cfDNA Levels Research Articles

Plasma cfDNA for the Diagnosis and Prognosis of Colorectal Cancer.

Objective To evaluate the value of cell-free DNA (cfDNA) for the diagnosis and prognosis of colorectal cancer (CRC). Methods Peripheral blood specimens of 120 CRC patients and 90 healthy volunteers (as a control cohort) were extracted. A quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the cfDNA expression. Following correlation analyses for cfDNA and clinical endpoints, a receiver operator characteristic (ROC) curve was established to assess the sensitivity and specificity of cfDNA, CEA, VEGF, and CA125 and for evaluating the disease-free survival (DFS) of patients. Results The plasma cfDNA level of colorectal cancer patients was significantly higher than that of healthy subjects (P < 0.05), and after chemotherapy, cfDNA level was significantly lower than that before chemotherapy (P < 0.05). CA125/CEA/VEGF expression significantly correlated with cfDNA level, but not with cfDNA integrity. There was also a significant correlation between tumor differentiation and the cfDNA level. cfDNA has a higher ROC value than the current tumor biomarkers. Survival analysis showed that the DFS of the low cfDNA expression group was longer (29.99 ± 0.78 months) than that of the high cfDNA expression group (27.66 ± 1.05 months, P=0.031). Conclusion The blood cfDNA is associated with the pathological features of CRC clinical cases and represents a possible indicator for CRC diagnosis and prognosis.

Clinical Value of Peripheral Blood Circulating Tumor Cells and Cell-Free DNA Combined Detection in Triple-Negative Breast Cancer

Objective: To determine the clinical value of combined detection of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in peripheral blood of patients with triple-negative breast cancer. Method: 41 patients with breast cancer admitted to the First Central Hospital of Baoding from January 2020 to December 2021 were selected and recruited into the experimental group, 42 patients with benign breast cancer admitted during the same period were recruited into the conditional control group, and 41 healthy patients admitted during the same period were recruited into the blank control group. The positive rate of peripheral blood CTCs, the level of cfDNA, and the diagnostic efficacy of peripheral blood CTCs, cfDNA alone and the combination thereof for breast cancer were analyzed. Result: The positive rates of peripheral blood CTCs in the experimental group, the conditional control group, and the blank control group were 43.90%, 11.90%, and 9.74%, respectively, and there was significant difference among the groups. The levels of cfDNA in peripheral blood of the experimental group, the conditional control group, and the blank control group were 0.26 ± 0.08 bp, 0.17 ± 0.03 bp, and 0.15 ± 0.04 bp, respectively, which were statistically significant. The detection levels of 100 bp hTERT/ng·ml-1 and 241 bp hTERT/ng·ml-1 in the experimental group were significantly higher than those in the conditional control group and the blank control group. The accuracy of peripheral blood CTCs detection in the three groups was 66.21%, the accuracy of cfDA241 bp / 100 bp hTERT detection was 80.41%, and the accuracy of combined detection of peripheral blood CTCs and cfDNA was 94.03%. Conclusion: The clinical application of peripheral blood CTCs combined with cfDNA level detection can increase detection accuracy, provide data support for clinicians, and improve the clinical diagnostic effect of triple-negative breast cancer.

C53 DONOR–DERIVED CELL–FREE DNA (DD–CFDNA)–BASED ASSESSMENT OF CORONARY ALLOGRAFT VASCULOPATHY IN HEART TRANSPLANTED PATIENTS

