The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial - PubMed (original) (raw)

Clinical Trial

The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial

Donald T Weed et al. Front Immunol. 2019.

Abstract

Myeloid Derived suppressor cells (MDSCs) play a key role in the progression and recurrence of human malignancies and in restraining the efficacy of adjuvant therapies. We have previously shown that Tadalafil lowers MDSCs and regulatory T cells (Treg) in the blood and in the tumor, primes a tumor specific immune response, and increases the number of activated intratumoral CD8+T cells in patients with primary Head and Neck Squamous Cell Carcinoma (HNSCC). However, despite these important immune modulatory actions, to date no clinically significant effects have been reported following PDE5 inhibition. Here we report for the first time interim results of our ongoing phase I clinical trial (NCT02544880) in patients with recurrent HNSCC to evaluate the safety of and immunological effects of combining Tadalafil with the antitumor vaccine composed of Mucin1 (MUC1) and polyICLC. The combined treatment of Tadalafil and MUC1/polyICLC vaccine was well-tolerated with no serious adverse events or treatment limiting toxicities. Immunologically, this trial also confirms the positive immunomodulation of Tadalafil in patients with recurrent HNSCC and suggests an adjuvant effect of the anti-tumor vaccine MUC1/polyICLC. Additionally, image cytometry analysis of scanned tumors indicates that the PDE5 inhibitor Tadalafil in conjunction with the MUC1/polyICLC vaccine effectively reduces the number of PDL1+macrophages present at the tumor edge, and increases the number of activated tumor infiltrating T cells, suggesting reversion of immune exclusion. However, this analysis shows also that CD163 negative cells within the tumor upregulate PDL1 after treatment, suggesting the instauration of additional mechanisms of immune evasion. In summary, our data confirm the safety and immunologic potential of PDE5 inhibition in HNSCC but also point to PDL1 as additional mechanism of tumor evasion. This supports the rationale for combining checkpoint and PDE5 inhibitors for the treatment of human malignancies.

Keywords: PDE5; PDL1; immune exclusion; mucin 1 vaccine; myeloid derived suppressor cells; poly-ICLC; recurrent HNSCC; tadalafil.

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Figures

Figure 1

Flow diagram and design of the phase 1 study.

Figure 2

Underglycosylated MUC1 is a common tumor specific antigen in stage 3/4 recurrent HNSCC. (A) MUC1 was evaluated by IHC on slides from the tumor specimens of enrolled patients. (B) Tumors and surrounding “normal” tissues were scored from 0 to 4 independently by four investigators based on the staining intensity and extension, and individual scores averaged. Examples of different scores are reported in Supplementary Figure 2. (C) ELISA for MUC1 specific antibodies was performed on the sera of healthy donors or enrolled patients at baseline.

Figure 3

Tadalafil and MUC1/polyICLC vaccine lowers circulating MDSCs and Treg and restores the expression of CD3 ζ-chain expression on CD8 T cells**. (A)** mMDSC, gMDSC, Treg, and the expression of CD3 ζ-chain expression on CD8 T cells was evaluated by multicolor flow cytometry on fresh blood of the enrolled patients or on age matched healthy donors. See Supplementary Figure 3 for gating strategies. Leukocyte subsets were enumerated with “123 beads” Two ways _T_-test value are reported. (B) The same subsets as in A were evaluated longitudinally in the patients enrolled in the treatment arm. The gray area correspond to the Tadalafil treatment. Significant Paired _T_-test value are indicated.

Figure 4

Immune response to the MUC1 and the influenza vaccines. (A) Anti-MUC1 or anti-flublock antibodies were evaluated longitudinally by ELISA in the plasma of the patients in the treatment arm. Arrowheads indicate the immunization time. MUC1 IHC score is indicated. (B) T cells from PBMCs drawn at baseline (before treatment initiation of course 1) and 2 weeks after completion of course 4 were stimulated with monocytes-derived autologous DC pulsed with MUC1 peptide. Four days later, CD8+ T-cell proliferation was evaluated by FACS. Background from parallel culture using unpulsed DC was subtracted.

Figure 5

Tumors from patients treated with Tadalafil and MUC1/polyICLC vaccine show a lower infiltration of MDSCs and Treg and a higher infiltration of activated CD8 in the tumor bed. Computer based image cytometry was performed to enumerate the number of (A) MDSC, (B) IL4Rα−myeloid cells, (C) CD4+T cell subsets, or (D) CD8+T cells. (E) CD69 expression within the CD8 is reported normalized on the CD69 expression on all the cells evaluated. Depending on the region of interest evaluated, at least 105-106 cells were analyzed. (F) The expression of CD69 in CD8+T cells was plotted against MUC1 IHC score of the corresponding tumor. Two ways _T_-test and relevant pearson correlation parameters are reported.

Figure 6

Tadalafil and MUC1/polyICLC vaccine treatments modulate the expression of PDL1 in the tumor microenvironment. The expression of PDL1 within the CD163+ (A) or the CD163−cells (B) was quantified by image cytometry in the tumor, at the tumor edge, or in “normal” surrounding tissue in the tumor specimen from the control (black filled circle) or Tadalafil and MUC1/polyICLC vaccine treated (gray filled circle) patients. Two way _T_-test _p_-value are reported. (C) Correlation between the expression of CD69 in the CD8+T cells and PDL1 expression on the CD163−cells. (D) Summary of the one way RM ANOVA analysis.

Figure 7

PDL1 expression limits the beneficial prognostic value of CD8a and CD69 in the tumor. KM plotter analysis (kmplot.com) was performed on tumor RNAseq data from patients with HNSCC (n = 499, all tumor stages) using the mean expression of the indicated genes with weight =1 and auto select best cutoff selected.

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The Reversal of Immune Exclusion Mediated by Tadalafil and an Anti-tumor Vaccine Also Induces PDL1 Upregulation in Recurrent Head and Neck Squamous Cell Carcinoma: Interim Analysis of a Phase I Clinical Trial - PubMed (original) (raw)