A comprehensive profiling of the immune microenvironment of breast cancer brain metastases

Abstract

Background: Despite potential clinical implications, the complexity of breast cancer (BC) brain metastases (BM) immune microenvironment is poorly understood. Through multiplex immunofluorescence, we here describe the main features of BCBM immune microenvironment (density and spatial distribution) and evaluate its prognostic impact.

Methods: Sixty BCBM from patients undergoing neurosurgery at three institutions (2003-2018) were comprehensively assessed using two multiplex immunofluorescence panels (CD4, CD8, Granzyme B, FoxP3, CD68, pan-cytokeratin, DAPI; CD3, PD-1, PD-L1, LAG-3, TIM-3, CD163, pan-cytokeratin, DAPI). The prognostic impact of immune subpopulations and cell-to-cell spatial interactions was evaluated.

Results: Subtype-related differences in BCBM immune microenvironment and its prognostic impact were observed. While in HR-/HER2- BM and HER2+ BM, higher densities of intra-tumoral CD8+ lymphocytes were associated with significantly longer OS (HR 0.16 and 0.20, respectively), in HR+/HER2- BCBMs a higher CD4+FoxP3+/CD8+ cell ratio in the stroma was associated with worse OS (HR 5.4). Moreover, a higher density of intra-tumoral CD163+ M2-polarized microglia/macrophages in BCBMs was significantly associated with worse OS in HR-/HER2- and HR+/HER2- BCBMs (HR 6.56 and 4.68, respectively), but not in HER2+ BCBMs. In HER2+ BCBMs, multiplex immunofluorescence highlighted a negative prognostic role of PD-1/PD-L1 interaction: patients with a higher percentage of PD-L1+ cells spatially interacting with (within a 20 µm radius) PD-1+ cells presented a significantly worse OS (HR 4.60).

Conclusions: Our results highlight subtype-related differences in BCBM immune microenvironment and identify two potential therapeutic targets, M2 microglia/macrophage polarization in HER2- and PD-1/PD-L1 interaction in HER2+ BCBMs, which warrant future exploration in clinical trials.

Keywords: brain metastases; breast cancer; immune biomarkers; immune microenvironment; multiplex immunofluorescence.

Fig. 1

mIF staining of BCBM. (a, b) Representative seven-color multispectral images of a BCBM sample stained with the first (a) and the second (b) mIF panels. Original magnification ×20. Immune markers and color codes are indicated in the legend. (c) Representative image of cell-cell distance analysis. Tumor cells (light blue dots) within a 10 µm radius from CD3+ cells (magenta dots) are represented. Abbreviations: BCBM, breast cancer brain metastases; mIF, multiplex immunofluorescence.

Fig. 2

Significant differences in immune infiltrate according to BCBM subtype and associations between BCBM immune cell composition and overall survival in the overall study cohort. (a–c) Significant differences in immune cell subpopulations infiltrating the stromal and the intra-tumoral regions according to BCBM subtype. Data are presented as (a, c) cell density (number of cells/mm²) or (b) as cell percentage among total CD8+ cells. (d) Mean distance (µm) between each PD-L1+ tumor cell and the nearest PD-1+ T lymphocytes within the tumor region according to BCBM subtype. (e) Mean distance (µm) between each PD-L1+ M2-polarized macrophage and the nearest PD-1+ T lymphocytes in the stromal compartment according to BCBM subtype. Floating box shows median, 25th to 75th percentiles, and smallest to largest values. Non-parametric Mann-Whitney statistical analysis was performed, and significantly different data are represented by *P < .05, **P < .01, and ***P < .001. (f–i) Kaplan-Meier curves for OS according to (f) stromal CD4+/CD8+ T-cell ratio, (g) stromal CD4+FoxP3+/CD8+ T-cell ratio, (h) intra-tumoral CD4+FoxP3+/CD4+ T-cell ratio, and (i) intra-tumoral density of CD163+ M2-polarized macrophages in BCBM of overall study cohort. Median value of each variable was used as cutoff. Log-rank P-values are reported in each graph. Abbreviations: BCBM, breast cancer brain metastases; OS, overall survival; PD-1, programmed cell death-1; PD-L1, programmed cell death ligand 1.

Fig. 3

Significant associations between immune contexture characteristics and overall survival and between cell-cell spatial interactions and overall survival in TNBC BMs cohort. Kaplan-Meier curves for OS according to (a) intra-tumoral density of total CD3+ T lymphocytes and (b) CD8+ T cells, (c) density of CD163+ M2-polarized macrophages in the intra-tumoral region, (d) percentage of CD163+ macrophages co-expressing PD-L1+ within the tumor region, (e, f) mean distance between (e) CD68+ macrophages and CD8+ T cells and (f) CD163+PD-L1+ M2-polarized macrophages and CD3+PD-1+ T lymphocytes. Median value of each variable was used as cutoff to identify high and low subgroups. Log-rank statistics were performed to determine significance; P-values are reported in each graph. (g–j) Univariate Cox model hazard ratios (HR), 95% confidence intervals (CI), and P-values for OS for percentage of tumor or immune cells present within 10, 15, 20, 25, and 30 µm radius from a cell with different phenotype in TNBC BMs. Median value of each variable was used as cutoff to identify high and low subgroups and HR for high vs low is reported. HR and 95% CI are represented. Bold characters indicate statistically significant cell interactions. Abbreviations: BMs, brain metastases; OS, overall survival; PD-1, programmed cell death-1; PD-L1, programmed cell death ligand 1; TNBC, triple-negative breast cancer.

Fig. 4

Significant associations between immune contexture characteristics and overall survival and between cell-cell spatial interactions and overall survival in HER2+ BCBM cohort. Kaplan-Meier curves for OS according to (a) stromal and (b) intra-tumoral densities of TIM-3+ CD163+ M2-polarized macrophages, (c) intra-tumoral density of CD8+ T lymphocytes and (d) mean distance between CD8+ Granzyme B+ T lymphocytes and tumor cells. Median value of each variable was used as cutoff to identify high and low subgroups. Log-rank statistics were performed to determine significance; P-values are reported in each graph. (e–h) Univariate Cox model hazard ratios (HR), 95% confidence intervals (CI), and P-values for OS for percentage of immune populations present within 10, 15, 20, 25, and 30 µm radius from a cell with different phenotype or tumor cells in HER2+ BCBM. Median cutoff of each variable was used to separate high and low groups and HR for high vs low percentage is reported. Abbreviations: BCBM, breast cancer brain metastases; HER2, human epidermal growth factor receptor type 2; OS, overall survival.

Fig. 5

Significant associations between immune cell infiltrate and overall survival in HR+/HER2− BCBM cohort. Kaplan-Meier curves for OS according to (a) intra-tumoral density of CD163+ M2-polarized macrophages, (b) stromal density of TIM-3+CD163+ macrophages, and (c) stromal CD4+FoxP3+/CD8+ cell ratio, in HR+/HER2− BCBM. Median value of each variable was used as cutoff. Log-rank P-values are reported in each graph. Abbreviations: BCBM, breast cancer brain metastases; HER2, human epidermal growth factor receptor type 2; HR, hormone receptor; OS, overall survival.