Purpose This phase I study assessed the safety, tolerability, pharmacokinetics, 
antitumor activity, and predictive biomarkers of pyrotinib, an irreversible 
pan-ErbB inhibitor, in patients with human epidermal growth factor receptor 2 
(HER2)-positive metastatic breast cancer. Patients and Methods Pyrotinib was 
administered continuously, orally, once per day to patients who did not have 
prior exposure to tyrosine kinase inhibitors of HER2. Planned dose escalation 
was 80, 160, 240, 320, 400, and 480 mg. For pharmacokinetic analysis, timed 
blood samples were collected on day 1 and day 28. Next-generation sequencing was 
performed on circulating tumor DNA and genomic DNA from tumor samples. Results 
Thirty-eight patients were enrolled. The dose-limiting toxicity was grade 3 
diarrhea, which occurred in two patients administered 480 mg of pyrotinib; thus, 
the maximum tolerated dose was 400 mg. Common pyrotinib-related adverse events 
included diarrhea (44.7% [17 of 38]), nausea (13.2% [five of 38]), oral 
ulceration (13.2% [five of 38]), asthenia (10.5% [four of 38]), and leukopenia 
(10.5% [four of 38]). The only grade 3 adverse event was diarrhea. 
Pharmacokinetic analyses indicated that pyrotinib exposure was dose dependent. 
The overall response rate was 50.0% (18 of 36), and the clinical benefit rate 
(complete response + partial response + stable disease ≥ 24 weeks) was 61.1% (22 
of 36). The median progression-free survival was 35.4 weeks (95% CI, 23.3 to 
40.0 weeks). The overall response rate was 83.3% (10 of 12) in trastuzumab-naive 
patients and 33.3% (eight of 24) in trastuzumab-pretreated patients. Preliminary 
results suggest that PIK3CA and TP53 mutations in circulating tumor DNA ( P = 
.013) rather than in archival tumor tissues ( P = .474) may predict the efficacy 
of pyrotinib. Conclusion Continuous once-per-day pyrotinib was well tolerated 
and demonstrated promising antitumor activity in HER2-positive patients with 
metastatic breast cancer. The maximum tolerated dose was established as 400 mg. 
Diarrhea was the dose-limiting toxicity. The promising antitumor activity and 
acceptable tolerability of pyrotinib warrant its further evaluation in a phase 
II study.