title: Sensitive detection of EML4-ALK fusion oncoprotein of lung cancer by in situ proximity ligation assay.
abstract: BACKGROUND
  EML4-ALK fusion oncogene has emerged as a novel molecular target in non-small cell lung cancer (NSCLC). Although break-apart fluorescent in situ hybridization (FISH) is the standard method for diagnosis, it is expensive, not readily available and sometimes difficult to interpret. In addition, ALK immunohistochemistry (IHC) may miss the diagnosis because of relatively low level of ALK transcription.
  
  
  METHODS
  In situ proximity ligation assay (PLA) originally developed for precise detection and quantification of proteins by dual recognition and amplification process was used for sensitive detection of EML4-ALK fusion oncoprotein in NSCLC cell lines (ALK negative cell: PC-9 and H460, ALK positive cell: H3122 and H2228). EML4-ALK oncogene and protein in lung cancer cells were confirmed by multiplex RT-PCR and Western blots.
  
  
  RESULTS
  We detected 117 kDa variant 1 of EML4-ALK in H3122 and 90 kDa variant 3 of EML4-ALK in H2228. These cells were more sensitive to crizotinib, an ALK inhibitor compared with PC-9 and H460 cells without EML4-ALK rearrangement. After fixing on glass slides by cytospin centrifuge, in situ PLA test was performed. Among four cell lines, distinct, tiny spots were visible only in H3122 and H2228 cell lines with ALK rearrangement. The same results were also obtained when paraffin-embedded cell blocks were used.
  
  
  CONCLUSIONS
  Highly specific and sensitive detection of EML4-ALK fusion oncoprotein is possible by in situ PLA method suggesting its clinical application.
authors.forename: Jin Kyung
authors.initials: JK
authors.lastname: Rho
authors.forename: Hyangsin
authors.initials: H
authors.lastname: Lee
authors.forename: Chan-Sik
authors.initials: CS
authors.lastname: Park
authors.forename: Chang-Min
authors.initials: CM
authors.lastname: Choi
authors.forename: Jae Cheol
authors.initials: JC
authors.lastname: Lee
mesh_terms: D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry* / Q000175:diagnosis / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry* / Q000175:diagnosis / Q000378:metabolism / Q000473:pathology; D008958:Models, Molecular; D015513:Oncogene Proteins / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C522504:EML4-ALK fusion protein, human; D015513:Oncogene Proteins; D015514:Oncogene Proteins, Fusion