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Context: Circulating free DNA (cfDNA) analysis has emerged as novel noninvasive diagnostic biomarker in several solid tumors. Raised levels have been reported in several malignancies and may correlate with clinicopathological and treatment response. The current study was designed to assess the diagnostics of cfDNA in different tumor types of malignancies correlating with tumor (T), nodes (N), and metastases (M) stage. Design: Serum samples were collected from treatment naïve cases with histologically diagnosed tumors including 23 brain tumors, 48 breasts, 50 gallbladder carcinoma (GBC), 13 lungs, 68 oral squamous cell carcinoma (OSCC), and 25 normal controls. CfDNA was quantified with real-time polymerase chain reaction (PCR), Invasive ductal carcinoma (IDC) using beta-globin gene amplification. Cut off values for diagnostics were calculated using receiver operating curve analysis. Results: Contrary to other cfDNA studies where it was postulated that cfDNA would not cross the blood–brain barrier and reach the systemic circulation, we found detectable cfDNA in glioma with median (Q1–Q3) of 349.22 ng/ml (19.87–1276.58). Median cfDNA concentration in breast, gallbladder, lung, oral and normal controls was 328.72 (128.38–624.44), 778.50 (589.88–1864.35), 348.73 (194.67–483.61), 386.27 (47.88–959.67), and 74.12 (49.66–120.00), respectively. Grades I and II glioma had significantly lower levels compared to Grades III and IV (P = 0.0001). Significant difference in median cfDNA values in IDC and GBC was observed with increasing tumor grades, stage, T stage, nodal stage and metastasis and with stage of OSCC cases. Conclusion: CfDNA levels showed good diagnostic discrimination in glioma, GBC, breast, lung carcinoma, and OSCC. Significant increase in titers was evident with increase in cancer stage from I to IV in breast, GBC and OSCC.
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Circulating cell-free DNA (cfDNA) is considered a promising entry point of liquid biopsy for the diagnosis of early-stage liver cancer; however, many studies have confirmed that the diagnostic efficacy of cfDNA alone is not stable, especially that quantitative test alone cannot well reflect the situation of tumor. More and more studies have shown that cfDNA is suitable for combined measurement of multiple indicators or combined measurement with other diagnostic markers for liver cancer. This article reviews the articles on combined liquid biopsy based on cfDNA published up to July 2021, summarizes the existing methods for combined measurement, and briefly describes the birth and research advances in combined diagnostic methods, so as to further clarify the significance and potential of combined liquid biopsy based on cfDNA in the diagnosis of early-stage liver cancer and thus provide ideas for taking better advantages of the diagnostic markers for liver cancer in the future.
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Objective:To verify the performance of the next-generation sequencing (NGS) platform and evaluate the application of NGS, droplet digital PCR (ddPCR) and super amplification refractory mutation system (super-ARMS) in the detection of circulating free DNA (cfDNA) mutations in patients with non-small-cell lung cancer (NSCLC).Methods:A total of 75 patients with NSCLC in the respiratory department of Zhongshan Hospital Affiliated to Fudan University were enrolled. The standards, cfDNA from 25 patients with newly diagnosed and untreated NSCLC, and self-made mixed samples mixed with hemoglobin (1 000 mg /dl), bilirubin (500 mg/l), fat emulsion (2%), enterococcus gDNA and Escherichia coli gDNA were used to verify the blank limit, analytical sensitivity, precision, accuracy and specificity of NGS platform. The cfDNA mutations of 75 NSCLC patients were detected by ddPCR and NGS, and the mutation positive rates of the two platforms were compared. The linear relationship between the two platforms was compared by Pearson correlation test. 12 patients were selected by simple random sampling for the detection of plasma super-ARMS platform. The performance of three platforms in the detection of plasma cfDNA mutation in patients with NSCLC was compared.Results:The blank limit of NGS platform was set to 0.00%, the analytical sensitivity was 0.2%, the intra-assay precision and inter-assay precision were 100%. The test results were not affected by endogenous hemoglobin, bilirubin or fat emulsion in plasma or exogenous DNA interference, and the analysis specificity was good. The mutation positive rates of plasma cfDNA in 75 NSCLC patients detected by ddPCR and NGS were 61.33% and 60.00%, respectively. The complete coincidence rate was 89.33%, which suggests there was a positive correlation between the mutation abundance of NGS and ddPCR ( r=0.984, P=0.001). Among the plasma of 12 NSCLC patients, the results of NGS, ddPCR and super-ARMS were completely consistent in 7 cases, including 2 wild-types and 5 mutants. Conclusion:The NGS platform was verified to be useful for cfDNA mutation detection in patients with NSCLC. The ddPCR, NGS and super-ARMS have their own advantages in detecting cfDNA mutations in patients with NSCLC.
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Imaging and serological approaches play an important role in the diagnosis and treatment of alveolar echinococcosis; however, they also suffer from some problems during their applications in clinical practices, which urges the identification of potential diagnostic markers. Novel serological, genomics and proteomics diagnostic markers alone or in combination may increase the sensitivity and specificity in early diagnosis of alveolar echinococcosis, which play vital roles in monitoring of disease courses and prognostic evaluation. This review mainly presents the advances in the studies on novel diagnostic markers for alveolar echinococcosis.
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Liquid biopsy is a new noninvasive detection method and is also the main molecular detection method to guide the application of precision medicine. Liquid biopsy mainly includes the measurement of circulating free DNA (cf-DNA), circulating tumor cells, and exosomes, and in particular, cf-DNA is becoming a valuable molecular detection tool, especially in the early detection and diagnosis of liver cancer and parasitic infections involving the liver. This article reviews the clinical application of cf-DNA in liver cancer and liver-related parasitic diseases.
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Neutrophil extracellular trap nets (NETs) are complex networks of proteins and DNA. Many studies have shown that NETs are important participants in the pathophysiological processes of various immune inflammatory diseases, thrombosis, cancer and so on. The dynamic changes of NETs at each stage can predict the course of the disease to a certain extent. This article systematically reviews the current detection methods for ef-fectively quantifying NETs in order to provide the latest theoretical reference for the early detection of chronic inflammation and targeted therapy of cancer.
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Circulating free DNA is DNA fragments in the blood,mainly from cell apoptosis,necrosis and release.Its content is significantly different in the bood of healthy human and malignant tuimor patients,which is associated with tumor characteristics.This review will summarize the research progress of CfDNA quantitative analysis for malignant solid tumor and prospect the application value for children's solid tumors.
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Circulating free DNA (cfDNA) are short fragments of nucleic acids present in circulation outside of cells.In patients with cancer,some portion of cfDNA is derived from tumor cells,termed circulating tumor DNA (ctDNA),and contains the same mutations and genetic changes as the cancer.The development of new and more effective methods to detect these changes has led to increased interest in developing ctDNA as a biomarker for cancer.Here we will review current literatures on the use of ctDNA,with an emphasis on pancreatic cancer,for cancer detection,prognosis,monitoring response to therapy and tracking the rise of new mutant subclones.
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PURPOSE: The aim of this study is to determine whether levels of circulating free DNA (cfDNA) increase according to cancer progression, whether they are restored after surgical resection, and to evaluate cfDNA in gastric cancer patients as a useful biomarker. METHODS: A case-control study design was used. Thirty gastric cancer patients and 34 healthy subjects were enrolled from two hospitals in South Korea. The plasma cfDNA of patients with gastric cancer were obtained before surgery and 24 hours after surgery, and then analyzed by a quantitative, real-time polymerase chain reaction. Plasma samples were also obtained from the control group. RESULTS: The mean levels of cfDNA in the healthy control group, patients with early gastric cancer, and with advanced gastric cancer were 79.78 +/- 8.12 ng/mL, 106.88 +/- 12.40 ng/mL, and 120.23 +/- 10.08 ng/mL, respectively (P < 0.01). Sensitivity was 96.67% and specificity was 94.11% when the cutoff value was 90 ng/mL. Variables representing the tumor burden such as tumor size, T stage, TNM stage, and curative resection are also associated with the levels of cfDNA. The levels of cfDNA in the 24-hour-after-surgery group decreased significantly (112.17 +/- 13.42 ng/mL vs. 77.93 +/- 5.94 ng/mL, P < 0.001) compared to the levels of cfDNA in the preoperation group. CONCLUSION: The changes in the levels of cfDNA can act as reliable biomarkers to detect cancer early, to predict tumor burden, estimate curative resection and even prognosis.
Subject(s)
Humans , Biomarkers , Case-Control Studies , DNA , Plasma , Prognosis , Real-Time Polymerase Chain Reaction , Republic of Korea , Sensitivity and Specificity , Stomach Neoplasms , Tumor BurdenABSTRACT
PURPOSE: Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol. MATERIALS AND METHODS: The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell lung cancer (NSCLC) patients was compared with that by QIAamp MinElute Virus Spin kit (Qiagen kit) as control, using the Wilcoxon rank-sum test. TaqMan quantitative polymerase chain reaction (qPCR) assays were used to quantify the plasma cfDNA extracted. Both Mutant-enriched PCR (ME-PCR) coupled sequencing and DxS EGFR mutation test kit were used to evaluate the impact of extraction method on EGFR mutation analysis. RESULTS: MPC method extracted more plasma cfDNA than Qiagen kit method (p=0.011). The proportion of longer fragment (> or =202 bp) in cfDNA extracted by MPC method was significantly higher than by Qiagen kit method (p=0.002). In the sequencing maps of ME-PCR products, a higher mutant peak was observed on plasma cfDNA extracted by MPC method than by Qiagen kit method. In DxS EGFR mutation test kit results, plasma cfDNA extracted by MPC method contained more tumor-origin DNA than by Qiagen kit method. CONCLUSION: An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.
Subject(s)
Humans , Base Sequence , Carcinoma, Non-Small-Cell Lung/genetics , Chloroform , DNA Mutational Analysis/methods , DNA, Neoplasm/blood , Genetic Testing/methods , Lung Neoplasms/genetics , Molecular Sequence Data , Phenol , Polymerase Chain Reaction/methods , ErbB Receptors/geneticsABSTRACT
Liquid biopsy is a new noninvasive detection method and is also the main molecular detection method to guide the application of precision medicine. Liquid biopsy mainly includes the measurement of circulating free DNA (cf-DNA), circulating tumor cells, and exosomes, and in particular, cf-DNA is becoming a valuable molecular detection tool, especially in the early detection and diagnosis of liver cancer and parasitic infections involving the liver. This article reviews the clinical application of cf-DNA in liver cancer and liver-related parasitic diseases.