Abstract 1914: Phenotypic characterization of a comprehensive set of HER2 missense mutants in ER+ breast cancer
Authors: Nayar Utthara , Piccioni Federica , Yang Xiaoping , Root David , Neal J T. , Eli Lisa D. , Diala Irmina , Lalani Alshad S. , Wagle Nikhil 
Published: September 2020

Abstract
Purpose: Resistance to endocrine therapies in estrogen receptor positive (ER+) metastatic breast cancer (MBC) is widespread, and understanding the mechanisms whereby these tumors acquire resistance is a critical need. We and others previously described acquired activating hotspot HER2 (ERBB2) mutations in ~5% of ER+ MBC that conferred resistance to multiple ER-targeting therapies, including the selective estrogen receptor degrader fulvestrant. These tumors could be re-sensitized to fulvestrant in vitro through addition of the irreversible pan-HER tyrosine kinase inhibitor neratinib, suggesting a possible clinical combination strategy for patients. Although some HER2 mutations are relatively more frequent in tumors, there is a “long tail” of rare HER2 mutations that have not been characterized but remain clinically important for patients whose tumors harbor them. Therefore, there is biological and clinical value in prospectively characterizing all possible missense mutations in HER2.
Methodology: Since only activating HER2 mutations conferred resistance to fulvestrant (and not passenger or inactivating mutations), resistance to fulvestrant in ER+ breast cancer cells can be used as a surrogate kinase assay for HER2 activity. Therefore, we are performing a saturation mutagenesis screen of HER2, using fulvestrant resistance as a readout for activating mutants. Screen optimization included: a) testing selected mutants cloned into a custom vector to identify appropriate positive and negative controls for screen QC, b) custom screen design (transduction under ER inhibition, and an empirically-determined ratio of growth in fulvestrant vs DMSO) to enable recovery of mutants of varying growth phenotypes. We also designed PCR conditions to enable efficient amplification of HER2 (the largest ORF ever tested by saturation mutagenesis) from genomic DNA, and custom-designed and built a comprehensive HER2 library that includes built-in controls such as stop codons. The saturation mutagenesis screen is currently underway, and will generate putative activating HER2 mutations (i.e., not growth-inhibited in fulvestrant versus complete media), that will be validated in a “minipool” screen. Validated hits will be further tested for sensitivity to the combination of fulvestrant+neratinib or other kinase inhibitors.
Summary and conclusions: We have designed a saturation mutagenesis screen to recover a spectrum of activating HER2 mutations, the largest such library generated to date, and using resistance to the ER inhibitor fulvestrant as a novel surrogate kinase assay. This screen will generate a comprehensive reference table of HER2 mutant phenotypes in terms of response and resistance to ER and HER2 targeting agents. These findings will have translational applicability, and may suggest promising precision medicine approaches for clinical management of patients harboring somatic HER2 mutations.
Citation Format: Utthara Nayar, Federica Piccioni, Xiaoping Yang, David Root, J T. Neal, Lisa D. Eli, Irmina Diala, Alshad S. Lalani, Nikhil Wagle. Phenotypic characterization of a comprehensive set of HER2 missense mutants in ER+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1914.