title: TaqMan based real time PCR assay targeting EML4-ALK fusion transcripts in NSCLC.
abstract: OBJECTIVE
  Lung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE).
  
  
  METHODS
  This assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH).
  
  
  RESULTS
  EML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing.
  
  
  CONCLUSIONS
  Our variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified.
authors.affiliation: Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic. Electronic address: blanka.robesova@fnbrno.cz.
authors.forename: Blanka
authors.initials: B
authors.lastname: Robesova
authors.affiliation: Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic.
authors.forename: Monika
authors.initials: M
authors.lastname: Bajerova
authors.affiliation: Department of Pathology, Masaryk University and University Hospital Brno, Brno, Czech Republic.
authors.forename: Kvetoslava
authors.initials: K
authors.lastname: Liskova
authors.affiliation: Department of Respiratory Diseases and Tuberculosis, Masaryk University and University Hospital Brno, Brno, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Skrickova
authors.affiliation: Department of Respiratory Diseases and Tuberculosis, Masaryk University and University Hospital Brno, Brno, Czech Republic.
authors.forename: Marcela
authors.initials: M
authors.lastname: Tomiskova
authors.affiliation: Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic; CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Sarka
authors.initials: S
authors.lastname: Pospisilova
authors.affiliation: Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic; CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Mayer
authors.affiliation: Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic; CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Dana
authors.initials: D
authors.lastname: Dvorakova
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000235:genetics*; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000737:chemistry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics*; D025202:Molecular Diagnostic Techniques; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D015342:DNA Probes; C522504:EML4-ALK fusion protein, human; D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger
keywords: EML4-ALK; FISH; Lung cancer; NSCLC; Real time PCR; Targeted therapy