The immune microenvironment seems to contributes to the aggressive and unique biological features associated with inflammatory breast cancer (IBC). In this study we assessed the spatial associations between immune cells in IBC.
Serial slides of 51 patients (= 104 tissue samples) were stained according to a validated protocol. We used five antibodies: PD-L1 (SP142 AB), CD79a (B cell lineage), CD8 (cytotoxic T cells), FOXP3 (Tregs) and CD163 (TAMs, Tumor-associated macrophages). Subsequently, slides were scanned, virtually aligned and evaluated using VISIOPHARM® software. To narrow down the number of stainings, Affymetrix gene expression data of the IBC patients were analyzed using the CIBERSORT module.
Using specific image analysis algorithms for every staining, we located each positive cell using XY coordinates. Spatial co-localization was examined using point pattern (effector indexm, El) and quadrant analysis (Morisita-Horn index, MHI), developed for ecological studies. The El is based on the number of cells within a circle of 30 μm surrounding a CD8+ cell and the MHI measures the dissimilarity in species between two quadrants.
Complete pathological response (pCR) after neo-adjuvant chemotherapy was achieved in 23.5% of our patients. A negative HR-status (P= 0.01) and a the presence of more CD8+ cells (P= 0.04) predicted pCR. Interestingly, a higher number of CD79a+(P= 0.005) or FOXP3+ cells (P= 0.02) in close distance of CD8+ T cells was associated with pCR (using both El and MHI), while solely the number of CD79α+ or FOXP3+ cells did not predict prognosis nor pCR.
PD-L1 positivity and the number of CD8+ cells were not associated with OS, but patients with more PD-L1+ cells (> 0.7 cells/30 μm) in close contact with the CD8+ cells had a worse survival outcome (5y OS: 50% vs 68%, P=0.03). TAMs near CD8+ cells also seem to inhibit a good cytotoxic immune response as the El for CD163+TAMs was also prognostic (P=0.02), while the absolute number of TAMs was not.
In a validation cohort of 51 patients we showed that not only the presence, but also the spatial localization of immune cells predicts prognosis and response to therapy in IBC.