title: Comparison study of the performance of the QIAGEN EGFR RGQ and EGFR pyro assays for mutation analysis in non-small cell lung cancer.
abstract: OBJECTIVE
  To compare 2 laboratory assays commonly used in the evaluation of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC).
  
  
  METHODS
  Fifty-three formalin-fixed, paraffin-embedded NSCLC specimens were selected. Extracted DNA was analyzed using the EGFR RGQ Amplification Refractory Mutation System Scorpions probe-based real-time polymerase chain reaction (PCR) assay and the EGFR Pyro pyrosequencing assay.
  
  
  RESULTS
  Fourteen EGFR mutations were identified in 13 specimens using at least 1 of the assays, with a mutation concordance rate of 92.9%. Using dideoxy sequencing as the gold standard, clinical sensitivity was 73.7% and 68.4% by the RGQ and Pyro assays, respectively, but 100% by both for common drug sensitivity mutations. Performance observations included the following: the RGQ system requires higher DNA input, the RGQ system is a single-step procedure, the EGFR Pyro assay is a 2-step procedure, only the RGQ system can identify exon 20 insertions, the RGQ system is more sensitive, and the Pyro system can specify exact mutations for all interrogated sites.
  
  
  CONCLUSIONS
  Both the RGQ real-time PCR and Pyro assays adequately detect common EGFR mutations; however, the RGQ system is more clinically and analytically sensitive. Performance characteristics should be considered when evaluating these EGFR mutation assays for clinical adoption.
authors.affiliation: Dept of Pathology and Microbiology, University of Nebraska Medical Center, 985454 Nebraska Medical Center, Omaha, NE 68105-5454, USA. acushman@unmc.edu
authors.forename: Allison M
authors.initials: AM
authors.lastname: Cushman-Vokoun
authors.forename: Ann M
authors.initials: AM
authors.lastname: Crowley
authors.forename: Sharleen A
authors.initials: SA
authors.lastname: Rapp
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Greiner
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D059645:Mutation Rate; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012907:Smoking
chemical_list: D004273:DNA, Neoplasm; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: ARMS; Amplification Refractory Mutation System; Epidermal growth factor receptor; Non–small cell lung cancer; Pyrosequencing; Real-time PCR