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analysis, all the six pair-wise comparison showed that the consistency of DE-miRNA profiling at pathway level is significantly higher than that at miRNA level. CONCLUSIONS: We identified 155 DE-miRNAs related to prostate tumor, some of them are experimentally reported and the others related to the enriched pathways common to all datasets deserve further experimental verification. In addition, we also confirmed that the consistency of prostate tumor expression profiles at pathway level is higher than that at microRNA level. Source of Funding: National Nature Science Foundation of China (31040020)

1613 GENETIC VARIATION IN OBESITY-RELATED GENES ADBR2, ADBR3, GHRL, HSD11B1, AND SHC1 AND RISK OF PROSTATE CANCER: A VA CASE-CONTROL STUDY Elizabeth M. Masko*, Durham, NC; Daniel M. Moreira, New Hyde Park, NY; Lionel L. Ban˜ez, Cathrine Hoyo, Delores J. Grant, Elizabeth E. Calloway, Kathleen H. Shuler, Kathryn A. Newman, Alexis R. Gaines, Loretta A. Taylor, Cary N. Robertson, Stephen J. Freedland, Durham, NC INTRODUCTION AND OBJECTIVES: Single nucleotide polymorphisms (SNPs) are single nucleotide variations that occur within alleles among individuals. It is believed that SNPs may predispose a person to diseases such as obesity or cancer. One study showed that SNPs within genes linked with obesity were also associated with breast cancer risk. As prostate cancer (PC) mortality is linked with obesity, we determined if SNPs within 5 obesity-related genes were also linked to PC risk in a similar manner to the breast cancer study. METHODS: A total of 53 SNPs were tagged in the genes ADBR2, ADBR3, GHRL, HSD11B1, and SHC1 using the program HAPMAP. DNA collected from 216 subjects (108 incident cases, 108 “healthy” controls) from the Durham VA Hospital were then genotyped for these SNPs. Cases and controls were frequency matched on age and BMI. Logistic regression models were used to determine the risk of obesity (BMI ⬎30 kg/m2) and PC (and high-grade defined as Gleason ⬎7 vs. low-grade/control) for each SNP, adjusting for age and race. RESULTS: In our study population, 83 men were Caucasian and 133 African American. There was no significant differences in age (p⫽0.18), race (p⫽0.96), and BMI (p⫽0.12) for cases and controls. Among the 53 SNPs genotyped, 5 SNPs in ADRB2, ADRB3, and SHC1 were associated with an increased risk of PC (OR 1.88 –2.21; p⫽0.01– 0.04), particularly high-grade disease (OR 2.50 –3.26; p⫽0.0004 – 0.05). Another 4 SNPs in ADRB2 and HSD11B1 were associated with a decreased risk of PC (p⫽0.01– 0.05). A total of 7 SNPs were associated with an increased risk of obesity (p⫽0.004 – 0.02). CONCLUSIONS: In this small pilot study, we found 14 of the 53 analyzed single SNPs in genes previously linked with obesity that were associated with PC risk, both positively and negatively at an alpha of ⬍0.05. This is more than the 3 SNPs that would be predicted based upon random chance. Future studies will validate these results in an independent cohort, with the hope of further confirming SNPs that may be used for personalized medicine as well as providing a genetic link between obesity and PC. Source of Funding: The Ernest & Mildred Mario Endowment

1614 INCREASED EXPRESSION OF NUSAP GENE IN RECURRENT PROSTATE CANCER IS MEDIATED BY E2F Zulfiqar Gulzar, James Brooks*, Stanford, CA INTRODUCTION AND OBJECTIVES: Increasing evidence suggests prostate cancer is over diagnosed and over treated. Identification of prognostic biomarkers would aid in treatment selection. METHODS: To define prognostic biomarkers for aggressive prostate cancer, we carried out gene expression profiling of 100 pros-

