PHILA 研究表明，与安慰剂、曲妥珠单抗和多西他赛相比，吡咯替尼、曲妥珠单抗和多西他赛可显着改善未经治疗的 HER2 阳性转移性乳腺癌患者的无进展生存期 (PFS)。在本研究中，我们旨在研究吡咯替尼加曲妥珠单抗的协同机制，并为 PHILA 试验提供进一步的见解。通过细胞生物学和生化实验评估联合治疗的体外活性。在细胞来源的异种移植物、TUBO 肿瘤模型和一个临床病例中评估了体内疗效。对 PHILA 试验中患者的循环肿瘤 DNA (ctDNA) 进行了新一代测序。在 HER2 依赖性乳腺癌模型中，吡咯替尼和曲妥珠单抗的组合比单独使用吡咯替尼或曲妥珠单抗更有效地抑制细胞生长。它增强了膜 HER2 泛素化和下调，从而导致 HER2 信号通路的全面阻断。吡咯替尼改变的膜 HER2 水平对曲妥珠单抗介导的抗体依赖性细胞介导的细胞毒性 (ADCC) 无显着影响。我们进一步验证了 TUBO 肿瘤和一个临床病例的协同机制，而不是在含有 PIK3CA H1047R 突变的 HCC1954 细胞模型中。同样，在我们的 PHILA 研究中心队列中，HER2 信号级联通路发生基因改变的患者的中位 PFS 显着短于具有野生型通路的个体。我们的研究结果强调了吡咯替尼和曲妥珠单抗在克服 HER2 依赖性方面的强大协同作用，并提供了吡咯替尼、曲妥珠单抗和多西他赛作为未经治疗的 HER2 阳性转移性乳腺癌患者的最佳选择之一的理由，这些患者依赖于 HER2 信号级联。这项工作得到了中国国家重点研发计划 ( 2021YFF1201300）、国家自然科学基金（82172875）、中国医学科学院医学科学创新基金（2022-I2M-2-001）、北京市自然科学基金与昌平区联合创新基金（L234004） .版权所有 © 2024 作者。由 Elsevier B.V. 出版。保留所有权利。

The PHILA study suggests that pyrotinib, trastuzumab, and docetaxel significantly improved progression-free survival (PFS) compared with placebo, trastuzumab, and docetaxel in patients with untreated HER2-positive metastatic breast cancer. In this study, we aimed to investigate the synergistic mechanisms of pyrotinib plus trastuzumab and provide further insights for the PHILA trial.The in vitro activity of combination treatments was assessed through cell biological and biochemical experiments. The in vivo efficacy was evaluated in cell-derived xenografts, a TUBO tumour model, and one clinical case. Next-generation sequencing was performed on circulating tumour DNA (ctDNA) from patients in the PHILA trial.The combination of pyrotinib and trastuzumab more effectively inhibited cell growth than pyrotinib or trastuzumab alone in models of HER2-dependent breast cancer. It potentiated membrane HER2 ubiquitination and downregulation, which resulted in a comprehensive blockade of the HER2 signalling pathway. The pyrotinib-altered membrane HER2 levels had no significant effect on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). We further validated the synergistic mechanisms in TUBO tumours and one clinical case, rather than models of HCC1954 cells harbouring the PIK3CA H1047R mutation. Similarly, in our centre cohort of the PHILA study, patients with genetic alterations in the HER2 signalling cascade had significantly shorter median PFS than individuals with the wild-type pathway.Our findings underscore the robust synergy between pyrotinib and trastuzumab in overcoming HER2 dependency and provide a rationale for pyrotinib, trastuzumab, and docetaxel as one of the optimal choices for patients with untreated HER2-positive metastatic breast cancer, who are dependent on the HER2 signalling cascade.This work was supported by the National Key Research and Development Program of China (2021YFF1201300), the National Natural Science Foundation of China (82172875), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2022-I2M-2-001), and the Joint Innovative Fund of Beijing Natural Science Foundation and Changping District (L234004).Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.