Window-of-opportunity clinical trial of pembrolizumab in patients with recurrent glioblastoma reveals predominance of immune-suppressive macrophages

Abstract

Background: We sought to ascertain the immune effector function of pembrolizumab within the glioblastoma (GBM) microenvironment during the therapeutic window.

Methods: In an open-label, single-center, single-arm phase II "window-of-opportunity" trial in 15 patients with recurrent (operable) GBM receiving up to 2 pembrolizumab doses before surgery and every 3 weeks afterward until disease progression or unacceptable toxicities occurred, immune responses were evaluated within the tumor.

Results: No treatment-related deaths occurred. Overall median follow-up time was 50 months. Of 14 patients monitored, 10 had progressive disease, 3 had a partial response, and 1 had stable disease. Median progression-free survival (PFS) was 4.5 months (95% CI: 2.27, 6.83), and the 6-month PFS rate was 40%. Median overall survival (OS) was 20 months, with an estimated 1-year OS rate of 63%. GBM patients' recurrent tumors contained few T cells that demonstrated a paucity of immune activation markers, but the tumor microenvironment was markedly enriched for CD68+ macrophages.

Conclusions: Immune analyses indicated that pembrolizumab anti-programmed cell death 1 (PD-1) monotherapy alone can't induce effector immunologic response in most GBM patients, probably owing to a scarcity of T cells within the tumor microenvironment and a CD68+ macrophage preponderance.

Keywords: clinical trial; glioblastoma multiforme; immune suppression; macrophages; pembrolizumab.

Fig. 1

Trial design, enrollment, and survival. (A) Summary of trial schema. (B) Profile of the trial patient composition. LMD, leptomeningeal disease; cycle (c); and day (d). (C) PFS based on radiographic evidence of tumor recurrence in terms of gadolinium contrast enhancement seen on MRI. (D) OS.

Fig. 2

GBM tumors are poorly infiltrated with T cells but are enriched in CD68+ myeloid cells. Single cell suspensions from resected GBM tumors were stained and analyzed using CyTOF. (A) Visualization of t-distributed stochastic neighbor embedding (ViSNE) plot (gated on total live cells) from all 5 pembrolizumab-treated GBM tumors showing CD45+ leukocytes. The majority of those leukocytes are CD68+ macrophages, and only a small proportion are CD3+ T cells. The color bar on the right shows range of expression of the indicated markers from blue to red representing zero to high expression, respectively. (B) Dot plot showing frequencies of leukocytes (CD45), T cells (CD3, CD4, CD8), macrophages (CD68), and NK cells (CD56) as a percentage of total cells in the tumors *P < 0.05. (C) ViSNE plot (gated on total live cells, bottom panel) and ViSNE plot (gated on the CD45+ leukocytes, top panel) showing total CD45+ immune cell composition analysis of the GBM microenvironment. The immune subset segregation was based on all 36 parameters studied. Shown are the expression of CD68, CD3, CD4, CD8, and CD56 over the different tumors overlayed together. Each individual tumor is color coded as shown. (D) ViSNE plot (gated on the CD45+ leukocytes) showing expression of specific markers on the total leukocyte population. Immune checkpoint ligands such as PD-L1, B7H3, and VISTA are associated with the CD68+ compartment. Class II MHC is preferentially expressed in the CD68 compartment. The color bar on the right shows the range of expression of the indicated markers from blue to red representing zero to high expression, respectively. *P < 0.05. (E) A cluster scatter (ViSNE) plot demonstrating the CD45+ population from the GBM patients’ tumors after treatment with pembrolizumab (n = 5) and analyzed by CyTOF. A total of 18 cell clusters were found using PhenoGraph and each cluster is color coded as shown in the figure. (F) Heat map showing expression of the 18 clusters in descending order of frequency. The color bar on the right, white to brown (low to high expression, respectively) demonstrates expression of the different markers within each cluster.

Fig. 3

The tumor microenvironment in GBM is poorly infiltrated with T effector cells and consists predominated of immunosuppressive CD68+ macrophages. IHC analysis of tumor tissues from GBM patients. Patients with newly diagnosed GBM without any prior treatment were designated as “untreated” (n = 5). Glioblastoma patients being actively treated with pembrolizumab are designated as “Pembro-treated” (n = 12). Bar plots showing the density of cells (number of positive cells/mm²) expressing each marker as shown in the figure. Plots for PD-L1 expression are based on either the absolute number of positive PD-L1 cells in the immune subset (PD-L1 density, 1 mm) or the relative percentage of PD-L1 expression on tumor cells (percent, PD-L1 tumoral).

Fig. 4

Immunofluorescent staining of GBM-associated macrophages. Multiplex immunofluorescence staining on GBM tumor tissues was performed for the following markers: CD68 (yellow) and the M2 marker CD163 (red), and the nuclei were counterstained with DAPI (blue). A representative image from one GBM tumor is shown. (A) Diffuse macrophage infiltration within the GBM is shown at 20x magnification. The inset shows the single stains of the square area marked in the composite image for CD68 (top) and CD163 (bottom). (B) 20x magnification image of a GBM with a blood vessel showing the enrichment of CD68+CD163+ macrophages present in the wall of the vessel. Bottom images show the singly stained images of CD68 (yellow, left) and CD163 (red, right) at 20x magnification. Bar = 200 µm.