Abstract Introduction Cardiac Allograft Vasculopathy (CAV) is a leading cause of long–term graft dysfunction and failure after Heart Transplant (HTx). Early diagnosis is crucial to tailor therapeutic strategies. Despite advancements in cardiac imaging, the standard for CAV diagnosis remains coronary angiography. Donor–derived cell free DNA (dd–cfDNA) is a novel marker of graft injury in solid organ transplants. Elevations in dd–cfDNA levels may reflect CAV development during subclinical inflammation, thus it could represent a non–invasive method of surveillance for CAV rejection in apparently stable post–transplant patients. Given these premises, we decided to evaluate the potential of dd–cfDNA as alternative diagnostic biomarker for CAV. Materials and Methods 31 blood samples from patients who underwent heart transplant at our institution in the last 10 years, were analyzed by Next Generation Sequencing (NGS) according to CareDx Alloseq protocol. The cfDNA was extracted starting from 2mL of plasma (Qiagen). Sequencing was performed on the Illumina Miseq platform. Troponin T (hs–TnT) and NT–proBNP (Brain Natriuretic Peptide) were measured by automated immunoassay analysers. All statistics were performed by STATA software and significance was set at p &amp;lt; 0.05. Results Heart transplant recipients were divided into 2 groups: 16 patients with known CAV status and 15 patients who had not (no–CAV). No significant differences between the two populations were observed, aside from time from HTx (177.28± 84.99 months in CAV vs 93.20 ± 84.39 months in no–CAV; p = 0.033). A significant difference in dd–cfDNA fraction was found between CAV and no–CAV patients (p = 0.03); with mean values of 0,41% e 0,13%, respectively. Neither NT-proBNP (p = 0.08) nor hs–TnT (p = 0.31) concentrations were significantly different between the two groups, but a positive correlation was found between the two biomarkers (r 0.6966; p = 0.0013). A trend of linear relationship emerged between cfDNA and NT-proBNP levels (r 0.4155; p = 0.08). Conclusions With this study, we demonstrated the feasibility of evaluating dd–cfDNA in long–term heart transplanted patients and its possible association with CAV development. Further investigations are warranted to explore a possible association between dd–cfDNA levels other circulating biomarkers and CAV progression. The definition of “homogeneous” subgroups of patients according to a “risk score” will allow to better manage their follow up and improve their prognosis.

Circulating Cell-free DNA in Serum as a Marker for the Early Detection of Tumor Recurrence in Breast Cancer Patients.

Circulating cell-free DNA (cfDNA) isolated from serum by noninvasive procedures can serve as a potential biomarker for the early detection of many cancers. The aim of this study was to implement a simple, yet effective quantitative method for measuring the cfDNA in serum and to investigate the relationship between cfDNA and the occurrence of recurrence in breast cancer (BrCa) patients. A total of 240 cases were selected, which comprised different subtypes of BrCa patients and control individuals. We selected 20 serum samples from patients which showed recurrence after 4-7 years of disease-free survival. SYBR green was used as a reporter molecule to estimate the amount of cfDNA in these serum samples. A global Wilcoxon analysis was performed to compare the cfDNA abundance between non-recurrent and recurrent patients. The amount of cfDNA was higher in recurrent patients (recurrent vs. non-recurrent ratio=1.3; p=0.03; AUC=0.76) compared to non-recurrent patients. The data between normal/healthy controls and non-recurrent patients indicated no significant differences (n=20 in each group, healthy to non-recurrent ratio=1.03; p=0.20; AUC=0.61). We implemented a straightforward one-step technique to measure the amount of cfDNA in serum, which can translate into a clinical diagnostic tool in the near future. The high levels of cfDNA in the serum of recurrent BrCa patients compared to non-recurrent BrCa patients indicates a possible uncovered role for circulating genetic information, which either contributes to the cancer recurrence phenomenon or at the very least, serves as an identifier for the potential of recurrence.

Cell-Free DNA, Neutrophil extracellular traps (NETs), and Endothelial Injury in Coronavirus Disease 2019- (COVID-19-) Associated Acute Kidney Injury.

