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. 2014 Jun;84(3):215-21. doi: 10.1016/j.lungcan.2014.03.002. Epub 2014 Mar 13.


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.

Keywords: 454 massive parallel sequencing; ALK rearrangement; EML4-ALK; Molecular diagnostic; NSCLC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • Female
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Lung Neoplasms / genetics*
  • Male
  • Middle Aged
  • Multiplex Polymerase Chain Reaction / methods
  • Oncogene Proteins, Fusion / analysis*
  • Oncogene Proteins, Fusion / genetics*
  • Sensitivity and Specificity
  • Sequence Analysis, RNA / methods


  • EML4-ALK fusion protein, human
  • Oncogene Proteins, Fusion