8.3 Commercial Agent: N/A 9. BIOMARKER, CORRELATI VE, AND SPECIAL STUD IES 9.1 Biomarker Studies The major focus will be to determine the relative level and persistence of vaccine induced immune response to PA2024 and PAP in patients receiving sipuleucel -T with and without CYT107. 9.2 Laboratory Correlative Studies 9.2.1 T-cell interferon -gamma responses to PA2024 – Laboratory Correlative Study #1 The primary endpoint will be the T -cell response to PA2024 measured at day 70 (week 11), 7 weeks after last CYT107 injection. T -cell responses to PA2024 and PAP will be measured by a direct 48 hour interferon -gamma ELISPOT assay utilizing recombinant proteins PA2024 and PAP (provided by Dendreon Corp.). Cellular immune responses to other ongoing and nascent antitumor responses (PRA ME, NY -ESO -1 and /or p53), additional tumor antigens specific to prostate cancer (e.g., PSA and /or PSMA), and memory viral responses (influenza A , CEF ) will also be assessed. Longitudinal responses to PA2024 and PAP will be considered in the primary analysi s; however power calculations are based on the magnitude of T cell ELISPOT responses measured at week 11. NCI Protocol #: CITN12 -03 Version Date: July 26, 2017 33 9.2.1.1 Collection of Specimen(s): Whole blood will be collected into heparinized (green top) tubes. 9.2.1.2 Handling of Specimens(s): Samples will be collected at room temperature and shipped to the CITN Central Laboratory the same day as the blood draw, so that the sample is received at the central laboratory within 24 hours of blood draw. PBMC will be isolated and cryopreserved prior to testing. 9.2.1.3 Shipping of Specimen(s): Samples will be shipped at ambient temperature for overnight FEDEX delivery. Clinical research sites will utilize a web -based specimen tracking system (BSI -II) to communicate with the CITN Cen tral Laboratory . 9.2.1.4 Site(s) Performing Correlative Study: The interferon -gamma ELISPOT assay will be conducted at the CITN Central Laboratory at the University of Washington (UW) Immunologic Monitoring Laboratory (IML), Seattle, WA. 9.2.2 T-cell proliferative responses – Laboratory Correlative Study #2 Peripheral blood lymphocytes will be analyzed directly ex vivo for the presence of proliferating T cells using a flow cytometric Ki -67 assay. Other surface markers will include CD3, CD4, CD8, CD45RA and CCR7 (among others) to identify T cell memory subsets. In addition, T cell proliferative responses to protein antigens PA2024 and PAP (from Dendreon Corp.) will also be tested in vitro . Results from this latter assay should corroborate the antigen -specific data obtained from Correlative Study #1. Longitudinal responses to PA2024 and PAP will also be evaluated. Cellular immune responses to other ongoing and nascent antitumor responses (PRAME, NY -ESO -1 and p53), additional tumor antigens specific to prostate can cer (e.g., PSA and PSMA), memory viral responses (influenza A and CEF ) may also be tested. 9.2.2.1 Collection of Specimen(s): Whole blood will be collected into heparinized tubes. 9.2.2.2 Handling of Specimen(s): Samples will be collected at room temperature and shipped to the CITN Central Laboratory the same day as the blood draw, so that the sample is received at the central laboratory within 24 hours of blood draw. PBMC will be isolated and cryopreserved prior to testing. 9.2.2.3 Shipping of Specimen(s): Samples will be shipped at ambient temperature for overnight FEDEX delivery. Clinical research sites will utilize a web -based specimen tracking system (BSI -II) to communicate with the CITN Cent ral Laboratory . 9.2.2.4 Site(s) Performing Correlative Study: The proliferation assays will be conducted at the CITN Central Laboratory at the University of Washington Immunologic Monitoring Laboratory, Seattle, WA. NCI Protocol #: CITN12 -03 Version Date: July 26, 2017 34 9.2.3 Quantification of PBMC and T cell subsets – Laboratory Correlative Study #3 The effect on the number and percentage of PBMC subsets and T lymphocyte subsets (i.e., CD4+ and CD8+ T cell naïve, effector memory, and central memory subsets, CD4+ Treg and activated CD4+ and CD8+ T cells) will be assess ed using multiparameter flow cytometric analysis of whole blood. The PBMC immunophenotyping assay will quantify the absolute number and proportion of T cells (both CD8+ and CD4+), NK cells (CD56+CD3 -), NKT cells (CD56+CD3+), B cells (CD19+), monocytes (CD1 6+), dendritic cells (HLA -DR+ CD123+ or CD11c+), and CD122 (IL15R -beta) -expressing cells. An additional panel will quantify the absolute number and proportion of memory and naïve T cell subsets using antibodies to CD45RA, CCR7, CD28, of regulatory T cells using antibody to CD25 (IL2R -alpha), the activation state of T cells using antibodies to PD-1, ICOS, and HLA -DR, among others and CD -127 (IL7R -alpha). The absolute change in each parameter as well as variance in change over time for each patient (mean, median, and SE/SD) will be evaluated. 9.2.3.1 Collection of Specimen(s): Whole blood will be collected into heparinized tubes. 