comparisons. With 24 evaluable patients with paired data, the trial had 80% power to detect a change from baseline equal to 0.67 SD of the change using a 0.025 significance level paired t- test for each of the two primary measures. In practice, a Wilcoxon signed rank test was to be used if the ratios were not normally distributed. To allow for a small number of non- evaluable patients, the accrual ceiling for the trial was 27 patients. Between October 2014 and May 2016, 27 patients were enrolled in a phase II study at the Center for Cancer Research, National Cancer Institute (table 1). The first four patients received monthly boosting vaccinations; all subsequent patients received boosts on weeks 3, 5 and 9. All patients completed the full series of vaccinations, 26/27 subsequently underwent radical prostatectomy (RP) and 1 patient elected to come off- study prior to surgery. ClInICAl outCoMesThe treatment course was well- tolerated, with no serious adverse events (AEs) or toxicities >grade 2 attributed to vaccine (online supplementary table 1). The most common AE attributed to vaccine was a self- limited injection- site reaction (28 events in 18 patients). Two grade 3 serious AEs occurred: an infected lymphocele and a thromboembolic event, both attributed to surgery. There were no significant changes in baseline and postvaccination PSA values (online supplementary figure 1); 26/27 patients underwent base-line MRI of the prostate, and 24 had follow- up imaging. There were no significant differences between baseline and postvaccination parameters (online supplementary table 2). Apparent diffusion coefficient (ADC) changes were further examined on a per- lesion basis and found to vary in degree and even direction in different lesions from the same subject (online supplementary figure 2). Patients were seen for the final protocol- required follow- up visit 12–15 months postoperatively, after which they had the option of transferring to the care of their local physician. Therefore, long- term data on biochemical or radiographic time to recurrence are limited. A total of 4/26 evaluable patients had biochemical recurrence within 2 years of prostatectomy. PrIMAry end PoInts tumor immune response Matched prevaccination (biopsy) and postvaccination (RP) prostate samples from 26 patients were available for evaluation, all of which provided adequate tumor for analysis by two approaches: non- compartmentalized analysis (NCA) and compartmentalized analysis (CA) (see ‘Methods’ section). Two RP cases and two biopsies were missing benign lymph glands (NL), and one biopsy was missing infiltrative margins (IM) due to complete involvement by tumor. Tumor CD4 T- cell infiltrates were significantly increased in postvaccination RP specimens compared with baseline biopsies in NCA (median 176/mm² vs 152/mm²; IQR 136–317/mm² vs 69–284/mm²; p=0.0249; median ratio 1.21; IQR 0.64–2.25). Using the same analysis, CD8 T- cell infiltrates did not significantly increase postvaccination compared with baseline (median 147/mm 2 vs 107/mm²; IQR 111–226/mm² vs 94–203/ mm²; p=0.23 (figure 1); median ratio 1.03; IQR 0.66–1.71 (figure 1, online supplementary figure 3)). The CA showed that both CD4 and CD8 T- cell infiltrates were seen more at the IM compared with the tumor core (CT) (figure 2). In this analysis, increased CD4 T- cell infiltrates at IM (median 198/mm² vs 151/mm²; IQR 123–500/mm² vs 85–256/mm²; p=0.042; median ratio 1.44; IQR 0.59–4.17) and CD8 T- cell infiltrates at CT (median 140/mm² vs 105/ mm²; IQR 91–175/mm² vs 83–163/mm²; p=0.036; median ratio 1.25; IQR 0.88–2.09) were noted in postvaccination RP specimens compared with baseline biopsies (figure 2). Similarly, an increase in CD4 T- cell infiltrates was seen postvaccination in RP specimens at NL (median 263/mm² vs 86/mm²; IQR 93–531/mm² vs 41–225/mm²; p=0.0002; median ratio 2.33; IQR 1.05–5.22) compared with baseline biopsies (figure 2, online supplementary figure 3). In fact, 21/25 patients (84%) had a >1.5- fold increase in CD4 T cells and 18/25 patients (72%) had a >1.5- fold increase in CD8 T cells in at least one of three compartments (online supplementary table 3). T- cell infiltrates were increased in the tumor core (CT) in postvaccination RP specimens compared with untreated control RP specimens (n=5) for both CD4 (median 65.17/mm²; IQR 11.79–110.4/mm²; p=0.0132) and CD8 (median 18.3/mm²; IQR 14.29–53.61/mm²; p=0.0007) (online supplementary figure 4). 