Background: Human epidermal growth factor receptor 2 (HER2, ERBB2) amplification occurs in 5% of non-breast non-gastric solid tumors. Ado-trastuzumab emtansine (T-DM1) showed preliminary efficacy in patients with HER2 amplified lung, endometrial, salivary gland, biliary tract and ovarian cancers, but the extent of its differential effects across histologies is unknown. Methods: Patients with HER2 amplified solid tumors were enrolled and received treatment 3.6mg/kg IV every 3 weeks. The primary endpoint was overall response rate (ORR) using RECIST v1.1 or PERCIST. A basket trial expansion used a Simon two stage optimal design applied to each of 5 histology cohorts with type I error rate under 2%, power of 90%, H0 10%, H1 40%, with family-wise error rate at 10%. After first stage, lung, salivary gland and endometrial cohorts were expanded to 23 patients. The null hypothesis was rejected for each cohort separately, if at least 6 responses were observed in each cohort. Secondary endpoints included duration of response (DOR), progression-free survival (PFS) and toxicity. HER2 amplification was identified by fluorescence in situ hybridization (FISH), or next generation sequencing (NGS). Correlative studies were performed using tissue immunohistochemistry (IHC). Plasma cell-free DNA (cfDNA) was collected throughout study treatment. Results: 88 patients with 8 unique cancer types were treated across 5 cohorts of HER2 amplified lung, salivary gland, colorectal, endometrial and other cancers. The median age was 66 (26-90). Median line of prior therapy was 2 (1-7). ORR was 33% (29/87 including 11 CRs, 95% CI 24-44%), including 47% (9/19) for lung cancers, 87% (13/15, 8 CRs) for salivary gland cancers, 22% (5/23, 3 CRs) for endometrial cancers, 12% (1/8) for biliary cancers, 14% (1/7) for ovarian cancers. Median DOR was 9.7 months (95% CI 4.8, 20.2), median PFS was 2.76 months (95% CI 2.53, 5.39). There were 8 (9%) G3 treatment related toxicities. HER2 fold change corrected for purity and ploidy by FACETS algorithm correlated with response (p=0.04) and PFS>=3 months (p=0.0037). HER2 amplification by NGS correlated with HER2/CEP17≥2 by FISH (67/71 tested) and IHC3+ (54/68 tested). We observed persistent HER2 amplification in plasma cfDNA during acquired resistance and progression on T-DM1 in patients with salivary gland cancers. Conclusions: Ado-trastuzumab emtansine showed promising efficacy in patients with HER2 amplified lung and salivary gland cancers as identified by NGS, meeting the primary endpoint. However, its efficacy did not meet prespecified response rate in patients with HER2 amplified endometrial, colorectal and other cancers. Histologic lineage differences in HER2 amplified cancers affect response and translational research is critical for further drug development. Clinical trial information: NCT02675829.