Abstract 4639: Single-cell immunoprofiling and spatial analysis of hormone receptor subtypes in HER2+ and HER2low breast tumors using multiplexed immunofluorescence
Authors: Juncker-Jensen Anna , Tallman David , Nunns Harry , Lefebvre Heather , Yamamoto Karen , Collier Katharine A. , Vater Mark , Strahan Ava , Willoughby Ava , Bouchard Olivia , Kingsbury Madison , Cherian Mathew , Pariser Ashley C. , Sudheendra Preeti K. , Ramaswamy Bhuvaneswari , Gatti-Mays Margaret , Kyshtoobayeva Ainura , Li Zaibo , Stover Daniel G. 
Published: April 2023

Abstract
Background: Breast cancer is characterized by distinct molecular subtypes based on expression of estrogen and progesterone hormone receptors (ER and PR), and epidermal growth factor receptor 2 (HER2). To investigate presence and location of distinct immune cell populations on single cell level we utilized the multiplexed immunofluorescence (mIF) platform MultiOmyx to investigate the tumor immune microenvironment (TME) in HER2+ breast cancer. We have built on growing data implicating distinct immunophenotypes in the breast cancer TME with breast cancer outcomes by profiling 1) prevalence; 2 location (e.g. intratumoral, stromal); 3) phenotype (e.g. activated, exhausted) of infiltrating immune cells.
Methods: We optimized a custom 26-marker MultiOmyx panel interrogating HER2 IF expression, HER2 signaling, and immune markers. This mIF platform leverages serial IF image capture to allow concurrent profiling of all 26 markers on a pathologic section at single cell resolution. We applied the 26-marker panel to a tissue microarray of 208 unique patients with matched tumor/normal tissue cores (1-4 cores/patient; total 333 tumor and 307 normal cores). HER2-positive was defined via ASCO/CAP guidelines; HER2-low was defined as HER2 immunohistochemistry (IHC) 1+/2+ but HER2 in-situ hybridization (ISH) negative.
Results: The 208 unique tumors profiled included 88.9% (185/208) HER2-positive and 11.1% (23/208) HER2-low; 62.5% (130/208) hormone receptor (HR) positive and 37.5% (78/208) HR negative. Median follow-up from diagnosis was 143 months. Among HER2-positive patients, 98.9% (n=183/185) received HER2-directed therapy in the (neo)adjuvant or metastatic setting or were diagnosed prior to FDA approval of trastuzumab for early stage disease. In sum, from 1166 regions of interest in 640 total cores, a total of 1,076,700 single cells were profiled via the 26-marker panel. Tumors categorized based on HR and HER2 reflected distinct immunophenotype. Relative to HR+HER2low, HR-HER2low tumors had a significant increase in the density of T cells (CD3) and tumor-associated macrophages (TAMs) (CD68). The increase in T cells was observed for T helper (CD3+CD4+), T regulatory (CD3+CD4+FoxP3+), as well as T cytotoxic cells (CD3+CD8+). Observed increases in pro-tumorigenic M2 TAM density (CD68+CD163+) were observed for both HR negative subtypes when compared to HR+HER2+ and HR+HER2low groups, indicating a negative correlation between M2 TAM infiltration and ER/PR status. HER2low tumors had significantly lower tumor PDL1+ than HER2+ tumors. Additionally, 18 of the 208 patients with equal distribution across the hormone-receptor subtypes underwent whole slide multiplexed analysis to perform a deeper spatial analysis.
Conclusions: In a large, clinically annotated cohort of breast cancers distinct immunophenotypes are evident among HR and HER2+ subsets.
Citation Format: Anna Juncker-Jensen, David Tallman, Harry Nunns, Heather Lefebvre, Karen Yamamoto, Katharine A. Collier, Mark Vater, Ava Strahan, Ava Willoughby, Olivia Bouchard, Madison Kingsbury, Mathew Cherian, Ashley C. Pariser, Preeti K. Sudheendra, Bhuvaneswari Ramaswamy, Margaret Gatti-Mays, Ainura Kyshtoobayeva, Zaibo Li, Daniel G. Stover. Single-cell immunoprofiling and spatial analysis of hormone receptor subtypes in HER2+ and HER2low breast tumors using multiplexed immunofluorescence. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4639.