Abstract Background: Approximately 10% of patients with HER2-positive breast cancer have primary resistance to trastuzumab, leading to poor prognosis. Although several trials enrolled those hard-to-treat patients, there has been no strong evidence available for the clinical decision making. This multicenter phase 2 trial aimed to investigate the activity and safety of pyrotinib plus capecitabine only in those patients with trastuzumab-resistant, HER2-positive advanced breast cancer. Methods: Patients from 17 sites in China received pyrotinib 400 mg once a day and capecitabine 1000 mg/m2 twice a day on days 1-14 every 21 days until disease progression or intolerable toxicity. Based on the definitions used in prior clinical trials, primary trastuzumab resistance was defined as progression during trastuzumab treatment (Group 1) or within 12 months after completing trastuzumab treatment in the (neo)adjuvant setting (trastzuzumab should have been for ≥9 weeks, Group 2), or progression within 6 months after the initiation of trastuzumab treatment in the advanced setting (treatment should have been for ≥6 weeks, Group 3). The primary endpoint was progression-free survival (PFS). The study is registered with ClinicalTrials.gov, NCT04001621. Results: Between June 2019 and September 2021, a total of 100 patients enrolled; 35 (35.0%) patients had hormone receptor (HR)-positive disease, and 65 (65.0%) had HR-negative disease. Prior use of trastuzumab, pertuzumab and antibody-drug conjugate was reported in 100%, 21.0% and 2.0% of patients, respectively. By the data cutoff on July 10, 2022, the median follow-up duration was 23.4 months (95%CI, 20.5-25.6) with 66 PFS events documented. Median PFS was 11.8 months (95%CI, 8.4-15.1) in the overall population. Patients in Group 2 (n=49) had the longest median PFS of 17.8 months (95%CI, 13.8-not reached), which was significantly different from either 8.2 months (95%CI, 3.0-20.7; p = 0.001) in Group 1 (n=21) or 5.6 months (95%CI, 4.1-6.9; p < 0.001) in Group 3 (n=30). No significant difference in median PFS was observed in subgroup by HR status (HR-positive: 9.7 months [95%CI, 6.4-18.4]; HR-negative: 12.3 months [95%CI, 8.2-17.8]; p = 0.764). Objective response rate was 70.0% (95%CI, 60.0%-78.8%). Overall survival data was immature. The most common grade ≥3 treatment-emergent adverse events included diarrhea (24.0%), palmar-plantar erythrodysaesthesia syndrome (9.0%), neutrophil count decreased (7.0%), hypokalemia (5.0%), and decreased appetite (5.0%). No treatment-related deaths occurred. Conclusions: Pyrotinib plus capecitabine resulted in a promising PFS that crossed the pre-specified efficacy boundary in patients with HER2-positive advanced breast cancer who met the traditional definition of primary trastuzumab resistance. Patients in Group 2 had a significant longer PFS than those in either Group 1 or Group 3, highlighting the need to re-define primary trastuzumab resistance and to clarify efficacy of new anti-HER2 biologicals for each subpopulation. Citation Format: Xichun Hu, Jun Cao, Yue’e Teng, Hui-Ping Li, Lili Zhang, Quchang Ouyang, Weimin Xie, Yueyin Pan, Zhenchuan Song, Xiaoling Ling, Xiaohong Wu, Jingwei Xu, Li Li, Liping Ren, Hong Wang, Dongxian Zhou, Jing Luo. PyrotInib in combination with Capecitabine for trasTUzumab-REsistant, HER2-positive advanced breast cancer (PICTURE): a multicenter phase 2 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-43.