21 While drawing the invasive margin for some cancer types such as PC could be challenging due to the intertwining, invasive nature of PC glands, it is clear that such a border is critical. 22 This is even more challenging when exam- ining biopsy specimens due to the limited availability of tissue. Our findings show that PROSTVAC vaccine increases CD4 T cells at the invasive margin and cytotoxic CD8 T cells in the tumor core. Such a pattern has been shown to be positively prognostic in other types of cancer. In a recent study of breast cancer using multiplexed ion beam imaging, tumors with compartmentalized immune- cell infiltrates at the IM (rather than mixed) had a better prognosis. 23 Nonetheless, up to ~70% and 57.6% of patients in our study showed a >twofold increase in CD4 and CD8 T cells in at least one of the three compart-ments, respectively. This suggests that PROSTVAC has a modulating effect on the spatial distribution of both CD4 and CD8 T- cell subsets within the prostate TIME. The frequency of these immune infiltrates varied within each of the three tumor compartments (intracompart-mental), suggesting a spatially heterogeneous response in the TIME. Focally diffuse infiltrates (peaks) different from tertiary lymphoid structures were also noted within each compartment. Notably, this heterogeneity can be seen within the same tumor, whether primary or meta-static, as previously described. 24 In this study, we proposed a non- standard TIME heterogeneity score that encom- passes and compares peak densities (focal) to average density (diffuse) of these immune infiltrates within each compartment. A peak density potentially reflects the maximum activity within the TIME, despite the presence of sampling size bias when comparing a biopsy with a whole section. Increases in both peak densities and PARIS scores of both CD4 and CD8 T- cell infiltrates were seen in the vast majority of the three compartment analyses (figure 3). This highlights a heterogeneous response to PROSTVAC in the TIME compared with baseline not seen with traditional measures. The importance of such scores remains to be proven by further TIME studies with associated clinical outcomes. The hypothesized benefit of this neoadjuvant vaccine strategy is to induce an inflammatory milieu that even-tually leads to a specific immune response (memory) capable of preventing metastases. While the optimal concentration threshold of immune cells needed to elicit a translatable immune response is not known, the odds of recognizing and killing cancer cells increases proportion-ally with immune- cell infiltration. 25 TIME studies histori- cally used similar cutoffs ranging from around a 1.5- fold26 to a ≥3- fold increase in IC. This is particularly important when this vaccine strategy is combined with other anti-tumor modalities such as checkpoint inhibitors. While PD- L1 expression in prostate cancer is generally low, 27 the 11% increase in tumor- cell PD- L1 after PROSTVAC remains low compared with other treatments.28 29 It is hard to compare PD- L1 status across studies in a non- assay setting, but this should not underscore the other limiting factors of this test. In addition, other immune regulatory changes in the TIME might be more rele-vant, as suggested by other studies. 29 30 In this study, 7/25 analyzed patients (28%) developed antigen- specific T- cell responses to PSA, the vaccine’s target antigen. This PSA- targeted response seen primarily in CD4 cells may partially explain the increase in T- cell infiltrates in the NL compartment, given that PSA is expressed in normal and tumor cells. Furthermore, 10/25 patients (40%) mounted postvaccination T- cell responses to other TAAs (MUC-1, brachyury) not present in the vaccine, highlighting the importance of antigen spreading, which is potentially more clinically relevant and effective than the original targeted response. 6 Interestingly, the four patients who experienced BCR did not develop any peripheral immune response, a distinction from the recurrence- free group (online supplementary table 5). No correlation with tumor immune response (tIR) or meaningful short- term clinical impacts on PSA or MRI features such as prostate volume, largest lesion size or Prostate Imaging Reporting & Data System (PI- RADS) score were seen. PROSTVAC failed to demonstrate improved overall survival in a randomized phase III study. However, this study suggests that PROSTVAC induces systemic immune responses, and concomitant increases in immune- cell infiltrates in and around the tumor. Further studies will focus on expanding the cells and promoting their func-tion in the TIME. 31 ConClusIonsThis study shows that neoadjuvant PROSTVAC vacci-nation can induce both tumor immune response and peripheral immune response. However, the heteroge-neity of the immune infiltrate in both cell subtypes and compartmental distribution highlights the complexity of analyzing the clinically relevant impact of immuno-therapy in tissue. It is far from a binary process where an immune infiltrate is simply increased or not, and benefi-cially inflammatory or detrimentally suppressive. A T- cell infiltrate at the IM may indicate a reactive suppressive mechanism of effector cells by exclusion or a positive prognostic sign of increased T helper- cell activity. Further study of various subset markers of exhaustion, activation 8 Abdul Sater H, et al. J Immunother Cancer 2020;8:e000655. doi:10.1136/jitc-2020-000655Open access and checkpoint expression by multiplex immunofluores- cence, along with RNA and T- cell receptor sequencing, will help to characterize these infiltrates more thor - oughly, providing a better understanding of the benefits and limitations of these immune responses. online supplementary methods Author affiliations 1Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 2Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 3Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 4Precision Biosciences Inc, Durham, North Carolina, USA 5Biostatistics and Data Management Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 6Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 7Clinical Research Directorate, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland, USA 8Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA twitter Christopher R Heery @chrisheery, Marijo Bilusic @mbilusic and James L Gulley @gulleyj1 Acknowledgements The authors would like to thank the patients involved in this study and their families. The authors would like to thank the nurses, medical/ surgical oncology fellows and consultation services at the National Institutes of Health Clinical Center for their excellent patient care. The authors would also like to thank the study team at the Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute for technical assistance with immune assays (Angie Schwab). Funding The control group of untreated RP specimens used in this study is supported by the Department of Defense Prostate Cancer Research Program, Award Nos. W81XWH-18-2-0013, W81XWH-18-2-0015, W81XWH-18-2-0016, W81XWH-18-2-0017, W81XWH-18-2-0018 and W81XWH-18-2-0019, Prostate Cancer Biorepository Network. This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health. Competing interests The NCI has a collaborative research and development agreement (CRADA) with Bavarian Nordic, the manufacturer of the vaccine used in this study. Under this CRADA, resources to develop agents are provided, including the vaccine. Patient consent for publication Not required. ethics approval The study protocol was approved by the National Cancer Institute’s Institutional Review Board, and all patients gave written informed consent according to institutional and federal guidelines. Provenance and peer review Not commissioned; externally peer reviewed. data availability statement Data are available in a public, open- access repository (https:// clinicaltrials. gov/ ct2/ show/ NCT02153918). open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY- NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non- commercial. See http:// creativecommons. org/ licenses/ by- nc/ 4.