title: A sensitive and high throughput TaqMan-based reverse transcription quantitative polymerase chain reaction assay efficiently discriminates ALK rearrangement from overexpression for lung cancer FFPE specimens.
abstract: OBJECTIVE
  ALK fusion gene is an oncogenic driver in lung cancer with low prevalence, which can be ameliorated by crizotinib. Currently, ALK fusion gene can be diagnosed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), but inconstistnt results between the two methods are encountered regularly. To make the ALK fusion gene screening more efficient and to provide a simple solution to clarify the discrepancy between FISH and IHC results, a sensitive TaqMan-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was established.
  
  
  METHODS
  The 3-plex TaqMan-based RT-qPCR assay was established and performed on 102 archived formalin-fixed, paraffin-embedded (FFPE) NSCLC samples to detect ALK rearrangement and overexpression. Break-apart FISH and automatic immunohistochemistry based ALK assays were performed side by side using tissue microarray.
  
  
  RESULTS
  The RT-qPCR was performed successfully for 80 samples and 10 of them showed positive signals. Three out of the 10 qPCR positive cases were further confirmed by FISH and IHC test. Two others were IHC positive and FISH negative, and expressed full-length ALK transcript. The rest were neither FISH nor IHC positive and their ALK expression level was significantly lower than those FISH or IHC positive cases.
  
  
  CONCLUSIONS
  Our RT-qPCR assay demonstrates that the capability and reliability of ALK detection is comparable to FISH and IHC, but it is more effective at discriminating ALK rearrangement from overexpression. The RT-qPCR assay easily clarifies the discrepancy between FISH and IHC, and can be incorporated into routine ALK screening for lung cancer.
authors.affiliation: Department of Medical Research and Development, Chang Gung Memorial Hospital, Chiayi branch, Taiwan.
authors.forename: Jrhau
authors.initials: J
authors.lastname: Lung
authors.affiliation: Division of Thoracic Oncology, Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Chiayi branch, Taiwan; Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Respiratory Care, Chang Gung University of Science and Technology, Chiayi Campus, Chiayi, Taiwan.
authors.forename: Yu-Ching
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Division of Thoracic Oncology, Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Chiayi branch, Taiwan; Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Respiratory Care, Chang Gung University of Science and Technology, Chiayi Campus, Chiayi, Taiwan.
authors.forename: Ming-Szu
authors.initials: MS
authors.lastname: Hung
authors.affiliation: Division of Thoracic Oncology, Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Chiayi branch, Taiwan.
authors.forename: Yuan Yuan
authors.initials: YY
authors.lastname: Jiang
authors.affiliation: Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Hematology and Oncology, Chang Gung Memorial Hospital, Chiayi branch, Taiwan.
authors.forename: Kuan-Der
authors.initials: KD
authors.lastname: Lee
authors.affiliation: Department of Pathology, Chang Gung Memorial Hospital, Chiayi branch, Taiwan.
authors.forename: Paul Yann
authors.initials: PY
authors.lastname: Lin
authors.affiliation: Division of Thoracic Oncology, Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Chiayi branch, Taiwan; Department of Respiratory Care, College of Medicine, Chang Gung University, Taoyuan, Taiwan. Electronic address: chestmed@cgmh.org.tw.
authors.forename: Ying Huang
authors.initials: YH
authors.lastname: Tsai
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000077548:Anaplastic Lymphoma Kinase; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D015321:Gene Rearrangement; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D059467:Transcriptome
chemical_list: D015514:Oncogene Proteins, Fusion; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases
keywords: Anaplastic lymphoma kinase; FISH; Immunohistochemisty; Lung cancer; Real time PCR