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tate tumors and normal prostate tissue samples with detailed clinical annotation and a median follow up of 5 years. NuSAP gene was functionally validated using invitro proliferation and invasion assay. Promoter analysis was performed by cloning 431bp NuSAP promoter region and ligating in pGL4.11 luciferase vector. RESULTS: Using Significance Analysis of Microarrays (SAM) we identified 28 transcripts significantly associated with recurrence after radical prostatectomy, including several that have been reported previously. Several transcripts were noted to function in mitotic assembly including NuSAP, which encodes for a protein that binds DNA to the mitotic spindle. NuSAP was found highly prognostic in 3 independent datasets. To characterize the role and regulation of NuSAP in prostate cancer, we studied the expression of NuSAP in the LNCaP and PC3 human prostate cancer cell lines. Using siRNA, we found that post transcriptional silencing of the NuSAP gene severely hampers the ability of PC3 to invade and proliferate in vitro. We cloned 431bp of human NuSAP promoter to understand its regulation. Sequence analysis of the promoter region suggests that NuSAP promoter contains 2 CCAAT boxes, Sp1 box and binding sites for several proteins including proliferation master regulator E2F. E2F has previously been shown to be over-expressed during prostate cancer progression. Transient transfection of E2F and the NuSAP promoter with a luciferase reporter construct identify E2F as a powerful regulator of NuSAP expression. Promoter deletion experiments demonstrate that E2F directly binds to NuSAP promoter at nt -243 to drive expression. Deletion of the E2F binding site negates the effects of E2F on NuSAP expression. CONCLUSIONS: NuSAP is a novel biomarker for prostate cancer recurrence after surgery and its over-expression might be largely driven by E2F activation. Source of Funding: None

1615 TADALAFIL INHIBITS PROSTATE CANCER CELL GROWTH IN A DOSE-DEPENDENT MANNER Niels Johnsen*, New Orleans, LA; Limin Ma, Nantong, China, People’s Republic of; Asim Abdel-Mageed, Wayne Hellstrom, New Orleans, LA INTRODUCTION AND OBJECTIVES: Erectile dysfunction (ED) following radical prostatectomy (RP) is commonly managed postoperatively with long⫺term phosphodiesterase type 5 (PDE5) inhibitors. The aim of this study was to examine the growth modulatory effect of tadalafil on various prostate cancer (PC) cell lines cultured in vitro. METHODS: Determination of drug concentrations was based on previously published data on steady⫺state plasma levels occurring with once daily 20mg dosing. Three androgen-independent PC lines, DU⫺145, PC⫺3 and C4⫺2B, were cultured at varying concentrations of purified tadalafil (1.5 ⫻ 10⫺3 mM to 1.5 ⫻ 10⫺9 mM) and cell proliferation was assessed using the WST⫺8 cell counting kit at 24, 48, and 72 hrs. RESULTS: PC-3 cells showed significant decreases in growth at 24 hours in 1.5 ⫻ 10-3 mM to 1.5 ⫻ 10-9 mM treatment groups, at 48 hours in the 1.5 ⫻ 10-3 mM, 1.5 ⫻ 10-4 mM, 1.5 ⫻ 10-6 mM, and 1.5 ⫻ 10-9 mM treatment groups, and at 72 hours in the 1.5 ⫻ 10-3 mM and 1.5 ⫻ 10-4 mM treatment group (p⬍0.05). DU-145 cells showed a significant dose-dependent decrease in cell growth at 24 hours in the 1.5 ⫻ 10-3 mM to 1.5 ⫻ 10-8 mM treatment groups, as well as at 48 hours in the 1.5 ⫻ 10-3 mM to 1.5 ⫻ 10-6 mM treatment groups and at 72 hours in the 1.5 ⫻ 10-3 mM treatment group (p⬍0.05). DU-145 cells did also show a time-dependent inhibition of growth over the first 48 hours at doses of 1.5 ⫻ 10-3 mM to 1.5 ⫻ 10-6 mM, with an average decrease in growth of 11.8% relative to control. C4-2B cells showed a decrease in cell growth of statistical significance (p⬍0.05) at 24 hours in the 1.5 ⫻ 10-4 mM and 1.5 ⫻ 10-5 mM treatment groups, and at 48 hours in the 1.5 ⫻ 10-3 mM to 1.5 ⫻ 10-7 mM treatment groups, as well as in the 1.5 ⫻ 10-9 mM group. C4-2B cells did not show a statistically significant growth inhibition at 72 hours at any tadalafil concentration tested. Time-dependent inhibition of growth, however, was also seen

