ORIGINAL ARTICLE Oncology & Hematology http://dx.doi.org/10.3346/jkms.2015.30.12.1784 • J Korean Med Sci 2015; 30: 1784-1792

Urinary Nucleic Acid TSPAN13-to-S100A9 Ratio as a Diagnostic Marker in Prostate Cancer

Chunri Yan,1 Ye-Hwan Kim,1 The potential use of urinary nucleic acids as diagnostic markers in prostate cancer (PCa) Ho Won Kang,1 Sung Phil Seo,1 was evaluated. Ninety-five urine samples and 234 prostate tissue samples from patients Pildu Jeong,1 Il-Seok Lee,1 Dongho Kim,2 with PCa and benign prostatic hyperplasia (BPH) were analyzed. Micro-array analysis was Jung Min Kim,3 Yung Hyun Choi,4 used to identify candidate , which were verified by the two- expression ratio 5 1 Sung-Kwon Moon, Seok Joong Yun, and validated in tissue mRNA and urinary nucleic acid cohorts. Real-time quantitative 1 and Wun-Jae Kim polymerase chain reaction (qPCR) was used to measure urinary nucleic acid levels and 1Department of Urology, College of Medicine, tissue mRNA expression. The TSPAN13-to-S100A9 ratio was selected to determine the Chungbuk National University, Cheongju; diagnostic value of urinary nucleic acids in PCa (P = 0.037) and shown to be significantly 2Bio-Medical Science Co. Ltd, Daejeon; 3Nucleic higher in PCa than in BPH in the mRNA and nucleic acid cohort analyses (P < 0.001 and Acid Research Center, Inc., Daejeon Oriental Hospital P = 0.013, respectively). Receiver operating characteristic (ROC) analysis showed that the of Daejeon University, Daejeon; 4Department of Biochemistry, Dong-Eui University, Busan; area under the ROC curve was 0.898 and 0.676 in tissue mRNA cohort and urinary nucleic 5Department of Food and Biotechnology, Chung- acid cohort, respectively. The TSPAN13-to-S100A9 ratio showed a strong potential as a Ang University, Seoul, Korea diagnostic marker for PCa. The present results suggest that the analysis of urine supernatant can be used as a simple diagnostic method for PCa that can be adapted to the Received: 29 May 2015 Accepted: 2 September 2015 clinical setting in the future.

Address for Correspondence: Keywords: Prostatic Neoplasms; Cell-free Nucleic Acid; Urine; Two-gene Expression Ratio; Wun-Jae Kim, MD S100A9; TSPAN13; Diagnostic Marker Department of Urology, Chungbuk National University, College of Medicine and Institute for Tumor Research, 776 1Sunhwan-ro, Seowon-gu, Cheongju 28644, Korea Tel: +82.43-269-6371, Fax: +82.43-269-6144 E-mail: [email protected]

Funding: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. NRF-2014R1A2A1A09006983) and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2013R1A1A2004740).

INTRODUCTION (cell-free) nucleic acids (CNA) are released from apoptotic and necrotic cells, although their exact origin is poorly understood Prostate cancer (PCa) is the most commonly diagnosed malig- (9). Despite the fact that their source remains unclear, CNAs re- nancy in Western men (1), and the number of newly diagnosed flect the molecular changes occurring in specific tissues (10). PCa cases in Korea is increasing (2). The widespread use of Furthermore, they can be isolated from body fluids using non- prostate-specific antigen (PSA) testing led to a dramatic in- invasive procedures, which make them superior as cancer bio- crease in the incidence and survival of PCa patients (3). How- markers to tissue markers that are obtained by invasive proce- ever, PSA is an excellent organ-specific but not cancer-specific dures. marker, and its specificity for the detection of PCa is low (4). Despite their value as cancer biomarkers, the quantification Transrectal prostate biopsy, which is necessary to confirm the of CNAs is difficult. Therefore, we adopted the two-gene ex- diagnosis of PCa, has a limited value because of its low detec- pression ratio method previously described by Ma et al. (11). tion rate and uncomfortable nature of the procedure. Therefore, The two-gene expression ratio consists of the comparison of accurate, specialized and convenient diagnostic biomarkers for one up-regulated gene to one down-regulated gene. In our pre- PCa are needed. vious study (10), urinary expression levels of S100A9 nucleic Cell-free nucleic acid-based biomarkers in body fluids have acids were significantly lower in PCa than in benign prostatic recently become a topic of interest (5). Fragmented DNAs and hyperplasia (BPH). Therefore, S100A9 was selected as the RNAs exist as circulating nucleic acids and can be isolated from down-regulated gene for the two-gene expression ratio. body fluids such as blood, feces and urine (6-8). Circulating In the present study, the up-regulated gene for the two-gene

© 2015 The Korean Academy of Medical Sciences. pISSN 1011-8934 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. eISSN 1598-6357 Yan C, et al. • Urinary Nucleic Acid TSPAN13-to-S100A9 Ratio in Prostate Cancer expression ratio was identified, and the mRNA expression lev- ≥ 2; PCa-associated genes were obtained from NCBI’s els were assessed. The ratio of up-regulated to down-regulated Gene (http://www.ncbi.nlm.nih.gov/gene/) and GeneCards gene was assessed in PCa and BPH tissues to confirm our find- (http://www.genecards.org/) databases. ing at the tissue mRNA level, and in urine samples from PCa and BPH to determine the value of urinary nucleic acids as di- Study population and samples agnostic biomarkers of PCa. A total of 95 urine samples and 234 prostate tissue samples ob- tained from patients with PCa and BPH treated at our institute MATERIALS AND METHODS were used in the study. In detail, 12 PCa cases and 5 BPH con- trols were used in the two-gene expression ratio selection study; Study design and 129 PCa and 105 BPH tissue samples and 37 PCa and 31 A schematic of the study design, which included four different BPH urine samples were used in the validation cohort (tissue stages, is shown in Fig. 1. Candidate genes were selected from mRNA validation cohort and urinary nucleic acid validation tissue mRNA micro-array data and evaluated using the two- cohort) (Fig. 1). All of the urine samples were collected prior to gene expression ratio method in urinary nucleic acids. The surgery on the first morning and centrifuged at 25,000 rpm for genes selected for two-gene expression ratio were validated in 15 min, and the supernatants were stored at -20°C until use. tissue mRNA and urinary nucleic acid cohorts. The tissue study included patients with PCa who underwent palliative transurethral resection (TUR) or radical prostatectomy, Candidate gene selection from micro-array data and patients with BPH who underwent TUR. All prostate tissue The gene expression profiles for GSE2618 (12), GSE6099 (13), samples were macro-dissected within 15 min of surgical resec- GSE6608 (14), GSE6919 (14), and GSE23388 (15) were down- tion. Each prostate specimen was confirmed by pathological loaded from the Gene Expression Omnibus (GEO; http://www. analysis of fresh-frozen sections, and the remaining tissue was ncbi.nlm.nih.gov/geo/) database and differentially expressed frozen in liquid nitrogen and stored at -80°C until use. The con- genes (DEGs) were selected using Gene Spring GX 7.3 software trols were matched by age; and subjects were screened to en- (Agilent Technology, Santa Clara, CA, USA). The DEGs were sure that their laboratory values were within the normal range identified using a t-test with a threshold P value < 0.05 (Ben- and that they had no history of cancer. Controls with serum jamini and Hochberg False Discovery Rate) and a fold-change PSA levels ≥ 3 ng/mL und