Immunological characterization of a long-lasting response in a patient with metastatic triple-negative breast cancer treated with PD-1 and LAG-3 blockade

Abstract

In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3⁺ and CD8⁺ cell infiltrate, with few PD1⁺ cells, rare CD4⁺ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B⁺ NK and T cells, including CD4⁺ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4⁺ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade.Trial registration: NCT02460224. Registered 02/06/2015.

Figure 1

Dynamics of response to dual PD-1/LAG-3 blockade and immune-molecular tumor characterization. (A) Shows the timeline of response dynamics together with the time of blood sampling for the immune monitoring. A rapid antitumor activity with a radiological partial response (as illustrated by the computed tomography images of a lymph node metastases in B) was achieved at week + 9 and was paralleled by the regression of most of the skin involvement. A complete clinical response of the extensive skin localization was observed at week + 18 (C). A complete radiological response (as per RECIST v.1.1) was achieved at week + 20. The immunohistochemical characterization of the tumor microenvironment showed a zonal CD3⁺ infiltrate mainly composed of CD8⁺ cells with rare CD4⁺ and rare PD-1⁺ cells (D, images at 20 × magnification). Tumor cells stained positive for the three main known LAG-3 ligands (HLA-DR, FGL-1 and galectin-3) and for the cancer testis antigen NY-ESO-1 (E, images at 20 × magnification).

Figure 2

Immunohistochemical staining at 4 × magnification. Immunohistochemical staining for PD-1 (A) and PD-L1 (B) at 4 × magnification. The dashed line rectangle indicates the area showed at 20 × magnification in Fig. 1D.

Figure 3

Immunohistochemical staining for NY-ESO-1 at 4 × magnification. The dashed line rectangle indicates the area showed at 20 × magnification in Fig. 1E.

Figure 4

Effects of PD-1/LAG-3 dual blockade on blood immune cell frequency and induction of a lymphocyte-mediated antitumor response soon after treatment initiation. Freshly thawed PBMCs were analysed by flow cytometry. (A) Percentage of LAG-3 (red) and PD-1 (blue) positive cells at the baseline in CD4⁺, CD8⁺, Treg (CD4⁺CD25⁺Foxp3⁺) and NK cells (CD16⁻CD56^hi and CD16⁺CD56^dim) are reported in graph. (B) Frequency of lymphoid (CD3⁺, CD4⁺, CD8⁺, Treg, NK cells) and myeloid (CD14⁺ monocytes, M-MDSC and PMN-MDSC) populations at the baseline (pre) and after treatments (post 1,2, 7) are shown. (C) overlay dot plots show the increased proliferation of GrZB⁺ cells (rectangle gate) after the first treatment (red) compared to the baseline (green) in CD4⁺ and CD8⁺ T cells (upper row) and in NK CD56^hi and NK CD56^dim (lower row). The frequency of cells expressing both the intracellular markers, Ki67 and GrZB, are reported in the graph as percentages (red = first treatment, green = baseline). (D) The presence of NY-ESO-1 specific CD8⁺ T cells was evaluated comparing PBMCs at the baseline and after the first treatment using HLA-A*0201/NY-ESO-1 multimer staining. The percentage of CD8⁺ multimer positive cells was calculated in the CD8⁺CD19⁻ gate and reported in the figure. (E) PMBCs were stained with a combination of mAbs to define naïve and memory T cell subsets. Histograms show the percentage of naïve (CD45RA⁺CCR7⁺, grey), effector memory (EM, CD45RA⁻CCR7⁻, blue), central memory (CM, CD45RA⁻CCR7⁺, yellow) and terminally effector (TE, CD45RA⁺CCR7⁻, red) cells in CD4⁺ and CD8⁺ lymphocytes at the baseline (pre), after the first treatment (post 1) and late in the treatment (post 48).