Effect of trastuzumab on antibody-dependent cellular cytotoxicity (ADCC) in HER2 nonamplified (non-amp) breast cancer (BC) cells.

Abstract

659 Background: Trastuzumab (T) is currently indicated for the treatment of Her-2 positive breast cancers. The NSABP B-31 adjuvant trial suggested a possible clinical benefit for the addition of T to chemotherapy in patients whose tumors were Her-2 negative (low Her-2 expressing, non-amplified by FISH). ADCC mediated by CD56+ natural killer (NK) cells is known to play a role in response to T in Her-2 positive tumors. To investigate the possibility that T may induce ADCC against Her-2 negative tumors, we examined NK cell-mediated ADCC in T-treated, non-amp, low Her-2 expressing BC cell lines. Methods: A membrane/cytosol separation kit and immunoblotting were used to assess surface Her-2 levels in the non-amp BC cell lines CAMA-1, T47D, EFM19, MCF-7 and CAL-51. The positive control for Her-2 expression and amplification was HCC1954 and MDA-MB-468 was the negative control. PBMCs were isolated from whole blood of 8 healthy volunteers using a Ficoll-Plaque method. CD56+ NK cells were isolated from the PBMC fraction using magnetic bead based positive selection. Direct NK activity against the K562 cell line and the ability of the NK cells to induce T-related ADCC were measured using flow cytometry. Results: Membrane expression of Her-2 was detected in all cell lines examined except the MDA-MB-468 cell line. The direct cytotoxicity of NK cells ranged from 42% to 73% (ratio 10:1, NK: K562). T induced a significant ADCC response in the positive control HCC1954 (p value 0.002) and in each of the five non-amp, low Her-2 expressing cell lines, but not in the negative control MDA-MB-468 (Table). Conclusions: These results suggest that Her-2 non-amp BC cells, with low but detectable levels of Her-2 protein, can bind T and initiate ADCC. Future research will include determination of T-induced ADCC response by NK cells from patients with Her-2 non-amp BC. % increase in ADCC in response to 200 nM T (Ratio 1:1, NK: Target cell) HCC1954 CAMA-1 T47D EFM19 MCF-7 CAL-51 MDA-MB-468 Total Her-2 +++ + + + + + - % Increase ADCC ± std. dev. 29 ± 3 43 ± 13 21 ± 3 25 ± 7 31 ± 15 33 ± 8 0.6 ± 1 p value* 0.002 0.019 0.037 0.015 0.026 0.005 0.627 * p value – Student's t test, % ADCC for each cell line ± T in triplicate p<0.05 significant. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Roche Roche