Immune monitoring of the circulation and the tumor microenvironment in patients with regionally advanced melanoma receiving neoadjuvant ipilimumab

Abstract

We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks ×2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2-19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin1-/HLA-DR-/CD33⁺/CD11b⁺ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4⁺CD25hi⁺Foxp3⁺) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4⁺ and CD8⁺ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8⁺ T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69⁺) CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells with evidence of induction/potentiation of memory T cells (CD45RO⁺). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1-/HLA-DR-/CD33⁺/CD11b⁺ was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy.

Conflict of interest statement

Competing Interests: AAT has acted as a consultant (advisory board participation) for Bristol-Myers Squibb. All other authors have no conflicts of interest to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. The authors confirm that the funding from received from “Bristol-Myers Squibb” along with any other relevant declarations as noted does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Forest plot of the multicolor flow cytometry data comparing the cell surface marker expression of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) on peripheral blood mononuclear cells (PBMC) at baseline and following treatment with ipilimumab (6 weeks).

The plot represents average within-patient changes from baseline to 6 weeks (with corresponding 95% confidence intervals). Treg were defined as cells expressing (1) CD4+CD25hi+FOXP3+ or (2) CD4+CD25hi+CD39+ activated T cells (CD3+CD4+CD25+). MDSC were defined as cells expressing (1) Lin1−/HLA-DR−/CD33+/CD11b+ lymphoid gate, (2) Lin1−/HLA-DR−/CD33+/CD11b+ monocyte gate or (3) HLA-DR+lo/CD14+ monocyte gate (N = 27 patients). Examples of raw data are provided in Figures S3 (T-reg gating) and S4 (MDSC gating).

Figure 2. Kaplan-Meier plot of progression free survival (PFS) by the dichotomized (at median) change in the percentage of circulating regulatory T cells (Treg) between baseline and week 6.

Greater increase in circulating Treg (CD4+CD25hi+Foxp3+%) was associated with improved PFS (HR = 0.57, p = 0.034; N = 27 patients). Example of raw data is provided in Figure S3 where the gating strategy used for Treg is shown.

Figure 3. Kaplan-Meier plot of progression free survival (PFS) by the dichotomized change in the percentage of circulating MDSC between baseline and week 6.

Greater decrease in circulating monocyte gate MDSC (Lin1−/HLA-DR−/CD33+/CD11b+%) was associated with improved progression free survival (PFS; p = 0.03; N = 27 patients). Example of raw data is provided in Figure S4 where the gating strategies used for MDSC subsets are shown.

Figure 4. Immunohistochemistry (IHC) of CD8+ tumor infiltrating lymphocytes (TIL).

There was a significant increase in CD8+ T cells from baseline to week 6 (Wilcoxon signed-rank test p = 0.02; N = 24 patients). (A) Boxplots of IHC scores of CD8+ TIL at baseline and week 6. Total counts from the same patient at the two time points are connected by light gray lines. (B) Plot of the median change in CD8+ TIL at week 6 (compared to baseline) with corresponding 95% confidence intervals. (C–D) Example of baseline and week 6 tumor CD8+ TIL (stained brown) by IHC. Magnification: 20×.