Concomitant Association of T-DM1 and Radiation: An In Vitro Study on HER2 Positive Cells of Breast Cancer

Abstract

The better understanding of the EGFR receptors involved in the oncogenesis of HER2 positive breast cancers has led to the emergence of new therapeutic classes: the Antibodies Drug-Conjugate (ADC). Trastuzumab-DM1 (T-DM1) is an ADC combining the anti-HER2 monoclonal antibody trastuzumab with molecules of DM1, a powerful mitotic spindle inhibitor. In HER2 positive breast cancer, T-DM1 is a validated treatment but the effects of concomitant association with radiation are not known. The objective of this study is to determine in vitro the effects of concomitant irradiation with T-DM1 on five different HER2 positive breast cancer cell lines. Five cell lines of human breast cancer with different levels of HER2 expression (HCC-1954, BT-474, SKBR-3, MDA-MB-453, ZR-75). T-DM1 was provided by Roche®/Genetech upon MTA. Expression levels were determined by western blot. T-DM1 toxicity was assessed using the CellTiter-Glo® assay. Cell cycle analysis was performed by flow cytometry after incorporation of BrdU. HER2 cells were irradiated (GSR 137Cs 662 keV) at different dose levels (from 1 to 8 Gy) after exposure to T-DM1. Survival fractions were determined based on clonogenic assays or on cell survival after 5 doubling times. Radiosensitivity parameter (D10 and D37) were calculated using the mean values of a and b determined from the curves drawn for best fit to the experimental data. The action of T-DM1 on HER2 cells showed significant lethality by deprivation of the HER2 signaling pathway and intracellular DM1 action on the cell cycle with significant G2/M phase blocking. After 72h treatment with T-DM1 alone, the median effective dose (ED50) determined for the six cell lines used is function of the expression of the receptor HER2 except for one cell line (BT474). Using sub-toxic doses of T-DM1 and short-term drug exposure (6 or 12h), the cell viability still decreases dramatically after several days. After irradiation alone, D37 and D10 were significantly higher for the three high-HER2 expressing cells lines (HCC1954, BT474 and SKBr3) compared to the low-HER2 expressing cells (MDA-453 and ZR-75-1), with a linear increasing relationship between the radioresistance and the level of HER2 expression (D37: r2=0.9; p<0.0001 and D10: r2=0.7; p<0.0001). In combination with radiation, T-DM1 elicited strictly additive interaction. There was no significant difference on D10 compared to control for all cell lines. HCC1954 : D10 T-DM1 = 6.15±0.64 vs 6.0±0.24 for control; BT-474: D10 T-DM1 = 7.86±0.22 vs 7.6.0±0.43 for control; SKBr3 : D10 T-DM1 = 5.68±0.29 vs 5.43±0.14 for control; MDA-MB-453 : D10 T-DM1 = 4.82±0.07 vs 4.02±0.17 for control and ZR-75-1 = D10 T-DM1 = 3.3±0.3 vs 3.02±0.3 for control. Although HER2 expression is a radioresistant factor, T-DM1 did not have any radiosensitizing impact on HER2 positive breast cancer cells in this in vitro study. The major toxicity of T-DM1 alone could explain this phenomenon but further explorations are needed.