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increased B-cell TILs has not been demonstrated in a systematic fashionin prostate cancer (CaP). Animal models have shown B-cell interactionsmay be a harbinger of hormone-refractory CaP. We investigate thedensity of B-cells within the CaP tissue utilizing a reproducible andquantitative computational method of identifying B-cells.

METHODS: 53 paraffin-embedded radical prostatectomyspecimens underwent CD20 immunohistochemical staining to identifyB-cells. CaP tumors were identified and marked by a genitourinarypathologist manually (figure 1a,b). Slides were digitally scanned and acomputer algorithm quantified the area of stained B-cells within the CaPand surrounding tissue (figure 1b,d). Patient clinicopathological featuresof each specimen were obtained. The primary outcome was B-celldensity within CaP versus outside the cancer. Secondary outcome wasB-cell density compared to outcomes including Gleason score, D’Amicorisk groups, and recurrence.

RESULTS: 8 (15.1%), 9 (17%) and 36 (67.9%) specimens werefrom patients with low, intermediate and high risk disease, respectively.19 (35.8%) specimenswere from any risk groupwith disease recurrence.For the entire cohort, the mean density (area of B-cells in mm2/area ofprostate analyzed in mm2) within the tumor was higher (3.22, SE¼0.29)than outside the tumor (2.24, SE¼0.19) (paired t test; P<0.001). D’Amicolow risk (0.0377 vs. 0.0246; p¼0.151) and intermediate risk (0.0260 vs.0.0214; p¼0.579) did not show significantly more B-cells within thetumor. The high risk group (0.0301 vs. 0.0197; p<0.001) and patientswho eventually had CaP recurrence (0.0343 vs. 0.0246; p¼0.019) didshow significantly more CD20+ B-cell staining within CaP tumors. B-celldensity did not correlate to any other patient clinical parameters.

CONCLUSIONS: B-cells are present in higher density withinCaP tissue than within benign prostate. The interaction of B-cells andCaP may serve as the basis of new therapeutic targets.

Source of Funding: Funding: Grant from Clinical TranslationalInstitute-Pfizer: UCSD (M. Karin, PI)

MP49-04ROLE OF NUCLEAR AND CYTOPLASMIC TBLR1 IN PROSTATECANCER

Garrett Daniels*, Yirong Li, New York, NY; Lan Lin Gellert, Nashville,TN; Jonathan Melamed, Xinyu Wu, David Zhang, Daniel Meruelo,Susan Logan, Ross Basch, Peng Lee, New York, NY

INTRODUCTION AND OBJECTIVES: TBLR1, a core compo-nent of thenuclear receptor corepressor (NCoR) complex, showsboth co-repressor andco-activator activitiesonnuclear receptors, although little isknown about its effects on Androgen Receptor (AR). AR coactivatorshave primarily been viewed as pro-proliferative, however increasing ev-idence shows the importance of AR coactivators in regulating growthsuppressiveAR target genes.Cofactorsmayalsohavedifferent functionsdependingon their cellular localization, switching from tumor suppressorsto oncogenes or vice versa. In our study, we aim to assess the cofactorTBLR1 function, expression, and localization in prostate cancer.

METHODS: Dual luciferase assays were performed using a re-porter plasmid containing 4Xandrogen responseelements. pBabe vectorcontaining TBLR1 fusion proteins with the strong NLS(PKKKRKV) orNES (MLQKKLEELE) fused to the N-terminus was used to create stablecell lines and proliferation was measured by WST-1 assay. RNA levelswere measured using quantitative RT-PCR and protein levels weremeasured by western blotting. ChIP was performed to determine theactivation of AR target genes by TBLR1. Immunohistochemistry usingTBLR1 antibody was performed on prostate cancer tissue microarray.

RESULTS: We showed TBLR1 functions as a coactivator of ARin prostate cancer cells and the activation is both phosphorylation and19S proteosome dependent. TBLR1 primarily localizes in the nucleus inbenign prostate cell lines and tissue. In contrast, there is a significantreduction in nuclear TBLR1 expression in prostate cancer (PCa) celllines and human tissue. Serum starvation induced growth arrest incancer cells leads to translocation of TBLR1 expression from cyto-plasmic to nuclear. Stable ectopic expression of nuclear TBLR1 leads toin vitro and in vivo growth suppression in AR-positive LNCaP cells byactivation of androgen regulated genes responsible for differentiation/growth suppression of the prostate (e.g. NKX3.1, KRT18). Interestinglyhowever, TBLR1 expressed in the cytoplasm promotes growth andgrowth in androgen free conditions.

CONCLUSIONS: There are distinct tumor suppressor andoncogenic functions of nuclear TBLR1 and cytoplasmic TBLR1respectively. Nuclear TBLR1 selectively activates AR target genesimportant for growth suppression, whereas cytoplasmic TBLR1 plays arole in promoting growth in the absence of androgens. Further char-acterizing the role of TBLR1 in prostate could provide novel insights indeveloping strategies for prostate cancer therapy.

