Patients receiving a t least one dose of the study drug(s) will be included in both safety and effica cy analyses. 10.2 Study Endpoints 10.2.1 Analysis of the primary endpoints 10.2.1.1 CD8+ T cell infiltration The primary efficacy objective of this study is to quantify the extent of CD8+ T cell infilt ration into the prostate gland in the two treatment arms, and to compare them. This outcome will be expressed as the mean CD8+ T cell staining per centage in harvested prostate tissues, and will be reported separately for each treatment arm. The standard deviation, 95% confidence interval, median , and range of values will also be reported where appropriate. Since the CD8 + T cell quantity is a ratio variable and the distribution is skewed, the log transformation will be used for the analysis . Tissue microarrays (TMAs) using the highest -grade/largest tumor per patient and sampling it with 100-fold redundancy will be used for CD8+ T cell quantification. We expect that 3 - 50% of the spots per patient will be assigned a carcinoma diagnosis by the study pathologist. Cores with both tumor and normal tissue will be excluded if they contain >10% of the other component. The percent posi tive staining score for CD8 + T cells in the spots classified as tumor will be used to quantify the primary outcome. The mean will be used to pool multiple spot measurements for each patient. Following a log transformation, means will be compared between t reatment arms using a two -way analysis of variance (ANOVA) with the stratification variable (Gleason score) treated as a block factor. Boxplots and descriptive summaries by treatment arm will also be provided as appropriate. To account for co - morbidities a nd other immunosuppressive factors as potential confounding factors for treatment comparisons, multivariable regression analysis will be performed. The model will consider Gleason score and disease stage as covariates . 10.2.1.2 Safety and feasibility An a dditional co-primary endpoint is safety. To this end, t he frequency of adverse events will be described separately for each treatment arm using summary statistics. The proportion of patients in each arm with an adverse event will be reported with an exact binomial 95% Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 50 of 68 J1265 PROT (Vers) 1.4 113012 confidence interval. All subjects receiving at least one dose of the study drug(s) will be evaluable for toxicity . Early stopping rules for safety: Safety will be monitored closely in the degarelix plus cyclophosphamide/GVAX arm of the study (arm B). The stopping rule for safety is based on the posterior probability that the adverse event (AE) rate is too high. Any systemic symptoms (fevers, rash, myelosuppression) compromising the planned surgery or any grade -3/4 local reactions (erythema, s welling, pain) attributable to the cyclophosphamide or GVAX immunotherapy will be considered AEs for purposes of this stopping rule. We expect that the adverse event rate will be low. Trial enrollment will be suspended for a safety evaluation if the AE rate convincingly exceeds 25%. This stopping rule will halt enrollment if the posterior probability of the AE rate exceeding 0.25 is 70% or higher. The prior probability for this toxicity monitoring rule will be a Beta -distribution with parameters of 2 and 8, assuming that 2 of 8 patients may experience an AE. This distribution corresponds to assuming a 1 -in-4 chance that the risk of AEs is 25% or higher and 90% certainty that the risk is between 4% and 43%. The stopping rule applies this prior distribution to the observed number of patients experiencing an AE and computes the resulting probability that the rate is too high. If the posterior certainty that the rate is too high based on Bayes’ rule and these assumptions is 70% or higher, the study should stop. The following table shows the resulting stopping rules. Study termination if: 2 AEs 3 AEs 4 AEs 5 AEs 6 AEs And number of patients between: 2 3 - 5 6 - 9 10 - 12 13 - 16 For example, the rule will call for stopping the study if 4 patients out of the first 6 experience AEs. The next table shows the percent age of the time that the stopping rule will terminate the study under different hypothetical risks of AEs, along with the average sample size (based on 5000 simulations). Risk of AE 0.10 0.20 0.25 0.30 0.35 0.40 % of Time study s tops 2.2% 16% 29% 45% 60% 75% Expected sample s ize 15.8 14.6 13.5 12.3 11.0 9.5 Early stopping rule for feasibility: Androgen deprivation therapy (ADT) does not independently alter the surgical approach to prostatectomy and we do not anticipate an increase in the surgical difficulty with the use of neoadjuvant degarelix. Men who have had acute (post- biopsy) and prolonged issues (chronic prostatitis) with prostate infection/inflammation are fairly routine in urologic surgical practice, and we anticipate that the immune infiltrate potentially induced by GVAX would not pose a substantial increase in surgical difficulties. However, i n addition to safety, feas ibility will be monitored separately in both arms of the study. The feasibility rule for this st udy will be based on a change in surgical outcomes beyond what may be expected for patients without presurgical interventions and that may be attributable to th e study drugs (degarelix, or th e combination of degarelix plus cyc lophosphamide/GVAX treatment). These events would include: (1) average blood loss in excess of 2500 mL, (2) average operative time in excess of 3.5 hours, and (3) average hosp ital stay in ex cess of 4 days. These values are approximately 2 standard deviations above the average surgical outcomes for men undergoing radical prostatectomy at Johns Hopkins Hospital. We require that the probability that the surgery will not be complicate d by these events to be high (≥90%). We will monitor this endpoint after every patient. If it becomes apparent that the surgeries are being negatively impacted by the pre- surgical investigational treatments, then the study will be suspended for review. This stopping r ule will halt Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 51 of 68 J1265 PROT (Vers) 1.4 113012 enrollment if the posterior pr obability of no complications <0.90 is greater than 80%. The prior proba bility for this rule has a Beta -distribution with parameters of 9 and 1, based on the expectation that these treatments will not impact the feasibility of surgery. Instances where the study would be temporarily suspended are listed below. Stopping rules : • Not f easible if both of the first 2 patients in an arm have surgical compli cations . • Not f easible if two of the first 3 patients in an arm have complications. • Not f easible if 2 out of the first 4 patients in an arm have complications. • Not f easible if 3 out of the first 6 patients in an arm have complications. • Not f easible if 4 out of the first 8 patients in an arm have complications. • Not f easible if 5 out of the first 10 patients in an arm have complications. • Not f easible if 6 out of the first 12 patients in an arm have complications. • Not f easible if 7 out of the first 14 patients in an arm have complicatio ns. • Not f easible if 8 out of 16 patients in an arm have complications. 