4 Abdul Sater H, et al. J Immunother Cancer 2020;8:e000655. doi:10.1136/jitc-2020-000655Open access Figure 1 Cell densities in prevaccination biopsies (Pre) and in radical prostatectomy sections postvaccination (Post) with PROSTVAC. (A) Representative image of multiplex immunofluorescence panel. Insets show a cell expressing four markers (CD4, FOXP3, Ki67 and DAPI). (B) Immune- cell infiltrates were quantified in both prevaccination and postvaccination sections using inForm software. NCA of both CD4 and CD8 immune- cell density ratios was assessed by the Wilcoxon signed rank test. Median±IQR shown with horizontal lines. (C) Exceptional case with CD4 and CD8 immune- cell infiltrates prevaccination and postvaccination. CTL, cytotoxic T lymphocyte; NCA, non- compartmentalized analysis; PD- L1, programmed death- ligand 1; Th, T helper; TIME: tumor immune microenvironment. Figure 2 Compartmental distribution of CD4 and CD8 T cells postvaccination in intraprostatic tissue. (A) Schematic representation of radical prostatectomy (RP) section with three virtually separate compartments: tumor core (CT), invasive margin (IM) and benign glands (NL). (B, C) CD4 and CD8 T- cell infiltrate average densities were quantified as previously described using inForm software in each compartment before and after treatment with PROSTVAC vaccine. CD4 and CD8 immune- cell density ratios were assessed by the Wilcoxon signed rank test. Median±IQR shown with horizontal lines. heterogeneity in immune cells, tumor-infiltrating lymphocytes and peak densityIntratumoral heterogeneity of various immune- cell infil- trate frequencies was more evident in RP specimens than in biopsies. Non- standard peak densities and a newly proposed heterogeneity score (peak to average immune score (PARIS), defined as peak density divided by average density, figure 3) were exploratory and used to assess the heterogeneity of immune infiltrates. The analysis of peak cell densities showed overall increases in CD4 and CD8 T- cell infiltrates in most compartments (p<0.05). This is particularly true for IM CD4 (median 533/mm² vs 174/ mm²; IQR 287–1221/mm² vs 100–433/mm²; p=0.0001; median ratio 2.82; IQR 1.08–9.06), and NL CD4 (median 5 Abdul Sater H, et al. J Immunother Cancer 2020;8:e000655. doi:10.1136/jitc-2020-000655Open access Figure 3 Heterogeneity scores of immune- cell infiltrates within prostatic tumor immune microenvironment (TIME) in prevaccination and postvaccination sections. (A, B) CD4 and CD8 T- cell infiltrate peak densities were quantified as previously described using inForm software in each compartment before and after immunotherapy. CD4 and CD8 immune- cell density ratios were assessed by the Wilcoxon signed rank test. Median±IQR is shown with horizontal lines. (C, D) Mean PARIS scores within each compartment in both prevaccination and postvaccination sections are shown. CD4 and CD8 cell density ratios were assessed by the Wilcoxon signed rank test. CT, tumor core; IM, infiltrative margins; NL, benign lymph gland; PARIS, peak to average immune score. 554/mm² vs 106/mm²; IQR 194–1431/mm² vs 67–276/mm²; p<0.0001; median ratio 4.65; IQR 1.99–9.85), as well as CT CD8 (median 281/mm² vs 159/mm²; IQR 186–429/mm² vs 98–243/mm²; p=0.001 (figure 3); median ratio 1.85; IQR 0.69–2.91 (figure 3)). PARIS score means for CD4 T cells postvaccination (2.26, 2.64, 2.43) were compared with prevaccination score means (2.21, 1.3, 1.25) in CT, IM and NL, respectively. PARIS score means for CD8 T cells postvaccination (2.11, 2.37, 2.10) were also compared with prevaccination score means (1.58, 1.33, 1.13) in CT, IM and NL, respectively. These scores were increased in all three compartments for both CD4 and CD8 T cells (p<0.05), except for CD4 T cells in CT (p=0.07) (figure 3). Immune-cell subsets In the NCA, an increase in effector (CD4+FOXP3–) T helper cell infiltrates (mean 307/mm² vs 194/mm²; 95% CI 184–430/mm² vs 117–271/mm²; p=0.03) was seen in postvaccination RP specimens compared with baseline biopsies (figure 4). Tumor- infiltrating T- regulatory cells (Tregs) (CD4+FOXP3+) tended to substantially decrease in postvaccination RP specimens compared with baseline biopsies (mean 5/mm² vs 17/mm²; 95% CI 4–7/mm² vs 5–29/mm²; p=0.06) (figure 4). Similarly, proliferative (Ki67+) effector cytotoxic CD8 T- cell infiltrates tended to have lower density in postvaccination RP specimens compared with baseline biopsies (mean 14/mm² vs 9/mm²; 95% CI 6–23/mm² vs 4–14/mm²; p=0.44). There was no change in CD8+Ki67– T- cell infiltrates (mean 165/mm² vs 156/mm²; 95% CI 121–209/mm² vs 90–222/mm²; p=0.56) in postvaccination versus baseline specimens. Pd-l1 expression Programmed death- ligand 1 (PD- L1) expression was focal (1%–10% on tumor cells) and only seen in 3/26 RP specimens (11.5 %) compared with 0/26 (0%) baseline specimens (online supplementary figure 5). Immune- cell PD- L1 expression was also focal and seemed to be present on macrophage- like cells. These cells were not stained with a dedicated marker or quantitated. On average, although variable by case and ROI, cases with PD- L1+ status had a higher post/pre ratio of immune- cell infil- trates (online supplementary figure 