HER2-positive breast cancer is heterogeneous. Some tumors express mutations, like activating PIK3CA mutations or reduced PTEN expression that negatively correlate with response to HER2-targeted therapies. In this exploratory analysis we investigated whether the efficacy of trastuzumab emtansine (T-DM1), an antibody-drug conjugate comprised of the cytotoxic agent DM1 linked to the HER2-targeted antibody trastuzumab, was correlated with expression of specific biomarkers in the phase III EMILIA study. Methods: Tumors were evaluated for HER2 (n = 866), EGFR (n = 832), and HER3 (n = 860) mRNA expression by quantitative reverse transcriptase-PCR; for PTEN protein expression (n = 271) by immunohistochemistry; and for PIK3CA mutations (n = 259) using a mutation detection kit. Survival outcomes were analyzed by biomarker subgroups.