Background: Human epidermal growth factor receptor 2 (HER2, ERBB2) amplification occurs in 2-5% of non-breast non-gastric cancers. Ado-trastuzumab emtansine is a HER2 targeted antibody drug conjugate that may have activity against a variety of cancers driven by HER2 amplification. Methods: Patients with HER2 amplified cancers were enrolled into a multi-histology basket trial of ado-trastuzumab emtansine, treated at 3.6mg/kg IV every 3 weeks. The primary endpoint was overall response rate (ORR) using RECIST v1.1 or PERCIST. A Simon two stage optimal design was applied to each histology cohort with type I error rate under 2.7%, power of 89%, H0 10%, H1 40%. Other endpoints include duration of response (DOR), progression-free survival (PFS) and toxicity. HER2 amplification was identified by next generation sequencing (NGS), and tumors with adequate tissue were subsequently tested by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Results: 58 patients were treated across 8 cohorts of advanced lung, endometrial, salivary gland, biliary tract, ovarian, bladder, colorectal and other cancers. The median age was 63 (range 34-90 years), 72% were female. The median lines of prior systemic therapy was 2 (range 1-7). ORR was 26% (14/53 confirmed, 95% CI 15-40%), including 50% (3/6) for lung cancers, 22% (4/18, 2 CR) for endometrial cancers, 100% (5/5, 3 CR) for salivary cancers, 17% (1/6) for biliary cancers, 17% (1/6) for ovarian cancers, not including partial responses awaiting confirmation. Median DOR was 6 months (range 2-22+), median PFS was 3 months (95% CI 2-6). There was 1 (2%) grade 3 febrile neutropenia, but no treatment related deaths. The degree of HER2 amplification (NGS fold change 1.7 to 27.9) did not predict response. HER2 amplification by NGS correlated well with HER2/CEP17≥2 by FISH (40/41 tested) or IHC3+ (31/40 tested), and tumor shrinkage was seen in 2 patients who were tested IHC negative. Conclusions: Ado-trastuzumab emtansine showed efficacy in patients with HER2 amplified lung, endometrial, salivary gland, biliary tract and ovarian cancers as identified by NGS. This study has met its primary endpoint. Further development is warranted. Clinical trial information: NCT02675829