positive neutrophils in asymptomatic patients were more dramatically increased than those in severe/critical patients, suggesting a decrease in neutrophil reserve that could undergo NETosis and fight for infection. Microfluidic shear-based assay and confocal imaging analysis further demonstrated an increased formation of NETs strings under flow in whole blood samples from severe COVID-19 patients compared with those from asymptomatic controls (p=0.012). The NETs strings were sensitive to digestion by DNase I (Fig. 2), suggesting the therapeutic efficacy of DNase I in severe COVID-19 patients. Finally, plasma levels of cell-free DNA and histone-DNA complexes (the soluble markers of NETs in blood) were also more significantly increased in patients with severe and critical COVID-19 than those in the asymptomatic controls. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Integrated GWAS and Gene Expression Suggest ORM1 as a Potential Regulator of Plasma Levels of Cell-Free DNA and Thrombosis Risk.

Plasma cell-free DNA (cfDNA) is a surrogate marker of neutrophil extracellular traps (NETs) that contribute to immunothrombosis. There is growing interest about the mechanisms underlying NET formation and elevated cfDNA, but little is known about the factors involved. We aimed to identify genes involved in the regulation of cfDNA levels using data from the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project. Imputed genotypes, whole blood RNA-Seq data, and plasma cfDNA quantification were available for 935 of the GAIT-2 participants from 35 families with idiopathic thrombophilia. We performed heritability and GWAS analysis for cfDNA. The heritability of cfDNA was 0.26 ( p = 3.7 × 10 −6 ), while the GWAS identified a significant association (rs1687391, p = 3.55 × 10 −10 ) near the ORM1 gene, on chromosome 9. An eQTL (expression quantitative trait loci) analysis revealed a significant association between the lead GWAS variant and the expression of ORM1 in whole blood ( p = 6.14 × 10 −9 ). Additionally, ORM1 expression correlated with levels of cfDNA ( p = 4.38 × 10 −4 ). Finally, genetic correlation analysis between cfDNA and thrombosis identified a suggestive association ( ρ g = 0.43, p = 0.089). All in all, we show evidence of the role of ORM1 in regulating cfDNA levels in plasma, which might contribute to the susceptibility to thrombosis through mechanisms of immunothrombosis.

Circulating Tumor DNA Profiling From Breast Cancer Screening Through to Metastatic Disease.

PURPOSEWe investigated the utility of the Oncomine Breast cfDNA Assay for detecting circulating tumor DNA (ctDNA) in women from a breast screening population, including healthy women with no abnormality detected by mammogram, and women on follow-up through to advanced breast cancer.MATERIALS AND METHODSBlood samples were taken from 373 women (127 healthy controls recruited through breast screening, 28 ductal carcinoma in situ, 60 primary breast cancers, 47 primary breast cancer on follow-up, and 111 metastatic breast cancers [MBC]) to recover plasma and germline DNA for analysis with the Oncomine Breast cfDNA Assay on the Ion S5 platform.RESULTSOne hundred sixteen of 373 plasma samples had one or more somatic variants detected across eight of the 10 genes and were called ctDNA-positive; MBC had the highest proportion of ctDNA-positive samples (61; 55%) and healthy controls the lowest (20; 15.7%). ESR1, TP53, and PIK3CA mutations account for 93% of all variants detected and predict poor overall survival in MBC (hazard ratio = 3.461; 95% CI, 1.866 to 6.42; P = .001). Patients with MBC had higher plasma cell-free DNA levels, higher variant allele frequencies, and more polyclonal variants, notably in ESR1 than in all other groups. Only 15 individuals had evidence of potential clonal hematopoiesis of indeterminate potential mutations.CONCLUSIONWe were able detect ctDNA across the breast cancer spectrum, notably in MBC where variants in ESR1, TP53, and PIK3CA predicted poor overall survival. The assay could be used to monitor emergence of resistance mutations such as in ESR1 that herald resistance to aromatase inhibitors to tailor adjuvant therapies. However, we suggest caution is needed when interpreting results from a single plasma sample as variants were also detected in a small proportion of HCs.