5). seCond Ary end PoInts Antigen-specific t -cell responses Of 25 patients evaluable for PSA- specific CD4+ and CD8+ T- cell responses, 7 (28%) developed measurable PSA- specific increases in T- cell cytokine production and/or CD107a positivity postvaccination (online supplementary table 4). Further evaluation showed that seven (28%) and six (24%) patients, respectively, developed measurable responses to the cascade antigens MUC-1 and brachyury. A total of 13/25 patients (52%) developed responses to any of the three TAAs tested, and 5 of the 13 had responses to more than one evaluated antigen (figure 5). None of the four patients who experienced early biochem-ical recurrence (BCR) developed antigen- specific T- cell 6 Abdul Sater H, et al. J Immunother Cancer 2020;8:e000655. doi:10.1136/jitc-2020-000655Open access Figure 4 Changes in immune- cell subsets within prostatic tissue before and after PROSTVAC vaccine therapy. All cell densities were quantified as previously described using inForm software. (A) Frequency of T- regulatory cells (Tregs) (CD4+FOXP3+) before and after PROSTVAC therapy in non- compartmentalized analysis (NCA). (B) Frequency of T helper cells (CD4+FOXP3–) before and after PROSTVAC therapy in NCA. (C, D) Activated (Ki67+) and non- activated (Ki67–) CD8 cytotoxic T lymphocytes were quantified using NCA in both prevaccination and postvaccination sections. For (A–D), Wilcoxon signed rank test was used. Median±IQR is shown with horizontal lines. (E, F) Representative images of all four immune- cell subsets (Tregs, T helper, activated cytotoxic T lymphocytes and non- proliferative cytotoxic T lymphocytes). Figure 5 Antigen- specific T- cell responses assessed by intracellular cytokine staining of peripheral blood mononuclear cells following in vitro stimulation (pIR). Responses to the vaccine target antigen prostate- specific antigen and the cascade antigens MUC-1 and brachyury were measured by increases in the number of CD4+ or CD8+ lymphocytes producing cytokines (interferon-γ, tumor necrosis factor-α or interleukin-2) or positive for CD107a. An increase of ≥twofold from baseline was considered positive. pIR, peripheral immune response.responses to any of the TAAs tested, compared with the 12/20 responses in the non- recurrence group (p=0.093 by Fisher’s exact test). Polyfunctional TAA- specific responses, defined as CD4+ or CD8+ T cells that express >2 of the markers interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2 or CD107a, were also measured before and after vaccination. Using the crite-rion of a >threefold increase postvaccination versus prevaccination, or the presence of >100 polyfunctional cells postvaccination per 1×10 6 peripheral blood mono- nuclear cells (PBMCs) (if negative at prevaccination), polyfunctional T cells specific for at least one of the TAAs tested were generated in 28% of patients (online supple-mentary figure 6A). Representative flow plots from a patient (#22) developing multifunctional MUC1- specific CD4+ T cells after vaccination is shown in online supple-mentary figure 6B. dIsCussIon The vaccine was well- tolerated, making it a low- risk inter - vention in patients with potentially curable cancer in the neoadjuvant setting. In addition, the results of this study show an increase in T- cell infiltrates, particularly CD4 T cells, into the tumor immune microenvironment (TIME) as a direct effect of PROSTVAC vaccine in PC. While cyto-toxic CD8 T cells remain the essential effector of tumor- cell killing, intratumoral CD4 T helper cells have been shown to play an essential antitumoral and immunomod-ulatory role. 15 16 Spitzer et al also describe an essential antitumor role for peripheral CD4 T- cell subsets rather than cytotoxic CD8 cells consistent with effective immune responses in a clinical trial with ipilimumab. 17 18 In our study, more than a third of patients doubled their average 7 Abdul Sater H, et al. J Immunother Cancer 2020;8:e000655. doi:10.1136/jitc-2020-000655Open access CD4 T- cell densities to a level similar to what is seen with other vaccines,3 but these densities remain low compared with hot tumors such as melanomas where average T- cell densities range from 500 to 2000/mm². Subset analysis showed a significant increase in T helper cells while Tregs constituted a minority of CD4 T cells. A trend to an abso-lute decrease in Treg density postvaccination is consistent with previously reported effects of PROSTVAC on Tregs peripherally 1 19 and intratumorally.20 Since the average density of infiltrating cytotoxic CD8 T cells and their subsets (Ki67+/–) did not change postvaccination, we asked whether this effect on CD4 T cells is accompanied by a more targeted CD8 T- cell response. The spatial distribution of certain immune subsets has been shown to be prognostic in various cancer types.