Investigation of Plasma Cell-Free DNA and MiRNA in Cholangiocarcinoma and Opisthorchiasis Viverrini Patients

Abstract

Objectives: This study aimed to assess the diagnostic potential of cell-free DNA (cfDNA) and cell-free miRNA (cf-miRNA) for distinguishing between Healthy, asymptomatic opisthorchiasis viverrini and cholangiocarcinoma in a preliminary manner.

Methods: In this study, 36 participants were enrolled into three health status groups: a healthy control group (HC), Opisthorchis viverrini-infected group (OV), and a cholangiocarcinoma group (CCA), each comprising 12 participants. Concentration measurements of cfDNA and cf-miRNA from plasma were conducted. Additionally, ultra-low-pass whole-genome sequencing (ULP-WGS) was employed to investigate DNA alterations.

Results: The study revealed a significant elevation in plasma cfDNA concentration in the cholangiocarcinoma (CCA) group compared to healthy controls (HC) and Opisthorchis viverrini-infected (OV) groups (P < 0.001). The cfDNA concentration demonstrated a sensitivity of 75.00% and specificity of 95.83% for differentiating cholangiocarcinoma, with a cut-off of > 30.50 ng/ml plasma. Likewise, the concentration of cf-miRNA in the CCA group significantly differed from that in the HC and OV groups, demonstrating a sensitivity of 83.33% and specificity of 95.83% with a cut-off set at > 70.50 ng/ml plasma. Furthermore, a positive correlation between plasma concentrations of cfDNA and cf-miRNA suggests a potential relationship between these two biomarkers. These findings indicated the diagnostic potential of cfDNA and cf-miRNA in distinguishing cholangiocarcinoma, emphasizing their role as promising biomarkers for further investigation and clinical applications.

Conclusion: Elevated plasma concentrations of cfDNA and cf-miRNA could serve as potential diagnostic tools for distinguishing cholangiocarcinoma from other conditions. cf-miRNA was superior to cfDNA in terms of sensitivity.

Keywords: Cell-free DNA; Diagnosis; Opisthorchis viverrini; cell-free miRNA; cholangiocarcinoma.

Figure 1

Scatter Plots of Plasma cfDNA (A) and cf-miRNA (B) Concentrations for Healthy Controls (HC) Subjects, O. viverrini-infected (OV) Subjects, and Cholangiocarcinoma (CCA) Subjects. A: concentration of plasma cfDNA in ng/ml plasma is on y-axis. B: concentration of plasma cf-miRNA in ng/ml plasma is on y-axis. Group is on x-axis. The green circle, blue triangle, and orange square indicate HC, OV, and CCA

Figure 2

The Correlation of Plasma cfDNA and cf-miRNA. Positive correlation between plasma cfDNA and cf-miRNA concentration shown by Pearson correlation coefficient (R² = 0.9740, P < 0.0001). The concentration of plasma cf-miRNA in ng/ml plasma is on y-axis and the plasma cfDNA is on x-axis. The orange circle indicates the cfDNA and cf-miRNA of each subject

Figure 3

Area under the Curve (AUC) of the Receiver Operating Characteristic (ROC) Curve for Plasma cfDNA (Green Line) and cf-miRNA (Orange Line). The % sensitivity is plotted against 100% - % specificity on the x-axis and y-axis, respectively. The AUC of ROC curves and 95% CI are calculated and indicated in each curve. The highest AUC of ROC curve is observed in plasma cfDNA concentration

Figure 4

The Somatic Copy Number Alteration (SCNA) Analysis of Plasma Cell-Free DNA (cfDNA) of Healthy Control (HC) Subjects, O. viverrini infected (OV) subjects, and cholangiocarcinoma (CCA) subjects. The log2 ratio of copy number (x-axis) indicates the degree of chromosomal gain or loss, with blue representing normal, brown representing gain, red representing amplification, and green representing deletion. The number of chromosomes is on the x-axis (Chromosome number 1-22 and X chromosome)