Utility of cell-free DNA concentrations and illness severity scores to predict survival in critically ill neonatal foals.

Plasma cell-free DNA (cfDNA) levels have been associated with disease and survival status in septic humans and dogs. To date, studies investigating cfDNA levels in association with critical illness in foals are lacking. We hypothesized that cfDNA would be detectable in the plasma of foals, that septic and sick-nonseptic foals would have significantly higher cfDNA levels compared to healthy foals, and that increased cfDNA levels would be associated with non-survival. Animals used include 80 foals of 10 days of age or less admitted to a tertiary referral center between January and July, 2020 were stratified into three categories: healthy (n = 34), sick non-septic (n = 11) and septic (n = 35) based on specific criteria. This was a prospective clinical study. Blood was collected from critically ill foals at admission or born in hospital for cfDNA quantification and blood culture. Previously published sepsis score (SS) and neonatal SIRS score (NSIRS) were also calculated. SS, NSIRS, blood culture status and cfDNA concentrations were evaluated to predict survival. Continuous variables between groups were compared using Kruskal-Wallis ANOVA with Dunn's post hoc test. Comparisons between two groups were assessed using the Mann-Whitney U-test or Spearman rank for correlations. The performance of cfDNA, sepsis score and NSIRS score to predict survival was assessed by receiver operator characteristic (ROC) curve analysis including area under the curve, sensitivity and specificity using cutoffs. Plasma cfDNA was detectable in all foals. No significant differences in cfDNA concentration were detected between healthy foals and septic foals (P = 0.65) or healthy foals and sick non-septic foals (P = 0.88). There was no significant association between cfDNA and culture status, SS, NSIRS or foal survival. SS (AUC 0.85) and NSIRS (AUC 0.83) were superior to cfDNA (AUC 0.64) in predicting survival. Although cfDNA was detectable in foal plasma, it offers negligible utility to diagnose sepsis or predict survival in critical illness in neonates.

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.

Clinical value of perioperative levels of DNA and mRNA in plasma of patients with renal cell carcinoma

IntroductionThe current challenge on renal cell carcinoma (RCC) is to finding a non-invasive biomarker for improving their diagnostic and therapeutic management. In the present study, we analyzed the clinical value of plasma levels of cell-free DNA (cfDNA) and RNA (cfRNA) of two genes: glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) and human telomerase reverse transcriptase (hTERT). Materials and methodsWe recruited 82 patients with RCC, and 20 healthy subjects. Using RT-PCR techniques, plasma levels of cfDNA and cfRNA from hTERT and GAPDH genes were quantified pre- and post-operatively, and one year after surgery. Relationships between such plasma levels and clinicopathological features and evolution of disease were analyzed. ResultsLevels of GAPDH cfDNA and cfRNA were significantly higher in patients than in healthy subjects. hTERT cfDNA was detected in plasma from 35% of RCC patients and in none healthy subject. At diagnosis, plasma levels of GAPDH cfDNA were higher in advanced pT and TNM stages, and hTERT cfDNA in patients with 3–4 Fuhrman grade and affected lymph nodes. Levels of cfNAs were not related to the presence of metastasis. Following nephrectomy, GAPDH cfDNA levels dropped, and patients with higher levels before and after nephrectomy, showed lower overall survival (OS). However, Cox's multivariate model did not prove any association of the cfNA levels with progression. ConclusionPlasma levels of cfDNA from GADPH and hTERT genes were correlated to tumor diagnosis and progression and, thus, such analyses might help to diagnosis and prognosis of RCC patients.