Introduction: Neutrophil extracellular traps (NETs) release (i.e., NETosis) has been recently implicated in the pathomechanism underlying severe end-organ damage in Coronavirus Disease 2019 (COVID-19) and could present a novel therapeutic target. We aimed to determine whether circulating levels of cell-free DNA (cfDNA), a surrogate for NETosis, may be associated with the development of acute kidney injury (AKI), a major contributor to poor outcomes and mortality in COVID-19. Methods: Blood samples were collected prospectively from adult patients infected with SARS-CoV-2 presenting to the emergency department (ED). Circulating levels of cfDNA were quantified from patients' serum. Further assessment of correlations between cfDNA levels and markers of AKI (i.e., serum creatinine (SCr), cystatin C, neutrophil gelatinase–associated lipocalin (NGAL)), biomarkers of thrombotic microangiopathy and of inflammation in patients' serum was performed. Results: Fifty-one COVID-19 patients were enrolled. cfDNA levels were found to be significantly higher in those who developed severe AKI (p < 0.001) and those needing renal replacement therapy (p = 0.020). cfDNA positively correlated with ED SCr, NGAL, cystatin C, neutrophil count, neutrophil-to-lymphocyte ratio, C3a, C5a, Scb5-9, IL-6, IL-8, IL-10, TNF-α, LDH, CRP, ferritin, and fibrinogen and negatively correlated with ADAMTS13/von-Willebrand factor ratio and lymphocyte count. In a multivariate logistic regression, a one-unit increase in cfDNA value was associated with 4.6% increased odds of severe AKI (OR = 1.046; p = 0.040). Finally, cfDNA significantly correlated with established NETs components, myeloperoxidase, and neutrophil elastase. Conclusion: Intravascular NETosis could be an important contributing factor in the development of microthrombosis and COVID-19-associated AKI. Further research is urgently needed to understand the role of NETosis in COVID-19 and evaluate therapeutic avenues for targeting this process.

Circulating cell free DNA and citrullinated histone H3 as useful biomarkers of NETosis in endometrial cancer

BackgroundCancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC).MethodsThe experiments were conducted on 21 healthy subjects (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features, IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and histone 2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondence analysis (MCA) between cfDNA, mtcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades.ResultsWe demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen.ConclusionOur data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.

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.

Cell-free DNA as a biomarker after lung transplantation: A proof-of-concept study.

BackgroundLung transplantation (LTx) is a lifesaving procedure burdened with limited long‐term survival. The most common cause of death after LTx is chronic lung allograft dysfunction (CLAD). Today, useful biomarkers for the detection of CLAD are lacking. Circulating cell‐free DNA (cfDNA) is released during cellular decay and can be detected using polymerase chain reaction (PCR). Thus, donor‐derived cfDNA in recipient serum indicates cellular decay in the transplanted organ. In the current study, we explore the possibility of using a novel PCR method to detect cfDNA as a biomarker for clinical events, especially CLAD.MethodsFour patients were retrospectively tested for levels of both donor and recipient‐derived cfDNA using digital droplet PCR after targeted preamplification. The results were correlated to recorded clinical events.ResultsAll available samples rendered results. Both patients that later developed CLAD showed a persistently elevated ratio between donor‐and recipient‐derived cfDNA. Also, the mean level of cfDNA was higher in the two patients who later developed CLAD than in patients who did not (p = .0015).ConclusionsThis proof‐of‐concept study suggests that cfDNA quantified with PCR may be used as a biomarker of significant clinical events such as CLAD.

Circulating Tumor DNA in Precision Oncology and Its Applications in Colorectal Cancer

Circulating tumor DNA (ctDNA) is a component of cell-free DNA (cfDNA) that is shed by malignant tumors into the bloodstream and other bodily fluids. ctDNA can comprise up to 10% of a patient’s cfDNA depending on their tumor type and burden. The short half-life of ctDNA ensures that its detection captures tumor burden in real-time and offers a non-invasive method of repeatedly evaluating the genomic profile of a patient’s tumor. A challenge in ctDNA detection includes clonal hematopoiesis of indeterminate potential (CHIP), which can be distinguished from tumor variants using a paired whole-blood control. Most assays for ctDNA quantification rely on measurements of somatic variant allele frequency (VAF), which is a mutation-dependent method. Patients with certain types of solid tumors, including colorectal cancer (CRC), can have levels of cfDNA 50 times higher than healthy patients. ctDNA undergoes a precipitous drop shortly after tumor resection and therapy, and rising levels can foreshadow radiologic recurrence on the order of months. The amount of tumor bulk required for ctDNA detection is lower than that for computed tomography (CT) scan detection, with ctDNA detection preceding radiologic recurrence in many cases. cfDNA/ctDNA can be used for tumor molecular profiling to identify resistance mutations when tumor biopsy is not available, to detect minimal residual disease (MRD), to monitor therapy response, and for the detection of tumor relapse. Although ctDNA is not yet implemented in clinical practice, studies are ongoing to define the appropriate way to use it as a tool in the clinic. In this review article, we examine the general aspects of ctDNA, its status as a biomarker, and its role in the management of early (II–III) and late (IV; mCRC) stage colorectal cancer (CRC).

