Data from Molecular Changes Associated with Acquired Resistance to Crizotinib in <i>ROS1</i>-Rearranged Non–Small Cell Lung Cancer

Abstract

<div>Abstract<p><b>Purpose:</b> Although <i>ROS1</i>-rearranged non–small cell lung cancer (NSCLC) is sensitive to crizotinib, development of resistance is inevitable. Here, we identified molecular alterations in crizotinib-resistant tumors from two NSCLC patients with the <i>CD74–ROS1</i> rearrangement, and in HCC78 cells harboring <i>SLC34A2–ROS1</i> that showed resistance to crizotinib (HCC78CR cells).</p><p><b>Experimental Design:</b> <i>ROS1</i> kinase domain mutations were examined in fresh tumor tissues from two NSCLC patients and HCC78CR1-3 cells by direct sequencing. Ba/F3 cells expressing <i>ROS1</i> secondary mutations were constructed to evaluate resistance to crizotinib. An upregulated pathway was identified using phospho-receptor tyrosine kinase array, EGFR signaling antibody array, and RNA sequencing (RNA-seq). Cell proliferation and <i>ROS1</i> downstream signaling pathways were compared between HCC78 and HCC78CR1-3 cells.</p><p><b>Results:</b> The <i>ROS1</i> G2032R mutation was identified in crizotinib-resistant tumors from one patient. Furthermore, HCC78CR1 and CR2 cells harbored a novel <i>ROS1</i> L2155S mutation (73.3% and 76.2%, respectively). <i>ROS1</i> G2032R and L2155S mutations conferred resistance to crizotinib in Ba/F3 cells. Evidence of epithelial-to-mesenchymal transition with downregulated E-cadherin and upregulated vimentin was observed in HCC78CR1-2 cells and in the other patient. RNA-seq and EGFR signaling antibody array revealed that the EGFR pathway was significantly upregulated in HCC78CR3 versus HCC78 cells. Cells with the <i>ROS1</i> mutation and upregulated EGFR were sensitive to foretinib, an inhibitor of c-MET, VEGFR2, and ROS1 and irreversible EGFR tyrosine kinase inhibitors plus crizotinib, respectively.</p><p><b>Conclusions:</b> Molecular changes associated with acquired crizotinib resistance in <i>ROS1</i>-rearranged NSCLC are heterogeneous, including <i>ROS1</i> tyrosine kinase mutations, EGFR activation, and epithelial-to-mesenchymal transition. <i>Clin Cancer Res; 21(10); 2379–87. ©2015 AACR</i>.</p></div>