Results were normalized against the constant gene segment of TCRA, which serves as endogenous reference gene. All experiments were done three times with similar results. Statistical analyses were done by Student’s t-test 1.2.6 The Importance of Evaluating the Immunologic Response Our group at in the GMB and LTIB has previously evaluated immunologic parameters in clinical trials with PSA-TRICOM among several immunotherapy 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 [17]. That same trial also suggested that changes in regulatory T-cell function were also associated with improved clinical outcomes [ 24]. While these findings are not surrogate markers of response, they have greatly improved our knowledge of a vaccine-generated immune response 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 parameters will be valuable in understanding the benefits of PSA- TRICOM with enzalutamide. Are the same associations seen or other changes in natural killer cells or cytokines of greater importance in this combination? Furthermore, baseline immune characteristics will be evaluated to determine if they have the potential to predict responders or likely non-responders to immune combinations. While these hypothesis generating data would have to 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 may have important ramifications 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 benefit 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 can enhance anti-tumor immune responses [25, 26]. With this new understanding, clinical trials are being 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. 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 treatments of prostate cancer. Few groups are as well positioned and have the experience as the GMB and LTIB to conduct this rigorous immune testing from clinical samples of patients treated with enzalutamide to gain a better understanding about its immune properties. 1.2.7 Rationale for Time to Progression as a Primary Endpoint Although vaccines have improved overall survival in mCRPC, they have not been shown to change short term TTP when used as monotherapy. Interestingly, similar findings were also seen with ipilimumab in melanoma [27].