Bladder cancer detection by urinary extracellular vesicle mRNA analysis

Abstract

Objective: Urinary extracellular vesicles (EV) could be promising biomarkers for urological diseases. In this retrospective feasibility study, we conducted biomarker screening for early stage bladder cancer using EV mRNA analysis.

Methods: Biomarker candidates were identified through RNA-seq analysis of urinary EV from patients with non-muscle invasive bladder cancer (N=3), advanced urothelial cancer (N=3), no residual tumor after TURBT (N=2), and healthy and disease controls (N=4). Diagnostic performance was evaluated by RT-qPCR in a larger patient group including bladder cancer (N=173), renal pelvis and ureter cancer (N=33), no residual tumor and non-cancer disease control (N=36).

Results: Urinary EV SLC2A1, GPRC5A and KRT17 were overexpressed in pT1 and higher stage bladder cancer by 20.6-fold, 18.2-fold and 29.5-fold, respectively. These genes allowed detection of non-muscle invasive bladder cancer (AUC: 0.56 to 0.64 for pTa, 0.62 to 0.80 for pTis, and 0.82 to 0.86 for pT1) as well as pT2 and higher muscle invasive bladder cancer (AUC: 0.72 to 0.90). Subgroup analysis indicated that these markers could be useful for the detection of cytology-negative/-suspicious and recurrent bladder cancers.

Conclusion: Three urinary EV mRNA were discovered to be elevated in bladder cancer. Urinary EV mRNA are promising biomarkers of urothelial cancer and worth further investigation.

Keywords: biomarker; bladder cancer; exosome; extracellular vesicles; mRNA.

Figure 1. RNA-seq analysis of urinary EV mRNA

(A) Unsupervised clustering analysis. Unsupervised clustering analysis of urinary EV mRNA profiles showed different clusters corresponding to healthy and disease controls (HC/DC, triangles and inverted triangle, respectively), no residual tumor (NRT, rhombuses), bladder cancer (BC, circles) and renal pelvis cancer (squares). (B) Molecular and cellular function annotations of BC urinary EV gene profiles. Ingenuity pathway analysis (IPA) determined the top molecular and cellular functions dysregulated in urinary EV from BC (circles) and NRT (squares) compared to HC/DC. Comparison between BC and NRT are also shown (triangles). Each activation/inactivation status is shown by color based on z score of IPA analysis. The functions activated in BC (z score > 2) but not in NRT (z score < 2) were highlighted in grey. (C) Volcano plot analysis. To identify bladder cancer biomarker candidates, volcano plot analysis of the differentially expressed genes was conducted. Top 12 candidates were selected from the genes up regulated in BC compared to HC/DC and NRT and their gene names are shown in red. Statistical analysis result is shown in Supplementary Table 1.

Figure 2. Urinary EV mRNA expression in various cancer types

Expression level of SLC2A1 (A), GPRC5A (B) and KRT17 (C) in urinary EV was quantified as shown in Materials and Methods, and compared by pT1 and higher stage cancer types shown in Table 2: healthy control (HC), disease control (DC), no residual tumor (NRT), bladder cancer (BC), renal pelvis cancer (RPC), ureter cancer (URC) and non-urothelial cancer (OT). Dots represent individual urine samples. Boxes indicate the first and third quartiles and the horizontal bar in each box represents median and the vertical lines represent minimum and maximum within 1.5 IQRs. Statistical significance was determined by Welchʼs t-test: p value < 0.05 (^*), < 0.005 (^**) and < 0.0005 (^***).

Figure 3. Urinary EV mRNA expression in various stages and grades of bladder cancer

Expression level of SLC2A1 (A, D), GPRC5A (B, E) and KRT17 (C, F) in urinary EV was compared among the bladder cancer stages (A – C) and grades (D – F) compared to the control groups (DC and NRT). Dots represent individual urine samples. Boxes indicate the first and third quartiles and the horizontal bar in each box represents median and the vertical lines represent minimum and maximum within 1.5 IQRs. Statistical significance was determined by Welchʼs t-test: p value < 0.05 (^*), < 0.005 (^**) and < 0.0005 (^***).

Figure 4. ROC curve analysis of urinary EV mRNA markers in various stages of bladder cancer

Diagnostic performance of urinary EV SLC2A1 (purple), GPRC5A (blue) and KRT17 (green) was evaluated against that of urine cytology (ocher) and BTA ELISA assay (red) for the detection of bladder cancer at various stages. (A) all stage bladder cancer (pTa, pTis, pT1 and > pT2), (B) pTa bladder cancer, (C) pTis bladder cancer, and (D) pT1 bladder cancer. Area under the curve (AUC) of ROC curve are shown in Table 3.