Cell-free DNA analysis in current cancer clinical trials: a review

Cell-free DNA (cfDNA) analysis represents a promising method for the diagnosis, treatment selection and clinical follow-up of cancer patients. Although its general methodological feasibility and usefulness has been demonstrated, several issues related to standardisation and technical validation must be addressed for its routine clinical application in cancer. In this regard, most cfDNA clinical applications are still limited to clinical trials, proving its value in several settings. In this paper, we review the current clinical trials involving cfDNA/ctDNA analysis and highlight those where it has been useful for patient stratification, treatment follow-up or development of novel approaches for early diagnosis. Our query included clinical trials, including the terms ‘cfDNA’, ‘ctDNA’, ‘liquid biopsy’ AND ‘cancer OR neoplasm’ in the FDA and EMA public databases. We identified 1370 clinical trials (FDA = 1129, EMA = 241) involving liquid-biopsy analysis in cancer. These clinical trials show promising results for the early detection of cancer and confirm cfDNA as a tool for real-time monitoring of acquired therapy resistance, accurate disease-progression surveillance and improvement of treatment, situations that result in a better quality of life and extended overall survival for cancer patients.

DNase 1 Protects From Increased Thrombin Generation and Venous Thrombosis During Aging: Cross‐Sectional Study in Mice and Humans

Background Human aging is associated with increased risk of thrombosis, but the mechanisms are poorly defined. We hypothesized that aging induces peroxide‐dependent release of neutrophil extracellular traps that contribute to thrombin generation and thrombosis. Methods and Results We studied C57BL6J mice and littermates of glutathione peroxidase‐1 transgenic and wild‐type mice at young (4 month) and old (20 month) ages and a healthy cohort of young (18–39 years) or middle‐aged/older (50–72 years) humans. In plasma, we measured thrombin generation potential and components of neutrophil extracellular traps (cell‐free DNA and citrullinated histone). Aged wild‐type mice displayed a significant increase in thrombin generation that was decreased in aged glutathione peroxidase‐1 transgenic mice. Both aged wild‐type and aged glutathione peroxidase‐1 transgenic mice demonstrated similar elevation of plasma cell‐free DNA compared with young mice. In contrast, plasma levels of citrullinated histone were not altered with age or genotype. Release of neutrophil extracellular traps from neutrophils in vitro was also similar between young and aged wild‐type or glutathione peroxidase‐1 transgenic mice. Treatment of plasma or mice with DNase 1 decreased age‐associated increases in thrombin generation, and DNase 1 treatment blocked the development of experimental venous thrombi in aged C57BL6J mice. Similarly, thrombin generation potential and plasma cell‐free DNA, but not citrullinated histone, were higher in middle‐aged/older humans, and treatment of plasma with DNase 1 reversed the increase in thrombin generation. Conclusions We conclude that DNase 1 limits thrombin generation and protects from venous thrombosis during aging, likely by hydrolyzing cell‐free DNA.

Increased Circulating Cell-Free DNA in Eosinophilic Granulomatosis With Polyangiitis: Implications for Eosinophil Extracellular Traps and Immunothrombosis

BackgroundEndogenous DNA derived from nuclei or mitochondria is released into the blood circulation as cell-free DNA (cfDNA) following cell damage or death. cfDNA is associated with various pathological conditions; however, its clinical significance in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) remains unclear. This study aimed to evaluate the clinical significance of cfDNA in AAV.MethodsWe enrolled 35 patients with AAV, including 10 with eosinophilic granulomatosis with polyangiitis (EGPA), 13 with microscopic polyangiitis, and 12 with granulomatosis with polyangiitis. Serum cf-nuclear DNA (cf-nDNA) and cf-mitochondrial DNA (cf-mtDNA) levels were measured by quantitative polymerase chain reaction before and after the initiation of immunosuppressive therapy. Tissue samples from EGPA patients were examined by immunofluorescence and transmission electron microscopy. The structure of eosinophil extracellular traps (EETs) and neutrophil extracellular traps (NETs) and stability against DNase were assessed in vitro. Platelet adhesion of EETs were also assessed.ResultsSerum cf-nDNA and cf-mtDNA levels were significantly higher in AAV than in healthy controls, with the highest levels in EGPA; however, serum DNase activities were comparable among all groups. cf-nDNA and cf-mtDNA decreased after treatment and were associated with disease activity only in EGPA. Blood eosinophil count and plasma D-dimer levels were significantly correlated with cf-nDNA in EGPA and cf-mtDNA. EGPA tissue samples showed lytic eosinophils and EETs in small-vessel thrombi. The structure of EETs showed bolder net-like chromatin threads in vitro and EETs showed greater stability against DNase than NETs. EETs provided a scaffold for platelet adhesion.ConclusioncfDNA was increased in EGPA, associated with disease activity. The presence of DNase-resistant EETs in small-vessel thrombi might contribute to higher concentration of cfDNA and the occurrence of immunothrombosis in EGPA.

