Abstract 6473: Determining the spatial composition of immune infiltrate in HER2+ breast cancerHER2+ breast cancer

Abstract Background: Immunotherapy has improved outcomes in triple negative breast cancer but there is less response in the other subtypes. Tumor infiltrating lymphocytes (TIL) infiltrated tumors respond better to immune therapy in other tumor types. We wanted to be able to identify tumor infiltrated verses sequestered tumors and then identify the immune populations of infiltrated/sequestered tumors using spatial localization of immune and breast cancer receptors. To do this we optimized digital spatial profiling using 2 methods (Nanostring GeoMx and 10X Genomics Xenium), immune protein expression using Lunaphore COMET, and hemoxylin & eosin (H&E) TIL evaluation on a single slide. Our goal was to identify an immune infiltrate biomarker that could predict patients that would respond to neoadjuvant immune therapy in HER2+ breast cancer. Methods: We evaluated 6 HER2+ breast cancer biopsies. Formalin fixed paraffin embedded (FFPE) 10 µM slides from untreated core biopsies were evaluated by the Xenium breast, GeoMx Cancer Transcriptome Atlas, and Comet Lunaphore 21 immune target panels. 4 of the 6 tumors were ER neg PR neg HER2 pos, one was ER neg PR pos HER2 pos, and one was ER pos PR neg HER2 pos. For GeoMX, each ROI was ≥ 250 nuclei and ≥ 400 µM diameter to ensure a sequencing depth of 30X and allow segmentation. Results: We sequentially stained and evaluated the entire specimen on a single FFPE slide using Xenium, Lunaphore, and H&E. The immune infiltrate for HER2 breast cancer was predominantly sequestered in stroma. In GeoMX, the highest expression in CK positive cells was ERBB2 (p=9.6X10-12), proliferation markers (CCND1 p=1.2X10-8), and cytokeratins (KRT7 p=-1.3X10-10) while in the stroma the highest expression was immune markers (CD163 p=6.1X10-12 and CD4 p=6.6X10-12). This was similarly seen in Xenium evaluation where the immune infiltration was predominantly CD163+CD68+ macrophages and FOXP3+CD4+ T cells in the stroma and ERBB2 and cytokeratins in the tumor. Lunaphore protein evaluation confirmed the findings on the same slide. Even with fewer targets in Xenium, similar results were seen between GeoMx and Xenium but the whole slide was able to be evaluated with Xenium. Conclusion: Sequential evaluation of Xenium, Lunaphore, and H&E is feasible and allows spatial identification of the TIL populations in HER2+ breast cancer. Consistent with previous studies, most immune infiltrate is localized in the stroma along the immune front and is predominantly CD4+ regulatory T cells and CD163+ macrophages. Citation Format: Jessica Linak, David Page, Jason Schlumbohm, Fred Robinson, Sasha Elizabeth Stanton. Determining the spatial composition of immune infiltrate in HER2+ breast cancerHER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6473.