Abstract 1470: Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small cell lung cancer.
Authors: Pailler Emma , Adam Julien , Barthelemy Amélie , Oulhen Marianne , Auger Nathalie , Valent Alexander , Planchard David , Taylor Melissa , Borget Isabelle , Andre Fabrice , Soria Jean-Charles , Vielh Philippe , Besse Benjamin , Farace Françoise 
Published: August 2014

Abstract
Purpose: The diagnostic test of ALK rearrangement in non-small-cell lung cancer (NSCLC) for crizotinib treatment is currently done on tumor biopsies or fine needle aspirations. The present study was designed to evaluate -1) whether ALK-rearrangement diagnosis could be performed using circulating tumor cells (CTCs), -2) whether CTCs harboring ALK-rearrangement could be monitored in ALK-positive patients treated by crizotinib. Patients and Methods: CTCs were isolated in 18 ALK-positive and 14 ALK-negative patients by blood filtration using ISET (Isolation by Size Epithelial Tumor cells) and tested by filter adapted-fluorescence in situ hybridization (FA-FISH), a FISH method optimized for filters. Numbers of ALK-rearranged cells and patterns of ALK-rearrangement were determined in CTCs and compared to those present in tumor biopsies. ALK-rearranged CTCs and tumor specimens were characterized for epithelial (cytokeratin, E-cadherin) and mesenchymal (vimentin, N-cadherin) markers expression. ALK-rearranged CTCs were monitored in ALK-positive patients treated by crizotinib. Results: ALK-rearranged CTCs [ranging from 4 to 34 CTCs /1mL] were detected in all ALK-positive patients, while no or only one ALK-rearranged CTCs was detected in blood samples obtained from ALK-negative patients. ALK-rearranged CTCs harboured a unique (3’ 5’) split pattern while heterogeneous patterns (3’ 5’, only 3’) of splits were present in tumors. ALK-rearranged CTCs exclusively expressed a mesenchymal phenotype and contrasted with heterogeneous epithelial and mesenchymal marker expressions in tumors. Variations in levels of ALK-rearranged CTCs and in CTCs harboring a gain of ALK native copies were detected under crizotinib treatment. Conclusion: We report that ALK-rearrangement can be detected in CTCs of ALK-positive NSCLC patients using ISET and FA-FISH, enabling diagnostic testing for crizotinib treatment. Our results suggest that CTCs harboring a unique ALK-rearrangement and mesenchymal phenotype may arise from the clonal selection of tumor cells that have acquired the potential to drive metastatic progression of ALK-positive NSCLC. The molecular characterization of CTCs in patients undergoing crizotinib treatment might provide new insights into mechanism of resistance to ALK tyrosine kinase inhibitors, and possible strategies to overcome this resistance.
Citation Format: Emma Pailler, Julien Adam, Amélie Barthelemy, Marianne Oulhen, Nathalie Auger, Alexander Valent, David Planchard, Melissa Taylor, Isabelle Borget, Fabrice Andre, Jean-Charles Soria, Philippe Vielh, Benjamin Besse, Françoise Farace. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1470. doi:10.1158/1538-7445.AM2013-1470