Background: Approximately 15-20% of women diagnosed with breast cancer have HER2+ disease. Trastuzumab is currently FDA approved for the treatment of HER+ breast cancer, based on assessment of HER2 status by the combination of IHC and FISH assays. We hypothesized that there are biomarkers besides HER2 that can help predict response, or resistance, to trastuzumab in HER2+ breast cancer.

Methods: We used the NanoString® GeoMx® Digital Spatial Profiler (DSP) to measure 58 protein targets in three different enriched compartments (tumor [PanCK+], leukocyte [CD45+/CD68-] and macrophage [CD68+]) in a cohort of 151 breast cancer patients that received trastuzumab in the adjuvant setting, represented in a tissue microarray. To assess stromal protein expression, leukocyte and macrophage compartments were analyzed in aggregate. Spatially-resolved proteins were correlated with disease-free survival (DFS). Then, we used multiplexed quantitative immunofluorescence (QIF), performed on the AQUA platform, to validate our findings. Statistical analyses were performed using a two-sided test (α=0.05) and multiple testing correction (Benjamini-Hochberg method, FDR<0.10).

Results: We assessed 58 individual protein targets in four compartments resulting in 282 candidate biomarkers per patient. After adjusting for multiple testing, high expression of alpha-smooth muscle actin (a-SMA) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), both measured in the leukocyte compartment, were associated with shorter DFS in univariate analysis (p=0.045 and p=0.084, respectively); high expression of a-SMA in the stroma was also associated with worse outcome (HR, 4.94; 95% CI, 1.07-22.86; unadjusted p=0.023). Digital counts of a-SMA were inversely correlated with immune cell infiltration in the stroma. Using QIF, and after adjusting for four clinical prognostic factors (stage, grade, ER status and PR status), we validated that high a-SMA expression in the stroma was predictive for shorter DFS (HR, 3.34; 95% CI, 1.18-9.48, p=0.023) in the same cohort.

Conclusions: This work supports the role of cancer-associated fibroblasts in the tumor microenvironment in orchestrating the immune response and mediating resistance to trastuzumab for patients with HER2+ breast cancer. In light of the many new HER2 targeted therapies, this observation identifies a-SMA as a potential biomarker to augment the predictive value of the current standard of care HER2 assay and justifies further validation.

Citation Format: Ioannis A. Vathiotis, Myrto K. Moutafi, Prajan Divakar, Thazin Nwe Aung, Aileen Fernandez, Vesal Yaghoobi, Saba Shafi, Konstantinos N. Syrigos, George Fountzilas, Lajos Pusztai, Sarah Warren, David L. Rimm. Resistance to trastuzumab is associated with alpha-smooth muscle actin expression in the stroma of patients with HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 339.