Prognostic Impact of Total Plasma Cell-free DNA Concentration in Androgen Receptor Pathway Inhibitor–treated Metastatic Castration-resistant Prostate Cancer

Total plasma cell-free DNA (cfDNA) levels were recently shown to be prognostic in two large phase III trials of taxane chemotherapy in metastatic castration-resistant prostate cancer (mCRPC). However, whether cfDNA concentration is predictive of treatment outcomes with androgen receptor pathway inhibitors (ARPIs) is unknown. We quantified plasma cfDNA levels at baseline (n = 74) and 4 weeks on treatment (n = 56) in a prospective cohort of mCRPC patients treated with the ARPIs abiraterone acetate or enzalutamide. Elevated total cfDNA concentration (log10) at both baseline (hazard ratio [HR] 5.5, p < 0.001) and week 4 (HR 7.5, p < 0.001) was a significant negative prognostic factor for overall survival (OS), a finding maintained after adjustment for plasma circulating tumour DNA fraction. Unexpectedly, a rise in cfDNA concentration from baseline to week 4 was also associated with significantly improved OS (HR 0.14, p = 0.003). Conversely, patients with ≥29.8% decrease in cfDNA from baseline (optimal cut-point) had significantly shorter median OS than the rest of the cohort (10.5 vs 25.7 mo, p = 0.03). Collectively, our findings point to the potential prognostic utility of quantifying cfDNA in mCRPC and in particular suggest that dynamic changes in total cfDNA levels may be a novel early predictive biomarker for therapeutic outcome in ARPI-treated patients. Patient summaryWe measured the levels of total cell-free DNA (cfDNA) in the plasma of patients with metastatic prostate cancer prior to and 4 weeks after starting new hormonal drugs. We found that patients with higher levels of cfDNA or a higher proportion of tumour-derived DNA at baseline had worse outcomes on hormonal therapies. Similarly, higher levels of cfDNA at 4 weeks into therapy were also associated with worse outcomes. However, a rise in total cfDNA levels at 4 weeks compared with baseline was linked with better outcomes. Measuring changes in cfDNA concentration may be a useful and technically straightforward early way to predict how patients will respond to treatment in metastatic prostate cancer.

Increased plasma concentration of cell-free DNA precedes disease recurrence in children with high-risk neuroblastoma

BackgroundNeuroblastoma is the most common extracranial solid tumor of childhood. The high rate of recurrence is associated with a low survival rate for patients with high-risk neuroblastoma. There is thus an urgent need to identify effective predictive biomarkers of disease recurrence.MethodsA total of 116 patients with high-risk neuroblastoma were recruited at Beijing Children’s Hospital between February 2015 and December 2017. All patients received multidisciplinary treatment, were evaluated for the therapeutic response, and then initiated on maintenance treatment. Blood samples were collected at the beginning of maintenance treatment, every 3 months thereafter, and at the time of disease recurrence. Plasma levels of cell-free DNA (cfDNA) were quantified by qPCR. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the ability of plasma cfDNA concentration to predict recurrence.ResultsOf the 116 patients, 36 (31.0%) developed recurrence during maintenance treatment. The median time to recurrence was 19.00, 9.00, and 8.00 months for patients who had achieved complete response (n = 6), partial response (n = 25), and stable disease (n = 5), respectively, after multidisciplinary treatment. The median plasma cfDNA concentration at the time of recurrence was significantly higher than the concentration in recurrence-free patients throughout maintenance treatment (29.34 ng/mL vs 10.32 ng/mL). Patients recorded a plasma cfDNA level ≥ 29 ng/mL an average of 0.55 months before diagnosis of disease recurrence. ROC analysis of the power of plasma cfDNA to distinguish between patients with or without recurrence yielded an area under the curve of 0.825, with optimal sensitivity and specificity of 80.6 and 71.3%, respectively, at a cfDNA level of 12.93 ng/mL.ConclusionsHigh plasma cfDNA concentration is a potential molecular marker to signal disease recurrence in patients with high-risk neuroblastoma.

Non-invasive Molecular Detection of Minimal Residual Disease in Papillary Thyroid Cancer Patients.

