Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing

Moskalev E, Frohnauer J, Merkelbach-Bruse S, Schildhaus HU, Dimmler A, Schubert T, Boltze C, Koenig H, Fuchs F, Sirbu H, Rieker RJ, Agaimy A, Hartmann A, Haller F (2014)

Publication Type: Journal article

Publication year: 2014

Journal

Lung Cancer Elsevier

Publisher: Elsevier

Book Volume: 84

Pages Range: 215-21

Journal Issue: 3

DOI: 10.1016/j.lungcan.2014.03.002

Abstract

Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in ~5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs.We developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5' and 3' ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analyzed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR.The H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5' and 3' ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3' ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples.Multiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing.

Authors with CRIS profile

Evgeny Moskalev Institute of Pathology Horia Sirbu Department of Thoracic Surgery Arndt Hartmann Lehrstuhl für Allgemeine Pathologie und Pathologische Anatomie

Involved external institutions

St. Vincentius-Kliniken / ViDia Kliniken DE Germany (DE) Universitätsklinikum Köln DE Germany (DE) SRH Wald-Klinikum Gera DE Germany (DE)

How to cite

APA:

Moskalev, E., Frohnauer, J., Merkelbach-Bruse, S., Schildhaus, H.-U., Dimmler, A., Schubert, T.,... Haller, F. (2014). Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing. Lung Cancer, 84(3), 215-21. https://doi.org/10.1016/j.lungcan.2014.03.002

MLA:

Moskalev, Evgeny, et al. "Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing." Lung Cancer 84.3 (2014): 215-21.

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