Abstract Purpose/Objective(s): To determine the effects of concurrent irradiation and T-DM1 on HER2-positive breast cancer cell lines. Materials/Methods: Five human breast cancer cell lines (in vitro study) presenting various levels of HER2 expression were used to determine the potential therapeutic effect of T-DM1 combined with radiation. The toxicity of T-DM1 was assessed using CellTiter-Glo® assays and cell cycle analysis was performed by flow cytometry after BrdU incorporation. HER2 cells were irradiated at different dose levels after exposure to T-DM1. Survival fractions were determined by cell survival after 5 population doubling times. Results: The results revealed that T-DM1 induced significant lethality due to the intracellular action of DM1 on the cell cycle with significant G2/M phase blocking. Even after a short time incubation, the potency of T-DM1 was maintained and even enhanced over time, with a higher rate of cell death. After irradiation alone, the D10 (dose required to achieve 10% cell survival) was significantly higher for high HER2-expressing cell lines than for low HER2-expressing cells, with a linearly increasing relationship. In combination with irradiation, using conditions that allow cell survival, T-DM1 does not induce a radiosensitivity, both effects are strictly additive. Conclusion: Although intrinsic HER2 expression is a factor of radioresistance, the results indicated that T-DM1 is not a radiation-sensitizer under the experimental conditions of this study. However, further investigations are needed, in particular in vivo studies before reaching a final conclusion. Citation Format: Fabien Mignot, Youlia Kirova, Pierre Verrelle, Marie-Paule Teulade-Fichou, Frédérique Megnin-Chanet. Trastuzumab Emtansine (T-DM1): No radiosensitizing effect in vitro on HER2-positive breast cancer cells (study in vitro) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-24.