Characterization of the immune microenvironment and spatial phenotypes across HER2 subtypes in advanced or metastatic breast cancer.

Abstract

1037 Background: Breast cancer is defined by HER2 and hormone receptors (HR) status, which influence the clinical outcomes. HER2-positive has been traditionally defined as HER2 overexpression on immunohistochemistry (IHC score of 3+) or 2+ and ERBB2 amplification on in situ hybridization (ISH). HER2 low (IHC 1+ or 2+ and non-amplified ISH) accounts for nearly half of tumors. There is a paucity of data regarding immune subpopulations and spatial phenotypes in HER2 subtypes. We have investigated the characteristics of tumor immune microenvironment contexture across HER2 groups (HER2 + vs HER2 low vs HER2 - (0 by IHC)) focusing tumor infiltrating lymphocytes (TIL) and on immune cell dynamics, including the distribution and spatial proximity to tumor cells to potentially inform treatment selection. Methods: Formalin-fixed paraffin-embedded (FFPE) samples of patients with metastatic breast cancer who had HER2 IHC/ISH testing according to ASCO-CAP guidelines were stained and analyzed using an 8-plex immunofluorescence (mIF) panel (CD3, CD8, CD69, FOXP3, Ki67, PD-L1, PD1, PanCK). For neighborhood analysis, samples with an area > 2 mm 2 and a phenotypes density with > 2 cells/mm² were considered. A novel spatial analysis method was used to quantify the Euclidian distance between tumor cells and surrounding immune cell populations. The clustering coefficient was used to determine the connectivity of immune cell node neighbors. These findings were analyzed in relation to the clinical characteristics. Results: Tumor and stromal compartment analysis was done on 44 FFPE samples (10 HER2 - , 19 HER2 low , and 15 HER2 + ) with 84% collected from metastatic sites. HER2 status was not significantly associated HR status or overall TIL infiltration into the tumor compartment. The dominant TIL subset identified was non-regulatory CD3+ T cells as defined as CD3 + /FOXP3 - /CD8 - . HER2 - samples were more associated with lack of PD-L1 expression on intratumoral myeloid cells and PD-L1 low expression on tumor cells as compared with HER2 low and HER2 + (p = 0.06). For spatial analysis, 33 samples (6 HER2 - , 16 HER2 low and 11 HER2 + ) were considered. Macrophages and proliferating tumor cells were more abundant in HER2 - samples than HER2 low or HER2 + (p = 0.006 and p-0.027, respectively). Median distances from tumor cells to macrophages and T regs were shorter in HER2 - cases compared to HER2l ow (p < 0.001). Although the clustering coefficient were similar between HER2 groups, HER2 low group clustered mostly around macrophages while HER2 + group preferred cytotoxic T cells (CD8 + ). Conclusions: The spatial organization and density of immune cells in the HER2 low and HER2 + breast cancer microenvironment may provide insight into prognosis and guide therapeutic approaches for combination therapies and HER2-targeted immunotherapies.