Abstract PS3-12-10: Enhancing responses to trastuzumab deruxtecan using tucatinib in HER2-negative breast cancer

Abstract Background: HER2 status in breast cancer (BC) is determined by a combination of immunohistochemistry (IHC) and in situ hybridization. HER2-positive (HER2+) tumors overexpress the marker, whereas HER2-negative tumors do not. However, approximately ½ of all BCs express modest levels of HER2, defined as HER2-low BC, with the remaining tumors expressing no detectable levels of HER2, considered HER2-0 disease. The antibody drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) has been shown to improve progressive-free and overall survival in metastatic HER2-low BC, leading to its FDA approval for the treatment of this population. Unfortunately, patients with HER2-0 BC are not eligible to receive T-DXd, despite trials demonstrating a subset of these patients respond to the ADC. Recent studies have shown that reversible tyrosine kinase inhibitors (TKIs) like tucatinib can increase membrane HER2 expression, potentially enhancing the efficacy of HER2-targeted ADCs. We hypothesize that tucatinib may increase HER2 expression in the HER2-0 BC, enhancing patient responses to T-DXd with low target expression. Methods: Baseline HER2 expression was assessed using IHC and flow cytometry (FC) across BC cell lines and patient-derived organoids (PDOs). CRISPR HER2-KO variants were created from HER2-low (MCF-7, T47D) and HER2-0 (MDA-MB-231, SUM149) cell lines. To assess T-DXd efficacy with or without tucatinib, growth assays were performed in vitro using Incucyte live-cell imaging and ATP quantification. Tucatinib’s effect on HER2 protein levels, stability, and downstream signaling were examined by FC, cycloheximide chase experiments, and western blot/qPCR analysis, respectively, to assess protein levels, stability, and downstream signaling. T-DXd uptake rates were measured using fluorescently tagged T-DXd by flow cytometry. Additionally, mass spectrometry quantified the deposition of the chemotherapy payload, deruxtecan (DXd). Results: T-DXd efficacy correlated with HER2 levels, as determined by FC and IHC, with membrane HER2 expression being required for response. Tucatinib increased membrane HER2 levels across all parental and CAS9-control cell lines, with no effect on the HER2-KO variants. Pulsing with tucatinib achieved similar results to continuous co-treatment in terms of membrane HER2 levels. The combination of T-DXd and tucatinib had little effect on cell growth in T-DXd-sensitive cell lines. However, it significantly enhanced efficacy in de novo resistant cell lines. These results correlated with increased T-DXd uptake and DXd deposition in vitro. Conclusions: The combination of T-DXd and tucatinib represents a novel treatment strategy that may expand the patient population that responds to T-DXd, particularly those with HER2-0 BC who tend to have highly variable responses to T-DXd at baseline. Given the increased side effect burden associated with tucatinib when used in combination with HER2-targeted ADCs, further studies evaluating tucatinib pulsing strategies are warranted and ongoing. This approach could optimize treatment efficacy while minimizing toxicity, improving outcomes for BC patients, particularly those with HER2-0 disease. Citation Format: D. S. Peiffer, N. Thompson, L. Nguyen, N. Chen, F. M. Howard, H. Shah, K. Cole, R. Nanda, G. L. Greene. Enhancing responses to trastuzumab deruxtecan using tucatinib in HER2-negative breast cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-12-10.