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. 2022 May 18;68(5):668-679.
doi: 10.1093/clinchem/hvac021.

ALK-Fusion Transcripts Can Be Detected in Extracellular Vesicles (EVs) from Nonsmall Cell Lung Cancer Cell Lines and Patient Plasma: Toward EV-Based Noninvasive Testing

Estela Sánchez-Herrero  1   2 , Carmen Campos-Silva  3 , Yaiza Cáceres-Martell  3 , Lucía Robado de Lope  1 , Sandra Sanz-Moreno  1 , Roberto Serna-Blasco  1 , Alejandro Rodríguez-Festa  1 , Dunixe Ares Trotta  1 , Paloma Martín-Acosta  4 , Cristina Patiño  5 , María José Coronado  6 , Alexandra Beneitez  7 , Ricardo Jara  7 , Nerea Lago-Baameiro  8 , Tamara Camino  8 , Alberto Cruz-Bermúdez  1 , María Pardo  8 , Víctor González-Rumayor  2 , Mar Valés-Gómez  3 , Mariano Provencio  1   9 , Atocha Romero  1   9
Affiliations

ALK-Fusion Transcripts Can Be Detected in Extracellular Vesicles (EVs) from Nonsmall Cell Lung Cancer Cell Lines and Patient Plasma: Toward EV-Based Noninvasive Testing

Estela Sánchez-Herrero et al. Clin Chem. .

Abstract

Background: ALK rearrangements are present in 5% of nonsmall cell lung cancer (NSCLC) tumors and identify patients who can benefit from ALK inhibitors. ALK fusions testing using liquid biopsies, although challenging, can expand the therapeutic options for ALK-positive NSCLC patients considerably. RNA inside extracellular vesicles (EVs) is protected from RNases and other environmental factors, constituting a promising source for noninvasive fusion transcript detection.

Methods: EVs from H3122 and H2228 cell lines, harboring EML4-ALK variant 1 (E13; A20) and variant 3 (E6a/b; A20), respectively, were successfully isolated by sequential centrifugation of cell culture supernatants. EVs were also isolated from plasma samples of 16 ALK-positive NSCLC patients collected before treatment initiation.

Results: Purified EVs from cell cultures were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Western blot and confocal microscopy confirmed the expression of EV-specific markers as well as the expression of EML4-ALK-fusion proteins in EV fractions from H3122 and H2228 cell lines. In addition, RNA from EV fractions derived from cell culture was analyzed by digital PCR (dPCR) and ALK-fusion transcripts were clearly detected. Similarly, plasma-derived EVs were characterized by NTA, flow cytometry, and the ExoView platform, the last showing that EV-specific markers captured EV populations containing ALK-fusion protein. Finally, ALK fusions were identified in 50% (8/16) of plasma EV-enriched fractions by dPCR, confirming the presence of fusion transcripts in EV fractions.

Conclusions: ALK-fusion transcripts can be detected in EV-enriched fractions. These results set the stage for the development of EV-based noninvasive ALK testing.

Keywords: ALK-TKI; EML4-ALK; NSCLC; extracellular vesicles; liquid biopsy.

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