Intra-tumor heterogeneity defines treatment-resistant HER2+ breast tumors

Targeted therapy for patients with HER2 positive (HER2+) breast cancer has improved the overall survival, but many patients still suffer relapse and death of the disease. Intra-tumor heterogeneity of both estrogen receptor (ER) and HER2 expression has been proposed to play a key role in treatment failure, but little work has been done to comprehensively study this heterogeneity at the single-cell level. In this study, we explored the clinical impact of intra-tumor heterogeneity of ER protein expression, HER2 protein expression, and HER2 gene copy number alterations. Using combined immunofluorescence and in situ hybridization on tissue sections followed by a validated computational approach, we analyzed more than 13,000 single tumor cells across 37 HER2+ breast tumors. The samples were taken both before and after neoadjuvant chemotherapy plus HER2-targeted treatment, enabling us to study tumor evolution as well. We found that intra-tumor heterogeneity for HER2 copy number varied substantially between patient samples. Highly heterogeneous tumors were associated with significantly shorter disease-free survival and fewer long-term survivors. Patients for which HER2 characteristics did not change during treatment had a significantly worse outcome. This work shows the impact of intra-tumor heterogeneity in molecular diagnostics for treatment selection in HER2+ breast cancer patients and the power of computational scoring methods to evaluate in situ molecular markers in tissue biopsies.