Abstract 5196: Feasibility investigation of EML4-ALK rearrangements in mNSCLC CTCs using a new in vivo procedure
Authors: Gallerani Giulia , Delmonte Angelo , Cocchi Claudia , Bocchini Martine , Piccinini Filippo , Burgio Marco Angelo , Casadei Carla , Rocca Andrea , Fabbri Francesco 
Published: August 2018

Abstract
Background - Circulating tumor cells (CTCs) are the liquid phase of a solid tumor. An advanced CTC phenotyping and genotyping procedure could allow a superior understanding of tumor heterogeneity aimed at improving personalized treatment selection. However, these investigations are hampered by the difficulty to catch and analyze CTCs. EML4-ALK chromosomal rearrangement is a fundamental marker of sensitivity in metastatic non-small cell lung cancer (mNSCLC), but conventional tissue-based ALK scrutiny is not always possible. A CTC-based ALK evaluation may be an easy and not invasive method for identifying rearrangements and hence patients eligible for specific treatments.
Methods - Analyses were performed via the Gilupi CellCollector CANCER01 (DC01), a device that allows the in vivo isolation of CTCs from a high volume of peripheral blood. The DC01 is a CE-approved functionalized 16 cm long medical wire that includes a functionalized 2 cm long gold-coated tip covalently coupled with antibodies against EpCAM. The DC01 can be inserted through a standard venous cannula into the cubital vein of cancer patients for 30 minutes where it captures CTCs directly from the bloodstream of cancer patients. CTC presence was investigated in 18 mNSCLC patients and CTC-ALK-chromosomal status was studied by an innovative method, the 3D-immuno-FISH, that combined immuno-fluorescence and FISH approaches on a 3D structure (the DC01). Two DC01 per patients were utilized. After removal, on one wire, CTCs were identified and counted by nuclei, EpCAM and cytokeratins staining. On the second wire, CTCs were recognized by EpCAM staining and analyzed for ALK alterations by FISH. Formalin-fixed paraffin-embedded primary tumor tissues from the same patients were analyzed for comparison. Immortalized cell lines attached to DC01 devices were exploited as positive controls.
Results - Using the DC01 it was feasible to isolate and detect CTCs. Among the 18 enrolled patients, 7 presented tumor-tissue ALK-translocation, whereas 11 were wild type. Overall, 61.1% patients were CTC positive (median CTC number, 3.0 per patient). Amid those that owned ALK-translocation, 28.6% were CTC-positive, whereas 81.8 % patient without ALK-translocation were CTC positive. ALK rearrangements were detectable directly on cells attached to the wires. However, the 3D-immuno-FISH analysis in patients was hampered by the low median number of detected CTCs.
Conclusions - The detection of ALK-chromosomal rearrangements with the 3D-immuno-FISH analysis in cells recovered by DC01 was feasible. This method is valid for tracking positive CTC patients, but the few cells retrieved per patients still limit CTC full exploitation. Although further studies are needed to validate our data and to improve DC01 capture efficiency, our results pave the way to CTC-based chromosomal rearrangement identification and therapy personalization.
Citation Format: Giulia Gallerani, Angelo Delmonte, Claudia Cocchi, Martine Bocchini, Filippo Piccinini, Marco Angelo Burgio, Carla Casadei, Andrea Rocca, Francesco Fabbri. Feasibility investigation of EML4-ALK rearrangements in mNSCLC CTCs using a new in vivo procedure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5196.