See rights and permissions. Published by BMJ.AbstrACt background Clinical trials have shown the ability of therapeutic vaccines to generate immune responses to tumor- associated antigens (TAAs). What is relatively less known is if this translates into immune- cell (IC) infiltration into the tumor microenvironment. This study examined whether neoadjuvant prostate- specific antigen (PSA)- targeted vaccination with PROSTVAC could induce T- cell immunity, particularly at the tumor site.Methods An open- label, phase II study of neoadjuvant PROSTVAC vaccine enrolled 27 patients with localized prostate cancer awaiting radical prostatectomy (RP). We evaluated increases in CD4 and CD8 T- cell infiltrates (RP tissue vs baseline biopsies) using a six- color multiplex immunofluorescence Opal method. Antigen- specific responses were assessed by intracellular cytokine staining after in vitro stimulation of peripheral blood mononuclear cells with overlapping 15- mer peptide pools encoding the TAAs PSA, brachyury and MUC-1. results Of 27 vaccinated patients, 26 had matched prevaccination (biopsy) and postvaccination (RP) prostate samples available for non- compartmentalized analysis (NCA) and compartmentalized analysis (CA). Tumor CD4 T- cell infiltrates were significantly increased in postvaccination RP specimens compared with baseline biopsies by NCA (median 176/mm² vs 152/mm²; IQR 136–317/mm² vs 69–284/mm²; p=0.0249; median ratio 1.20; IQR 0.64–2.25). By CA, an increase in both CD4 T- cell infiltrates at the tumor infiltrative margin (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 in CD8 T- cell infiltrates at the tumor core (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. A total of 13/25 patients (52%) developed peripheral T- cell responses to any of the three tested TAAs (non- neoantigens); five of these had responses to more than one antigen of the three evaluated.Conclusion Neoadjuvant PROSTVAC can induce both tumor immune response and peripheral immune response. trial registration number NCT02153918. bACkground Therapeutic vaccines have been investigated in patients with cancer. Clinical trials have shown their ability to generate immune responses to tumor- associated antigens (TAAs), 1 2 but relatively little is known about whether this translates into more tumor- targeted immune cells getting into the tumor and, if it does, what the other effects are of this new balance of cells in the tumor micro-environment. One prior study in patients with localized prostate cancer (PC) suggested an increase in immune infiltrates at the inter - face of tumor and normal tissue following intravenous administration of the therapeutic vaccine sipuleucel- T. 3 PROSTVAC is a therapeutic cancer vaccine designed to activate T cells specific against prostate- specific antigen (PSA). 4 Previous trials have shown PROSTVAC vaccine to be safe and associated with peripheral immune responses. 1 5–7 The focus of this study was to determine whether neoadjuvant vaccination with PROSTVAC could induce T- cell immu- nity, particularly at the tumor site, as well as any correlations with peripheral and clinical outcomes. This study was designed before the results of the recently reported phase III study of PROSTVAC in the metastatic setting with no survival benefit were known. 8 CD4 and CD8 T- cell infiltrates were chosen as primary outcome measures. 2 Abdul Sater H, et al. J Immunother Cancer 2020;8:e000655. doi:10.1136/jitc-2020-000655Open access Methods eligibilityEligible patients had histopathologically verified local-ized PC and were surgical candidates for RP based on a standard workup of PSA measurement, biopsy results and imaging as needed. Patients must have chosen RP as their definitive treatment and needed to have evaluable biopsy tissue available for analysis or be willing to undergo a targeted prevaccination prostate biopsy. Patients were ≥18 years of age, had an ECOG performance status of 0–1 and had adequate organ function as defined by liver, kidney and hematological laboratory tests. Patients could have no evidence of immunocompromise or significant autoimmune disease that was active or potentially life- threatening if activated. study design This phase II, single- arm study evaluated the impact of PROSTVAC vaccination in the neoadjuvant PC setting. Patients received recombinant vaccinia (rV)- PSA(L155)- TRICOM (2×10 8 IU subcutaneously) as a priming vacci- nation, followed by monthly boosts on weeks 5, 9 and 13 with recombinant fowlpox (rF)- PSA(L155)- TRICOM (1×10 9 IU subcutaneously). After completing the vacci- nation series, patients