Cell-Free DNA Promotes Inflammation in Patients With Oral Lichen Planus via the STING Pathway.

BackgroundDamaged and dead cells release cell-free DNA (cfDNA) that activates cyclic GMP–AMP (cGAMP) synthase (cGAS), which leads to the activation of stimulator of interferon genes (STING) via the second messenger cGAMP. STING promotes the production of inflammatory cytokines and type I interferons to induce an inflammatory response. Oral lichen planus (OLP), a chronic autoimmune disease involving oral mucosa characterized by the apoptosis of keratinocytes mediated by T-lymphocytes, is related to the activation of multiple inflammatory signaling pathways. Currently, the relationship between cfDNA and OLP has not been confirmed. We hypothesized that cfDNA may be a potential therapeutic target for OLP.MethodscfDNA was extracted from the saliva and plasma of OLP patients; its concentration was measured using the Quanti-iT-PicoGree kit and its relationship with OLP inflammation was assessed. cfDNA of OLP patients (cfDNA-OLP) was transfected into THP-1 macrophages and the expression of inflammatory factors was investigated by performing quantitative real time PCR (qRT-PCR), western blotting, and enzyme-linked immunosorbent assay (ELISA). STING expression was analyzed in the tissues of OLP patients and healthy controls using immunohistochemical staining and western blotting. siRNA was used to knockdown STING expression in THP-1 macrophages, and the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secreted by cells following cfDNA-OLP transfection were detected using ELISA. Finally, the effect of the cationic polymer PAMAM-G3 was evaluated on the treatment of inflammation induced by cfDNA-OLP.ResultsThe concentration of cfDNA in the saliva and plasma of OLP patients was considerably higher than that of healthy controls, and it positively correlated with the levels of inflammatory cytokines and clinical characteristics. cfDNA-OLP induced an inflammatory response in THP-1 macrophages. STING expression was significantly higher in OLP tissues than in the gingival tissues of healthy controls. STING knockdown suppressed cfDNA-OLP-induced inflammation in THP-1 macrophages. PAMAM-G3 inhibited the inflammatory response caused by cfDNA-OLP.ConclusionThe cfDNA level is increased in OLP patients, and the STING pathway activated by cfDNA-OLP might play a critical role in OLP pathogenesis. Treatment with PAMAM-G3 reduced the inflammation induced by cfDNA-OLP, and therefore, may be a potential treatment strategy for OLP.

Osteosarcoma: Novel prognostic biomarkers using circulating and cell-free tumour DNA

AimOsteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Circulating free (cfDNA) and circulating tumour DNA (ctDNA) are promising biomarkers for disease surveillance and prognostication in several cancer types; however, few such studies are reported for OS. The purpose of this study was to discover and validate methylation-based biomarkers to detect plasma ctDNA in patients with OS and explore their utility as prognostic markers. MethodsCandidate CpG markers were selected through analysis of methylation array data for OS, non-OS tumours and germline samples. Candidates were validated in two independent OS datasets (n = 162, n = 107) and the four top-performing markers were selected. Methylation-specific digital droplet PCR (ddPCR) assays were designed and experimentally validated in OS tumour samples (n = 20) and control plasma samples. Finally, ddPCR assays were applied to pre-operative plasma and where available post-operative plasma from 72 patients with OS, and findings correlated with outcome. ResultsCustom ddPCR assays detected ctDNA in 69% and 40% of pre-operative plasma samples (n = 72), based on thresholds of one or two positive markers respectively. ctDNA was detected in 5/17 (29%) post-operative plasma samples from patients, which in four cases were associated with or preceded disease relapse. Both pre-operative cfDNA levels and ctDNA detection independently correlated with overall survival (p = 0.0015 and p = 0.0096, respectively). ConclusionOur findings illustrate the potential of mutation-independent methylation-based ctDNA assays for OS. This study lays the foundation for multi-institutional collaborative studies to explore the utility of plasma-derived biomarkers in the management of OS.

