Background: T-DM1, an antibody–drug conjugate comprising trastuzumab, DM1 (microtubule inhibitor), and a stable linker, is approved for patients (pts) with HER2-positive metastatic BC previously treated with trastuzumab and a taxane. In two phase 3 studies, T-DM1 prolonged progression-free survival (PFS; EMILIA, TH3RESA) and overall survival (EMILIA) v control arms. Here we examine the relationship between tissue BM related to the HER2 pathway and PFS in TH3RESA (NCT01419197). Methods: Pts had prior taxane therapy and ≥2 HER2-directed regimens, including trastuzumab and lapatinib, and were randomized 2:1 to T-DM1 (3.6 mg/kg q3w) v TPC. qRT-PCR for HER2, HER3, and G6PDH (ref) mRNA was performed with available pt tumor tissue. Analyses of tumor DNA for PIK3CA mutations (exons 1, 4, 7, 9, 20) and cytoplasmic PTEN expression by IHC were performed as optional research. PFS was analyzed for each BM subgroup using the Kaplan-Meier method and a Cox regression model. Results: Overall, median HER2 mRNA, HER3 mRNA, and PTEN levels and PIK3CA mutation status were consistent across arms and with previous results. For all BM subgroups, median PFS was longer with T-DM1 v TPC (Table). Relative risk reduction for PFS was numerically greater for T-DM1 v TPC for >median HER2 mRNA levels than ≤median levels but similar within HER3 mRNA, PIK3CA, and PTEN (not shown) subgroups. Conclusions: Similar to previous results, T-DM1 prolonged PFS in all BM subgroups analyzed with a greater benefit observed for pts with tumors expressing >median HER2 mRNA levels. Although PIK3CA mutation status was not associated with decreased PFS in the control arm, benefit was seen with T-DM1 regardless of mutation status. Clinical trial information: NCT01419197.