Application of Circulating Tumor Cells and Circulating Free DNA from Peripheral Blood in the Prognosis of Advanced Gastric Cancer

Objective. To explore the application value of circulating tumor cells (CTCs) and circulating free DNA (cfDNA) from peripheral blood in the prognosis of advanced gastric cancer (AGC). Here, we measured CTCs and cfDNA quantity for predicting the outcome of patients. Patients and Methods. Forty-five patients with advanced gastric cancer who underwent neoadjuvant chemotherapy and surgical treatment were enrolled in this study. All patients received neoadjuvant chemotherapy with paclitaxel + S-1 + oxaliplatin (PSOX) regimen, and CTCs and cfDNA of the peripheral blood were detected before and after neoadjuvant therapy. Relationships between the number/type of CTC or cfDNA and the efficacy of neoadjuvant chemotherapy were analyzed. Results. Among 45 patients, 43 (95.6%) were positive, and the positive rate of mesenchymal CTC was increased with the increase in the T stage. The proportion of mesenchymal CTC was positively correlated with the N stage ( P < 0.05 ), and the larger N stage will have the higher proportion of mesenchymal CTC. Patients with a small number of mesenchymal CTC before neoadjuvant chemotherapy were more likely to achieve partial response (PR) with neoadjuvant therapy. Patients with positive CA-199 were more likely to achieve PR with neoadjuvant therapy ( P < 0.05 ). Patients in the PR group were more likely to have decreased/unchanged cfDNA concentration after neoadjuvant therapy ( P = 0.119 ). After neoadjuvant therapy (before surgery), the cfDNA concentration was higher and the efficacy of neoadjuvant therapy (SD or PD) was lower ( P = 0.045 ). Conclusions. Peripheral blood CTC, especially interstitial CTC and cfDNA, has a certain value in predicting the efficacy and prognosis of neoadjuvant chemotherapy in advanced gastric cancer.

5-Hydroxymethylcytosine Profiles in Plasma Cell-free DNA Reflect Molecular Characteristics of Diabetic Kidney Disease

Background: Complications of diabetes mellitus (DM) are the leading cause of DM-related disability and mortality. Notably, diabetic kidney disease (DKD), one of the main complications of DM, has become a frequent cause of end-stage renal disease. A clinically convenient, non-invasive approach for monitoring the development of DKD would benefit the overall life quality of patients with DM and contribute to lower medical burdens through promoting preventive interventions.Methods: We utilized 5hmC-Seal to profile genome-wide 5-hydroxymethylcytosines in plasma cell-free DNA (cfDNA). Candidate genes were identified by intersecting the differentially modified 5hmC marker genes (DMGs) and differentially expressed genes (DEGs) from the GEO datasets GSE30528 and GSE30529. Cytoscape software was used to construct the protein-protein interaction (PPI) network and identify the hub genes.Results: The final gene panel of 9 hub genes, including (CTNNB1, PTEN, MYD88, ITGAM, CD28, ITGB2, VCAM1, CXCR4, CD44) were confirmed. Further analysis indicated that this 9-gene signature showed a good capacity to distinguish between DKD and DM. Conclusions: The 5hmC-Seal assay was successfully applied to the cfDNA samples from a cohort of DM patients with or without DKD. Altered 5hmC signatures in plasma cfDNA indicate that 5hmC-Seal has the potential to be a non-invasive epigenetic tool for monitoring the development of DKD and be a part of diabetic care.