Detection of Circulating Tumor Cells Harboring a Unique ALK Rearrangement in ALK-Positive Non–Small-Cell Lung Cancer

Abstract

Purpose The diagnostic test for ALK rearrangement in non–small-cell lung cancer (NSCLC) for crizotinib treatment is currently done on tumor biopsies or fine-needle aspirations. We evaluated whether ALK rearrangement diagnosis could be performed by using circulating tumor cells (CTCs). Patients and Methods The presence of an ALK rearrangement was examined in CTCs of 18 ALK-positive and 14 ALK-negative patients by using a filtration enrichment technique and filter-adapted fluorescent in situ hybridization (FA-FISH), a FISH method optimized for filters. ALK-rearrangement patterns were determined in CTCs and compared with those present in tumor biopsies. ALK-rearranged CTCs and tumor specimens were characterized for epithelial (cytokeratins, E-cadherin) and mesenchymal (vimentin, N-cadherin) marker expression. ALK-rearranged CTCs were monitored in five patients treated with crizotinib. Results All ALK-positive patients had four or more ALK-rearranged CTCs per 1 mL of blood (median, nine CTCs per 1 mL; range, four to 34 CTCs per 1 mL). No or only one ALK-rearranged CTC (median, one per 1 mL; range, zero to one per 1 mL) was detected in ALK-negative patients. ALK-rearranged CTCs harbored a unique (3′5′) split pattern, and heterogeneous patterns (3′5′, only 3′) of splits were present in tumors. ALK-rearranged CTCs expressed a mesenchymal phenotype contrasting with heterogeneous epithelial and mesenchymal marker expressions in tumors. Variations in ALK-rearranged CTC levels were detected in patients being treated with crizotinib. Conclusion ALK rearrangement can be detected in CTCs of patients with ALK-positive NSCLC by using a filtration technique and FA-FISH, enabling both diagnostic testing and monitoring of crizotinib treatment. Our results suggest that CTCs harboring a unique ALK rearrangement and mesenchymal phenotype may arise from clonal selection of tumor cells that have acquired the potential to drive metastatic progression of ALK-positive NSCLC.