Perioperative changes in cell-free DNA for patients undergoing surgery for colon cancer

BackgroundVarious conditions with cellular decay are associated with elevated cell-free DNA (cfDNA). This study aimed to investigate if perioperatively measured cfDNA levels were associated with the surgical approach, complications, or recurrence.MethodsPlasma was obtained from patients who underwent surgery for colon cancer at admission and at the time of discharge. Quantitative measurement of cfDNA was performed by amplifying two amplicons of 102 base pairs (bp) and 132 bp of Beta-2-Microglobulin (B2M) and Peptidyl-Prolyl cis–trans Isomerase A (PPIA), respectively.ResultscfDNA was measured in 48 patients who underwent surgery for colonic cancer. Sixteen patients had recurrence during the follow-up period, fifteen developed a postoperative complication, and seventeen patients developed neither, acting as the control group. Postoperative cfDNA levels were significantly elevated from baseline samples, across all groups, with a median preoperatively B2M level of 48.3 alleles per mL and postoperatively of 220 alleles per mL and a median preoperatively level PPIA of 26.9 alleles per mL and postoperatively of 111.6 alleles per mL (p < 0.001 for B2M and p < 0.001 for PPIA). Postoperative levels of PPIA, but not B2M, were significantly higher in patients experiencing complications than in the control group (p = 0.036). However, a tendency towards an association between the surgical approach and the changes in cfDNA levels was found for PPIA (p = 0.058), and B2M (p = 0.087).ConclusionsPlasma cfDNA was increased after surgery in all patients with colon cancer. Postoperative PPIA levels were significantly higher in patients experiencing surgical complications but not in B2M levels.

CfDNA Levels in Ex Vivo Lung Perfusate Are Associated with the Prognosis of Donor Lungs

<h3>Purpose</h3> The detection of cell-free DNA (cfDNA) in EVLP perfusate has been shown to be a good indicator of apoptosis and potentially reflects lung injury. Biomarker scoring algorithms have been developed that predict donor lung injury and outcomes following ex vivo lung perfusion (EVLP); however, the mechanisms behind what is driving the poor algorithm scores remains unknown. In this study, we investigated the relationship between cfDNA in EVLP perfusate and donor lung suitability as predicted by biomarker algorithms. <h3>Methods</h3> A total of n=42 clinical EVLP cases were used in this single-center, retrospective cohort study; n=23 were EVLP cases that were transplanted and n=19 were EVLP cases deemed unsuitable for transplantation. Perfusate samples were collected after 1h and 4hr of perfusion. Perfusate supernatants following centrifugation were collected, snap frozen, and stored at -80C prior to cfDNA testing. cfDNA was extracted and quantified using Agilent Bioanalyzer. Results from a biomarker scoring algorithm were obtained and used for comparison with cfDNA data. <h3>Results</h3> The probability that a donor lung will be declined after EVLP correlated with cfDNA levels measured at 4h as well as the change in cfDNA levels between 1 and 4h (Figure 1A; 4h p<0.0001, r=0.59; delta p<0.001, p=0.56). Conversely, a negative correlation was observed between the amount of cfDNA and the probability that a recipient would be extubated in less than 72h post-transplant (Figure 1B; 4h p<0.001, r=-0.56; delta p<0.001, r=-0.54). A cfDNA cut-off of 463ng/mL after 4h of perfusion or an increase of 345ng/mL of cfDNA between 1 and 4h of perfusion were significant prognostic indicators of donor lung decline or extubation >72h post-transplant (Figure 1C and 1D). <h3>Conclusion</h3> The amount of cfDNA in EVLP perfusate correlates with the prognosis of lungs on EVLP using biomarker algorithms. This study indicates that apoptotic cell death may be an explanatory feature for algorithms that predict lung injury and outcomes following EVLP and transplantation.

Cell-Free DNA in the Early Course After Heart Transplantation

<h3>Purpose</h3> Cell-free DNA (cfDNA) has been used as a marker of rejection after heart transplantation (HTx). The commonly reported outcome is donor fraction (DF). However, cfDNA from donor (dd-cfDNA) and recipient (rd-cfDNA) can be quantified separately. We aimed to investigate the influence of donor and recipient-derived factors as well as intraoperative measures on levels of cfDNA. <h3>Methods</h3> Blood samples were collected in parallel to the first endomyocardial biopsy in 45 patients (8 children, 37 adults) after HTx. Levels of rd- and dd-cfDNA and DF were determined using digital PCR. Regression analyses were performed with respect to the influence of donor- (age, body surface area (BSA), gender) and recipient-derived factors (age, BSA, gender, kidney function pre-HTx, ventricular assist device (VAD) pre-HTx, previous heart operation) as well as perioperative measures (operation time, ischemic graft time, early rejection, presence of acute kidney injury). <h3>Results</h3> The first biopsy was taken on a mean of 11 days after HTx (median 10, range 5-14, IQR 9-12). Mean ischemic time was 180 min (median 180, range 62-394, IQR 118-215), mean operation time was 376 min (median 333, range 174-745, IQR 239-510). Mean rd-cf-DNA levels were 190,000 copies/μl (median 140,000, range 33,000-1,400,000, IQR 83,000-200,000), mean dd-cfDNA-levels 700 copies/μl (median 400, range 150-7000, IQR 270-620). Mean DF was 0.48 (median 0.33, range 0.09-5.3, IQR 0.2-0.5). In a multiple regression model, ischemic graft time was the only factor with a statistically significant impact on levels of dd-cfDNA, but not on DF. The biggest influence on rd-cfDNA-levels was the use of a VAD pre-HTx, but this was not statistically significant. Strong correlations were seen between age and BSA of donor and recipient. <h3>Conclusion</h3> The separate quantification of both rd- and dd-cfDNA, in contrast to solely reporting DF, allows for the distinction of factors mainly influencing the recipient (such as the pre-HTx use of VAD) and the donor (such as ischemic graft time). In this proof-of-concept study, most of the investigated measures did not reach statistical significance. The correlation of ischemic graft time and dd-cfDNA (but not DF), however, shows the possibility of precisely detecting graft injury

Successful Use of Belatacept in Three Lung Transplant Recipients for Unique Indications

<h3>Introduction</h3> Belatacept is a selective costimulatory blocker approved for immunosuppression after kidney transplantation. Data for its use in other solid organ transplants shows mixed results. We present 3 cases of its efficacy in lung transplant as an alternative agent for maintenance therapy in unique contexts. <h3>Case Report</h3> Patient #1 is a 40 year old male who received bilateral lung transplant for ILD from scleroderma complicated by AKI requiring hemodialysis with partial renal recovery at discharge. Over the next 7 months, his renal function declined and with a creatinine of 3.9 mg/dL, belatacept was initiated in combination with a lowered tacrolimus goal of 5 - 6 ng/mL to minimize further nephrotoxicity. 2 months into this course, his creatinine improved to 3.2 mg/dL without untoward effect on the allograft. Patient #2 is a 47 year old female who underwent a third retransplant for CLAD. Prior to the third transplant, her immunosuppression consisted of sirolimus, tacrolimus, and prednisone. Mycophenolate mofetil was avoided due to prior skin cancer. Given her history of rejection and high PRA, she was started on belatacept to supplement tacrolimus and prednisone post-op. 22 months post-transplant she has low level DSA and normal cfDNA levels with a normally functioning graft. Patient #3 is a 66 year old male with a right lung transplant for IPF. His course was complicated by TTP and HUS secondary to calcineurin inhibitors, which reoccurred on rechallenge with mTOR inhibitors. He was subsequently started on belatacept along with mycophenolate mofetil and prednisone. He has been on this regimen for 2 years without recurrent side effects, cytopenia, or renal dysfunction. <h3>Summary</h3> Belatacept is renal sparing, which is beneficial as standard immunosuppression regimens include calcineurin inhibitors that are nephrotoxic. Its utility to prevent rejection was shown when evaluated by DSA and cfDNA levels. In the rare case of TTP and HUS, these phenomena did not reoccur. It is imperative to recognize the risks with this agent. PTLD has occurred in EBV-seronegative recipients. There has been an association of viral infections, including CMV. ACR has been documented in heart and kidney recipients. In conclusion, belatacept has a role as an alternative immunosuppressive component in the appropriate clinical context.

Absolute Quantification of Donor Derived Cell Free DNA in Heart Transplant Patients

PurposeCirculating donor-derived cell-free DNA (dd-cfDNA) is a validated biomarker of allograft rejection. Current assays express dd-cfDNA as a percentage of total cell-free DNA (donor fraction). However, a number of factors can influence recipient cfDNA levels (e.g. BMI, infection) unrelated to the allograft. Thus, we assessed the performance of absolute quantification to detect acute rejection (AR) in heart transplant recipients.MethodsWe developed a method to quantify absolute levels of dd-cfDNA expressed as copies/mL. We measured both dd-cfDNA fraction (%) and absolute quantity (copies/mL) using a clinically available SNP-based massively multiplexed PCR dd-cfDNA assay (ProsperaTM, Natera, San Carlos, CA). These values were compared with diagnosis of AR by histology (defined as ACR >1R and pAMR >0) and test performance was assessed for sensitivity, specificity, negative predictive value and positive predictive value.ResultsOut of 447 samples collected from 150 heart-transplant only patients, 29 had AR and 418 did not. Both dd-cfDNA fraction and absolute quantity were significantly higher in samples with AR compared to samples with no AR. Table 1 characterizes the performance of donor fraction and quantification to distinguish AR. Median dd-cfDNA fraction for AR was 0.62% (IQR: 0.09-1.43) and significantly higher than no AR at 0.04% (0.01-0.11, p < 0.01). Median absolute dd-cfDNA for AR was 78.8 copies/mL (5.3-125.6) and significantly higher than no AR at 2.7 copies/mL (1.37-6.90, p=1.67e-09).ConclusionAbsolute quantification of dd-cfDNA may increase the accuracy to discriminate rejection when compared with dd-cfDNA expressed as a percentage of total cfDNA. Circulating donor-derived cell-free DNA (dd-cfDNA) is a validated biomarker of allograft rejection. Current assays express dd-cfDNA as a percentage of total cell-free DNA (donor fraction). However, a number of factors can influence recipient cfDNA levels (e.g. BMI, infection) unrelated to the allograft. Thus, we assessed the performance of absolute quantification to detect acute rejection (AR) in heart transplant recipients. We developed a method to quantify absolute levels of dd-cfDNA expressed as copies/mL. We measured both dd-cfDNA fraction (%) and absolute quantity (copies/mL) using a clinically available SNP-based massively multiplexed PCR dd-cfDNA assay (ProsperaTM, Natera, San Carlos, CA). These values were compared with diagnosis of AR by histology (defined as ACR >1R and pAMR >0) and test performance was assessed for sensitivity, specificity, negative predictive value and positive predictive value. Out of 447 samples collected from 150 heart-transplant only patients, 29 had AR and 418 did not. Both dd-cfDNA fraction and absolute quantity were significantly higher in samples with AR compared to samples with no AR. Table 1 characterizes the performance of donor fraction and quantification to distinguish AR. Median dd-cfDNA fraction for AR was 0.62% (IQR: 0.09-1.43) and significantly higher than no AR at 0.04% (0.01-0.11, p < 0.01). Median absolute dd-cfDNA for AR was 78.8 copies/mL (5.3-125.6) and significantly higher than no AR at 2.7 copies/mL (1.37-6.90, p=1.67e-09). Absolute quantification of dd-cfDNA may increase the accuracy to discriminate rejection when compared with dd-cfDNA expressed as a percentage of total cfDNA.

Stimulator of Interferon Genes (STING) Promotes Staphylococcus aureus-Induced Extracellular Traps Formation via the ROS-ERK Signaling Pathway.

Stimulator of interferon genes (STING) is a cytosolic DNA sensor or directly recognizes bacterial cyclic dinucleotides, which is required for the detection of microbial infection. Extracellular traps (ETs) are known to be part of the antimicrobial defense system. However, the implication of STING in ETs formation during microbial infection remains unknown. Here, we showed that STING contributed to Staphylococcus aureus (S. aureus)-induced ETs formation through the ROS-ERK signaling. STING deficiency exhibited decreased cell-free DNA (cfDNA) level, reduced expression of citrullinated histone H3 (CitH3), and diminished DNA colocalization with CitH3 and myeloperoxidase (MPO). Interestingly, NADPH oxidase-derived reactive oxygen species (ROS) promoted ETs formation, accompanied by increased activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in S. aureus-stimulated bone marrow-derived macrophages (BMDMs). Corresponding to less ROS production, decreased ERK1/2 activation was shown in STING-/- BMDMs after S. aureus infection. Importantly, inhibiting the ROS-ERK signal reduced the ETs formation and the differences disappeared between WT and STING-/- BMDMs after S. aureus infection. Moreover, STING-/- BMDMs exhibited significantly increased levels of extracellular bacteria compared to WT BMDMs regardless of phagocytosis. In addition, such differences disappeared after DNase I treatment. DNase I treatment also facilitated pathogen colonization without affecting the inflammatory cells infiltration and pro-inflammatory factors secretion following pulmonary S. aureus infection. Furthermore, STING-/- mice presented decreased levels of cfDNA and CitH3, along with increased bacterial colonization compared to WT mice. Altogether, these findings highlighted that STING promoted ETs formation via the ROS-ERK signal for host defense against S. aureus infection.

DETECT: Development of Technologies for Early HCC Detection

DETECT: Development of Technologies for Early HCC Detection

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.

Changes in Specific Biomarkers Indicate Cardiac Adaptive and Anti-inflammatory Response of Repeated Recreational SCUBA Diving.

ObjectiveRecreational SCUBA (rSCUBA) diving has become a highly popular and widespread sport. Yet, information on molecular events underlying (patho)physiological events that follow exposure to the specific environmental conditions (hyperbaric conditions, coldness, immersion, and elevated breathing pressure), in which rSCUBA diving is performed, remain largely unknown. Our previous study suggested that repeated rSCUBA diving triggers an adaptive response of cardiovascular and immune system. To elucidate further molecular events underlying cardiac and immune system adaptation and to exclude possible adverse effects we measured blood levels of specific cardiac and inflammation markers.MethodsThis longitudinal intervention study included fourteen recreational divers who performed five dives, one per week, on the depth 20–30 m that lasted 30 min, after the non-dive period of 5 months. Blood samples were taken immediately before and after the first, third, and fifth dives. Copeptin, immunoglobulins A, G and M, complement components C3 and C4, and differential blood count parameters, including neutrophil-to-lymphocyte ratio (NLR) were determined using standard laboratory methods. Cell-free DNA was measured by qPCR analysis and N-glycans released from IgG and total plasma proteins (TPP), were analyzed by hydrophilic interaction ultra-performance liquid chromatography.ResultsCopeptin level increased after the first dive but decreased after the third and fifth dive. Increases in immunoglobulins level after every dive and during whole studied period were observed, but no changes in C3, C4, and cfDNA level were detected. NLR increased only after the first dive. IgG and TPP N-glycosylation alterations toward anti-inflammatory status over whole studied period were manifested as an increase in monogalyctosylated and core-fucosylated IgG N-glycans and decrease in agalactosylated TPP N-glycans.ConclusionrSCUBA diving practiced on a regular basis promotes anti-inflammatory status thus contributing cardioprotection and conferring multiple health benefits.