XWH - 06 - 1 - 0116 TITLE : Role of Caveolin - 1 in Prostate Cancer Angiogenesis

Purpose: Up-regulation of caveolin-1 (cav-1) is associated with virulent prostate cancer, and serum cav-1levels are elevated in prostate cancer patients but not in benign prostatic hyperplasia. In this study, we evaluated the potential of high preoperative serum cav-1 levels to predict biochemical progression of prostate cancer. The value of the combined preoperative markers, prostate-specific antigen (PSA), biopsy Gleason score, and serum cav-1for predicting biochemical recurrence was also investigated. Experimental Design: Serum samples taken from 419 prostate cancer patients before radical prostatectomy were selected from our Specialized Programs of Research Excellence prostate cancer serum and tissue bank. Serum samples were obtained 0 to 180 days before surgery and all patients had complete data on age, sex, race, stage at enrollment, and follow-up for biochemical recurrence. Serum cav-1 levels were measured according to our previously reported ELISA protocol. Results:Cav-1levels weremeasured in the sera of 419 prostate cancer patients; themean serum level was 4.52 ng/mL (median1.01ng/mL). Patients with high serum cav-1levels had a 2.7-fold (P = 0.0493) greater risk of developing biochemical recurrence compared with those with low serum cav-1levels. Importantly, patientswith serumPSAz10 ng/mL andelevated levels of serum cav-1had 2.44 times higher risk (P = 0.0256) of developing biochemical recurrence compared withpatientswithlowlevelsofcav-1. Inaddition,highserumcav-1levels combinedwithincreasing biopsyGleasonscorepredictedmuchshorter recurrence-free survival in the groupofpatientswith PSA z10 ng/mL (P = 0.0353). Cav-1was also able to distinguish between highand lowrisk patientswithbiopsyGleason score of seven, after adjusting, for patients PSA levels (P =0.0429). Conclusions: Overall, elevated preoperative levels of serum cav-1predict decreased time to cancer recurrence. In the subset of patients with serum PSA ofz10 ng/mL, the combination of serum cav-1andbiopsyGleason scorehas the capacity topredict time tobiochemical recurrence. In 2005, f90% of newly diagnosed prostate cancer patients had clinically localized disease (1). Consequently, the majority of patients are treated with curative intent by either radical prostatectomy or radiation therapy. It is well established, however, that 10% to 50% of patients who undergo radical prostatectomy will show biochemical evidence of disease recurrence [prostate-specific antigen (PSA) recurrence] within 5 years of surgery (2, 3). Various clinical variables have been used, singly and in combination (nomograms, tables, etc.), to predict, preoperatively, which patients are likely to fail definitive therapy (4). However, the predictive value of these variables has been thwarted by the vexing biological diversity of clinical prostate cancer. New markers are needed, preferably serum markers that have been mechanistically implicated in the progression of virulent disease. We believe that serum caveolin1 (cav-1) may be such a marker. Cav-1 is an important structural/regulatory molecule involved in many aspects of molecular transport and cell signaling (5, 6). Tissue cav-1 is overexpressed in metastatic and in hormone-resistant prostate cancer (7). Overexpression correlates with a shortened interval to disease recurrence following therapy for localized disease and tends to be associated with a high Gleason score pathologically (8–10). Interestingly, cav-1 is secreted by prostate cancer cells (11) and we have developed a sensitive ELISA immunoassay for the detection of cav-1 in the serum (12). In a preliminary study, we documented that prostate cancer patients have a higher serum cav-1 level when compared with age-matched controls with benign prostatic hyperplasia (12). We report here the utility of a single preoperative measurement of serum cav-1 for predicting disease recurrence in a cohort of 419 prostate cancer patients undergoing radical prostatectomy at our institution. Imaging, Diagnosis, Prognosis Authors’ Affiliations: Scott Department of Urology and Departments of Pathology, Radiology, Medicine, and Molecular and Cellular Biology, Baylor College of Medicine, Houston,Texas Received 2/21/06; revised 3/31/06; accepted 4/20/06. Grant support: National Cancer Institute Specialized Programs of Research Excellence grants P50-58204 and RO1-68814. 