Moskalev, Evgeny A., Frohnauer, Judith, Merkelbach-Bruse, Sabine, Schildhaus, Hans-Ulrich, Dimmler, Arno, Schubert, Thomas, Boltze, Carsten, Koenig, Helmut, Fuchs, Florian, Sirbu, Horia, Rieker, Ralf J., Agaimy, Abbas, Hartmann, Arndt and Haller, Florian (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). S. 215 - 222. CLARE: ELSEVIER IRELAND LTD. ISSN 1872-8332
Full text not available from this repository.Abstract
Objectives: Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in similar to 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. Materials and methods: 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. Results: 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. Conclusion: 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. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
| Item Type: | Journal Article | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| URN: | urn:nbn:de:hbz:38-437580 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| DOI: | 10.1016/j.lungcan.2014.03.002 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Journal or Publication Title: | Lung Cancer | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Volume: | 84 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Number: | 3 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Page Range: | S. 215 - 222 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Date: | 2014 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Publisher: | ELSEVIER IRELAND LTD | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Place of Publication: | CLARE | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| ISSN: | 1872-8332 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Language: | English | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Faculty: | Unspecified | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Divisions: | Unspecified | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Subjects: | no entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| URI: | http://kups.ub.uni-koeln.de/id/eprint/43758 |
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