Background: Papillary thyroid cancer (PTC) is the most common type of thyroid malignancy. Serum thyroglobulin (Tg) levels are used to monitor PTC treatment response and recurrences however, in about 25% of the cases the sensitivity of this method is compromised due to either the presence of neutralizing anti-Tg antibodies (TgAb) or the absence of Tg in less differentiated tumors. Up to 80% of PTC tumors harbor the c.1799T>A hotspot mutation in the BRAF gene (BRAFV600E). Here, we assessed the potential use of plasma cell-free BRAFV600E mutant tumor DNA (ctDNA) levels in determining the minimal residual tumor status of PTC patients.Methods: Patients were classified as either having persistent disease (PD) or no evidence of disease (NED) based on clinicopathological assessments. Tumor BRAFV600E status was determined by both direct sequencing and digital PCR. Plasma total cell-free BRAFV600 wild type DNA (cfDNA) and ctDNA fractions circulating in the plasma of PTC patients were determined by an emulsion based-digital PCR and total ctDNA was quantified by 3D digital PCR. The total ctDNA levels (copies/ml) were then compared to patients' clinicopathological features.Results: About 74% (28/38) of tumors harbored the BRAFV600E mutation. Percent plasma ctDNA fractions for PD patients with BRAFV600E tumors ranged from 0 to 2.07%, whereas absolute plasma ctDNA copies ranged from 0 to 62 copies. The ctDNA levels accurately detected tumor burden of PTC patients whose tumors harbored BRAFV600E; median plasma ctDNA copy numbers were significantly higher (Wilcoxon test, p = 0.03) in patients with metastasis (MET) (20 copies/ml) compared to patients with non-metastatic (non-MET) tumors (1 copy/ml). The plasma ctDNA levels (copies/ml) accurately determined the disease status of PTC patients with sensitivity of 86% and specificity of 90% as compared to 78% sensitivity and 65% specificity determined by serum Tg levels (ng/ml) with areas under the curves (AUC) of 0.88 and 0.71, respectively. Intriguingly, plasma total cfDNA levels were significantly higher in patients with no evidence of residual disease (NED) compared to persistent disease (PD) patients.Conclusions: Our study supports the clinical applicability of plasma ctDNA as biomarker to determine the residual tumor status and tumor burden of PTC patients.

Plasma Levels of Histone-DNA Complex and Cell-Free DNA Predict Mortality in Patients with Immune Thrombotic Thrombocytopenic Purpura

Background: Deficiency of plasma ADAMTS13 activity resulting from acquired autoantibody against ADAMTS13 is the primary cause of immune thrombotic thrombocytopenic purpura (iTTP). Therapeutic plasma exchange plus corticosteroids and immunosuppression is the standard of care for patients with iTTP. However, mortality rate remains high and clinical factors or laboratory biomarkers that predict mortality are not fully established. Previous studies have demonstrated that plasma levels of histone-DNA complexes and cell-free DNA are dramatically elevated in patients with acute disease but reduced during remission. We hypothesize that elevated histone-DNA complexes or cell-free DNA may have a predictive role for mortality in patients with acute iTTP. Methods: 102 unique patients with a confirmed diagnosis of iTTP who underwent therapeutic plasma exchange (TPE) at UAB-Medical Center from April 2006 to December 2018 were enrolled into the study. Demographic information and laboratory parameters were collected on admission and during the follow-up. Plasma levels of histone-DNA complexes and cell-free DNA were determined by an enzyme-linked immunosorbent assay and PicoGreen dsDNA assay, respectively. Mann-Whitney, Fisher's exact, t test, log-rank test, and Cox proportional hazard regression analysis were performed to determine the significance of each marker in predicting death. Results: In this cohort, age was 44.7 ± 1.3 (mean ± SD) years old, 56.9% were female, 82.4% were African Americans, 61.8% had an initial episode, the median (IQR) body mass index (BMI) was 33.6 (29.3-41.5). 52.9% of patients had hypertension, 21.6% diabetes, 18.6% systemic lupus erythematous and 8.7% HIV. The overall mortality rate was 10.8% (11/102). Plasma levels, median (IQR), of histone-DNA complexes and cell-free DNA on admission were 56.3 (35.8-136.4) AU/mL and 952.2 (799.7-1431.4) ng/mL, respectively. Mann-Whitney test revealed that high plasma levels of histone-DNA complex (≥134.9 U/mL) (p=0.007) and cell-free DNA (≥952.2 ng/mL) (p=0.009) were associated with the increased risks of myocardial injury (e.g. elevated troponin). Cox proportional hazard regression analysis demonstrated that the increased levels of histone-DNA complexes and cell-free DNA were also predictive for the increased mortality rates in patients with iTTP with hazard ratios (HRs) of 4.1 (95% CI, 1.2-13.4) (p=0.02) and 8.5 (95% CI, 2.2-33.3 (p=0.002), respectively. Kaplan-Meier survival analysis revealed significant differences in the disease-free survival rates of patients with high levels (≥75 percentile) vs. low levels (&lt;75 percentile) of plasma histone-DNA complexes and cell-free DNA, respectively (see Fig. 1). Conclusion: Our results demonstrate that the elevated plasma levels of histone-DNA complexes and cell-free DNA predict the worse outcome (e.g. the increased mortality rates) in patients with iTTP. *SD, standard deviation; IQR, interquartile range; 95% CI, 95% confidential interval. Disclosures Zheng: Ablynx/Sanofi: Consultancy, Speakers Bureau; Alexion: Speakers Bureau; Shire/Takeda: Research Funding; Clotsolution: Other: Co-Founder.

