A non-invasive miRNA based assay to detect bladder cancer in cell-free urine

Abstract

RNA from cell-free urine was analyzed in an attempt to identify a microRNA (miRNA) profile that could be used as a non-invasive diagnostic assay to detect the presence of urothelial carcinoma of the bladder (UCB) and provide a discriminatory signature for different stages of progression. In addition, the presence of specific miRNAs co-isolating with urinary extracellular vesicles/exosomes was investigated. RNA was isolated from cell-free urine of patients diagnosed with UCB (TaG1, T1G3, ≥T2, CIS) and control patients (healthy control and UCB patients with no evidence of disease). MiRNAs were profiled by qRT-PCR array on pooled samples within each group. Validation of the miRNAs was performed on individual samples using qRT-PCR. Extracellular vesicles were isolated via ultracentrifugation. 236 miRNAs were detected in at least one of the pooled samples. Seven of the miRNAs validated on individual samples had significantly higher levels in the cancer group. A panel of miRNAs discriminated between cancer and cancer-free patients with a sensitivity of 88% and specificity of 78%, (AUC=88.8%). We recorded a sensitivity of 80% for TaG1, 95% for T1G3, 90% for ≥T2 with specificity of 77% for healthy controls and 80% for no evidence of disease. Select miRNAs were detected in extracellular vesicles of UCB patients and healthy controls, albeit at different levels. Utilizing this non-invasive assay, we identified miRNA capable of detecting UCB and distinguishing different stages of progression, providing evidence that miRNA profiling in cell-free urine holds promise for the development of valuable clinical diagnostic tools.

Keywords: cell-free system; exosomes; microRNA; urinary bladder neoplasm; urine.

Figure 1

miRNA qRT-PCR array results. The number of microRNAs detected by qRT-PCR array analysis, per sample type, using a cycle threshold (Ct) cutoff of 35.

Figure 2

A: Mean miRNA levels in cancer and cancer-free samples. Error bars represent standard error above and below the mean. B: Mean fold change in cancer versus cancer-free samples. The 7 microRNAs with significantly higher levels in the cancer group (p<0.05) expressed as a mean fold increase relative to cancer-free samples. Error bars represent standard error above and below the mean.

Figure 3

MiRNA-940 levels concordant with disease progression. The mean levels of miRNA-940 correlates with disease progression with significant differences between stages: as determined by one-way ANOVA [F(4,125)=26.936, p<0.001]. NC=no cancer.

Figure 4

MiRNA-26a levels. Expression of miR-26a is lower in non-invasive UCB patients with significant differences between stages: as determined by one-way ANOVA [F(4,125)=4.902, p=0.001]. NC=no cancer.

Figure 5

A: ROC Curve for the classifier using miR-26a, miR-93, miR-191, and miR-940: including CIS Samples. B: ROC curve for the classifier using miR-26a, miR-93, miR-191, and miR-940: excluding CIS samples.

Figure 6

Western Blot Analysis of Alix and TSG101. Exosomal markers Alix and TSG 101 are concentrated in the EV pellets compared to the cell free urine supernatant (CFS) in both healthy control (HC) and cancer samples (≥T2). Increased levels of both markers are present in cancer samples compared to healthy control.

Figure 7

A: Mean fold level differences of miR-940 in urine samples before and after EV isolation. The levels of miR-940 are similar before (cell free supernatant (CFS)) and after (high speed supernatant (HSS) and pellet) EV isolation, expressed as a mean fold increase relative to healthy control. Error bars represent standard error above and below the mean. B: Mean fold level differences of miR-93 in urine samples before and after EV isolation. The relative level of miR-93 is considerably higher in the depleted high speed supernatant after EV isolation for cancer samples compared to healthy controls. This indicates a greater proportion of miR-93 is located outside of EVs in the urine of UCB patients. Error bars represent standard error above and below the mean.