Background: T-DM1 is an antibody-drug conjugate composed of trastuzumab and the maytansinoid antimicrotubule, DM1. T-DM1 was granted FDA approval in 2nd-line MBC after prior trastuzumab (T) and taxane. Current practice in USA is for pts to receive T and pertuzumab (P) as neoadjuvant or as 1st-line therapy. Retrospective analysis of T-DM1 after T-P suggests a lower response rate (17%) than T-DM1 after T and taxane (EMILIA 43%). This study investigates the safety and efficacy of T-DM1 + N. Methods: Eligible pts had prior T-P as neoadjuvant therapy or in 1st-line, measurable disease, ECOG PS ≤2, adequate hematologic, renal, and liver function. Pts with stable brain metastases (CNS) were eligible. Treatment was T-DM1 at 3.6 mg/kg iv q 3 wk and N at escalating doses of 120, 160, 200, and 240 mg/d using 3+3 design. HER2 + was required on primary tissue but was not reassessed at entry. Blood samples were required upon entry. Pharmacokinetic (PK) studies were performed on a limited number of pts. Primary diarrhea prophylaxis with intensive loperamide was mandated. Results: Twenty-seven T-P resistant pts were enrolled. 26 were evaluable for toxicity, and 20 were evaluable for efficacy. A dose-limiting toxicity occurred in 6 during cycle 1. The RP2D was N 160 mg/d. Treatment-related grade 3 toxicities included diarrhea (5 pts), thrombocytopenia (4), nausea (3), and ALT elevation (1). Of 20 pts evaluable after 2 cycles, 3 had CRs and 9 had PRs (ORR 64%). The duration of response ranged from 42 d to 650+ d. New CNS disease occurred in 1 (8/27 at entry). Nine pts had PK determinations during the first 24h, and 13 had steady-state neratinib level on cycle 2 d1. There was no correlation between N dose and peak or steady-state levels; responses were seen at all doses. Data from blood samples collected on d 1 for HER2 amplification by ctDNA will be presented. Conclusions: Full-dose T-DM1 + N at 160 mg/d was well tolerated with notable activity. Responses were seen at all dose-levels of N. Limited PK analysis did not show that peak or steady-state concentration of N was related to response. Baseline peripheral blood samples are being assessed for HER2 amplification. Support: Puma Biotechnology Clinical trial information: NCT02236000