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over the first 48 hours in the C4-2B line at doses of 1.5 ⫻ 10-3 mM to 1.5 ⫻ 10-7 mM, with a mean decrease in growth relative to control of 16.3%. CONCLUSIONS: Once daily dosing of 20mg tadalafil attains a steady⫺state concentration that is within the treatment range shown to decrease PC cell growth. Though 20mg dosing is rare, this preliminary study suggests that routine administration of tadalafil for ED may prove to have the additional benefit of inhibiting residual metastatic PC cell growth after RP, even at plasma concentrations attained with lower daily doses. Further studies are needed to examine the growth modulatory effect in normal prostate and androgen-dependent cancer cells, as well as to elucidate the mechanism of growth inhibition involved. Source of Funding: Niels V. Johnsen supported by the SMSNA Scholars in Sexuality Research Grants Program, SMSNA; Tadalafil provided by Eli Lilly and Company

1616 RATIONAL BASIS FOR THE COMBINATION OF PCA3 AND TMPRSS2:ERG GENE FUSION IN PROSTATE CANCER DIAGNOSIS Gre´goire Robert*, Bordeaux, France; Sander Jannink, Tilly Aalders, Peter Mulders, Jack Schalken, Nijmegen, Netherlands INTRODUCTION AND OBJECTIVES: The prostate cancer gene 3 (PCA3) and TMPRSS2:ERG gene fusion are promising prostate cancer (PCa) specific biomarkers. Our aim was to simultaneously quantify the expression levels of PCA3 and TMPRSS2:ERG in a large panel of benign prostatic hyperplasia (BPH), normal prostate adjacent to PCa (NP) and PCa tissue samples, to provide a rational basis for the understanding of the false-positive and false-negative results of the urine assays. METHODS: The tissue samples were carefully histopathologically characterized to obtain homogeneous groups. The mRNA was isolated, transcribed into cDNA and the expressions of PCA3 and TMPRSS2:ERG were measured using a quantitative real-time polymerase chain reaction. The relative expression levels of the markers were normalized for the housekeeping gene HPRT and were compared between the different groups. RESULTS: We included 48 BPH, 32 NP and 48 PCa. The PCA3 test had a sensitivity of 85.4% but led to the identification of 1 false-positive and 7 false-negative samples. The TMPRSS2:ERG gene fusion test was positive in 8.3% of the BPH, 15.6% of the NP and 50% of the PCa samples. We had to define a cut-off value to avoid 8 false-positive results with the TMPRSS2:ERG test. The combination of the PCA3 test with the TMPRSS2:ERG test had the highest sensitivity and the best diagnostic accuracy. In case of a negative PCA3 test, the addition of the TMPRSS2/ERG test allowed to diagnose 4 of the 7 false-negative samples and added only 1 false-positive. CONCLUSIONS: Most of the false-negative results of the PCA3 test were corrected by the combination with TMPRSS2:ERG (57%). The combination had a higher sensitivity and a better accuracy. Some of the control samples did express TMPRSS2:ERG and a cut-off value had to be defined to avoid false-positive results. Second Line -

Se (%) 85.4

Sp (%) 98.7

Youden Index 0.84

TMPRSS2:ERG

-

45.8

98.75

0.45

PCA3 ⫹ TMPRSS2:ERG

-

91.7

97.5

0.89

TMPRSS2:ERG

93.75

97.5

0.9

First Line PCA3

PCA3

For PCA3, the cut-off value was 19.6; For TMPRSS2 :ERG, the cut-off value was 2.71

Source of Funding: Gre´goire Robert was granted by the EAU scholarship Program and by the French Urological Association (AFU)