9.2.3.2 Handling of Specimen(s): Samples will be collected at room temperature and shipped to the CITN Central Laboratory the same day as the bl ood draw, so that the sample is received at the Central Laboratory within 24 hours of blood draw. 9.2.3.3 Shipping of Specimen(s): Samples will be shipped at ambient temperature for overnight FEDEX delivery. Clinical research sites will utilize a web -based specimen tracking system (BSI -II) to communicate with the CITN Central Laboratory . 9.2.3.4 Site(s) Performing C orrelative Study: The immunophenotyping assays will be conducted at the CITN Central Laboratory at the University of Washington Immunologic Monitoring Laboratory, Seattle, WA. 9.2.4 PA2024 and PAP -specific Antibody Responses – Laboratory Correlative Study #4 IgM and IgG responses to PAP and PA2024 will be quantified using standard ELISA. The level of antibody responses to these antigens will be compared between the groups. The absolute change in each parameter as well as variance in change over time for each patient (mean, median, and SE/SD) will also be evaluated. 9.2.4.1 Collection of Specimen(s): Whole blood will be collected into red top (serum) tubes. 9.2.4.2 Handling of Specimen(s): Samples will be centrifuged, and serum aliquoted at each collection site within 2 -4 hours of blood draw. Samples will be frozen at -80C and stored for batch shipment to the CITN Central Laboratory . NCI Protocol #: CITN12 -03 Version Date: July 26, 2017 35 9.2.4.3 Shipping of Specimen(s): Batched samples will be shipped on dry ice for overnight FEDEX delivery. Clinical research sites will utilize a web -based specimen tracking system (BSI -II) to communicate with the CITN Central Laboratory . The Central Laboratory will ship coded , batched specimens to Dendreon Corp. for testing. 9.2.4.4 Site(s) Performing Correlative Study: The ELISA assays will be conducted at Dendreon Corp. , Seattle, WA. 9.2.5 T-cell receptor rearrangement DNA excision circles (TRECs) – Laboratory Correlative Study #5 Thymic function will be assessed by measuring serum levels of T -cell receptor rearrangement DNA excision circles (TRECs), in order to determine the contribution of new thymic emigrants to the T cell receptor repertoire. TREC levels will be compared between the two arms. The absolute change in each parameter as well as variance in change over time for each patient (mean, median, and S E/SD) will be evaluated. 9.2.5.1 Collection of Specimen(s): Whole blood will be collected into red top (serum) tubes. 9.2.5.2 Handling of Specimen(s): Samples will be centrifuged, and serum aliquoted at each collection site within 2 -4 hours of blood draw . Samples will be frozen at -80C and stored for batch shipment to the CITN Central Laboratory . 9.2.5.3 Shipping of Specimen(s): Batched samples will be shipped on dry ice for overnight FEDEX delivery. Clinical research sites will utilize a web -based specimen tra cking system (BSI -II) to communicate with the CITN Central Laboratory . 9.2.5.4 Site(s) Performing Correlative Study: The TREC assays will be conducted at the laboratory of Dr. Bruce Blazar, CITN site at the University of Minnesota. 9.2.6 T-cell receptor deep sequen cing – Laboratory Correlative Study #6 The T -cell repertoire will be characterized by T -cell receptor (TCR) deep sequencing (Harlan Robins, PhD, with Adaptive Biotechnologies, Seattle, WA) and compared between the two arms. These assays will evaluate the extent to which polyclonal or oligoclonal expansion of T cells has occurred with CYT107 therapy. 9.2.6.1 Collection of Specimen(s): Whole blood will be collected into ACD tubes. 9.2.6.2 Handling of Specimen(s): Samples will be collected at room temperature and shipped to the CITN Central Laboratory the same day as the blood draw, so that the sample is received at the Central Laboratory within 24 hours of blood draw. PBMC will be isolated and cryopreserved prior to isolation of genomic DNA. NCI Protocol #: CITN12 -03 Version Date: July 26, 2017 36 9.2.6.3 Shipping of Specimen(s): Sa mples will be shipped at ambient temperature for overnight FEDEX delivery. Clinical research sites will utilize a web -based specimen tracking system (BSI -II) to communicate with the CITN Central Laboratory . Processed samples will be batch -shipped by the Ce ntral Laboratory to Adaptive Biotechnologies for testing. 9.2.6.4 Site(s) Performing Correlative Study: The TCR deep sequencing assay will be conducted at the Adaptive Biotechnologies, Seattle, WA. 9.2.7 Peripheral Blood Kyn/Trp Ratios – Laboratory Correlative Study #7 Kyn/Trp ratios will be assayed in peripheral blood at baseline and at specified intervals throughout the trial. IDO catabolizes the conversion of Tryptophan to Kynurenine, which has potent immunosuppressant properties. An increase in Kynurenine level s over time, relative to Tryptophan levels, in patients who are failing therapy would indicate that IDO expression may be leading to treatment failure. This mechanism of resistance could potentially be overcome with an IDO inhibitor.