Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts

Clin Cancer Res. 2008 Oct 15;14(20):6618-24. doi: 10.1158/1078-0432.CCR-08-1018.


Purpose: EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2)(p21p23) in the genome of non-small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes.

Experimental design: Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs.

Results: From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens.

Conclusions: These data reinforce the importance of accurate diagnosis of EML4-ALK-positive tumors for the optimization of treatment strategies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / diagnosis
  • Adenocarcinoma / genetics
  • Carcinoma, Adenosquamous / diagnosis
  • Carcinoma, Adenosquamous / genetics
  • Carcinoma, Large Cell / diagnosis
  • Carcinoma, Large Cell / genetics
  • Carcinoma, Non-Small-Cell Lung / diagnosis
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • Carcinoma, Squamous Cell / diagnosis
  • Carcinoma, Squamous Cell / genetics
  • Cell Transformation, Neoplastic
  • Chromosome Inversion
  • DNA Primers / chemistry
  • Exons / genetics*
  • Gene Rearrangement
  • Humans
  • Immunoenzyme Techniques
  • In Situ Hybridization, Fluorescence
  • Lung Neoplasms / diagnosis
  • Lung Neoplasms / genetics
  • Oncogene Proteins, Fusion / genetics*
  • RNA, Neoplasm / genetics
  • RNA, Neoplasm / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction / methods*


  • DNA Primers
  • EML4-ALK fusion protein, human
  • Oncogene Proteins, Fusion
  • RNA, Neoplasm