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

// Cheng-Han Tsai 1, * , Yi-Ting Chen 1, 2, 3, 4, * , Ying-Hsu Chang 5, 6, * , Chuen Hsueh 2, 7 , Chung-Yi Liu 5 , Yu-Sun Chang 1, 2, 8 , Chien-Lun Chen 9, 10 and Jau-Song Yu 1, 2, 11, 12 1 Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 2 Molecular Medicine Research Center, College of Medicine, Chang Gung University, Taoyuan, Taiwan 3 Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 4 Department of Nephrology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan 5 Division of Urology, Department of Surgery, LinKou Chang Gung Memorial Hospital, Taoyuan, Taiwan 6 Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 7 Department of Pathology, Chang Gung Memorial Hospital, Linkou, Chang Gung University, College of Medicine, Taoyuan, Taiwan 8 Department of Otolaryngology - Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan 9 Department of Urology, Chang Gung Memorial Hospital, Taoyuan, Taiwan 10 College of Medicine, Chang Gung University, Taoyuan, Taiwan 11 Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan 12 Research Center for Food and Cosmetic Safety, Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan * These authors contributed equally to this work Correspondence to: Yi-Ting Chen, email: ytchen@mail.cgu.edu.tw Jau-Song Yu, email: yusong@mail.cgu.edu.tw Chien-Lun Chen, email: clc2679@cgmh.org.tw Keywords: bladder cancer; biomarker verification; MRM; targeted proteomics; protein quantification Received: July 02, 2017      Accepted: February 20, 2018      Published: July 20, 2018 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.