Capturing Age-Related Changes in the Tumor Microenvironment of Luminal Breast Cancer Using the Multiplex Immunohistochemistry Technique, MILAN.

e15089 Background: The ‘immune landscape’ of luminal breast tumors is poorly described to date. We applied the ‘multiple iterative labeling by antibody ‘neodeposition’ (MILAN) technique for in-depth characterization of the tumor microenvironment of luminal-like breast cancer (BC) in relation to age. Methods: Newly diagnosed patients with early luminal BC (grade 2/3, ER+, HER2-, tumor size ≥1,5 cm) were prospectively included in UH Leuven between March 2014 and November 2015 and divided into 3 distinct age categories (young: 35-45 yrs.; middle: 55-65 yrs.; old: ≥70 yrs.). 55 patients (young: n = 12; middle: n = 16; old: n = 27) were included in the cohort. From each patient up to 2 cores from the tumor center (TC) and 2 cores from the invasive front (IF) were sampled from formalin-fixed and paraffin-embedded tumor resection specimens and embedded in tissue microarrays. The MILAN workflow included 20 rounds of three-color indirect immunofluorescence (FITC, TRITC, and Cy5 channel combined with 58 antibodies), image acquisition, and removal/stripping of the antibodies before any other stain. The MILAN data analysis pipeline was applied to study the expression of phenotypic and functional proteins at single-cell level including spatial resolution. These were compared between the different age groups using Wilcoxon's test (FDR correction). Results: We identified 1,5 million cells, of which 59% were epithelial cells (cancer and normal), 29% were immune cells, and the remaining 12% were endothelial or stromal cells. Within the immune cell fraction, T-helper (Th) cells, B-cells, T-cytotoxic (Tcy) cells, and NK-cells significantly decreased with aging while monocytes significantly increased in TC and IF (Table 1). The evaluation of the functional markers in the inflammatory cells revealed a decreased expression with aging of PD-L1 in Tcy cells (p < 0.001 for TC and IF), Th cells (p = 0.003 for IF), T-regulatory (Treg) cells (p = 0.038 for TC), monocytes (p < 0.001 for TC and IF), and B-cells (p = 0.023 for TC). Perforin expression significantly decreased with aging in Th cells (p < 0.05 for TC and IF) and monocytes (p < 0.001 for TC and IF). OX-40 expression significantly decreased with aging in Th cells (p = 0.001 for TC), Treg cells (p = 0.002 for TC), and B-cells (p = 0.012 for TC). Conclusions: Our results suggest important compositional and functional changes in the tumor inflammatory microenvironment during aging in patients with luminal BC. Further analysis on other cell types is warranted. [Table: see text]