The expression and clinical significance of neutrophil extracellular trapping nets in neonatal sepsis / 中国新生儿科杂志

Objective:To study the expression and significance of neutrophil extracellular traps (NETs) in neonatal sepsis.Methods:Prospective research were used in this study. Term infants with neonatal sepsis hospitalized for the first time in the Department of Neonatology, Children's Hospital of Soochow University from June 2020 to November 2020 were selected as the sepsis group. According to a ratio of about 1∶1, term infants with mild hyperbilirubinemia who were admitted in the same period, with gestational age difference less than 1 week from those in the sepsis group, and whose parents agreed to participate in the study were selected as the control group. On admission, clinical data as well as blood samples of the two groups were collected. Levels of NETs marker citrulline histone H3-DNA (CitH3-DNA) were detected by Enzyme-linked immunosorbent assay, and circulating cell-free DNA (cfDNA) was tested by the fluorescence microplate reader. General data, white blood cell (WBC), neutrophil count (NE), platelet (PLT), C- reactive protein (CRP), blood culture, CitH3-DNA and cfDNA were compared between the two groups. The diagnostic value of CITH3-DNA and cfDNA in neonatal septicemia was analyzed by the receiver operating characteristic (ROC) curve.Results:A total of 74 infants were included in the study, including 39 cases in the sepsis group and 35 cases in the control group. CitH3-DNA and cfDNA in the sepsis group were significantly higher than those in the control group [CitH3-DNA (optical density): 0.85±0.05 vs. 0.48±0.03, cfDNA (mg/L): 0.90±0.05 vs. 0.56±0.03] ( P<0.01). There was no significant correlation between CitH3-DNA and cfDNA. The level of CitH3-DNA had no correlation with gender, gestational age, age, birth weight, WBC, NE, PLT and CRP ( P>0.05). cfDNA was positively correlated with age and NE ( P<0.05), and negatively correlated with PLT ( P<0.05). Combined with CRP, the area under the ROC curve of CitH3-DNA+CRP, cfDNA+CRP, and CitH3-DNA+cfDNA+CRP were 0.947, 0.947 and 0.970 respectively, and the sensitivity to predict neonatal sepsis were 92.3%, 84.6% and 94.9% respectively, the specificity were 94.3%, 97.1% and 100% respectively, all higher than the predictive value of each index alone. Conclusions:The plasma NETs levels increase significantly in neonatal sepsis patients, especially CitH3-DNA with a strong specificity, and can be considered as a biomarker for early diagnosis of neonatal sepsis. NETs together with CRP, could drastically improve the predictive value of neonatal sepsis.

Influence of Different Therapies on EGFR Mutants by Circulating Cell-free DNA of Lung Adenocarcinoma and Prognosis / 中国肺癌杂志

BACKGROUND@#Epidermal growth factor receptor (EGFR) gene mutation is closely related to the EGFR-TKI target treatment and prognosis of lung adenocarcinoma patients. The mutation status of EGFR is limited by tissue detection. The purpose of this study was to investigate the difference of EGFR mutants in plasmacirculating cell-free DNA (cfDNA) obtained from patients with non-small cell lung cancer (NSCLC) in three groups: pre-therapy, after traditional chemotherapy and targeted therapy. The aim of this study was to analyze whether the plasma cfDNA could effectively determine the EGFR mutations and monitor the drug resistant gene T790M, as well as its prognostic prediction value in patients with targeted therapy.@*METHODS@#ARMS (amplification refractory mutation system)-PCR was used to detect EGFR mutations in 107 (50 of pre-therapy, 29 after traditional chemotherapy and 28 after targeted therapy) cases of paired plasma and tumor tissue specimens, followed by comparing their concordance. The sensitivity, specificity and the prognostic value of plasma cfDNA detection were also observed.@*RESULTS@#The total rate of EGFR mutation was 56% (60/107) in all plasma samples and 77.6% (83/107) in corresponding tumor tissues. Completely the same mutants and wild-type EGFR were found in 68.2% cases of paired specimens. The sensitivity of plasma cfDNA detection was 72.3% and the specificity was up to 100%. Patients were sub-categorized according to therapy. The results showed that the highest consistent rate of cfDNA and tumor tissues was found in the group of pre-therapy (74%, 37/50). Whereas, the lowest consistent rate was observed in the targeted therapy group (57.1%, 16/28). It indicated that the targeted treatment could change the EGFR status in plasma cfDNA. Further analyses on inconsistent cases in this group revealed that 50% of them were compound EGFR mutations with T790M. Thereby, it suggested that targeted therapy might induce the emergence of drug resistance gene T790M. This speculation was confirmed by survival analyses. Based on plasma cfDNA results, patients with T790M mutant had significantly worse progression-free survival (PFS) and overall survival (OS).@*CONCLUSIONS@#For EGFR testing, ARMS-PCR on plasma cfDNA is a promising methodology with the highest specificity and effective sensitivity. It is useful for EGFR testing in patients before treatment, especially the late-stage patients. Simultaneously, plasma cfDNA could be used to monitor the drug resistant mutation, T790M status and predict prognosis after targeted therapy.

