[CANCER RESEARCH 64, 5882–5890, August 15, 2004] Three Biomarkers Identified from Serum Proteomic Analysis for the Detection of Early Stage Ovarian Cancer Zhen Zhang, 1 Robert C. Bast, Jr., 2 Yinhua Yu, 2 Jinong Li, 1 Lori J. Sokoll, 1 Alex J. Rai, 1 Jason M. Rosenzweig, 1 Bonnie Cameron, 1 Young Y. Wang, 1 Xiao-Ying Meng, 3 Andrew Berchuck, 4 Carolien van Haaften-Day, 5 Neville F. Hacker, 5 Henk W. A. de Bruijn, 6 Ate G. J. van der Zee, 6 Ian J. Jacobs, 7 Eric T. Fung, 3 and Daniel W. Chan 1 1 Department of Pathology, Biomarker Discovery Center, Johns Hopkins Medical Institutions, Baltimore, Maryland; 2 M. D. Anderson Cancer Center, Houston, Texas; 3 Ciphergen Biosystems, Inc., Fremont, California; 4 Duke University Medical Center, Durham, North Carolina; 5 The Royal Hospital for Women, Randwick, New South Wales, Australia; 6 University Hospital Groningen, Groningen, the Netherlands; and 7 Bart’s and The London, Queen Mary’s School of Medicine, London, United Kingdom ABSTRACT Early detection remains the most promising approach to improve long-term survival of patients with ovarian cancer. In a five-center case- control study, serum proteomic expressions were analyzed on 153 patients with invasive epithelial ovarian cancer, 42 with other ovarian cancers, 166 with benign pelvic masses, and 142 healthy women. Data from patients with early stage ovarian cancer and healthy women at two centers were analyzed independently and the results cross-validated to discover poten- tial biomarkers. The results were validated using the samples from two of the remaining centers. After protein identification, biomarkers for which an immunoassay was available were tested on samples from the fifth center, which included 41 healthy women, 41 patients with ovarian cancer, and 20 each with breast, colon, and prostate cancers. Three biomarkers were identified as follows: (a) apolipoprotein A1 (down-regulated in can- cer); (b) a truncated form of transthyretin (down-regulated); and (c)a cleavage fragment of inter--trypsin inhibitor heavy chain H4 (up-regu- lated). In independent validation to detect early stage invasive epithelial ovarian cancer from healthy controls, the sensitivity of a multivariate model combining the three biomarkers and CA125 [74% (95% CI, 52– 90%)] was higher than that of CA125 alone [65% (95% CI, 43– 84%)] at a matched specificity of 97% (95% CI, 89 –100%). When compared at a fixed sensitivity of 83% (95% CI, 61–95%), the specificity of the model [94% (95% CI, 85–98%)] was significantly better than that of CA125 alone [52% (95% CI, 39 – 65%)]. These biomarkers demonstrated the potential to improve the detection of early stage ovarian cancer. INTRODUCTION Despite progress in cancer therapy, ovarian cancer mortality has remained virtually unchanged over the past two decades (1). Annually in the United States alone, 23,000 women are diagnosed with the disease and almost 14,000 women die from it (1). Given our knowl- edge about the steep survival gradient relative to the stage at which the disease is diagnosed, it is reasonable to suggest that early detection remains the most promising approach to improve the long-term sur- vival of ovarian cancer patients. The relatively low prevalence (40 out of 100,000) of ovarian cancer among postmenopausal women in the general population, the lack of a clearly defined precursor lesion, and the high cost and possible complications associated with surgical confirmatory procedures have placed stringent requirements on any test intended for general popu- lation screening. Currently, none of the existing serum markers, such as CA125, CA 72– 4, or macrophage colony-stimulating factor, can be used individually for screening (2). Longitudinal studies are under way in Europe, Japan, and the United States to evaluate screening strategies using CA125 and/or transvaginal sonography (3–5) and their impact on overall cancer mortality (6). Preliminary results have shown encouraging evidence of a survival benefit among patients diagnosed through a screening regimen (3). Reports from retrospective studies have shown that multivariate predictive models combining existing tumor markers improve cancer detection (7, 8). Recent advances in genomic and proteomic profiling technology have made it possible to apply computational methods to detect changes in protein expressions and their association to disease conditions, thereby hastening the identification of novel markers that may contribute to multimarker combinations with better diagnostic performance (9 –13). In this study, we hypothesized that comparison of protein expres- sions of serum specimens from patients with early stage ovarian cancer with those from healthy women could lead to the discovery of candidate biomarkers for the detection of early stage ovarian cancer. To ensure that the discovered biomarkers are truly associated with ovarian cancer rather than the result of biases in samples, profiling data of specimens from multiple institutions were used for cross- comparison and independent validation. We additionally determined the protein identities of the discovered biomarkers to allow for addi- tional validation with independent methods and as a first step toward understanding the pathways in which they may function. MATERIALS AND METHODS Samples. The study involved a retrospective sample of 645 serum speci- mens. All were collected with institutional approval. Proteomic profiles were obtained from 503 specimens collected at four medical centers (M. D. Ander- son Cancer Center, Duke University Medical Center, Groningen University Hospital, the Netherlands, and the Royal Hospital for Women, Australia). Among them, the cancer group consisted of 65 patients with stages I/II invasive epithelial ovarian cancer, 88 patients with stages III/IV invasive epithelial ovarian cancer, 28 patients with borderline tumors, and 14 patients with recurrent disease, all optimally staged by pathologists based on Fe ´de ´ration Internationale des Gynaecologistes et Obstetristes criteria. Among the stages I/II invasive cases, 20 were serous, 17 were mucinous, 15 were endometrioid, 8 were clear cell, 1 was carcinosarcoma, and 4 were mixed epithelial carci- noma. The samples also included 166 patients with benign pelvic masses and 142 healthy donors as controls. All of the samples were collected before the day of surgery or treatment, stored at -70°C, and thawed immediately before assay. CA125 levels had been obtained previously using a CA125II radioim- munoassay (Centocor). The clinical characteristics and age distribution of the proteomic profiling study population are summarized in Table 1. In addition to the 503 specimens for proteomic profiling, 142 independent, archived serum specimens collected for routine clinical laboratory testing at the Johns Hopkins Medical Institutions were tested for levels of the identified biomarkers for which an immunoassay test was available. The sample included 41 healthy women, 41 patients with late-stage ovarian cancer, and groups of 20 patients each with breast, colon, and prostate cancers. All of the samples were processed promptly after collection and stored at 2– 8°C for a maximum of Received 3/1/04; revised 5/17/04; accepted 5/26/04. Grant support: National Cancer Institute Grant 1P50 CA83639, UTMDACC Spe- cialized Programs of Research Excellence in Ovarian Cancer (R. Bast, Jr., and Z. Zhang), and funding from Ciphergen Biosystems, Inc. (Z. Zhang, J. Li, A. Rai, J. Rosenzweig, B. Cameron, Y. Wang, and D. Chan). 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. Requests for reprints: Zhen Zhang, Center for Biomarker Discovery, Johns Hopkins Medical Institutions, 419 N. Caroline Street, Room 200, Baltimore, MD 21231. E-mail: zzhang7@jhmi.edu. 5882