Abstract 561: ALK fusions can be clearly detected in extracellular vesicles from NSCLC cell lines: A become of hope for non-invasive ALK testing
Authors: Sánchez-Herrero Estela , Campos-Silva Carmen , Martell Yaiza Cáceres , Sanz-Moreno Sandra , Serna-Blasco Roberto , Festa Alejandro Rodríguez , Molina-Vila Miguel Ángel , Provencio Mariano , Valés-Gómez Mar , Romero Atocha 
Published: July 2021

Abstract
Background: Identification of ALK rearrangements in NSCLC patients has important implications as it significantly expands their therapeutic opportunities. However, the availability of tumors biopsies is sometimes limited in the clinical setting. The analysis of liquid biopsies can potentially overcome such limitation. Nevertheless, the identification of ALK rearrangements using liquid biopsies remains challenging as fusion transcripts are rapidly degraded by RNAses present in the blood. Nonetheless, encapsulated RNA inside extracellular vesicles (EVs) is protected from RNAses. The main objective of this study is to explore the potential value of exosomes for non-invasive ALK testing.
Methods: H3122 and H2228 cell lines, derived from NSCLC patients with EML4-ALK variant 1 (E13;A20) and variant 3 (E6a/b;A20) respectively, were used to isolate exosomes by the sequential centrifugation of cell culture supernatants. Briefly, supernatants were centrifuged twice for 10 min at 200xg, twice for 500xg for 10 min, once for 30 min at 10,000xg and a final ultracentrifugation at 100,000×g for 2 h at 4 °C. Purified exosomes were verified in terms of morphology and size by transmission electron microscopy (TEM). In addition, specific EVs markers, namely CD63, CD81 and CD9, were measured by flow cytometry and Western-Blot. EML4-ALK variants were analyzed at the protein level by Western blot using 5A4 antibody, and RNA level by digital PCR (dPCR), using a QuantStudio® 3D Digital PCR System and specific TaqMan® assays for EML4-ALK variant 1 and 3. Samples were also submitted to NGS analysis using the Oncomine™ Pan-Cancer Cell-Free Assay kit.
Results: TEM showed spherical vesicles around 100 nm in both cell lines. Flow cytometry and Western blot confirmed expression of the exosomal specific markers CD63, CD81 and CD9 tetraspanins. Western blot also showed EML4-ALK variant 1 (120 kDa) and variant 3 (87 kDa) in H3122 and H2228 purified exosomes, respectively. Isolated exosome RNA profile was composed of small RNA (&lt; 200 nt), although 18S and 28S rRNA peaks were also present. dPCR and NGS confirmed EML4-ALK rearrangements in exosome derived from both cell lines.
Conclusions: EML4-ALK fusions can be detected at the RNA levels and at the protein levels analyzing EVs derived from H3122 and H2228 cell lines. These results set the stage for the development of EV-based non-invasive ALK testing in NSCLC patients.
Citation Format: Estela Sánchez-Herrero, Carmen Campos-Silva, Yaiza Cáceres Martell, Sandra Sanz-Moreno, Roberto Serna-Blasco, Alejandro Rodríguez Festa, Miguel Ángel Molina-Vila, Mariano Provencio, Mar Valés-Gómez, Atocha Romero. ALK fusions can be clearly detected in extracellular vesicles from NSCLC cell lines: A become of hope for non-invasive ALK testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 561.