All experiments were done three times with similar results. Statistical analyses were done by Student’s t-test Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 181.2.7 Rationale for conducting th e study in nmCSPC patients The standard of care for patients with nm CSPC includes ADT and active surveillance. Generally, these patients can be followed without therapy until their PSA doubling time escalates at which time ADT is often starte d. All patients in the proposed tr ial will receive enzalutamide. Based on previous trials with high dose bical utamide, 34 of 36 evaluable patients had PSA declines by a median of 96.5% and these nadirs were achieved within the first 4 weeks of treatment ( 10). We would expect similar if not better results with enzalutamide. Therefore, a clinical trial in this population is ethical and appropriate. The NC I has conducted several trials in this population previously. This population will also offer unique opportuni ty to evaluate the independent impact (in the absence of ADT) of enzalutamide on both PS A as well as the immune system. Much like previous studies with high-dose bicalutamide in this population, s imilar trials are likely to be done to see if disease control with enzalutamide is possible in nmCSPC, with less side effects relative to ADT ( 10). Given the previous experience with high dose bicalutamide in this population, a study with time to progression as an end point with c ontinuous enzalutamide would be expected to take well over 2 years ( 10). We feel that using a short course of enzalutamide therapy of 3 months we can assess the immunologic impact and determine the impact of vaccine on tumor re-growth rates. Therefore, for practical considerations (number of patients required and length of time to conduct the trial with a clinical progressi on endpoint) this trial will be conducted over a minimum of 6-12 months, including a 3 month course of therapy of enzalutamide. This proposed brief 3 month enza lutamide course of therapy is consistent with a standard approach of intermittent ADT ( 36,37). In the intermittent ADT approach in nmCSPC, one or two doses of ADT are given and then PSA is followed for recovery at which time subsequent ADT dosing is considered. The same intermittent strategy will be done in this trial, substituting enzalutamide for ADT. Furthermore, this popula tion allows for the unique opportunity to follow PSA/tumor re-growth rate/recovery once a standard therapy is discontinued. Consistent with common clinical practice no immediate re-treatment will be required in the months following discontinuation of enzalutamide. Patients would be started on ADT as clinically appropriate, as suggested by their PSA doubling time. 1.2.8 The Importance of Evaluating the Immunologic Response Our group at in the LTIB has previously ev aluated immunologic parameters in clinical trials with PSA-TRICOM among several immunothe rapy studies. A previous trial in mCRPC patients with PSA-TRICOM alone suggested that patients with greatest magnitude of T-cell- specific response against PSA had favorable clinical outcomes ( 21). That same trial also suggested that changes in regulatory T-cell function were also associated with improved clinical outcomes ( 38). While the these findings are not surrogate markers of response, they have improved our knowledge of a va ccine-generated immune res ponse and provided a better understanding of what factors are potentially important in mounting a sufficient anti-tumor immune response that could be associated with improved clinical outcomes. These and other data have allowed us to optimize vaccines in subsequent clinical trials. Similarly, immunologic paramete rs will be valuable in unde rstanding the benefits of PSA-TRICOM with enzalutamide. Are the same a ssociations seen or other changes in natural killer cells or cytokines of greater importance in this combination? Furthermore, baseline Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 19immune characteristics will be evaluated to determine if they have the potential to predict responders or likely non-responders to immune comb inations. While this hypothesis generating data would have to be prospectively evaluated in future clinical trials, it may improve our understanding about how best to deploy vaccines in combination with enzalutamide and other forms of hormonal therapy. If enzalutamide increases naïve T-cell production from the thymus as suggested by the pre-clinical data, this ma y have important ramifi cations in the timing of vaccine in future clinical trials. This and other data may also provide information early on in the therapeutic regimen about which patients would benef it from continued vaccine in combination with enzalutamide. It will also be of great importance to understand what the immunologic impact is of enzalutamide alone. New data from commonly used cytotoxic agents has suggested that some chemotherapy agents can enhanc e anti-tumor immune responses (34, 39). With this new understanding, clinical trials are be ing design to exploit this aspect of these respective therapies. Enzalutamide and other modern androgen receptor-targeting agents will be the mainstay of prostate cancer therapy for the foreseeable future. Future trials are likely to evaluate enzalutamide monotherapy in men with nmCSPC and this trial will be ab le to evaluate the specific immunologic impact of enzalutamide in men with normal testoste rone levels. With sipuleucel-T already approved and ipilimumab and PSA-TRICOM in phase III testing, it is likely that immunologic optimization will be important in the future trea tments of prostate cancer. Few groups are as well positioned and have the experience as the LTIB to conduct this rigorous immune testing from clinical samples of patients treated with enzalutamide to gain a better understanding about its immune properties. 