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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Dov Kadmon, Scott Department of Urology, Baylor College of Medicine, 6560 Fannin, Suite 2100, Houston, TX 77030. E-mail: [email protected]. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-0417 www.aacrjournals.org Clin Cancer Res 2006;12(16) August15, 2006 4872 Materials andMethods Study population. The sera of 419 prostate cancer patients were obtained from the Specialized Programs of Research Excellence prostate cancer blood and tissue bank at Baylor College of Medicine. Entry into the study required availability of preoperative serum samples obtained within 6 months of surgery and complete data on age, race, stage at enrollment and follow-up, as well as availability of postoperative serum samples, as this is a part of a larger ongoing investigation. In addition, patients could have had no preoperative therapy. After completion of cav-1 measurements in the serum, it was discovered that seven patients were missing reliable data on their preoperative PSA and/or biopsy Gleason score and/or follow-up information. These patients were included in all analyses that did not require missing data. The preoperative serum collected from 355 patients was at a time period between prostate biopsy and surgery. No information was available in our database on the exact preoperative serum collection timing with respect to biopsy for 64 patients. The mean age of this patient group was 62.6 years (range 42.6-78.9 years); 91.4% were White males, with Hispanics, African-Americans, and Asians comprising 6.0%, 2.4%, and 0.2%, respectively. Mean follow-up time among this group of patients was 52 months, with a median follow-up time of 48 months. Biochemical recurrence is defined throughout this study as serum PSA level of z0.2 ng/mL on two consecutive measurements, using the firstgeneration postresection PSA assay (Hybritech, Beckman Coulter, Inc., Fullerton, CA). Patient data were gathered from the Informatics Core using the Specialized Programs of Research Excellence in Prostate Cancer Information System. Determination of serum cav-1. Cav-1 was determined in the serum samples by the sandwich ELISA protocol developed in our laboratory (12). Briefly, Costar microplate wells were coated with 0.5 Ag cav-1 polyclonal antibody (Transduction Laboratories, San Diego, CA) and blocked with TBS buffer containing 1.5% bovine serum albumin and 0.05% v/v Tween 20. Serum samples, calibrators, and controls (50 AL) were added to the well, and 50 AL TBS containing 0.5% v/v Tween 20 was added to each well. The plate was incubated at room temperature for 2 hours with shaking and after extensive washing, 100 AL horseradish peroxidase–conjugated cav-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200 in blocking buffer was added to each well. The microplate was incubated for 90 minutes at room temperature with shaking, the wells were then washed extensively, and 100 AL 3,3¶,5,5¶-tetramethylbenzidine substrate solution (Sigma-Aldrich, St. Louis, MO) was added and the blue color was allowed to develop for 20 minutes in the dark. The reaction was stopped by adding 50 AL of 2 N H2SO4, and the absorbance was read at 450 nm using a microplate reader (Sunrise Microplate Reader, Tecan US, Inc., Charlotte, NC). Statistical analysis. Correlations of preoperative serum cav-1 levels with clinical and pathologic variables were evaluated using the Spearman correlation. The predictive value of cav-1 univariately and multivariately with other preoperative clinical and pathologic variables, such as preoperative PSA and biopsy Gleason score, as well as of the interactive terms, were analyzed using the Cox proportional hazards regression model. The minimum P value method was used to group patients into ‘‘low-level’’ and ‘‘high-level’’ cav-1 categories (13). The hazard ratio (HR) and 95% confidence intervals (95% CI) were computed for each marker. Kaplan-Meier survival curves were plotted for each risk category. P < 0.05 was considered statistically significant. All analyses were done using the SPSS 12.0 software package (SPSS,