Prostate Specific or Enriched Genes as Composite Biomarkers for Prostate Cancer PRINCIPAL INVESTIGATOR:

Purpose: Prostate cancer is the third leading cause of cancer death in the United States, following lung and colorectal cancer.We previously identifiedWDR19 as a prostate-specific, androgenregulated gene. Here, we evaluate its utility as a prostate cancer tissue marker for diagnosis and prognostic evaluation. Experimental Design: Real-time quantitative PCR was done on a panel of prostate tissue isolated by laser capture microdissection. After generating antibodies against WDR19, tissue microarrays (TMA) were employed to compareWDR19 expression between normal, benign prostatic hyperplasia, and prostate cancer tissue. Results: Using microarrays and real-time quantitative PCR, we showed thatWDR19 mRNA expression was increased in cancer.We further showed thatWDR19 protein is localized to cytoplasmic subcellular granules and is expressed exclusively in prostate epithelia. Large-scale immunohistochemical staining using TMAs reveals a significant percentage of increase in intensely staining tissue cores in cancer tissue when compared with normal or benign prostatic hyperplastic tissue. Based on the analysis of a separateTMA for which clinical follow-up information was available, low-intensityWDR19 staining was significantly associated with decreased time to biochemical failure (P = 0.006) and with decreased time to locoregional recurrence (P = 0.050). Conclusions: WDR19 should be added to the list of prostate cancer tissue markers. The continued expansion of a multiple-marker panel will conceivably increase the sensitivity and specificity of prostate cancer diagnosis and prognosis. Prostate cancer is the third leading cause of cancer death, following lung and colorectal cancer, in the United States. The American Cancer Society estimated 218,890 new cases and f27,050 deaths in the United States in 2007 (1). Early diagnosis is important because low-stage cancer is more effectively treated by surgery or radiation compared with high-stage tumors. However, early diagnosis can generate lead-time bias, and consequently, improved 5-year survival of patients with prostate cancer (2). After diagnosis, the key question is how the patient will respond to treatment and how long the patient will survive. However, our capacity to predict the prognosis of patients with prostate cancer is limited. Better markers are therefore needed to both diagnose and predict disease course more accurately. Recent progress has led to the identification of many candidate markers for prostate cancer, including NKX3.1, KLK2, KLK3 (PSA), FOLH1 (PSMA), STEAP2, PSGR, PRAC, RDH11, Prostein, Hepsin, a-methylacyl CoA racemase (AMACR), FASN, EZH2 (3), and Huntingtininteracting protein 1 (4). Effective markers need not only be tumor antigens, but could also be autoantibodies, such as those for Huntingtin-interacting protein 1 and those for 22 peptides Imaging, Diagnosis, Prognosis Authors’Affiliations: Zhejiang-California International Nanosystems Institute, Hangzhou, China; The Institute for Systems Biology, Department of Urology, University of Washington, Fred Hutchinson Cancer Research Center, Seattle, Washington; and The Gade Institute, Section for Pathology, University of Bergen, and Haukeland University Hospital, Bergen, Norway Received 6/20/07; revised11/13/07; accepted11/29/07. Grant support: This publication was made possible by grants 5P50GM076547 and 5U54CA119347 from NIH, DOD grant W81XWH-07-1-0108, and grants from The Cancer Society of Norway (to L.A. Akslen) and the Research Council of Norway (to L.A. Akslen and K-H. Kalland), and MOST grant 2006AAO2Z4A2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Biaoyang Lin, Department of Urology, University of Washington, Seattle,WA 98195. Phone: 206-543-3640; Fax: 206-543-3272; E-mail: [email protected]; or Lars A. Akslen or Karl-Henning Kalland, Department of Pathology, The Gade Institute, University of Bergen, Haukeland University Hospital, N-5021Bergen, Norway. Phone: 47-5597-3182; Fax: 475597-3158; E-mail: [email protected]. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-1535 www.aacrjournals.org Clin Cancer Res 2008;14(5) March1, 2008 1397 identified by phage display (5, 6). Despite this progress, there is still no individual marker that can segregate tumors with a clinically benign behavior. It is generally believed that a multiple biomarker panel is important to increase the sensitivity and specificity in early diagnosis, and to increase the accuracy in predicting disease outcome and monitoring treatment efficacy. We report here the identification of WDR19 as another tissue marker for prostate cancer. We show, using microarrays and real-time quantitative PCR (qPCR), that WDR19 mRNA expression increases in prostate cancer. Generating antibodies against WDR19 permitted us to determine that WDR19 protein is expressed solely in prostate epithelia and is localized to subcellular granules. Large-scale immunohistochemical staining using tissue microarrays (TMA) revealed a significantly increased percentage of tissue cores with intense WDR19 staining in cancer compared with normal or benign prostatic hyperplasia (BPH) tissue. Finally, we found that low-intensity WDR19 staining is significantly associated with decreased time to biochemical failure (P = 0.006) and with decreased time to locoregional recurrence (P = 0.050) within the group of prostate cancers. This protein may be added to the list of prostate cancer tissue markers, thereby helping expand the multiple-marker panel needed to increase diagnostic and prognostic sensitivity and specificity. Materials andMethods cDNA microarray analysis. A custom-built cDNA microarray (PEDB Array; refs. 7, 8) was used. This array contains 6,000 cDNAs printed in four replicates on the same slide. The cDNA was labeled with Cy5 as we described previously (8, 9). Spotfinding was done using AnalyzerDG software (MolecularWare, Inc.). Real-time qPCR. The RNA and cDNA preparation from prostate cancer tissues and cells were previously described (10). TaqMan Gene Expression Assay kits for WDR19 and HPRT were obtained from Applied Biosystems, Inc. The PCR reactions were done using the ABI Prism 7900HT Sequence Detection System and SDS Enterprise Database (Applied Biosystems Inc.). PCR variables were 50jC for 2 min, 95jC for 10 min, then 40 cycles with each cycle at 95jC for 15 s and 60jC for 1 min. Relative quantification for real-time qPCR was done using the mathematical model and formula published by