1.2.9 Change in regrowth rate as endpoint Although vaccines have improved overall survival in mCRPC, they have not been shown to change short term TTP when used as monothe rapy. Interestingly, similar findings were also seen with ipilumimab in melanoma ( 40). Although this is not what is customarily seen with cancer treatments, it may be a characteristic of modern immune therapeutics ( 41). An emerging mechanism to evaluate tumor growth rates using mathematical models may provide additional insight into this phenomenon( 42,43). In a recent review of 5 NCI mCRPC trials including a vaccine trial, there appeared to be a sustained decrease in tumor growth rate in vaccine patients as compared to those treated with chemotherapy , who had transient reductions in tumor size, but then growth rate resumed at the same pre-treatment rate once cytotoxic therapy was discontinued. ( 44) This data suggests that a vaccine i nduced immune response may not decrease tumor size (or change TTP) but may lead to a ltered tumor growth rates which could have more substantial impact on overall survival ( 41). (See Figure 9) Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 20Figure 8 . PSA-TRICOM can affect growth rate in 80 days. An analysis of the ECOG trial 9802, which treated castration-sensitive non-metastatic prostate cancer patients (n=29) suggests PSA- TRICOM alters tumor growth rate, as measured by PSA (log g (growth rate) +/- SE (standard error)), within 80 to100 days. (45)This retrospective analysis used the tumor growth rate equation (44)determined that by day 80 after PSA-TRICOM therapy was initiated a statistically significant change in tumor growth rate was detectable compared to baseline. This altered growth rate was sustained at day 100 as well. These data supports the hypothesis that vaccines exert their therapeutic effect not by reducing tumor burden, but by altering tumor growth rate. Furthermore, unpublished data from an EC OG PSA-TRICOM trial in non-metastatic prostate cancer demonstrated a reduced growth rate within 100 days of vaccine initiation ( 45). (Figure 8 ) Similarly, Sipuleucel-T has significantly prolonged PSA doubling time (another way to measure growth rate) after initial hormona l therapy in the same patient population ( 46). Together, these data suggest that vaccine can impact growth rate in the nmCSPC population within 80 to 100 days. Furthermore, for patients getting combination therapy, disease can be controlled by standard therapies in the short term, while vaccines may impact tumor re-growth rates in the long term, ultimately resulting in prolonged TTP compared to standard therapy alone, (Figure 10). This trial will provide an opportunity to pr ospectively define the mechanism by which vaccines in combination with standard therapies ca n result in TTP benefit compared to that same standard therapy alone. By discontinuing the enzalutamide after 3 months and allowing PSA values to recover to baseline, we will have the opportunity to determine if a vaccine-mediated immune response can alter that tumor re-growth rate . As demonstrated by Drs. Fojo and Stein previously, 80-100 days is sufficient time to calc ulate a meaningful change in growth rate in patients with nmCSPC. (Figure 8 ) If this study can prospectively demonstrat e a difference, measured by one standard deviation in tumor re-growth rate, it would pr ovide an important proof of concept for the mechanism by which vaccines may improve standard therapies. Furthermore, it would better define the potential role of vaccines (as part of a therapeutic combination) in the treatment of prostate cancer and other malignancies. Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 211.2.10 Tumor Growth Kinetics 1.2.10.1 The regression-growth equation Drs. Stein and Fojo at the NCI have developed an equation based on the assumption that the change of a tumor's quantity during therapy results from 2 independent component processes: an exponential (first-order kinetics) decrease/regression and an exponential regrowth of the tumor. (44) The equation is f(t) = exp(-d * t)+exp(g * t) - 1 (A) where exp is the base of the natural logarithm, e= 2.7182, and f(t) is the tumor (or in MTC calcitonin) measurement at time tin days, normalized to (divided by) the tumor measurement at day 0, the time at which treatment is commenced. Rate constant d(decay, in daysí) represents the exponential decrease/regression of the serum tumor marker (i.e. PSA) signal during therapy. Rate constant g(growth, also in daysí) represents the exponential growth/regrowth of th e tumor during treatment. These rate constants may be expressed in terms of ha lf-lives and doubling times. Thus, dequals ln 2(0.693) divided by the time it takes for the regressing part to shrink by half, whereas g equals ln 2divided by the time for the growing component to double. ( 44) Two earlier papers depict theoretical curves de picting the separate components of Equation (A) and how these combine together to give the time dependence of the tumor size, f.(44) When the data showed a continuous decrease from the time of treatment start, so that only the regression parameter dwas found to differ significantly from zero with P< 0.05, Equation (A) was replaced by the following reduced form, with the growth rate constant eliminated: f(t) = exp(-d * t ) (B) When tumor measurements showed a continuous increase, so that only the growth parameter gwas found to differ significantly from zero with P< 0.05, Equation (A) was replaced by the following reduced form, with the decay constant eliminated: f(t) = exp(g *t) . 1.2.10.2 Data Analysis For each patient an attempt to fit Equation (A) to each data set for which more than one data point is available. Curve fitting will be performed using Sigmaplot (Systat Software), or by using the Solver routine in an Excel spreadsheet. We will extract parameters g and d with their associated Student's t and P values. ( 44) Data will be analyzed in Excel (Microsoft) and in Sigmaplot 9.0. Linear regressions to evaluate the relationship between the growth rate constant, g, or other parameters will be imp lemented using the polynomial li near routine of Sigmaplot 9.0. Sample comparisons were performed by Student's t-test, using SigmaStat 3.5 (Systat Software), with Pset at 0.05 for significance.