Mechanism Of Pertuzumab In Combination With Trastuzumab Plus Docetaxel Therapy In A Breast Cancer Xenograft Model

Background: Pertuzumab is a first-in-class humanized monoclonal antibody that prevents HER2-dimerization with ligand-activated HER family receptors. In a Phase III trial for HER2-positive breast cancer (CLEOPATRA), the triple-drug combination arm of trastuzumab (Tras), docetaxel (DTX), and pertuzumab (Per) showed significantly longer progression-free survival and overall survival compared to the Tras + DTX arm. In this study, we investigated the mechanism of action for the triple-drug combination therapy in vivo. Methods: A mouse model xenografted with KPL-4, a human breast cancer cell line that over-expresses HER2, was used for examining the efficacy. Tras (10 mg/kg) and Per (20 mg/kg) were intraperitoneally administered once a week for 3 weeks, and DTX (10 mg/kg) was intravenously injected once every 3 weeks. The tumor tissues excised 4 days after the initial treatment were analyzed in the following ways: counting the number of cells in the mitotic phase on Hematoxylin-Eosin stained specimens; assessing the apoptosis and growth phase of the tumor cells by TUNEL assay and Ki-67 immunohistochemistry, respectively; and examining the signal transduction of HER2 by Western blotting. Results: Tumor volume 21 days after treatment started was 560 ± 206 mm 3 (Control group), 574 ± 262 mm 3 (Per group), 133 ± 102 mm 3 (Tras + DTX group) and 11 ± 27 mm 3 (Tras + DTX + Per group). Tumor growth inhibition by Per + DTX (106 ± 95 mm 3 ) was similar to Tras + DTX. Furthermore, the triple-drug combination showed significantly stronger antitumor activity than the combination of 30 mg/kg of Tras (equal amount with the triple-drug combination as an anti-HER2 antibody) and DTX. Thus, our preclinical model accurately reflects the results shown by the two different anti-HER2 antibodies in clinical treatment. Using this model, we examined how Per enhanced the antitumor activity of Tras + DTX treatment. The number of cells in the mitotic phase in tumors treated with Tras + DTX and Tras + DTX + Per was equivalent to DTX alone, indicating that Per and Tras did not enhance the mitotic arrest induced by DTX. On the other hand, in the Tras + DTX + Per group compared to the Tras + DTX group, Ki-67 staining was significantly less and apoptotic tumor cells significantly enhanced, even though neither Tras nor Per increased apoptosis as a single agent. These results were supported by Western blot analysis showing that Tras + DTX + Per decreased phosphorylation of HER2, HER3, and Akt more strongly than Tras + DTX. These results suggest that Per in combination with Tras + DTX accelerated apoptosis by DTX after enhancing Tras-mediated HER2-HER3-AKT signal inhibition. Conclusion: The mechanism of the synergistic efficacy of Per in combination with Tras + DTX is at least in part attributed to the acceleration of DTX-mediated apoptosis after mitotic arrest. Citation Format: Yoriko Yamashita-Kashima, Sei Shu, Keigo Yorozu, Yoichiro Moriya, Naoki Harada. Mechanism of pertuzumab in combination with trastuzumab plus docetaxel therapy in a breast cancer xenograft model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2637. doi:10.1158/1538-7445.AM2015-2637