title: Comparison of reverse transcription-polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization methodologies for detection of echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase fusion-positive non-small cell lung carcinoma: implications for optimal clinical testing.
abstract: BACKGROUND
  Echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non-small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection.
  
  
  OBJECTIVE
  To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions.
  
  
  METHODS
  Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n  =  42; mutant, n  =  4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b).
  
  
  RESULTS
  EML4-ALK variant 3a/b was detectable by RT-PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation.
  
  
  CONCLUSIONS
  The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions.
authors: Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah, USA.
authors: Michelle L
authors: ML
authors: Wallander
authors: Katherine B
authors: KB
authors: Geiersbach
authors: Sheryl R
authors: SR
authors: Tripp
authors: Lester J
authors: LJ
authors: Layfield
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D015514:Oncogene Proteins, Fusion; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases; C415585:EML4 protein, human; D012697:Serine Endopeptidases