The spatial relations among immune cells are prognostic in inflammatory breast cancer
Authors: C. Van Berckelaer, C.G. Colpaert, C. Rypens, K. Marien, M. Kockx, Y. Waumans, P. Vermeulen, L.Y. Dirix, S.J. Van Laere, P. Van Dam
Journal: Annals of Oncology (2019)

Background: The mechanisms contributing to the aggressive biology of inflammatory breast cancer (IBC) are still under investigation. Our lab reported a 79-gene signature that is shaped by specific immune response programs and discriminates between IBC and non-IBC (nIBC). In this study we assessed the spatial associations between immune cells in IBC. Methods: Affymetrix gene expression data of 105 IBC patients were analyzed using the CIBERSORT module to narrow down the number of stainings. Serial slides were stained according to a validated protocol, scanned, virtually aligned and evaluated using VISIOPHARM® software. We used five validated antibodies: PD-L1 (SP142 AB), CD79α (B cell lineage), CD8 (cytotoxic T cells), FOXP3 (Tregs) and CD163 (TAMs, Tumor-associated macrophages). Using staining-specific image analysis algorithms, we located each positive cell using XY coordinates. Spatial co-localization was examined using point pattern (effector index, EI) and quadrant analysis (Morisita-Horn index, MHI), developed for ecological studies. The EI is based on the number of cells within a circle of 30 μm around a CD8+ cell and the MHI measures the dissimilarity in cells between two quadrants. Results: A total of 51 patients (= 104 tissue samples) were analysed and 23.5% achieved complete pathological response (pCR) after neo-adjuvant chemotherapy. A negative HR-status (P= .01) and a the presence of more CD8+ cells (P= .04) predicted pCR. Interestingly, a higher number of CD79α + (P= .005) or FOXP3+ cells (P= .02) in close distance of CD8+ T cells was associated with pCR (using both EI 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 CD8+ cells had a worse survival outcome (5y OS: 50% vs 68%, P= .03).TAMs surrounding CD8+ cells also seem to inhibit a good cytotoxic immune response as the EI for CD163+ TAMs was also prognostic (P= .02), while the absolute number of TAMs was not. Conclusions: In a discovery 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. By May we will have data of 148 IBC cases. Legal entity responsible for the study: Translational Cancer Research Unit, GZA Hospitals & CORE, MIPRO, University of Antwerp, Antwerp, Belgium. Funding: PhD grant for CVB, from the Research Foundation - Flanders (FWO) [grant number 1189617N]. Disclosure: K. Marien, Y. Waumans: Employee: HistoGeneX NV, which performs immunohistochemistry and molecular testing for pharmaceutical companies as part of (pre)clinical studies that evaluate new anticancer drugs. M. Kockx: CEO: HistoGeneX NV, which performs immunohistochemistry and molecular testing for pharmaceutical companies as part of (pre)clinical studies that evaluate new anticancer drugs. All other authors have declared no conflicts of interest.