title: Large-scale screening and molecular characterization of EML4-ALK fusion variants in archival non-small-cell lung cancer tumor specimens using quantitative reverse transcription polymerase chain reaction assays.
abstract: BACKGROUND
  The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non-small-cell lung cancer (NSCLC) patients.
  
  
  METHODS
  We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients.
  
  
  RESULTS
  Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23-89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001).
  
  
  CONCLUSIONS
  This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.
authors.affiliation: *University of California Davis Comprehensive Cancer Center, †Department of Internal Medicine, Division of Hematology & Oncology, University of California, Davis, School of Medicine, Sacramento, California; ‡Department of General-, Visceral- and Tumor Surgery, University of Cologne, Cologne, Germany; §Response Genetics, Inc., Los Angeles, California; ‖Department of Public Health Sciences, University of California, Davis, Davis, California; ¶Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California; and #formerly Response Genetics, Inc., Los Angeles, California.
authors.forename: Tianhong
authors.initials: T
authors.lastname: Li
authors.forename: Martin K H
authors.initials: MK
authors.lastname: Maus
authors.forename: Sonal J
authors.initials: SJ
authors.lastname: Desai
authors.forename: Laurel A
authors.initials: LA
authors.lastname: Beckett
authors.forename: Craig
authors.initials: C
authors.lastname: Stephens
authors.forename: Eric
authors.initials: E
authors.lastname: Huang
authors.forename: Jack
authors.initials: J
authors.lastname: Hsiang
authors.forename: Gary
authors.initials: G
authors.lastname: Zeger
authors.forename: Kathleen D
authors.initials: KD
authors.lastname: Danenberg
authors.forename: Stephanie H
authors.initials: SH
authors.lastname: Astrow
authors.forename: David R
authors.initials: DR
authors.lastname: Gandara
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000077548:Anaplastic Lymphoma Kinase; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics; D005260:Female; D005838:Genotype; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D016612:Paraffin Embedding; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: C522504:EML4-ALK fusion protein, human; D015514:Oncogene Proteins, Fusion; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases