key: cord-0828771-esf3ty6o authors: Hoeter, K.; Neuberger, E.; Fischer, S.; Herbst, M.; Juskeviciute, E.; Rossmann, H.; Sprinzl, M. F.; Simon, P.; Bodenstein, M.; Schäfer, M. K. E. title: Evidence for the utility of cfDNA plasma concentrations to predict disease severity in COVID-19 date: 2021-05-02 journal: nan DOI: 10.1101/2021.04.29.21256291 sha: 4230d2e54747edbab4961aac63899e36d22a975f doc_id: 828771 cord_uid: esf3ty6o COVID-19 is a pandemic caused by the highly infective SARS-CoV-2. There is a need for biomarkers not only for overall prognosis but also for predicting the response to treatments and thus for improvements in the clinical management of patients with COVID-19. Circulating cell-free DNA (cfDNA) has emerged as a promising biomarker in the assessment of various disease conditions. The aim of this retrospective and observational pilot study was to examine the potential value of cfDNA plasma concentrations as a correlative biomarker in hospitalized COVID-19 patients. Lithium-Heparin plasma samples were obtained from twenty-one COVID-19 patients during hospitalization in the University Medical Center of Mainz, Germany, and the cfDNA concentrations were determined by quantitative PCR yielding amplicons of long interspersed nuclear elements (LINE-1). cfDNA plasma concentrations of COVID-19 patients ranged between 247.5 and 6346.25 ng/ml and the mean concentrations were 1831 {+/-} 1388 ng/ml ({+/-} standard deviation). Correlations were found between cfDNA levels and the occurrence of acute respiratory distress symptom (ARDS), acute kidney injury (AKI), myositis, neurological complications, bacterial superinfection and disease severity as defined by sepsis-related organ failure assessment score (SOFA) score. D-Dimer and C-reactive-protein (CRP), determined by clinical laboratory analysis, showed the highest correlations with cfDNA levels. The results of this observational study suggest that cfDNA plasma concentrations may serve as a predictive biomarker of disease severity in COVID-19. Prospective studies enrolling larger patient cohorts are ongoing to test this hypothesis. Coronavirus disease 2019 (COVID-19) is a pandemic caused by the highly infective SARS-CoV-2. To date, SARS-CoV-2 has caused more than 130 million cases worldwide. Although COVID-19 vaccines are being developed rapidly, compared to traditional vaccines, and have been approved worldwide (1) , the ongoing outbreak of COVID-19 puts an enormous strain on health resources and represents an extraordinary threat to global public health (2) . In addition, new SARS-CoV-2 variants with increased transmission rate have emerged in the past months which further complicated the situation (3) . Disease caused by SARS-CoV-2 infection ranges from mild course of illness to severe respiratory diseases, multiple organ failure and death. Amongst others, pulmonary manifestations are common and range from cough to pneumonia and acute lung failure. Hematological and immune systemrelated changes such as thrombocytopenia and dysregulation of blood clotting have been reported (4) . In addition, neurological manifestations (5), acute kidney failure (6) and symptoms of the gastrointestinal tract such as nausea and vomiting, diarrhea, and gastrointestinal bleeding occur in the context of COVID-19 disease (7) . Encouraging effects on the course of the disease have been reported after corticosteroid treatment (8) , however, to date, there are no generally proven effective therapies for COVID-19 or antivirals against SARS-CoV-2 (2) . Thus, the only life-saving therapy is bridging-torecovery which means in the case of organ failure by organ support or replacement. The increasing incidence of SARS-CoV-2-infections with possible serious consequences in almost every age group, makes optimal biomarkers necessary. Biomarkers are not only necessary for prognosis, but also for predicting the response to treatments and thus for improvements in the clinical management of patients with COVID-19. Recent studies associated laboratory measures of hyperinflammation such as macrophage chemoattractant protein 1 (MCP-1), C-reactive-protein (CRP), and interleukin-6 (IL-6) as strong predictors of disease severity in hospitalized patients with COVID-19 (9, 10) . Another promising, non-invasive biomarker from liquid biopsy is cell-free DNA (cfDNA), which is passively released after cell damage and/or actively released from hematopoietic (immune) cells (11, 12) . Elevated concentrations of cfDNA have been detected under various pathological conditions (13) . In tumour diseases, cfDNA levels have been utilized to evaluate tumour burden, progression, and treatment responses (14) (15) (16) (17) . Elevated levels of cfDNA were also found in patients with severe bacterial infections or viral infections and correlated with the course and severity of diseases, respectively (18, 19) . Most notably, recent studies (20, 21) report on the value of cfDNA as a predictive biomarker for COVID-19 severity. Here, we present data from a retrospective observational study to further assess the value of cfDNA as a potential biomarker in hospitalized COVID-19 patients. Twenty-one patients hospitalized between March and June 2020 at the University Medical Center, Mainz, Germany, were assessed in this explorative study. Patients` clinical data and laboratory findings were reviewed retrospectively through the electronic hospital information systems (i.s.h.med ® , SAP, Weinheim Germany, Nexus Swisslab, Berlin, Germany). The study was approved by German law Chemistry and Laboratory Medicine of the University Medical Center, Mainz, as described (22) . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. family 2 (L1PA2). The assay shows a limit of quantification < 1ng/ml, a sufficient dynamic range, as well as repeatability < 12%. To determine storage dependent changes in cfDNA levels in lithium heparin syringes, EDTA or Lithium-Heparin syringes containing plasma from three different subjects were collected and processed directly, or stored for five days at 4°C, respectively. As expected, in EDTA samples the cfDNA concentration increased significantly ~60 fold during the extended storage time (P=0.004). In Lithium-Heparin samples no significant differences were determined between direct processing and extended storage (P=0.5). The samples showed a concentration of 71.8 ± 33.1 and 80.2 ± 32.1 ng/ml, respectively (mean ± SD) demonstrating that Lithium-Heparin rather than EDTA plasma samples are suitable for the analysis of cfDNA. Data analysation is descriptive. Mean and standard deviation or absolute and relative frequency were calculated. Student`s T-test was used to describe association between clinical complication of patients and the concentration of cfDNA in plasma samples. Spearman's correlation was used to describe association between cfDNA concentration (grouped as low, moderate, or high) and laboratory parameters. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2021. ; Twenty-one hospitalized patients with positive proof of SARS-CoV-2 were assessed in this study, twelve of whom were male and nine were female. The patients were 68 ± 17 years and on average overweight (BMI 28.8 ± 6.6 kg/m 2 , table 1). Most of the patients had at least one pre-existing risk-factor for a severe course of COVID-19 comprising cardiac (19%), renal (33%), pulmonary (38%), or immunological conditions (14%), arterial hypertension (57%), diabetes (19%) or adipositas (33%, table 2). Several laboratory parameters of the patients were elevated during their course of COVID-19 (table 3) . Increased levels of creatinine, urea or LDH and decreased estimated glomerular filtration rate (eGFR) were indicative for organ dysfunction, i.e. acute kidney injury (AKI). Inflammatory parameters CRP and procalcitonin (PCT) were markedly present in the majority of patients, reflecting hyperinflammation and/or secondary bacterial infection. Increased D-Dimer concentration was considered as an indicator for COVID-19 associated coagulopathy (table 3) . The patients suffered from several complications, most frequently AKI (62%) followed by pulmonary complications including invasive ventilation and ARDS (43%), anemia and secondary infections (table 4) . COVID-19 was potentially fatal, 2 of 21 patients died during the hospital stay (table 5) . Association between plasma cfDNA concentration and complications, risk factors or outcome revealed that the occurrence of myositis was associated with higher cfDNA concentration (table 6) . Interestingly, thromboembolism was found in patients with relatively low cfDNA concentrations and higher cfDNA concentrations were associated with a lack of thromboembolism (table 6). The average cfDNA plasma concentration was 1831 ± 1388 ng/ml (table 7) . The lowest cfDNA concentration was 248 ng/ml, the highest cfDNA concentration was 6346 ng/ml. The patients were divided into three groups according to their plasma cfDNA concentrations: High concentration > 2000 ng/ml, moderate concentration 1000-2000 ng/ml and low concentration ≤ 1000 ng/ml. The group sizes (each consisting of n=6-8) were almost evenly distributed (table 7) . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Associations between these three groups, complications and outcome included predominantly renal, pulmonary, and neurological complications, among others, and respiratory complications were more frequently in the cohort with moderate and high cfDNA concentrations. Mortality only occurred in the group with highest cfDNA concentration (table 8) . Extreme laboratory parameters occurred in temporal association with the cfDNA concentration measurement depending on the group (table 9) . D-Dimer and CRP concentration were associated to cfDNA concentration (both p-value of Spearman's correlation were 0.08, table 9). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2021. (23) . As the relative range of cfDNA concentrations determined in COVID-19 patients in our study was broader than the range of LDH, cfDNA may be utilized to discriminate different grades of COVID-19 severity more accurately than LDH. Testing this hypothesis will be the subject of future studies. It should be noted that cfDNA is not only a biomarker for upstream pathophysiological mechanisms but has been also proposed to trigger specific downstream effects. For instance, cfDNA was shown to be an immune system regulator (24) with distinct immunoregulatory properties in healthy and diseased individuals (25). It has also become clear that cfDNA is among the factors contributing to neutrophil extracellular trap (NET) formation. NET plays a key role in immunothrombosis and was demonstrated to be consistently increased in COVID-19 and linked to disease severity (26, 27) . Indeed, complications such as acute arterial thromboembolism have been reported in COVID-19 (28) and inflammatory cells are prominent in arterial thromboembolic material All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2021. ; https://doi.org/10.1101/2021.04.29.21256291 doi: medRxiv preprint from COVID-19 patients (29) . However, recent data did not provide evidence for classic thrombotic microangiopathy in COVID-19 (22) which is in agreement with our observation that higher cfDNA concentrations were not associated with thromboembolism. One hypothesis is that laboratory hallmarks of thrombotic microangiopathy are lacking in COVID-19 due to its restriction to the pulmonary microcirculation (22) which distinguishes COVID-19 pulmonary pathology from that of equally severe influenza virus infection (30) . It has been hypothesized that neutrophils can amplify pathological damage and aggravate a hyperinflammatory state (31) . Moreover, SARS-CoV-2 was found to induce the release of neutrophil extracellular traps (NETs) by neutrophils (32) . It remains to be clarified whether specific pathways downstream of cfDNA are crucial for hyperinflammation and COVID-19-associated coagulopathy following SARS-CoV-2 infection. However, current knowledge is compatible with the hypothesis that cfDNA triggers both hyperinflammation and coagulation (Fig. 1 ). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 28.8 ± 6.6 Data are given as mean ± standard deviation. BMI body mass index All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2021. ; https://doi.org/10.1101/2021.04.29.21256291 doi: medRxiv preprint SD standard deviation, eGFR estimated glomerular filtration rate, CRP C-reaktive protein, PCT procalcitonin, LDH lactate dehydrogenase All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2021. ; https://doi.org/10.1101/2021.04.29.21256291 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2021. ; (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Data are given in n(%). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Spearman's rank correlation coefficient was calculated analyzing the laboratory parameters in three different cohorts of cfDNA concentration (low, moderate, high). The laboratory parameters evaluated here are maximum (max) or minimum (min) values during the next 2 or 7 days after cfDNA concentration was measured. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2021. ; https://doi.org/10.1101/2021.04.29.21256291 doi: medRxiv preprint Abbreviations: NETs, neutrophil extracellular traps; NF-ĸB (Nuclear Factor Kappa-light-chain-enhancer of activated B-cells); IL-6 (Interleukin 6), IL-1 (Interleukin 1), TNF-α (Tumor Necrosis Factor Alpha) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2021. ; https://doi.org/10.1101/2021.04.29.21256291 doi: medRxiv preprint COVID-19 vaccines: rapid development, implications, challenges and future prospects Characteristics of SARS-CoV-2 and COVID-19 COVID-19 evolution during the pandemic -Implications of new SARS-CoV-2 variants on disease control and public health policies COVID-19 and coagulation: bleeding and thrombotic manifestations of SARS-CoV-2 infection Clinical manifestations and evidence of neurological involvement in 2019 novel coronavirus SARS-CoV-2: a systematic review and metaanalysis Incidence and risk factors of kidney impairment on patients with COVID-19: A meta-analysis of 10180 patients Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples From a Hong Kong Cohort: Systematic Review and Meta-analysis A Review of the Evidence for Corticosteroids in COVID-19 Increased interleukin-6 and macrophage chemoattractant protein-1 are associated with respiratory failure in COVID-19 IL-6 and other biomarkers as predictors of severity in COVID-19 Fatal outcome in bacteremia is characterized by high plasma cell free DNA concentration and apoptotic DNA fragmentation: a prospective cohort study Prognostic utility and characterization of cell-free DNA in patients with severe sepsis Circulating (cell-free) nucleic acids--a promising, non-invasive tool for early detection of several human diseases Circulating nucleic acids (CNAs) and cancer--a survey Exploratory Analysis of TP53 Mutations in Circulating Tumour DNA as Biomarkers of Treatment Response for Patients with Relapsed High-Grade Serous Ovarian Carcinoma: A Retrospective Study Establishing PNB-qPCR for quantifying minimal ctDNA concentrations during tumour resection Detection of circulating tumor DNA in early-and late-stage human malignancies Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection Increased plasma cell-free DNA level during HTNV infection: correlation with disease severity and virus load Cell-free DNA maps COVID-19 tissue injury and risk of death, and can cause tissue injury. JCI insight Circulating mitochondrial DNA is an early indicator of severe illness and mortality from COVID-19 No Evidence for Classic Thrombotic Microangiopathy in COVID-19 Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study ):17478. 25. Brynychova I, Zinkova A, Hoffmanova I, Korabecna M, Dankova P. Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients -A pilot study Circulating Markers of Neutrophil Extracellular Traps Are of Prognostic Value in Patients With COVID-19. Arteriosclerosis, thrombosis, and vascular biology Vascular occlusion by neutrophil extracellular traps in COVID-19 Early experience with arterial thromboembolic complications in patients with COVID-19 Histopathologic Evaluation of COVID-19 Patients with Peripheral Arterial Thromboembolism: Does clot composition make any sense? Annals of vascular surgery Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19 COVID-19 and Neutrophils: The Relationship between Hyperinflammation and Neutrophil Extracellular Traps SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology