Abstract Background: T-DXd is FDA approved for patients with metastatic breast cancer (MBC) and HER2+ or HER2 low/ultra-low status. However, response and resistance mechanisms associated with T-Dxd are not well understood and how they differ across HER2 subtypes requires further interrogation. Cathepsins are a family of proteolytic enzymes that play crucial roles in cellular processes including protein degradation and turnover in endosomes/lysosomes and when secreted into the extracellular space can contribute to matrix remodeling. Proteases are also important for ADC linker cleavage and payload release. Recent preclinical data revealed that specific extracellular and intracellular proteases, including cathepsins, may alter the efficacy of ADCs with cleavable linkers, such as T-DXd, as opposed to ADCs with non-cleavable linkers like T-DM1 that depend on complete lysosomal degradation. Here, from a clinicogenomic database, we characterized the transcriptomic expression of proteases prior to T-DXd vs T-DM1 treatment (tx) to understand their impact on clinical outcomes in MBC. Methods: Using the Tempus database, the transcriptomic expression of two protease genes (cathepsin L “CTSL”, cathepsin B “CTSB”) was generated from pre-tx biopsies collected from patients with MBC (N = 453; 36% TNBC, 26% HR+/HER2-, 19.2% HER2+, 19% NOS) 1 year prior to or up to 15 days post-tx with SG (n = 204), T-DXd (n = 178), or T-DM1 (n = 71). RNA-sequencing data were generated and processed with the Tempus xR assay. Cox proportional hazards models with risk set adjustment were used to test for associations between pre-tx gene expression and overall survival (OS), where gene expression was modeled as a continuous linear predictor. The proportional hazards assumption for OS was tested. Given the exploratory nature of the analyses, all p-values are uncorrected and nominal statistical significance was set at p 0.05. Results: This cohort had a median age of 52 and was diverse (55% White, 14% Black, 7.1% Other, 24% Unknown). Among patients treated with T-DXd (n = 170), high expression of CTSB and CTSL was associated with significantly worse OS in the HER2+ subgroup (n = 35; HR 4.35; p = 0.013 and HR 6.04; p = 0.002, respectively) but correlated with improved outcomes in patients with HR+/HER2− disease (n = 75; HR 0.51; p = 0.024 and HR 0.56; p = 0.013, respectively). In the TNBC cohort (n = 28), we observed the following trends: CTSB: HR 0.63, p = 0.4 and CTSL: HR 0.75, p = 0.5. In contrast, in the T-DM1 cohort, no significant associations were found between protease expression and OS (entire cohort: n = 59, HR 1.03; p = 0.9 and HR 0.96; p = 0.9 for CTSB and CTSL, respectively; HER2+cohort: n=39, HR 0.55; p = 0.10 and HR 0.69; p = 0.2 for CTSB and CTSL, respectively). Conclusions: Using a real-world multimodal dataset, here we build upon recent in-vitro findings and demonstrate the potential role of proteases as biomarkers of response to T-DXd in HER2 low/ultralow, but not HER2 positive/amplified breast cancer. These findings highlight the potential differential dependence on cathepsins based on HER2 expression and impact on payload release modulating efficacy in MBC. These findings need to be validated in additional datasets and can guide biomarker-driven clinical application of ADCs. Citation Format: S. Alkassis, A. Dugan, U. Jariwala, B. Yilma, S. Fragkogianni, M. Lipsyc-Sharf, A. LeVee, Y. Yuan, F. Meric-Bernstam, H. Rugo, J. O’Shaughnessy, M. Rimawi, R. Schiff, D. Elashoff, J. Glaspy, M. McDermott, A. Cummings, J. Mercer, A. Bardia. Cathepsin Protease Expression and Therapeutic Outcomes to Trastuzumab Deruxtecan (T-DXd) in Metastatic 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 PS2-08-18.
Alkassis et al. (Tue,) studied this question.