Abstract Background: HR+/HER2+ breast cancer is a unique molecular subtype of HER2+ breast cancer, which is characterized by mild biological behavior and is sensitive to endocrine therapy. Although the standard treatment for HR+/HER2+ metastatic breast cancer is chemotherapy combined with anti-HER2 therapy, this combination is not ideal for patients because of the heavy side effects of chemotherapy. Previous studies have shown that endocrine therapy combined with anti-HER2 therapy can also bring survival benefits to these patients. However, due to the limitations of previous treatment, the optimal combination mode of endocrine and anti-HER2 therapies has not been found. Pyrotinib is the most efficient pan-HER tyrosine kinase inhibitor (TKI) with irreversible blocking of HER2, while fulvestrant is the most potent estrogen receptor (ER) inhibitor. Previous cell experiments showed the cross-talk between ER and HER2 receptor pathways, indicating that the two signal pathways were important mechanisms of drug resistance for each other. Further research showed that ER inhibitor fulvestrant had synergistic effect with HER2 inhibitor pyrotinib. Therefore, we firstly explored the efficacy and safety of pyrotinib combined with fulvestrant in the treatment of HR+/HER2+ metastatic breast cancer.Methods: Eligible patients had histologically confirmed HR+/HER2+ metastatic breast cancer with no more than one line of prior treatment for metastatic disease. Those with central nervous system metastases or any prior HER2 TKI were excluded. Patients were treated with oral pyrotinib 400 mg once daily plus intramuscular injection of fulvestrant 500 mg on days 1, 15, and 29, and once every 28 days thereafter. The primary endpoint was progression-free survival (PFS), as assessed by the data and safety monitoring committee. Secondary endpoints included objective response rate, disease control rate (DCR), overall survival and safety. We also explored the efficacy of subgroups defined by different gene signatures, which were identified using comprehensive genomic variation profiling (FoundationOne CDx). This study is registered with ClinicalTrials.gov, NCT04034589.Results: From July 9, 2019 to June 20, 2021, 34 patients were enrolled; 19 (55.8%) patients had visceral metastases. Of 14 patients with measurable disease according to RECIST 1.1, seven (50%) achieved objective response. The DCR was 84.6%. Nine of the 34 included patients discontinued treatment because of disease progression, and PFS was immature. Twelve patients had available data of comprehensive genomic variation profiling, and the results showed that six patients had PIK3CA mutation, ten had TP53 mutation, and 11 had ErbB2 overexpression. The most common treatment-related adverse events were diarrhea and fatigue. The incidence of grade 3 or greater diarrhea was 12.1% (4/33) in patients with available safety data. No patient discontinued treatment because of adverse events. Conclusions: The combination of pyrotinib and fulvestrant was convenient and effective with manageable toxicity, which may offer a chemotherapy-free alternative treatment option for patients with HR+/HER2+ metastatic breast cancer. Citation Format: Ying Wang, Jianli Zhao, Zhongyu Yuan, Guorong Zou, Haiyan Li, Linxiaoxiao Ding, Yaping Yang, Jie Chai, Donggeng Liu, Herui Yao. Pyrotinib combined with fulvestrant in women with hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-positive (HER2+) metastatic breast cancer: A single-arm phase II clinical trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-35.