Source of Funding: NYUMolecular Oncology and ImmunologyTraininggrant (T32CA009161) postdoctoral fellowshipsNationalInstitutes of Health to GD

MP49-05APPLICABILITY OF MACROPHAGE INHIBITORY CYTOKINEL1 ASA POTENTIAL BIOMARKER FOR RACIAL DISPARITY INPROSTATE CANCER

Seema Dubey, Jo Wick, Ossama Tawfik, Daniel Zainfeld*,Jeffrey Holzbeierlein, Peter Van Veldhuizen, Brantley Thrasher,Dev Karan, Kansas City, KS

INTRODUCTION AND OBJECTIVES: Prostate cancer is asignificant health problem for men in the United States that dispropor-tionately affects African American (AA) men in both incidence andmortality rate in comparison to Caucasians. Although there is no clearevidence for the cause of such disparity, it is likely that differences in thebiology of prostate tumor may contribute significantly to the aggressivenature of prostate cancer in AA men. In this pilot study, we sought toexamine if serum MIC�1 (macrophage inhibitory cytokine�1) providesany predictive capability for the severity of prostate cancer inpre�surgical diagnosed males.

METHODS: Serum samples for 40 Caucasians and 40 AA menwere obtained. Serum MIC�1 level was measured by sandwich ELISA.Due to the non�normality of MIC�1 and PSA, natural log trans-formations were used to meet the assumptions of correlation andregression analyses. Differences between AA and Caucasians wereidentified using Wilcoxon tests for continuous variables and Fisherexact tests for categorical variables. Pearson’s correlation coefficient,univariable linear regression, and analysis of covariance were used toidentify significant associations between continuous outcomes anddifferences among races. All p�values reported are two�sided, and ana priori 5% level of significance was used.

RESULTS: Forty Caucasian and forty AA men between theages of 43 and 75 years (Median ¼ 60 years) were analyzed. Highlysignificant differences among the two races were found in MIC�1 (p ¼0.0001) and Gleason scores (p ¼ 0.0009), with AA having higherMIC�1 expression (Median 1220.4 versus 790.8) and Gleason scores(Median 7 versus 6) than Caucasians, on average. PSA was also

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significantly higher in AA (Median 6.72 versus 6.35, p ¼ 0.04). No dif-ferences in age or stage of disease were detected between groups(p > 0.05). In Caucasians, MIC�1 expression was positively associatedwith PSA (p < 0.01), and age (p > 0.0001), while Gleason score waspositively associated with PSA (p < 0.05) and age (p < 0.05). Log�transformed PSA and MIC�1 were used for valid inferences. Thus,higher levels of MIC�1 expression and higher Gleason scores wereassociated with older patients when limiting our sample to Caucasians.In AA, however, both older and younger patients had highly expressedMIC�1 and high Gleason scores.

CONCLUSIONS: Although a detailed sample analysis isrequired, these observations indicate that addition of MIC�1 may helpto improve the diagnostic capability of an aggressive stage of prostatecancer at least in African American men.

Source of Funding: none

MP49-06GENOMIC ANALYSIS OF MULTIPLE PROSTATE CANCERMETASTASES HIGHLIGHTS METASTASIS FROM LOW-GRADECANCER FOCI AND DYNAMIC ADAPTATIONS TO THERAPY

Matthew Hong*, Geoff Macintyre, Clare Sloggett, Marek Cmero,Andrew Lonie, Haroon Naeem, Melbourne, Australia; David Wedge,Peter van Loo, Ludmil Alexandrov, Cambridge, United Kingdom;Nikhil Sapre, Pramit Phal, Xiaowen Chin, Michael Kerger,John Pedersen, Andrew Ryan, Anthony Costello, Melbourne, Australia;Ultan McDermott, Cambridge, United Kingdom; Niall Corcoran,Christopher Hovens, Melbourne, Australia

INTRODUCTION AND OBJECTIVES: Genomic studies of mul-tiple samples of metastatic prostate cancer matched with primary tumoursamples from the same individuals can allow inference of evolutionaryprocesses behind cancer progression and provide a biological rationalefor many clinical questions. In this genomics study of paired samples ofprimary and serial metastases, taken pre- and post-castration from thesame individual, we examine the response of a metastasis to androgendeprivation and the origin of metastases in multifocal prostate cancer.

METHODS: A 71 year-old patient underwent radical prostatec-tomy for organ-confined Gleason 3+4 prostate cancer. Within 18 monthshe suffered from local recurrence and metastatic relapse. Fresh frozentumour samples were taken from his original prostatectomy specimen.Local recurrence was sampled at transurethral resection. Percutaneousbiopsy of bone metastasis was performed prior to androgen deprivationand subsequently following treatment. Tissue samples were cryosec-tioned and tumour content confirmed by a pathologist prior to mechanicalhomogenisation and simultaneous DNA and RNA extraction. Othertumour foci from the archival prostatectomy specimen were macro-dissected andDNAextracted.Whole genomesequencingwas performedto 40x coverage and transcriptome sequencing to 120 million reads bothon the Illumina HiSeq2000 platform. The Illumina HumanOmniExpress-FFPE BeadChip was used for SNP analysis on archival tissue DNA.

