التفاصيل البيبلوغرافية
| العنوان: |
Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing. |
| المؤلفون: |
Moskalev, Evgeny A.1, Frohnauer, Judith1, Merkelbach-Bruse, Sabine2, Schildhaus, Hans-Ulrich2, Dimmler, Arno3, Schubert, Thomas4, Boltze, Carsten5, König, Helmut6, Fuchs, Florian7, Sirbu, Horia8, Rieker, Ralf J.1, Agaimy, Abbas1, Hartmann, Arndt1, Haller, Florian1 florian.haller@uk-erlangen.de |
| المصدر: |
Lung Cancer (01695002). Jun2014, Vol. 84 Issue 3, p215-221. 7p. |
| مصطلحات موضوعية: |
*LUNG cancer, *RNA sequencing, *GENE fusion, *GENE rearrangement, *ANAPLASTIC lymphoma kinase, *MICROTUBULE-associated protein kinase, *PROTEIN-tyrosine kinase inhibitors, *FLUORESCENCE in situ hybridization |
| مستخلص: |
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 ∼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. [Copyright &y& Elsevier] |
| قاعدة البيانات: |
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