Published OnlineFirst July 2, 2018; DOI: 10.1158/1078-0432.CCR-18-0991 Personalized Medicine and Imaging Clinical Cancer Research High-Throughput Functional Evaluation of Variants of Unknown Significance in ERBB2 Masaaki Nagano1,2, Shinji Kohsaka3, Toshihide Ueno1, Shinya Kojima1, Kanju Saka4, Hirotaro Iwase4, Masahito Kawazu3, and Hiroyuki Mano1,5 Abstract Purpose: The advent of next-generation sequencing tech- prevalence of concurrent ERBB2 mutation with gene amplifi- nologies has enabled the identification of several activating cation and found that approximately 30% of ERBB2-amplified mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) urothelial carcinomas simultaneously carried ERBB2 muta- among various cancers. However, the significance of infre- tions, altering their sensitivity to trastuzumab, an mAb against quent mutations has not been fully investigated. Herein, we ERBB2. Furthermore, the MANO method was applied to comprehensively assessed the functional significance of the evaluate the functional significance of 17 compound muta- ERBB2 mutations in a high-throughput manner. tions within ERBB2 reported in the COSMIC database, reveal- Experimental Design: We evaluated the transforming activ- ing that compound mutations involving L755S were sensitive ities and drug sensitivities of 55 nonsynonymous ERBB2 to osimertinib but insensitive to afatinib and neratinib. mutations using the mixed-all-nominated-in-one (MANO) Conclusions: Several ERBB2 mutations showed varying method. sensitivities to ERBB2-targeted inhibitors. Our comprehensive Results: G776V, G778_S779insG, and L841V were newly assessment of ERBB2 mutations offers a fundamental database revealed to be activating mutations. Although afatinib, ner- to help customize therapy for ERBB2-driven cancers. atinib, and osimertinib were shown to be effective against We identified several ERBB2 mutations as activating muta- most of the ERBB2 mutations, only osimertinib demonstrated tions related to tumorigenesis. In addition, our comprehensive good efficacy against L755P and L755S mutations, the most evaluation revealed that several ERBB2 mutations showed common mutations in breast cancer. In contrast, afatinib and varying sensitivities to ERBB2-targeted inhibitors, and thus, neratinib were predicted to be more effective than other the functional significance of each variant should be inter- inhibitors for the A775_776insYVMA mutation, the most preted precisely to design the best treatment for each patient. frequent ERBB2 mutation in lung cancer. We surveyed the Clin Cancer Res; 1–11. Ó2018 AACR. Introduction ERBB2-positive gastric and breast cancers (8, 9). ERBB2 mutations were first reported in 2% to 4% of lung adenocarcinomas (10), Erb-B2 receptor tyrosine kinase 2 (ERBB2) is a member of the and cancer genome resequencing with next-generation sequenc- ErbB tyrosine receptor family, which also includes EGFR, ERBB3, ing technologies has identified many ERBB2 mutations across a and ERBB4. ERBB2 dimerizes with itself or other ErbB members to variety of cancers, such as breast, lung, gastric, colorectal, liver, activate two major downstream signaling pathways: PI3K–AKT ovarian, and urothelial cancers (11–16). and MEK–ERK (1). ERBB2 gene amplification occurs in a wide Some ERBB2 mutations are known to be activating mutations variety of human cancers (2–4). ERBB2-targeted therapies, such as that induce oncogenic transformation (11, 17), but many other trastuzumab, lapatinib, and pertuzumab, have improved out- mutations are still variants of unknown significance (VUS; refs. 16, comes in patients with ERBB2 amplification-positive cancers 18). In fact, although over 300 ERBB2 nonsynonymous mutations (5–7), and these drugs have been approved by the FDA against are reported in the COSMIC database (http://cancer.sanger.ac.uk/ cosmic/), only 74 of these are annotated in the OncoKB database (http://oncokb.org/). Furthermore, only a few studies have exam- 1Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 2Department of General Thoracic Surgery, Graduate ined the sensitivity of these mutations to drugs (11, 18, 19). Phase School of Medicine, The University of Tokyo, Tokyo, Japan. 3Department of I and II clinical trials have shown that irreversible ErbB receptor Medical Genomics, Graduate School of Medicine, The University of Tokyo, Tokyo, family inhibitors, such as afatinib, neratinib, and dacomitinib, are Japan. 