8. OUTCOME ASSESSMENT 8.1 Radical Prostatectomy Specimen Most of the study outcomes in this immunologic trial will depend on collection of prostate tissue from prostatectomy specimens. All pathologic specimens will be handled in routine fashion by the operating room (OR) staff, except that as soon as the specimen is removed the OR nurse will directly page a tissue harvesting technician tha t is part of the Brady Urological Research Institute Prostate Specimen Repository Team. Accessioning of pathology specimens will be coordinated in the OR areas by the Departments of Urology and Pathology. A study technician will be available to the patholo gist receiving the specimen to assure that the tissue is handled appropriately for the intended bioassays. At the time of harvesting, the pathologist will apply ink and cut the prostate specimen in transverse sections. The specimen will be fixed in 10% phosphate buffered formalin, and tissue blocks will be paraffin -embedded and sectioned at 4 μm thickness for routine histologic evaluation and for immunohistochemical determinations. Fixation should occur as soon as possible after operative removal of the pro state, and ideally within 30 -60 minutes. Assessment of index tumors for Gleason grade, nodal involvement, and pathologic staging will be conducted in usual fashion and will be provided to the patient for prognostic information. After the pathology report i s available, a database will be established and all information from the pathology reports of all samples will be included. Tumor blocks and/or additional unstained slides will be collected for study -specific quantitative immunohistochemical evaluations . Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 43 of 68 J1265 PROT (Vers) 1.4 113012 8.2 Primary Endpoint s 8.2.1 CD8+ T cell infiltration The primary measure of treatment effect in this study will be achieved by quantify ing the extent of CD8+ T cell infiltration into the prostate from harvested prostate glands in men with high -risk localize d prostate cancer receiving neoadjuvant ADT alone or in sequence with low -dose cyclophosphamide and GVAX (administered three weeks prior to radical prostatectomy) . This will be done using immunohistochemical staining methods. We will also attempt to quant ify prostatic CD4+ T cell infiltration and Treg infiltration, as well as to determine the CD8 /T reg ratio and the CD4 /T reg ratio . The primary endpoint will be expressed as the mean CD8+ T cell stainin g percentage in harvested tumor tissue, and will be analyzed separately for each treatment arm. Analysis of the primary endpoint will be achieved by preparing tissue mic roarrays (TMAs) using the highest -grade/largest tumor per patient and sampling it with 100-fold redundancy. All tissues will first be fixed in 10% neutral buffered formalin and processed into paraffin blocks. For each immunohistochemical stain ( e.g. CD8+ T cell stain, CD4+ T cell stain, T reg stain ), TMA slides will be scanned using the Aperio ScanScope® CS instrument (Aperio Technologies , Vista , CA) and TMA cores will first be assigned a diagnosis by the study pathologist and will then be subjected to semi -automated image analysis using the Aperio system . For each biomarker, we will divide the area of brown DAB staining by the area of epit helial cells on the TMA co re, obtaining a staining percentage. The area of epithelium will be obtained on each TMA core by staining with cytokeratin -8 and using automated image analysis. Cores with both tumor and normal tissue will be excluded if they cont ained >10% of the other component. For CD8 staining , slides will be steamed for 20 minutes in citrate antigen retrieval solution (Vector Laboratories, Burlinga me, CA) followed by incubation with a mouse monoclonal anti -CD8 antibody for 45 minutes at room temperature (Dako, Carpinteria, CA ). For CD4 staining, slides will be steamed for 40 minutes in high pH antigen retrieval solution (Dako, Carp interia, CA) and then incubated with a mouse monoclonal anti -CD4 antibody for 45 minutes at room temperature (Sero tec, Kidlington, UK) . For T reg analysis, cells will be stained for the FoxP3 protein by steaming slides for 40 minutes in high pH antigen retrieval solution (Dako, Car pinteria, CA) and incubating them with a mouse monoclonal anti -FoxP3 antibody for 45 minu tes at room temperature (eBioscience, San Diego, CA, 1:1000 dilution). In all cases, p oly-HRP -conjugated anti -mouse IgG Ab (Dako, Carpinteria, CA ) will be used as the secondary antibody. Stai ning will be visualized with diaminobenzidine (Sigma, Saint Louis, MO) and slides will be counterstained with hematoxylin . Images of each TMA core will be captu red by automated scanning of TMA slides usi ng the Aperio ScanScope® CS instrument (Aperio Technologies , Vista , CA). Captured images will be imported into the TMAJ Images Application program (http://tmaj.pathology.jhmi.edu ). Histological diagnoses (normal, atrophy, pr ostatic intraepithelial neoplasia , cancer) will be applied to all images used for the analyses by a pathologist. In addition, for TMA spots containing more than one type of lesion, the percentage of each diagnosis will be noted . All images and data will be available for viewing/ download ing at http://demarzolab.pathology.jhmi.edu/Pubs.html . For image analysis , we will use a custom open source software package, FRIDA (Fr amework fo r Image Dataset Analysis; http://sourceforge.net/projects/fridajhu ), for the evaluation of red -green -blue (RGB) color image datasets, including those generated from scann ing of tissue microarray slides. To analyze CD4, CD8 and FoxP3 (Treg) staining, hue -saturation -brightness (HSB) segmentation ranges for brown DAB staining will be defined from the tissue microarray image set, and the total number of pixels in every image that fall within the defined parameters for brown DAB Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 44 of 68 J1265 PROT (Vers) 1.4 113012 staining will be calculated, reflecting the total area of b rown DAB staining for each spot. For every spot, a “staining ratio” for each of the three proteins will be calculated by dividing the total area (in pixels) of brown DAB staining by the average TMA spot area. Previous TMA studies conducted by Angelo DeMarz o, MD PhD and Charles G. Drake , MD PhD have defined the extent of T cell infiltration into human prostate glands using prostatectomy specimens . In normal prostate tissue, the mean staining percentage for CD8+ T cells is 0.29% (interquartile range , IQR, 0.13% - 0.39%) while in tumor tissue, the mean percentage of CD8+ T cells is 0.42% ( IQR 0.07% - 0.71%) . The corresponding values for CD4+ T cell infiltration in normal and tumor tissue are 0.16% ( IQR 0.04% - 0.18%) and 0.25% ( IQR 0.01% - 0.32%) , respectively . The corresponding values for T reg infiltration in normal and tumor tissue are 0.03% ( IQR 0.02% - 0.04%) and 0.06% ( IQR 0.02% - 0.08%) , respectively (Gurel and DeMarzo, unpublished data). These values are helpful for the determination of sample size/power calculations in the present study (see Section 10.1). The above an alyse s will be performed in the laboratory of Angelo DeMarzo, MD PhD. A detailed description of these methodologies has previously been published ( Zha et al 2001; Faith et al 2004; Gurel et al 2008). The contact information for the DeMarzo laboratory is listed below: Angelo DeMarzo, MD, PhD CRB 1 - Room 151 1650 Orleans Street Baltimore, MD 21231 Phone: (410) 614-5686 Fax: (410) 502- 9817 Email: ademarz@ jhmi.edu 8.2.2 Rate of adverse events All subjects receiving at least one dose of the study drug (s) will be evaluated for safety by monitoring symptoms, physical examinations, and laboratory tests. Adverse events will be classified and graded according to the NCI Common Toxicity Criteria version 4 .0 (see Appendix B).