ALK 融合转录本可在非小细胞肺癌细胞系和患者血浆的细胞外囊泡 (EVs) 中检测到：迈向基于 EV 的非侵入性检测。

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.

机构信息

Liquid Biopsy Laboratory, Medical Oncology Department, Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana, Majadahonda, Spain.

I+D Department, Atrys Health, Barcelona, Spain.

出版信息

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

DOI:10.1093/clinchem/hvac021

PMID: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.

摘要

背景

ALK 重排存在于 5%的非小细胞肺癌（NSCLC）肿瘤中，可识别出能从 ALK 抑制剂中获益的患者。使用液体活检进行 ALK 融合检测虽然具有挑战性，但可以大大扩展 ALK 阳性 NSCLC 患者的治疗选择。细胞外囊泡（EVs）内的 RNA 免受 RNase 和其他环境因素的影响，构成了非侵入性融合转录本检测的有前途的来源。

方法

通过连续离心细胞培养上清液，成功地从 H3122 和 H2228 细胞系中分离出含有 EML4-ALK 变体 1（E13；A20）和变体 3（E6a/b；A20）的 EVs。还从 16 名在开始治疗前采集的 ALK 阳性 NSCLC 患者的血浆样本中分离出 EVs。

结果

通过透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）和流式细胞术对培养的 EVs 进行了表征。Western blot 和共聚焦显微镜证实了 EV 特异性标志物以及 H3122 和 H2228 细胞系 EV 部分中 EML4-ALK 融合蛋白的表达。此外，通过数字 PCR（dPCR）分析了来自细胞培养物的 EV 部分的 RNA，并清楚地检测到 ALK 融合转录本。同样，通过 NTA、流式细胞术和 ExoView 平台对来自血浆的 EV 进行了表征，最后显示 EV 特异性标志物捕获了含有 ALK-融合蛋白的 EV 群体。最后，通过 dPCR 在 50%（8/16）的富含 EV 的血浆中鉴定出 ALK 融合，证实了 EV 部分中存在融合转录本。