Abstract 3099: Predicting response to trastuzumab deruxtecan in breast cancer using flow cytometry

Background: Historically, characterization of HER2 status has focused on stratifying tumors that over express HER2, known as HER2-positive (HER2+) breast cancer (BC), and those that do not, known as HER2-negative (HER2-) BC. Approximately half of all BCs, considered HER2-, express low levels of HER2 by immunohistochemistry (IHC). Although HER2-low BCs are resistant to traditional HER2-targeted therapies, recent trials have demonstrated they are sensitive to the antibody drug conjugate (ADC) trastuzumab deruxtecan (T-DXd). Tumor expression of HER2 was hypothesized to be required for response to T-DXd, however studies have demonstrated its efficacy in patients with tumors that stained <1+ on IHC, considered HER2-zero (HER2-0) disease. We hypothesize that the HER2 heterogeneity in HER2-low and HER2-0 tumors makes IHC a suboptimal prediction tool. Thus, there is a clinical need for other quantification methods to determine if HER2 represents a valid biomarker to predict response to T-DXd. Methods: We measured HER2 levels by flow cytometry (FC) in IHC-determined HER2-low and HER2-0 BC cell lines and patient-derived organoids (PDOs) and correlated the results to T-DXd sensitivity in vitro. To determine if HER2 is required for response to T-DXd, ERBB2 knocked down by siRNA and CRISPR-CAS9 HER2-knock out (HER2-KO) variants from the HER2-low and HER2-0 parental cell lines and PDOs were generated. To model intratumor HER2 heterogeneity, mixtures of HER2 depleted cells with control cells were made to assess T-DXd efficacy in tumors with decreasing amounts of HER2-expressing cells. To assess ADC uptake, cell lines and PDOs were treated with fluorescently tagged T-DXd and intracellular ADC levels were measured by immunofluorescence. Relative amounts of deruxtecan deposition in the TME were quantified by mass spectrometry. In vivo studies using mixtures of CAS9-control and HER2-KO HER2-low cells implanted into NSG mice are ongoing. Results: FC quantification of HER2 levels across BC cell lines and PDOs were reproducible and agreed with IHC results. Levels of HER2 expression directly correlated with sensitivity to T-DXd, and HER2 expression was required for response. Cell mixtures of HER2 knockdown + SCBi-control cells or CAS9-control and HER2-KO cells were more representative of PDO HER2 heterogeneity as quantified by FC. Growth assays of cell mixtures showed sensitivity to T-DXd was the same regardless of if there were 100%, 50% or <25% HER2-expressing cells. Conclusions: FC represents a novel method for predicting tumor cell response to T-DXd as it can reproducibly quantify HER2 heterogeneity within the TME. Our results suggest that small amounts of HER2-expressing cells are sufficient to elicit response to T-DXd, which may be missed on IHC. Thus, use of techniques like FC to quantify HER2 levels may identify T-DXd responders in the IHC-defined HER2-0 population, thus broadening the eligible patient population that benefits. Citation Format: Daniel S. Peiffer, Muriel Laine, Long Nguyen, Natalia Povarova, Frederick M. Howard, Nan Chen, Kimberly Cole, Olufunmilayo I. Olopade, Rita Nanda, Geoffrey L. Greene. Predicting response to trastuzumab deruxtecan in breast cancer using flow cytometry [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 3099.