1617 SUSTAINED AEROBIC EXERCISE ALONE DOES NOT COUNTERACT THE TUMOR-PROMOTING EFFECTS OF A WESTERNIZED DIET ON PROSTATE CANCER PROGRESSION IN VIVO Natalie Venier, Avi Vandersluis, Alexandra Colquhoun*, Alex Kiss, Neil Fleshner, Laurence Klotz, Vasundara Venkateswaran, Toronto, Canada INTRODUCTION AND OBJECTIVES: The influence of lifestyle behaviors on prostate cancer (PCa) is well established. Our lab has previously reported that diets high in both fat and carbohydrates – the “Westernized” diet – promote PCa tumor growth in vivo. Numerous studies have also found an inverse relationship between exercise and PCa progression. However, the exact relationships between diet, exercise and PCa, as well as their underlying mechanisms, are unclear. Herein we aim to investigate the effect of sustained aerobic exercise on PCa tumor growth in animals fed a “Westernized” diet or a normal diet. METHODS: Athymic nude mice (n⫽46) were inoculated subcutaneously with LNCaP cells. Mice were fed ad libitum with either a high-fat/high-carbohydrate diet (HFHC) or a standard normal diet (Normal), and randomized into exercising (Ex) and non-exercising groups (No-Ex). Exercise was undertaken 3 days per week (3 ⫻ 15 mins; 2-min breaks between cycles; 2.0 –7.0 m/min) for 8 weeks, using a forced exercise wheel. Body weights, tumor volumes, and food consumption were recorded tri-weekly. Comparisons between groups over time were performed using RANOVA Type 3 Tests of Fixed Effects. RESULTS: There were no significant differences in body weight between the groups over time. The HFHC-Ex group (n⫽10) had the highest rate of tumor growth compared to all other groups (p⬍⫽0.0007). No significant differences were observed between the rate of tumor growth of the HFHC-No Ex group (n⫽11) and the Normal-No Ex group (n⫽12) or between the Normal-No Ex group and the Normal-Ex group (n⫽10). However, the rate of tumor growth of the Normal-Ex group was reduced compared to the HFHC-No Ex group [p⬍⫽0.0008]. Food consumption analysis revealed significant differences (p⬍⫽0.012) in energy consumption (kcal) between each of the groups over time. Mice in HFHC-Ex group consumed the most energy compared to all other groups followed by HFHC-No Ex⬎NormalEx⬎Normal-No Ex groups. CONCLUSIONS: The results indicate that exercise stimulated an increase in food consumption. In mice fed a tumor-promoting “Westernized diet,” this enhanced the rate of tumor growth. However, in mice placed on a normal diet, this increase in energy consumption did not correspond with increased tumor growth over time, emphasizing the importance of both exercise and healthy diet in the progression of PCa. Further studies to examine the relationship between diet and exercise with respect to PCa progression are currently underway. Source of Funding: CIHR

1618 MULTIPHOTON MICROSCOPY PROVIDES VIRTUAL HISTOLOGY OF PROSTATIC AND PERIPROSTATIC STRUCTURES IN HUMAN RADICAL PROSTATECTOMY SPECIMENS Sonal Grover*, Prasanna Sooriakumaran, Abhishek Srivastava, Michael Herman, Joshua Sterling, Robert Leung, Maria Shevchuk, Sushmita Mukherjee, Ashutosh Tewari, New York, NY INTRODUCTION AND OBJECTIVES: This was a prospective study involving Multiphoton Microscopy (MPM) imaging and histopathologic correlations of human prostate and peri-prostatic tissue. The aim of this study was to assess the potential of MPM to provide virtual histology as a proof of principle for real-time imaging during nerve sparing radical prostatectomy and for MPM guided biopsy. METHODS: We used two types of specimens for imaging: (1) intraoperative margins and biopsies; (2) tissue sections obtained from the excised prostate. The imaging was carried out using intrinsic fluorescence and scattering properties of the tissues without any ex-