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.
Clin Chem
; 68(5): 668-679, 2022 05 18.
Article
em En
| MEDLINE
| ID: mdl-35348673
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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Carcinoma Pulmonar de Células não Pequenas
/
Vesículas Extracelulares
/
Neoplasias Pulmonares
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Clin Chem
Assunto da revista:
QUIMICA CLINICA
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Espanha