RESULTS: All the fresh frozen samples shared 223 mutations,with 504 unique to the sampled primary cancer, 3200 mutations uniqueto the mutations and the local recurrence. Subclonal analysis revealedthat the local recurrence sample was most closely linked with the post-androgen deprivation metastasis sample, suggestive of metastatic re-seeding from the local recurrence after commencement of androgendeprivation therapy. Analysis of the archival tissue DNA samples fromdifferent foci of primary cancer to determine the origin of metastasisrevealed evidence to suggest that a Gleason 6 focus of adenocarci-noma at least contributed subclones to the various metastatic sites.

CONCLUSIONS: Control of local disease may be important inthe treatment of metastatic prostate cancer, as it could serve as areservoir of resistant subclones capable of re-seeding distant sites. Thediscovery of metastatic subclones from Gleason 6 foci may have im-plications for focal therapy and active surveillance.

SourceofFunding:MHhasbeensupportedbyscholarships fromthe Royal Australasian College of Surgeons, the National Healthand Medical Research Council, Australia, and the Melville

Hughes Scholarship, Faculty of Medicine, Dentistry and HealthSciences, University of Melbourne. GM is supported by NICTA.NICTA is funded by the Australian Government as representedby the Department of Broadband, Communications and theDigital Economy and the Australian Research Council throughthe ICT Centre of Excellence program.

MP49-07THE ANDROGEN RECEPTOR IS A DRIVER OF DNABREAKPOINTS AND FUSION EVENTS IN PROSTATE CANCER

Matthew Hong*, Geoff Macintyre, Niall Corcoran, Clare Sloggett,Haroon Naeem, Marek Cmero, John Pedersen, Melbourne, Australia;Izhak Haviv, Safed, Israel; Andrew Ryan, Pramit Phal,Anthony Costello, Christopher Hovens, Melbourne, Australia

INTRODUCTION AND OBJECTIVES: The TMPRSS2-ERGfusion is the most well-known fusion event in prostate cancer. Sincespecific fusion genes are associated with specific tumour types, it islikely that tissue-specific mechanisms underlie the formation of struc-tural rearrangements in cancer genomes. Recent studies have sug-gested that androgen receptor (AR) may play a role in the formation ofTMPRSS2-ERG fusions, bringing the two loci in close proximity in thenucleus and facilitating DNA strand break and repair along with ARassociated enzymes. To explore this mechanism more comprehen-sively, we performed whole genome sequencing on 14 prostate cancersamples from 7 patients.

METHODS: Fresh frozen prostate cancer samples were takenfrom patients undergoing radical prostatectomy or biopsy. Tissue sam-ples were cryosectioned and tumour content confirmed by a pathologistprior to mechanical homogenisation and simultaneous DNA and RNAextraction (Allprep Micro Kit, Qiagen). Whole genome sequencing wasperformed to 40x coverage on the Illumina HiSeq2000 platform.

RESULTS: Across the 14 cancer genomes we identified abase-pair resolution high confidence set of 2,921 structural variations(some recurrent) at an average of 209 per sample and found that a largeproportion of these breakpoints were in close proximity to curatedandrogen receptor binding sites from high-confidence ChIP-Seq data.Furthermore, when we examined breakpoints in genome datasets from11 other cancers from the TCGA and ICGC projects, we identified asimilar association with androgen (and estrogen) receptor binding sitesspecifically in hormone-dependent tumour types including prostate,breast, squamous cell lung, and ovarian cancers, but no such correla-tion in glioblastoma, renal, lung, colon or lymphoma genome datasets.

CONCLUSIONS: These data suggest that the androgen re-ceptor drives genome wide breakpoints and novel fusion events inprostate cancer and potentially serves to explain why androgen expo-sure is necessary for prostate cancer development.

Source of Funding: MH has been supported by scholarshipsfrom the Royal Australasian College of Surgeons, the NationalHealth and Medical Research Council, Australia, and theMelville Hughes Scholarship, Faculty of Medicine, Dentistry andHealth Sciences, University of Melbourne. GM is supported byNICTA. NICTA is funded by the Australian Government asrepresented by the Department of Broadband, Communicationsand the Digital Economy and the Australian Research Councilthrough the ICT Centre of Excellence program.

MP49-08WNT/b-CATENIN SIGNALLING IS A POTENTIAL THERAPEUTICTARGET FOR CASTRATE-RESISTANT PROSTATE CANCER.

Yoshiaki Kawano*, Yoshihiro Maeda, Takanobu Motoshima,Takahisa Imamura, Wataru Takahashi, Yoshihiro Wada, Kumamoto,Japan; Robert Kypta, London, United Kingdom; Masatoshi Eto,Kumamoto, Japan

INTRODUCTION AND OBJECTIVES: Wnt/b-catenin pathwayis aberrantly activated in substantial fraction of castration-resistant