Quantification of plasma cell-free DNA levels in dogs with various tumors.

Circulating cell-free DNA (cfDNA) is extracellular DNA released into the bloodstream by apoptotic or necrotic tumor cells, with cfDNA determination proposed as a noninvasive, sensitive marker for the diagnosis of human cancer. We evaluated cfDNA quantification as a diagnostic and prognostic tool in dogs with various tumors. We quantified plasma cfDNA concentration by absolute real-time PCR of long interspersed nuclear elements in 50 dogs with malignant tumors, 13 dogs with benign tumors or nodules, and 11 healthy controls. Six patients with malignant tumors were followed-up, and plasma cfDNA was quantified throughout disease progression. We found that plasma cfDNA concentrations were significantly elevated in dogs with malignant tumors compared with dogs with benign nodules or healthy controls. The DNA integrity index (the ratio between long and short cfDNA fragments) was significantly lower in dogs with malignant tumors compared to healthy controls. Significantly higher cfDNA levels and a lower DNA integrity index were observed in dogs with lymphoma or leukemia, hemangiosarcoma, and distant metastasis; cfDNA levels correlated well with clinical stage and tended to increase during or before periods of disease progression, suggesting potential efficacy of cfDNA for the detection of distant metastasis and to monitor the clinical stage of neoplasia.

Early second-trimester plasma cell free DNA levels with subsequent risk of pregnancy complications

ObjectiveTo investigate the association between cfDNA levels measured during non-invasive prenatal testing (NIPT) and the risk of pregnancy complications in a Chinese population. MethodsThis was a retrospective cohort study of 831 pregnant women who underwent NIPT at 12–22 weeks of gestation. Maternal plasma cfDNA levels and pregnancy outcomes were obtained from NIPT Screening System and hospitalization records, respectively. Logistic regression analysis was performed to investigate the relationship between cfDNA levels and pregnancy complications (after adjusting for confounding factors). ResultsMaternal cfDNA levels were significantly higher in women diagnosed with intrahepatic cholestasis of pregnancy (ICP) and preeclampsia (PE) compared to pregnant women with non-pregnancy complications (NPC) (median cfDNA 7.07, 6.42 vs. 5.99 ng/mL). Increase in cfDNA levels were associated with an increased risk for ICP (adjusted-OR = 1.20, 95% CI: 1.07–1.34) and PE (adjusted-OR = 1.14, 95% CI: 1.02–1.26). In addition, increase in cfDNA levels were associated with risk of GDM, and was dependent on maternal age (maternal age ≥ 35 years: adjusted-OR = 1.16, 95% CI: 1.04–1.29; maternal age < 35 years: adjusted-OR = 0.85, 95% CI: 0.73–0.99). ConclusionMaternal plasma cfDNA levels measured during NIPT are associated with pregnancy complications (ICP, PE and GDM). Maternal age may be an important effect modifier for the association between plasma cfDNA levels and GDM.