Alpha-smooth Muscle Actin Expression in the Stroma Predicts Resistance to Trastuzumab in Patients with Early-stage HER2-positive Breast Cancer

Purpose: The companion diagnostic test for trastuzumab has not changed much in the last 25 years. We used high-plex digital spatial profiling to identify biomarkers besides HER2 that can help predict response to trastuzumab in HER2-positive breast cancer. Experimental design: Fifty-eight protein targets were measured in three different molecularly defined compartments by the NanoString GeoMx Digital Spatial Profiler (DSP) in a tissue microarray containing 151 patients with breast cancer that received adjuvant trastuzumab as part of the Hellenic Cooperative Oncology Group 10/05 clinical trial. Promising candidate biomarkers were orthogonally validated with quantitative immunofluorescence (QIF). RNA-sequencing data from the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimisation Study (NeoALTTO) were accessed to provide independent cohort validation. Disease-free survival (DFS) was the main outcome assessed. Statistical analyses were performed using a two-sided test (alpha = 0.05) and multiple testing correction (Benjamini-Hochberg method, FDR < 0.1). Results: By DSP, high expression of alpha-smooth muscle actin (alpha-SMA), both in the leukocyte and stromal compartments, was associated with shorter DFS in univariate analysis (P = 0.002 and P = 0.023, respectively). High alpha-SMA expression in the stroma was validated by QIF after controlling for estrogen receptor and progesterone receptor status [HR, 3.12; 95% confidence interval (CI), 1.12-8.68; P = 0.029] showing recurrence on trastuzumab in the same cohort. In the NeoALTTO cohort, elevated levels of ACTA2 were predictive for shorter DFS in the multivariate analysis (HR, 3.21; 95% CI, 1.14-9.05; P = 0.027). Conclusions: This work identifies a-SMA as a novel, easy-to-implement biomarker of resistance to trastuzumab that may be valuable in settings where trastuzumab is combined with other therapies.

Automated summary

Companion diagnostic test for trastuzumab has remained largely unchanged over the last 25 years. High-plex digital spatial profiling identified alpha-smooth muscle actin (alpha-SMA) as a potential biomarker of resistance to trastuzumab in HER2-positive breast cancer. Orthogonal validation confirmed the association of high alpha-SMA expression with shorter disease-free survival, suggesting its potential role in predicting response to trastuzumab in combination therapy settings.