Pyrotinib and trastuzumab combination treatment synergistically overcomes HER2 dependency in HER2-positive breast cancer: insights from the PHILA trial

Abstract

Background: 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.

Methods: 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.

Findings: 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.

Interpretation: 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.

Funding: 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).

Keywords: HER2-positive breast cancer; PHILA study; Pyrotinib; Trastuzumab.

Fig. 1

Pyrotinib plus trastuzumab shows superior inhibition of cell growth than pyrotinib or trastuzumab alone in HER2-dependent breast cancer. a–c, BT474 and SKBR3 cells were fixed and stained with 0.06% crystal violet. Representative images of breast cancer cells after various treatments are shown in a. Cell viability was quantified using ImageJ and normalised with vehicle controls (b and c). d and e, Cell viability of BT474 and SKBR3 cells upon treatment with increasing concentrations of pyrotinib as monotherapy or in combination with trastuzumab for 72 h. f, BT474 and SKBR3 cells were cultured with various anti-HER2 agents for 48 h. Phosphorylation and total levels of HER2, EGFR, HER3, AKT, and ERK were detected by Western blotting. Three independent experiments were performed. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, and ns not significant by Student’s t test. Data are represented as mean ± SEM. NC vehicle, pyro pyrotinib, H trastuzumab, pyroH pyrotinib plus trastuzumab, HP trastuzumab plus pertuzumab.

Fig. 2

Pyrotinib plus trastuzumab shows superior inhibition of HER2 signalling pathway than pyrotinib or trastuzumab alone in HER2-dependent breast cancer. The mice bearing BT474 xenografts were treated with various anti-HER2 agents for 7 days, and the tumours were collected. Protein levels of pHER2, HER2, pAKT, pERK, and Ki67 were detected by IHC. Representative images are shown. Scale bar, 50 μm. n = 5 animals per group. NC vehicle, pyro pyrotinib, H trastuzumab, pyroH pyrotinib plus trastuzumab, HP trastuzumab plus pertuzumab.

Fig. 3

The combination of pyrotinib and trastuzumab potentiates HER2 ubiquitination and downregulation. a and b, BT474 and SKBR3 cells were treated with increasing concentrations of pyrotinib with or without trastuzumab for 24 h. Membrane-bound HER2 was detected by Western blotting. c–e, BT474 and SKBR3 cells were treated with various anti-HER2 agents for 6 h. HER2 protein expression was examined by immunofluorescence. Representative images are shown in c. Scale bar, 5 μm. HER2 immunoprecipitations were performed using trastuzumab itself (or IgG control) as primary antibody in BT474 and SKBR3 cells (d and e). f and g, Computed tomography and HER2 IHC staining of a patient with untreated HER2-positive metastatic breast cancer who responded to pyrotinib, trastuzumab and taxane. Scale bar, 200 μm. Three independent experiments were performed. Data are represented as mean ± SEM. NC vehicle, pyro pyrotinib, H trastuzumab, pyroH pyrotinib plus trastuzumab.

Fig. 4

Pyrotinib synergises with anti-neu antibody in the TUBO tumour model. a, Treatment scheme of BALB/c mice bearing TUBO tumours. b–d, Tumour volume (b), tumour weight (c), and body weight (d) changes of the mice in various treatment groups over 21 days. e, Western blotting for pHER2, HER2, pAKT, AKT, pERK, ERK, Survivin, and cPARP in TUBO tumours. f, HER2 IHC staining of TUBO tumours. Representative images are shown. Scale bar, 50 μm g, Schematic representation for the synergistic mechanisms of pyrotinib plus trastuzumab in HER2 positive breast cancer cells. n = 7 animals per group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, and ns not significant by Student’s t test. Data are represented as mean ± SEM. NC vehicle, pyro pyrotinib, pyro+7.16.4 pyrotinib plus 7.16.4.

Fig. 5

Pyrotinib and trastuzumab combination treatment shows marginal efficacy in HER2-positive breast cancer with genetic alterations in HER2 signalling cascade. a and b, HCC1954 cells were fixed and stained with 0.06% crystal violet. Representative images are shown in a. Cell viability was quantified using ImageJ and normalised with vehicle controls (b). c, Cell viability of HCC1954 cells upon treatment with increasing concentrations of pyrotinib as monotherapy or in combination with trastuzumab for 72 h. d, HCC1954 cells were cultured with various anti-HER2 agents for 48 h. Phosphorylation and total levels of HER2, EGFR, HER3, AKT, and ERK were detected by Western blotting. e, The landscape of altered genes at baseline in a cohort of 18 patients at our centre from the PHILA trial. f, GO enrichment analysis of mutated genes in plasma from our centre cohort. g, The Kaplan–Meier curves of PFS in the 18-patient cohort by the alteration status of HER2 signalling pathway. Three independent experiments were performed. ns not significant by Student’s t test. Data are represented as mean ± SEM. NC vehicle, pyro pyrotinib, H trastuzumab, pyroH pyrotinib plus trastuzumab, HP trastuzumab plus pertuzumab. TpyroH pyrotinib, trastuzumab, and docetaxel. TH placebo, trastuzumab, and docetaxel.