Abstract P5-04-25: Detection of Human Epidermal Growth Factor Receptor 2 (HER2) in Cultured Circulating Tumor Cells (CTCs) Isolated from Peripheral Blood of Breast Cancer Patients

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Abstract Background: Overexpression of HER2 is indicative of a subset of breast cancers that are characterized by rapid growth and poor prognosis. The advent of HER2-targeted therapies has transformed the treatment landscape, making accurate detection and quantification of HER2 expression critical for guiding treatment decisions. Typically, HER2 status is determined through invasive tissue biopsies, which pose risks to patients and logistical and financial burdens. Analysis of CTCs from liquid biopsies offer a minimally invasive alternative, but the rarity of CTCs in peripheral blood and the technical challenges associated with their isolation and characterization have hindered the widespread adoption of this technique for protein biomarker analysis. In response to these challenges, we propose a novel approach to enhance the detection and quantification of HER2 using liquid biopsies by cultivating CTCs ex vivo, thereby amplifying their numbers to a level that allows for more reliable detection. Methods: Adult breast cancer patients at least 18 years of age with tumor size of at least 1 cm were prospectively enrolled for this study. Breast tumor tissue was acquired via core needle biopsy and tested for HER2 protein expression following the hospital’s standard procedures for immunohistochemistry (IHC) and, if needed, for HER2 gene amplification with fluorescence in situ hybridization (FISH) as recommended per the ASCO-CAP Guideline. The peripheral blood mononuclear cell (PBMC) layer was isolated from whole blood for culturing of CTCs on the 3D organoid culture platform, Rapid, Reproducible, Rare Cell 3D Expansion (R3CE) (AcroCyte Therapeutics, Inc., New Taipei City, Taiwan). After 7 days of culture, a multiplex immunofluorescence (IF) staining protocol targeting cell nuclei (DAPI), CD45, EpCAM, and HER2 was employed to identify and enumerate HER2-positive CTCs. Mean CTC counts were compared using Student’s t-test. Results: Ten female breast cancer patients with a mean (standard deviation) age of 55 (8) years were included in the study. One (10%), 5 (50%), and 4 (40%) patients had stage I, II, and III cancer, respectively. Two (20%) and eight (80%) patients had grade 2 and 3 cancer, respectively. Based on tumor tissue pathology, three (30%) were HER2-positive (IHC 3+). FISH was conducted for two patients with IHC 2+, and both were negative. For the remaining 5 patients, 4 were IHC 1+ and one was IHC 0. After 7 days of culturing, CTCs were identified for 8 (80%) of the patients. Mean CTC counts were higher for grade 3 cancer [261 (435)] than grade 2 cancer [80 (113)], but the difference was not statistically significant (p=0.33). Positive HER2 IF staining was observed for all cases in which CTCs were identified. The percentage of CTCs with positive HER2 staining ranged from 38% to 100%, with a median of 77%. No significant difference in percentage of CTCs with positive HER2 staining was observed between patients who were HER2-positive and HER-2 negative by tissue pathology. Conclusion: Our study demonstrated the feasibility of culturing and IF staining CTCs from breast cancer patients for HER2 analysis. Additional investigations of the association between HER2 expression in CTCs and tumor tissue are needed to realize the potential of CTCs for guiding treatment with HER2-targeted therapies. Citation Format: Ying Chang, Wen-Hung Kuo, Yen-Jang Huang, Jia-Yang Chen, Aurin E. Liu, J. Patrick Kampf, Yi-Chun Wu. Detection of Human Epidermal Growth Factor Receptor 2 (HER2) in Cultured Circulating Tumor Cells (CTCs) Isolated from Peripheral Blood of Breast Cancer Patients [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-04-25.

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