Abstract 2472: HER2 protein localization in super-resolution and anti-HER2 therapy response in breast cancer

Abstract Introduction: Human epidermal growth factor receptor 2 (HER2) in breast cancer (BC) is an important biomarker for a better decision making of anti-HER2 therapy. Trastuzumab has been the standard of care treatment for HER2 positive patients, but recently new antibody-drug conjugates (ADCs) have given an alternative option in clinical practice. Since ADCs efficacy is due to HER2 antibody localization and internalization in the cell, we hypothesize that cellular sensitivity to the drug may be linked to cellular vesicle trafficking systems and membrane protrusions which indicate the resistance of HER2 internalization. Super resolution fluorescence microscope named HM-1000 achieves a resolution less than 20 nm. With this new tool, we aim to study the intracellular distribution pattern of HER2 including vesicle-like structures and membrane protrusions to better identify responders of anti-HER2 therapies, especially ADCs. Method: A retrospective unicentric study is designed to recruit BC patients who have received ADCs treatment that have formalin fixed paraffin-embedded tissue samples stored for routine diagnosis. 5 baseline tumor samples were stained by commercialized HER2 in situ immunofluorescence, and O2 scavenging buffer was applied and sealed to capture the fluorescent blinking every 30 milliseconds by HM-1000. The 50, 000 image frames per microscopic field were processed using ImageJ software and created super-resolution images. Results: We observed that HER2 signals from ADCs responding patients exhibit predominant membrane localization, especially in those with 3+ immunohistochemistry staining status, compared to ADCs non-responding HER2 3+ cases which show low membrane localization of HER2 and more intracellular distribution. Additionally, this finding is highlighted by intense membrane protrusions in HER2 3+ tumor which present very good response of ADCs. Conclusion: We found different HER2 localization in ADC responding and non-responding patients. The presence of membrane protrusions may facilitate HER2 targeted antibody binding and induce both cellular cytotoxicity from immune effector cells and endocytosis of cytotoxic drug, which explains ADCs response. Further studies are needed to support this finding and better predict the effectiveness of ADCs therapies in HER2 positive tumors. Citation Format: Vicente Peg, Yuko Saruta, Yun Li, Santiago Ramon y Cajal. HER2 protein localization in super-resolution and anti-HER2 therapy response in 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 2472.