Abstract 4818: Detection of ROS1 rearrangement in circulating tumor cells in ROS1-positive non-small-cell lung cancer patients
Authors: Pailler Emma , Besse Benjamin , Zajac Olivier , Coudert Benoît , Soria Jean-Charles , Farace Françoise 
Published: May 2015

Abstract
Purpose:
Non-small-cell lung cancer (NSCLC) accounts for ∼80% of lung cancers and is the major cause of cancer-related death worldwide. NSCLC are subdivided in clinically relevant molecular subsets according to a classification based on specific so-called oncogenic driver mutations.
Recently, ROS1 gene rearrangement was identified in 1-2% of NSCLC resulting in the formation of fusion proteins in which the kinase domain of ROS1 is constitutively active. Targeting ROS1 proteins with crizotinib is showing promise as an effective therapy in ROS1-positive patients. The diagnostic test for ROS1 rearrangement is currently done on tumor biopsies or fine-needle aspirations. We evaluated whether ROS1 rearrangement could be detected and monitored using circulating tumor cells (CTCs) in ROS1-positive patients treated by crizotinib. Patients and Methods:
Blood samples were drawn in four ROS1-positive patients at baseline and different time-points under crizotinib treatment as well as in four ROS1-negative (ALK-positive) patients. CTC enrichment was performed by filtration (ISET, isolation by size of epithelial tumor cells). ROS1 gene alterations were examined in CTCs by combining four-color immunofluorescent staining and FA-FISH (filter-adapted fluorescent in situ hybridization) according to a method we recently reported (Pailler et al, J Clin Oncol. 2013) and compared with those present in tumor biopsies. Chromosomal instability (CIN) present in CTCs was characterized using multi color FA-FISH and DNA image cytometry.
Results:
All ROS1-positive patients had high levels (&gt;20 CTCs/3ml) of ROS1-rearranged CTCs. ROS1-rearranged CTCs consistently harbored high numbers of native ROS1 copies and a 3′5′ split contrasting with ROS1 gene patterns present within the corresponding tumors. A high level of cell-to-cell variability in ROS1 pattern abnormalities gene was observed in CTCs of ROS1-positive patients. Experiments are ongoing to characterize the CIN status of CTCs using both FA-FISH and DNA image cytometry in ROS1-positive patients. Changes in the levels of ROS1-rearranged CTCs were observed during crizotinib treatment.
Conclusion
ROS1 rearrangement can be detected in CTCs of patients with ROS1-positive NSCLC by using a filtration technique and FA-FISH combined with immunofluorescence staining, enabling both diagnostic testing and monitoring of crizotinib treatment. The high rate of cell-to-cell variability in ROS1 pattern abnormalities and tumor heterogeneity observed in ROS1-rearranged CTCs suggests that aneuploidy and genetic instability might have an important role in tumorigenesis of ROS1-positive NSCLC.
Citation Format: Emma Pailler, Benjamin Besse, Olivier Zajac, Benoît Coudert, Jean-Charles Soria, Françoise Farace. Detection of ROS1 rearrangement in circulating tumor cells in ROS1-positive non-small-cell lung cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4818. doi:10.1158/1538-7445.AM2014-4818