underwent RP. The boosting schedule was subsequently amended to weeks 3, 5 and 9 to allow for earlier surgeries. A final follow- up safety evaluation was performed 12–15 months postopera-tively. AEs were monitored using Common Terminology Criteria for Adverse Events V.4.0. The primary objective was to examine changes in CD4 and CD8 T- cell infiltrates in tumor tissue postvaccination. Secondary objectives included investigating changes in peripheral PSA- specific T- cell responses, intraprostatic Treg infiltrates, PSA and MRI parameters postvaccination. Multiplex immunofluorescence staining and multispectral imagingWe stained 5 µm- thick formalin- fixed paraffin- embedded sections from prostate biopsies and RP specimens using Opal multiplex 6- plex kits, according to the manufactur - er’s protocol (PerkinElmer), for a panel of DAPI, CD4, CD8, FOXP3, Ki67, PanCK and PD- L1 (online supple- mentary methods). H&E and multiplex immunofluorescence scans were captured by a PerkinElmer Vectra Polaris. Both biopsies and RP tissue slide scans were virtually segmented by a research pathologist into three distinct compartments: CT, tumor IM and NL. Five randomly selected regions of interest (ROI) (mm 2, original magnification 40×, 0.25 µm/pixel resolution) from each compartment were then selected from each slide for multispectral imaging (MSI). Vectra Polaris captures spectral information from an MSI image using a multispectral camera, and the intensity of each fluorescent target is extracted from the multispectral data using linear unmixing. Unmixed MSI images were analyzed using inForm OS 2.3.1 software (PerkinElmer). A common algorithm was built based on five representative images, with adjustment from one batch to another due to batch variation. The algorithm consisted of cell segmentation, phenotyping and thresh-olding. All immune- cell infiltrates were measured as cell counts/mm² of tissue (density). Antigen-specific t -cell responses Antigen- specific responses were assessed by intracellular cytokine staining following a period of in vitro stimulation (IVS) of PBMCs with overlapping 15- mer peptide pools encoding the TAA PSA. The PSA peptide pool contained a previously identified agonist epitope 9; pools encoding for human leukocyte antigen (HLA) and CEFT (a mixture of cytomegalovirus, Epstein- Barr virus, influenza and tetanus toxin) served as negative and positive controls, respectively. PBMCs from patients before and 60 days after therapy were stimulated by IVS and stained with antibodies to identify the absolute number of CD4+ or CD8+ lymphocytes producing cytokine (IFN-γ , TNF-α or IL-2) or positive for CD107a, as previously described. 10 The background signal (obtained with the HLA peptide pool) and values obtained prevac-cination were subtracted from those obtained postvaccina-tion. Values >250 were scored as positive for TAA- specific immune response postvaccination if they were also at least twofold greater than that obtained with HLA. To explore evidence of cross- priming and development of an immune response to TAAs not found in the vaccine (antigen cascade or antigen spreading), responses to the cascade antigens MUC-1 and brachyury were tested using the same method. Peptide pools of MUC-1 and brachyury contained previ-ously identified agonist epitopes. 11 12 MrI responsesWhen feasible, an MRI was performed at baseline and after the last vaccination to assess for changes in the pros-tate and PC tumors prevaccination and postvaccination, including prostate volume, number of lesions, largest lesion length, PI- RADS score and ADC mapping. 13 ADC values have been found to be decreased in various malig-nancies due to hypercellularity, and lower pretreatment ADC values in cervical cancer have been associated with a higher risk of recurrence. 14 statistical analysisThe primary objective was to determine whether there were changes in CD4 or CD8 cells overall, as well as within subsets based on compartments or peaks within ROIs. Ratios were formed to compare results post/pre. They were tested for whether the ratio=1 by the Wilcoxon signed rank test. Changes in averaged values over five randomly selected ROIs for the CD4 and CD8 T- cell infil- trates were considered the primary end point (p<0.025 would be significant following Bonferroni correction for these two tests, per protocol). Other measures were considered exploratory and were interpreted in the context of the number of tests performed, but without formal adjustment for multiple comparisons. All p values are reported as obtained, without adjustment for multiple 3 Abdul Sater H, et al.