Exploring microRNAs in Bile Duct Stents as Diagnostic Biomarkers for Biliary Pathologies

Abstract

Background/objectives: Obstruction of the biliary duct may be caused by various conditions ranging from chronic inflammation to neoplasia, including cholangiocarcinoma (CCA). While the definite histological diagnosis of intrahepatic lesions is relatively straightforward, the diagnostic workup of biliary duct stenosis can be challenging, despite the availability of novel tools for intraductal diagnosis. This proof-of-principle study aimed to investigate whether microRNAs (miRNAs) from bile duct stents may be used as biomarkers to differentiate between various bile duct diseases.

Methods: For this purpose, we included 100 patients with one or more bile duct stents for various reasons, including malignant disease (n = 40), stenosis due to liver transplantation or surgery (n = 60), and cholangitis (n = 42). During endoscopic retrograde cholangiography, the stents were collected, and miRNA analyses were performed to evaluate miR-16, miR-21, and miR-223.

Results: All studied miRNAs were successfully detected from the specimens obtained from the bile duct stents and were comparable in different stents from the same subjects. Following normalization, significant increases in miR-16, -21, and -223 levels were identified in patients with cholangitis compared to specimens from a non-inflammatory cohort. However, when comparing the data from patients in the malignant and non-malignant cohorts, the individual levels of miR-16, miR-21, and miR-223 showed high variation, without reaching a statistically significant difference.

Conclusions: In summary, bile duct stents can be considered as potential sources of intraductal biomarkers, specifically miRNAs. Further profiling and validation analyses are necessary to identify the most appropriate miRNA targets for differentiating bile duct diseases.

Keywords: bile ducts; cholangiocarcinoma; cholangitis; cholestasis; miRNA; stents.

Figure 1

Preanalytical analysis of miRNA in bile duct stents. (A) Overall comparison of the expression of the three studied miRNAs from all samples following normalization. Friedman’s test was used for analysis. (B) Comparison of the expressions of miRNAs based on the stent type. No significant differences could be shown. Kruskal–Wallis test was used to analyze this. (C) Illustration of the correlations between the miRNA expression levels and the indwelling time. No significant correlations could be shown. Analyses were performed using Spearman’s test. (D) Correlation of CT values for miR-16 and miR-21, miR-223 from the total cohort. The correlation analyses between miR-16 and miR-21 (r = 0.8042; 95%-CI: 0.7190 to 0.8656, p ≤ 0.0001), and miR-223 (r = 0.7672, 95%-CI: 0.6689 to 0.8392, p ≤ 0.0001) were performed using Spearman’s test. The miRNA were normalized to spiked-in cel-miR-39 using 2^−∆Ct-method. The values are shown using mean ± SD. **** p < 0.001.

Figure 2

Expression of miR-16 (A,B), miR-21 (C,D), and miR-223 (E,F) in comparison between the malignant cohort, the CCA subcohort and the non-malignant cohort. The miRNAs were normalized for better comparability with cel-miR-39 and 2^∆Ct-method. The values are shown as mean ± SD. Mann–Whitney test was used to analyze this.

Figure 3

Expression of miR-16, -21, and -223 (A) in comparison between the cholangitis cohort and the non-inflammatory cohort. Mann–Whitney test was used for analysis. Expression of miR-16, miR-21, and miR-223 and the correlation with laboratory values (B). The miRNAs were normalized for better comparability with cel-miR-39 and 2^−∆Ct-method. The values are shown using mean ± SD. Correlation analysis were performed using Spearman’s test. *** p < 0.001; ** p < 0.01; and * p < 0.05.