Progress of circulating cell-free DNA in B-cell neoplasms / 白血病·淋巴瘤

Circulating cell-free DNA (cfDNA) is a kind of free DNA in circulating blood, including circulating tumor DNA (ctDNA) deriving from tumor cells and DNA deriving from normal tissue cells. CtDNA is a specific tumor DNA in cfDNA. CfDNA could reflect the status of tumor to a certain extent, which plays an important role in tumor diagnosis, disease monitoring, treatment evaluation, prognosis and recurrence judgement, and has a potential clinical value as a simple, effective and noninvasive "liquid biopsy"technology. As a new concept, the number of cfDNA analysis in B-cell tumors is increasing, such as the relationship between tumor-specific gene mutations, methylation changes, minimal residual disease (MRD) and prognosis, recurrence, monitoring of treatment effects, targeted therapy by using cfDNA sequencing technology.

Circulating cell-free DNA as a biomarker in the diagnosis and prognosis of colorectal cancer

Abstract Colorectal cancer (CRC) is a disease without evident clinical symptoms in early stages, leading to late diagnosis and disease management. Current diagnostic and prognostic tools require invasive procedures and circulating molecular biomarkers fail to have optimal sensitivity and specificity. Circulating biomarkers with high clinical performance may be valuable for early diagnosis and prognosis of CRC. The purpose of this review was to investigate the application of circulating cell-free DNA (ccfDNA) in CRC diagnosis and prognosis and the analytical methods used in blood samples in articles published between 2005 and 2016. Based on specific inclusion and exclusion criteria, 26 articles were selected. Most studies used ccfDNA quantification as the molecular biomarker. The analytical method was mainly based on the quantitative polymerase chain reaction (qPCR). Biomarkers based on aberrantly methylated genes (n=6) and ccfDNA integrity/fragmentation (n=2) were also used for the CRC diagnosis. The CRC prognosis used the detection of oncogene mutations, such as KRAS and BRAF, in ccfDNA. Significant differences were found in variables among the studies revealing potential bias. ccfDNA quantification as a diagnostic biomarker for CRC has promising results but it lacks clinical specificity since other diseases present a similar increase in ccfDNA content. However, increasing research in the epigenomic field can lead the way to a clinically specific biomarker for the CRC early diagnosis. As for the analytical method, qPCR and derivatives seem to be a perfectly valid technique. The use of ccfDNA quantification in CRC prognosis seems promising. The attempt to use the ccfDNA quantification in clinical practice may reside in the prognosis using a qPCR technique.

Plasma cell-free DNA level and integrity as biomarkers to diagnose hepatocellular carcinoma / 解放军医学杂志

Objective To detect the circulating cell-free DNA (cf-DNA) level and integrity in plasma of the patients with hepatocellular carcinoma (HCC), HBV and healthy controls by real time PCR (RT-PCR), evaluate their diagnostic value in hepatocellular carcinoma. Methods Venous blood samples from 52 HCC patients, 58 HBV patients and 60 healthy controls were collected. The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin400 and β-actin167 in plasma cf-DNA and the integrity of cf-DNA (reflected by β-actin400/β-actin167) were determined by RT-PCR. The differences in different groups and between two groups were analyzed with SPSS software. Results The β-actin167 levels in plasma cf-DNA and the integrity of cf-DNA were significantly lower in healthy controls than in HCC patients (Z=–4.328, P=0.000; Z=–3.885, P=0.001) and HBV patients (Z=–2.473, P=0.005; Z=–3.295, P=0.013), and the integrity of cf-DNA was obviously lower in HBV patients than in HCC patients (Z=–4.836, P=0.002). Conclusion The integrity of circulating cf-DNA β-actin167 and β-actin400 detected by RT-PCR may be auxiliary biomarker in diagnosis of hepatocellular carcinoma.

