Urinary MicroRNAs as Potential Markers for Non-Invasive Diagnosis of Bladder Cancer

Abstract

Currently, voided urine cytology (VUC) serves as the gold standard for the detection of bladder cancer (BCa) in urine. Despite its high specificity, VUC has shortcomings in terms of sensitivity. Therefore, alternative biomarkers are being searched, which might overcome these disadvantages as a useful adjunct to VUC. The aim of this study was to evaluate the diagnostic potential of the urinary levels of selected microRNAs (miRs), which might represent such alternative biomarkers due to their BCa-specific expression. Expression levels of nine BCa-associated microRNAs (miR-21, -96, -125b, -126, -145, -183, -205, -210, -221) were assessed by quantitative PCR in urine sediments from 104 patients with primary BCa and 46 control subjects. Receiver operating characteristic (ROC) curve analyses revealed a diagnostic potential for miR-96, -125b, -126, -145, -183, and -221 with area under the curve (AUC) values between 0.605 and 0.772. The combination of the four best candidates resulted in sensitivity, specificity, positive and negative predictive values (NPV), and accuracy of 73.1%, 95.7%, 97.4%, 61.1%, and 80.0%, respectively. Combined with VUC, sensitivity and NPV could be increased by nearly 8%, each surpassing the performance of VUC alone. The present findings suggested a diagnostic potential of miR-125b, -145, -183, and -221 in combination with VUC for non-invasive detection of BCa in urine.

Keywords: miRNA; quantitative PCR; tumor marker; urothelial carcinoma; voided urine cytology.

Figure 1

Dependence of the relative miR-125b expression levels (normalized to the geometric mean of the reference RNAs RNU44 and RNU48) in urine sediments on histopathological factors. The relationships between urinary miR-125b expression levels and tumor stage (A), as well as tumor grade according to the WHO classifications from 1973 (B) and from 2004 (C) are shown. Differences were tested by the Mann–Whitney U test. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

The distribution of the relative expression levels of the significantly altered miRNAs in urine sediments from controls and BCa patients is presented by box plots. Differences in the relative expression levels of the miRNAs (normalized to the geometric mean of the reference RNAs RNU44 and RNU48) were assessed using the Mann–Whitney U test followed by Bonferroni’s correction for multiple comparisons.

Figure 3

The diagnostic value assessed by ROC curve analysis of the six most promising miRNAs, which were significantly up- (A) or down-regulated (B) in urine sediments from BCa patients in comparison with controls. AUC, area under the curve; ROC, receiver operating characteristic.

Figure 4

Diagnostic value of the miRNAs assessed by penalized linear regression. The deviance profile plot corresponding to standardized miRNAs as predictor variables in the glmnet model is shown. The slope of each path changes each time as another miRNA enters the model. The miRNAs, which explain a larger fraction of deviance, play a more important role in the prediction of the tumor status.