Neoadjuvant PROSTVAC prior to radical prostatectomy enhances T-cell infiltration into the tumor immune microenvironment in men with prostate cancer

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.

Keywords: clinical trials as topic; immunotherapy, active; tumor microenvironment; urologic neoplasms; vaccination.

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.

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.

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.