Systematic verification of bladder cancer-associated tissue protein biomarker candidates in clinical urine specimens

Abstract

Bladder cancer biomarkers currently approved by the Food and Drug Administration are insufficiently reliable for use in non-invasive clinical diagnosis. Verification/validation of numerous biomarker candidates for BC detection is a crucial bottleneck for novel biomarker development. A multiplexed liquid chromatography multiple-reaction-monitoring mass spectrometry assay of 122 proteins, including 118 up-regulated tissue proteins, two known bladder cancer biomarkers and two housekeeping gene products, was successfully established for protein quantification in clinical urine specimens. Quantification of 122 proteins was performed on a large cohort of urine specimens representing a variety of conditions, including 142 hernia, 126 bladder cancer, 67 hematuria, and 59 urinary tract infection samples. ANXA3 (annexin A3) and HSPE1 (heat shock protein family E member 1), which showed the highest detection frequency in bladder cancer samples, were selected for further validation. Western blotting showed that urinary ANXA3 and HSPE1 protein levels were higher in bladder cancer samples than in hernia samples, and enzyme-linked immunosorbent assays confirmed a higher urinary concentration of HSPE1 in bladder cancer than in hernia, hematuria and urinary tract infection. Immunohistochemical analyses showed significantly elevated levels of HSPE1 in tumor cells compared with non-cancerous bladder epithelial cells, suggesting that HSPE1 could be a useful tumor tissue marker for the specific detection of bladder cancer. Collectively, our findings provide valuable information for future validation of potential biomarkers for bladder cancer diagnosis.

Keywords: MRM; biomarker verification; bladder cancer; protein quantification; targeted proteomics.

Figure 1. Workflow for the methodological verification of protein biomarker candidates in clinical urine samples

(A) Strategy for biomarker discovery and methodological verification. I. Discovery phase: Up-regulated candidates for MRM-MS assay development were identified by an analysis of the BC tissue proteome. II. MRM-MS assay development: 130 up-regulated candidates (T/N ≥ 1.5) were selected for assay development. Dimethylated peptides and transitions of each protein candidate were predicted by Skyline software and used for the development of the MRM-MS method. III. Candidate verification methodologically: Protein targets in clinical urine samples were quantified using the MRM-MS assay. A total of 119 urine samples from BC, hernia, hematuria, and UTI patients were tested. All candidates were quantified and verified using MRM-MS based on the fixed amount of heavy-labeled dimethylated synthetic peptides. The diagnostic power of potential biomarkers was estimated by statistical analyses of multiplexed MRM-MS data. (B) Proteins in urine samples from individual BC patients (n = 30) and non-cancer patients (n = 89) were extracted for tryptic digestion and light isotopic dimethylation. Each individual sample was spiked with 122 heavy dimethyl-labeled peptide standards, which were subsequently detected by LC-MRM-MS.

Figure 2. Methodological verification of HSPE1 in individual urine specimens from BC and hernia patients by antibody-based analysis

(A) Expression of HSPE1 in individual clinical urine specimens from BC and hernia patients, detected by Western blot analysis using an anti-HSPE1 antibody. A pooled urine sample prepared from BC and hernia patients was used as control for comparison of multiple gels. Data were quantified using Image J software. (B–D) Results of statistical analyses of HSPE1 expression in urine from hernia and BC patients by ELISA. (B) Differences in HSPE1 concentrations in urine specimens from hernia (n = 142), BC (n = 59), hematuria (n = 67) and UTI (n = 59) patients. (C) Difference in HSPE1 concentration in urine specimens from hernia (n = 142), low-grade BC (n = 42) and high-grade BC (n = 84) patients. (D) Difference in HSPE1 concentration in urine specimens from hernia (n = 142), early-stage BC (n = 98), and advanced-stage BC (n = 28) patients. Differences between groups were determined using the Mann-Whitney test.

Figure 3. Up-regulation of HSPE1 in individual bladder tumor tissue specimens, determined by IHC

(A) IHC scores for normal tissue (n = 19) and tumor tissue (n = 92) in bladder tumor slides. (B) IHC score for adjacent normal tissue (n = 19) and paired tumor tissue (n = 19) in paired bladder tumor slides. HSPE1 expression data for normal and tumor tissue cells from a given individual are linked with dashed lines. Differences between groups were determined using the Mann-Whitney test for (A) and paired Student's t-test for (B).