10.2.2 Analysis of the secondary endpoints The seconda ry endpoints of this study have previously been defined (see Section 8.3). The statistical analysis of these endpoints is described below. Data transformation will be performed when they are not normally distributed. • Regulatory T cell (T reg) density . The methods for analyzing T reg infiltration will be similar to those desc ribe d for the primary endpoint. Descriptive statistics and graphical summaries by treatment arm for these outcomes will be provided. In addition, the CD8+/T reg ratio and the CD4+/T reg ratio will be computed, and reported using descriptive statistics. Compariso ns between treatment arms will be made using two-way ANOVA. • CD4+ T cell density. The methods for quantifying and analyzing CD4+ T cell infiltration will be similar to those described for the primary endpoint ( i.e. CD8+ T cell infiltration). Descriptive sta tistics and graphical summaries by treatment arm for these outcomes will be provided. Comparisons between treatment arms will be made using two -way ANOVA . • Tissue androgen concentrations . Intraprostatic androgen concentrations ( i.e. testosterone and dihydrotestosterone) will be summarized descriptively by study arm, and comparisons across arms will be made using two -way ANOVA. • Androgen receptor (AR) quantification . Androgen receptor (AR) staining concentrations will be summarized des criptively by study arm, and comparisons across arms will be made using two-way ANOVA. • Apoptotic markers . Caspase 3 staining (and/or TUNEL assay results) will be expressed as the mean staining percentage in tumor samples, and reported separately for each t reatment group. Standard deviations, 95% confidence intervals, and ranges will also be reported where appropriate. Means will be compared across treatment arms using two -way ANOVA. • Proliferation markers . Ki-67 staining will be expressed as the mean staining percentage in tumor samples, reported separately for each treatment arm. Standard deviations, 95% confidence intervals, and ranges will also be reported where appropriate. Means will be compared across treatment arm s using two -way ANOVA . • Pathological complete responses (pCR) . This will be defined as an absence of tumor identification on standard histological analysis of the resected prostate specimens. The proportion of patients achieving a pCR will be compared between treatment arms using the Mantel -Haenszel test . Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 52 of 68 J1265 PROT (Vers) 1.4 113012 • Novel anti-p rostate antibodies . Induction of novel anti -prostate antibodies will be assessed with a high -throughput immunoblot (HTI) assay, before and after treatment initiation. The change in immunoreactivity will be evaluated for each antigen and scored as a gain, loss, or no change. These data will be visualized per patient for both tr eatment groups using heatmaps. A comparison of the proportions of patients with these ordered outcomes between the treatment arms will be made using an e xact version of the Cochran -Armitage test for trend (Armitage 1955). Antibody responses will be dichotomized as “gain” versus “loss or no change” and correlated with PSA response and pathologic complete response (pCR) using χ2-tests or Fisher’s exact tests . Antibody responses will be correlated with time- to-PSA -recurrence using Kaplan- Meier curves and log -rank tests . The correlation of antibody responses with CD8 concentrations, CD4 concentrations, and T reg concentrations will be explored using Spearman’s c orrelation coefficient ( ρ). • PSA response rates. This will be defined as an undetectable PSA (<0.1 ng/ mL) at 3 months after prostatectomy. The proport ion of patients achieving a PSA response will be comp ared between treatment arms using the Mantel -Haenszel test . • Time to PSA recurrence . This will be defined as the interval from prostatectomy to the time when the serum PSA is ≥0.2 ng/mL. For subjects who have not yet demonstrated PSA recurrence at the time of censoring, patients will be censored at the date of the last assessment that show s a lack of PSA recurrence. For each treatment arm, t he med ian time to PSA recurrence after prostatectomy (i.e., the median PSA -recurrence -free survival) will be estimated with 95% confidence intervals using Kaplan-Meier survival analysis . Comparisons will be sought using the log -rank test, stratified by Gleason score . 10.3 Analysis Populations 10.3.1 Intention -to-treat population All patients who meet eligibility criteria and receive at least one dose of the study drug(s ) will be included in the analysis of the primary and secondary endpoints , even if there are subsequent protocol deviations. However, in cases where prostatectomy is not performed or if adequate surgical tissue is not collected, then determination of the primary endpoint (tissue CD8 + T cell analysis) and some secondary endpoints (apoptosis/proliferation marker analysis) will not be possible. Patients will be replaced if they are removed from the study after signing the informed consent but before receiving the study drug(s) . 10.3.2 Safety population All patients enrolled in the study will be included in the safety analysis population and conside red evaluable for toxicity from the time of their first dose of the study drug (s). Patients never receiving any of the study drugs will not be included in this analysis. Demographic and baseline characteristics for the safety population will be summarized by number and percent for categorical data and by descriptive statistics for continuous data. 10.4 Safety Analysis 10.4.1 Evaluation of adverse event s Treatment -emergent adverse events will be translated from investigator terms to MedDRA version 6.0 terminology and summarized (number and percentage of patients) for all patients who receive at least one dose of the study drug (s). Adverse event summaries will be organized by body system, frequency of occurrence, intensity (i.e. , severity grade), and causality or attribution. Patients who experience an adverse event more than once will be counted only once.