4Department of Forensic Medicine, Graduate School of Medicine, The effective for tumors expressing ERBB2 mutations (20–22). Recent- 5 University of Tokyo, Tokyo, Japan. National Cancer Center Research Institute, ly, the efficacy of neratinib monotherapy was tested across 21 Tokyo, Japan. cancer types in the SUMMIT trial (23), and the greatest clinical Note: Supplementary data for this article are available at Clinical Cancer activity was observed in breast cancer (objective response rate at Research Online (http://clincancerres.aacrjournals.org/). week 8, 32%). Moreover, a recent study has demonstrated osi- Corresponding Authors: Shinji Kohsaka, Department of Medical Genomics, The mertinib, a third-generation tyrosine kinase inhibitor (TKI) University of Tokyo, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, against EGFR T790M, as a potential ERBB2-targeting agent Tokyo 113-0033, Japan. Phone: 813-5841-0633; Fax: 813-5841-0634. E-mail: (24). However, there has been no large clinical trial of afatinib [email protected]; and Hiroyuki Mano, [email protected] or osimertinib conducted for ERBB2 mutation–positive cancers. doi: 10.1158/1078-0432.CCR-18-0991 Moreover, recent studies have also reported the emergence of Ó2018 American Association for Cancer Research. ERBB2 K753E or L755S mutation after trastuzumab treatment or www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst July 2, 2018; DOI: 10.1158/1078-0432.CCR-18-0991 Nagano et al. Virus production and infection Translational Relevance The recombinant plasmids were transduced together with The development of next-generation sequencing technolo- packaging plasmids (Takara Bio) into HEK293T cells to achieve gies has identified many Erb-B2 receptor tyrosine kinase 2 recombinant retroviral particles. The 3T3 cells were infected in (ERBB2) mutations across a wide range of cancers. Several 12-well plates with ecotropic recombinant retroviruses using ERBB2 mutations are known to be activating mutations, but 4-mg/mL Polybrene (Sigma-Aldrich) for 24 hours. Ba/F3 cells many other mutations are still variants of unknown signifi- were plated in retronectin-coated (Takara Bio) 12-well plates cance. Moreover, only a few studies have examined the sen- and infected with the retroviruses in RPMI1640 medium con- sitivity of the ERBB2 mutations to drugs. In this study, we taining 20 U/mL IL3. comprehensively assessed the transforming activities and drug sensitivities of 55 nonsynonymous ERBB2 mutations in a Focus formation assay high-throughput manner using the mixed-all-nominated-in- For the focus formation assay, 3T3 cells expressing various one (MANO) method. We identified several ERBB2 mutations ERBB2 mutants were cultured in DMEM-F12 supplemented with as activating mutations related to tumorigenesis and revealed 5% bovine calf serum for 2 weeks. The cells were then stained with that several ERBB2 mutations showed varying sensitivities to Giemsa solution. ERBB2-targeted inhibitors. Thus, the functional significance of each variant should be interpreted precisely to design the best The MANO method treatment for each patient, and the MANO method might be Schematic representation of the MANO method is shown in beneficial for the determination of the best treatment for Supplementary Fig. S1. This method uses a retroviral vector that cancers harboring ERBB2 mutations. enables the stable integration of individual genes into the genome of assay cells (such as mouse 3T3 fibroblasts or the IL3-dependent, murine pro–B-cell line Ba/F3) along with 6-basepair (bp) barcode sequences. Individually transduced assay cells are subsequently pooled and cultured in a competitive manner to evaluate their of ERBB2 T798I mutation after neratinib treatment, suggesting transforming potential or drug sensitivity, either in vitro or that acquired secondary mutations may be a mechanism of in vivo. At the end of expansion period, genomic DNA was resistance to ERBB2-targeted inhibitors (25, 26). obtained from cell lysates using the QIAamp DNA Mini Kit Herein, we searched for nonsynonymous ERBB2 mutations (Qiagen), followed by amplification by PCR using primers that are recurrently reported in the COSMIC database (v78) and 50-TGGAAAGGACCTTACACAGTCCTG-30 and 50-GACTCGTT- comprehensively evaluated their transforming activity and GAAGGGTAGACTAGTC-30. The obtained products were puri- drug sensitivity in a high-throughput manner using the mixed- fied using AMPure beads (Beckman Coulter), and the sequenc- all-nominated-in-one (MANO) method, which was recently ing libraries were prepared using the NEB NextUltra DNA developed in our laboratory (27). This method is beneficial in Library Prep Kit (NEB) according to the manufacturer's instruc- that it enables the assessment of not only the oncogenic potential tions. The library quality was evaluated using a Qubit 2.0 but also the drug sensitivity of hundreds of gene mutations within fluorometer (Thermo Fisher Scientific) and the Agilent 2200 a short period of time in a competitive