The immune micro-environment of inflammatory breast cancer is characterized by an influx of CD163+ tumor-associated macrophages.
Authors: Van Berckelaer Christophe , Van Laere Steven , Colpaert Cecile , Bertucci Francois , Kockx Mark , Dirix Luc Yves , Van Dam Peter 
Published: June 2022

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Background: Inflammatory breast cancer (IBC) is a rare form of breast cancer characterized by rapid progression. A specific immune response seems to be an important driver for the aggressive biological behavior. We previously demonstrated that the spatial composition of the tumor immune micro-environment (TIME) is associated with survival in IBC. However, it remains an enigma how the TIME can contribute to the IBC phenotype. Since the number of tumor-infiltrating lymphocytes (TILs) between IBC and non-inflammatory breast cancer (nIBC) is similar and PD-L1 expression is higher, the functional state or composition of the immune infiltrate might determine the fulminant course of IBC. In this study, we assess the composition of the TIME in both IBC and a cohort of subtype-matched nIBC patients. Methods: We collected clinicopathological variables, evaluated PD-L1 positivity (SP142, Ventana) and scored TILs in a cohort of 161 IBC and 115 molecular subtype-matched nIBC patients. Affymetrix data (for CIBERSORT analysis) was available for 30 IBC and 20 nIBC patients. Immunostainings for CD8+ cytotoxic T cells, FOXP3+ Tregs, CD79a+ activated B cells and CD163+ TAMs (Hematoxylin-DAB) were done according to validated protocols. All slides were were evaluated in Visiopharm to quantify the number (density) and area (relative marker area, RMA) of DAB+ immune cells in both the invasive margin (IM) and the tumor stroma (TS). Results: Patients with IBC presented with higher stage disease (P< 0.001), but there were no other significant differences in clinicopathological parameters. In both cohorts, TAMs were the most abundant immune subset followed by B cells, CD8+ T cells and Tregs. For every subset the number of immune cells was higher in the IM than in the TS. Independent of molecular subtype or stage, IBC patients had more infiltration with TAMs in the TS. This was shown using both density (Median IBC: 424/mm2 vs nIBC: 290/mm2, OR: 0.82, 95% CI 0.76 – 1.00, P= 0.008) and RMA (Median IBC: 1.02% vs nIBC: 0.73%, OR: 0.87, 95% CI 0.77 – 1.00, P= 0.04). As previously described, PD-L1 positivity was significantly higher in the IBC cohort (P= 0.005), but no other significant differences in TIME composition between IBC and nIBC were found. Gene expression of CD163 correlated with the number of CD163+ TAMs (R= 0.38, P= 0.005) and CIBERSORT analysis confirmed a profile enriched for macrophages in IBC. Interestingly, the number of M1 macrophages was also higher in IBC (P= 0.03) and there was a strong correlation between the number of CD163+ TAMs in the TS and the M1 macrophage CIBERSORT subset (R=0.48, P< 0.001), possibly indicating that not only the number but also the functional state of TAMs is different in IBC. Conclusions: Using an extensive immune-phenotyping protocol we demonstrate, in a large cohort of IBC patients, that IBC is characterized by a specific tumor micro-environment in which TAMs play an important role.