The antibody-drug conjugate trastuzumab emtansine (T-DM1) is approved for human epidermal growth factor receptor 2 (HER2/ERBB2)-positive breast cancer. We aimed to study tumor HER2 expression and its effects on T-DM1 responses in patients with HER2-positive urothelial bladder cancer (UBC) or pancreatic cancer (PC)/cholangiocarcinoma (CC). In the phase II KAMELEON study (NCT02999672), HER2 status was centrally assessed by immunohistochemistry, with positivity defined as non-focal homogeneous or heterogeneous overexpression of HER2 in ≥30% of stained cells. We also performed exploratory biomarker analyses (e.g., gene-protein assay) on tissue samples collected from study participants and consenting patients who failed screening. Of the 284 patients successfully screened for HER2 status (UBC, n = 69; PC/CC, n = 215), 13 with UBC, four with PC, and three with CC fulfilled eligibility criteria. Due to recruitment difficulty, the sponsor terminated KAMELEON prematurely. Of the five responders in the UBC cohort (overall response rate, 38.5%), HER2 expression was heterogeneous in two and homogeneous in three. The one responder in the PC/CC cohort had PC, and the tumor displayed homogeneous expression. In the biomarker-evaluable population, composed of screen-failed and enrolled patients, 24.3% (9/37), 1.5% (1/66), and 8.2% (4/49) of those with UBC, PC, or CC, respectively, had HER2-positive tumors. In a gene-protein assay combining in situ hybridization with immunohistochemistry, greater HER2 homogeneity was associated with increased ERBB2 amplification ratio. In conclusion, KAMELEON showed that some patients with HER2-positive UBC or PC can respond to T-DM1 and provided insight into the prevalence of HER2 positivity and expression patterns in three non-breast tumor types.