Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014 THE JOURNAL OF UROLOGY� e433

HSP90 inhibitor) as well as STA9090 and AUY922 (second genera-tion inhibitors). Cells were also treated with the HSP70 inhibitor VER155008 alone and in combination with a HSP90 inhibitor. Cell prolif-eration rates were determined by MTT assays, while the expressionof HSPs, apoptotic and cell signaling markers were determined byWestern analysis.

RESULTS: UMUC3 cells showed the most sensitivity of allcell lines. Fifty percent growth inhibition occurred within 48H oftreatment using 10 nM of the second generation inhibitors. Thissame degree of inhibition required 10-fold higher concentrations of17-AAG. STA9090 exhibited the highest efficacy showing 80%growth inhibition at 100 nM for all the cell lines tested. All cell linesshowed a 5-10 fold increase in the expression of HSP70 within 24H of treatment with HSP90 inhibitors. In addition there was also a50% increase in the expression of HSP40, 60 and 110. Since theoverexpression of other HSPs may compensate for the inhibition ofHSP90, we sequentially inhibited the expression of HSP90 withSTA9090, and HSP70 with VER 155008, and determined the effectson cell proliferation. The combination of drugs showed a synergisticinhibition of growth with 78% inhibition at 24H compared to 68%inhibition with VER155008 alone, or 43% inhibition with STA9090alone. HSP90 inhibition induced apoptosis as demonstrated byincreased expression of cleaved caspase 3 and downregulationof mediators of several oncogenic signaling pathways includingEGFR, ErbB2, cyclin D1, pAKT, pSTAT3/5, pS6, p4EBP1 in the celllines tested.

CONCLUSIONS: HSP inhibitors as monotherapy have shownpromising preclinical efficacy but this has not been replicated inclinical trials. Data from this study suggests that combinationHSP90/70 inhibition is superior to either alone in a bladder cancercytotoxicity model and the significance of this in the clinical settingremains to be seen.

Source of Funding: This work was supported by internal grantSRC-S-22 from the Geisinger Health System to HW.

MP39-19L-SELECTIN (CD62L) EXPRESSION IN HIGH GRADEUROTHELIAL CARCINOMA AS A POTENTIAL MARKER OFMETASTATIC DISEASE

Dharamainder Choudhary, Poornima Hegde, Shilpa Choudhary,Kevin Claffey, Pramod Srivastava, Carol Pilbeam, John Taylor III*,Farmington, CT

INTRODUCTION AND OBJECTIVES: L-selectin (CD62L) is avascular adhesion molecule constitutively expressed on leucocyteswith a primary function of directing leucocyte migration and homingof lymphocytes to lymph nodes (LNs). Microarray data from lasercaptured, micro-dissected human specimens of high grade muscleinvasive (MIBC) vs. low grade (LGBC) bladder cancers foundCD62L to be the highest differentially expressed gene between thegroups. We further characterized the mRNA and protein expressionof CD62L in high grade cancer and its potential as a marker formetastatic disease.

METHODS: MIBC and LGBC fresh frozen, paraffin embeddedand serum samples were obtained from the UCHC tumor bank. mRNAwas evaluated by quantitative polymerase chain reaction (qPCR) andprotein levels by immunohistochemistry (IHC) and enzyme linkedimmunosorbant assay (ELISA). Flow cytometry (FACS analysis) wasused to identify the relative number of cells expressing CD62L in freshtumor tissue. In silico studies were performed using the Onco-mine Database.

RESULTS: Quantification of CD62L transcripts in high grademetastatic MIBC vs. LGBC frozen specimens(microarray; 3.81 �2.94 vs 0.06 � 0.01 p¼0.04 ,confirmed by qPCR 0.14 � 0.04 vs.23.31 � 22.47) and immunohistochemistry on paraffin embeddedhigh grade metastatic MIBC vs. LGBC specimens confirmed theelevated expression of CD62L in MIBC samples. Localization of

CD62L was also confirmed in discrete foci of bladder tumor cells inLN specimens of high grade metastatic disease. Upregulatedexpression of CD62L (9.4-fold, p<0.01) was observed by FACSanalysis of freshly isolated tumor cells from high grade cancers vs.LGBC specimens. Circulating CD62L levels were also found to behigher (640 � 75 vs. 558 � 34 ng/ml) in serum samples from pa-tients with high grade metastatic vs. high grade non-metastaticMIBC. Furthermore, in silico analysis using the Oncomine microarraydatabase showed an association of CD62L expression with tumoraggressiveness and clinical outcomes.

CONCLUSIONS: Our preliminary findings are the first reportof increased expression of CD62L in MIBC and suggest a role in met-astatic spread to LNs. CD62L could act as a potential marker for pre-dicting patients who are at high risk of developing or harboringmetastatic disease.

Source of Funding: ACS-MRSG 08-270-01-CCE andThe Leo and Anne Albert Charitable Trust (JAT), NIHR01DK48361(CCP)

MP39-20ALEATORIC LARGE DATASET ANALYSIS FRAMEWORK:A PRACTICAL APPROACH TO MINING PUBLIC EXPRESSIONDATA FOR CANCER RESEARCHERS

Jonathan A. Ewald*, Howard H. Bailey, William A. Ricke,Tracy M. Downs, Madison, WI

INTRODUCTION AND OBJECTIVES: Public archives ofexpression microarray data provide a powerful resource for cancerresearch that can save valuable resources by allowing existing data tobe re-purposed. These results from previous and current studiesinclude data from in vitro experiments and analyses of clinical patient-derived tissues, including tumors. The availability of these data to the at-large cancer research community can promote the development ofinnovative ideas that otherwise may not occur to a professionaldata analyst. However, many investigators avoid this resource due to aperception that these data are inaccessible and too big for a cancerbiologist or clinician to manage and interrogate effectively. Recent ad-vances in bioinformatics software development have produced open-access tools more friendly to use by the general population. Ourobjective was to develop a practical, versatile workflow to guide cancerresearchers’ use of public expression microarray data for hypothesisdevelopment and validation.

METHODS: In our experiences exploring and interrogating thisresource, we have developed a simple, intuitive framework largelythrough trial-and-error to guide our selection, comparison, and analysis.

RESULTS: This approach, called ALDAF (Aleatoric LargeDataset Analysis Framework), provides a rules-based needs-drivenstrategy that is adjustable and adaptable to diverse research goals.This includes: (1) Exploring and Identifying Datasets; (2) CalculatingDifferences; (3) Selecting Data: Top-Down; (4) Selecting Data: Bot-tom-Up; (5) Bioinformatics Analysis and Interpretation; and (6) Vali-dation of Selected Data. Data is available through the NCBI GeneExpression Omnibus (GEO), which can be searched according tokeywords to identify applicable data. To compile, compare, identifyand select data, we use Microsoft Excel to automate many of thesetasks using formulas that simplify and accelerate this process.Resulting lists of genes can then be analyzed and interpreted usingopen-access web-based bioinformatics programs including Web-Gestalt, DAVID, Chilibot, and PubGene/Coremine. Gene lists canalso be verified by iterative analysis of additional independent data-sets to further select genes of interest, or validated by novel originalexperiments.

CONCLUSIONS: We conclude that ALDAF provides a simple,intuitive and practical means for cancer researchers to utilize publicexpression microarray data.

Source of Funding: None