Abstract P1-04-02: Exome sequencing identified emergence of HER2 kinase domain mutations in trastuzumab-resistant breast cancer
Authors: Jiang Yizhou , Yu Ke-Da , Zuo Wen-Jia , Wang Yu-Jie , Shao Zhi-Ming 
Published: May 2015

Abstract
Purpose
Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is primarily treated with trastuzumab and other HER2-targeted therapies. However, approximately 30% of HER2-positive tumors exhibit de novo resistance, whereas 40% acquire resistance to these therapies. The mechanisms underlying drug resistance are unclear, and a better understanding of acquired resistance to HER2-targeted therapy is essential for the development of more effective therapeutics.
Methods
We enrolled 11 patients with HER2-positive breast cancer, all of whom had been treated with trastuzumab for one year and developed lung metastasis in the follow-up. We conducted exome sequencing of the primary and paired metastatic tumors from four patients. We identified somatic mutations in HER2 (K753E and L755S) affecting the kinase domain only in the metastatic lesions of two cases. Sanger sequencing was performed to analyze all exons of the HER2 gene in the paired primary and metastatic tumors of the remaining seven cases. Furthermore, we enrolled 48 patients with HER2-positive disease who underwent trastuzumab-based neoadjuvant therapy. We performed Sanger sequencing of the HER2 gene in paired tumor DNA before and after neoadjuvant therapy. To determine the phenotype of these mutations, we functionally characterized the HER2 mutations using protein structure analysis, in vitro kinase assays, cell culture, and xenograft experiments.
Results
We identified somatic mutations in HER2 affecting the kinase domain in four of 11 metastatic tumors (one case with K753E and three cases with L755S). None of the primary tumors harbored HER2 mutations. Furthermore, Sanger sequencing in the neoadjuvant setting revealed two of 48 cases harboring HER2 mutations (K753E and L755S) in the post-treatment samples, none of whom had any HER2 mutation in the paired pre-treatment tumor. Protein structure visualization hinted that the HER2 L753 and L755 side chains were in close proximity to the binding site for small-molecule inhibitors, indicating mutations at this residue might produce drug resistance. In vitro kinase assays revealed that HER2 K753E had greater tyrosine kinase-specific activity than wild-type. HER2 autophosphorylation, phosphorylation of HER2’s dimerization partners (EGFR or HER3) and downstream signaling proteins were significantly upregulated in MCF10A and MCF7 cells overexpressing HER2 K753E or L755S. Trastuzumab or lapatinib treatment did not decrease the number of colonies formed in MCF10A cells transduced with K753E or L755S. MCF7 cells bearing K753E or L755S mutant could reverse the inhibition of lapatinib in xenografts. The MCF10A-HER2 K753E and L755S cells were resistant to trastuzumab (IC50&gt;1000μg/mL) and lapatinib (IC50&gt;10μmol/L) but could be readily inhibited by neratinib (IC50 of 15 nmol/L), an irreversible kinase inhibitor of HER2.
Conclusion
These data implicate that mutations in HER2 kinase domain are a key mechanism in resistance to HER2-targeted therapy. The selection of functional HER2 mutations might act as potential drivers of trastuzumab resistance during the progression of HER2-positive disease. Furthermore, more potent HER2 inhibitors such as neratinib might be of therapeutic benefit for trastuzumab-resistant breast cancer.
Citation Format: Yizhou Jiang, Ke-Da Yu, Wen-Jia Zuo, Yu-Jie Wang, Zhi-Ming Shao. Exome sequencing identified emergence of HER2 kinase domain mutations in trastuzumab-resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-04-02.