Circulating cell-free nucleic acids in cancer liquid biopsy and its clinical application / 肿瘤

As the development of current advanced biotechnologies, the detection of circulating cell-free nucleic acid (cfNA) as a noninvasive liquid biopsy biomarker has emerged recently. Compared with the traditional tissue biopsy for pathological diagnostics, cfNA as a noninvasive liquid biopsy biomarker has shown many advantages, such as overcoming the heterogenicity of cancer cells and noninvasive sampling procedure. Recently, the detection of cfNA has emerged as a promising noninvasive biomarker in cancer diagnosis, prognosis prediction, and treatment response evaluation. This review article is to summarize the application of cfNA, including circulating cell-free DNA (cfDNA) and circulating cell-free RNA (cfRNA) in cancer liquid biopsy field.

Clinical utility of circulating cell-free DNA in breast cancer / 中国肿瘤临床

Circulating cell-free DNA (cfDNA), which is released by normal cells and cancer cells, is defined as extracellular DNA in the blood. The cfDNA levels in breast cancer patients are higher than those in healthy control donors. cfDNA also carries the features of tumor tissue, such as mutations, methylations, copy number changes, and loss of heterozygosis. cfDNA is a potential bio-marker in the diagnosis, management, and prognosis of breast cancer. In this review, the authors briefly describe the biological features of cfDNA, and discuss its clinical utility as a blood biomarker in quantitative and qualitative research.

Elevated plasma level of circulating cell-free DNA may derive from NETosis and is associated with active lupus nephritis in systemic lupus erythematosus / 中华风湿病学杂志

Objective To explore the correlations between elevated cfDNA with lupus nephritis and indentify the influencing factors of cfDNA in systemic lupus erythematosus (SLE).Methods Fifty four patients with SLE [37 patients with lupus nephritis (LN) and 43 age-and sex-matched healthy controls] were included in the study.In 37 LN patients,26 patients were at active stage,and 11 patients were in remission.cfDNA concentration was measured with Picogreen Kit and low-density granulocytes (LDGs) was tested by flowcytometer.Correlation and regression analysis were performed to discover whether cfDNA is related to LN and identify the influencing factor of cffDNA.Results The cfDNA in SLE group was (237±40) ng/ml,which was significantly higher than that in healthy control group (188±41 ng/ml,P＜0.01).cfDNA in LN group was significantly higher than that in patients without LN (NLN) (247±47 ng/ml vs 214±31 ng/ml,P=0.028).cfDNA in patients with active LN was significantly higher than that in patient with inactive LN (RLN) (254±50 ng/ml vs 216±29 ng/ml,P=0.035).In SLE group,cfDNA was positively correlated with quantitative 24-hour urinary protein (r=0.350,P=0.013) and reversely correlated with albumin (r=-0.500,P＜0.01) and endogenous creatinine clearance rate (Ccr) (r=-0.354,P=0.044).Percentage of LDGs in peripheral blood mononuclear ceils (PBMCs) of the SLE group was (8.3± 12.9)％,significantly was higher than that in healthy controls [(1.2±0.7)％,P=0.004].The cfDNA was positively correlated with LDGs (r=0.636,P=0.002) and neutrophils (r=0.599,P＜0.01).Conclusion NETs excessively released by neutrophils as well as LDGs may be one of the main reasons for elevated cfDNA level in SLE.cfDNA level is associated with LN activity,suggesting that there is a intrinsic link between NETs-related biomarkers and active LN and that more specific biomarkers of NETs may become a clinical biomarker for active LN.

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100 percent efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.