title: Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.
abstract: BACKGROUND
  EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples.
  
  
  METHODS
  A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples.
  
  
  RESULTS
  All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%.
  
  
  CONCLUSIONS
  We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.
authors.affiliation: .
authors.forename: Saeam
authors.initials: S
authors.lastname: Shin
authors.affiliation: .
authors.forename: Juwon
authors.initials: J
authors.lastname: Kim
authors.affiliation: .
authors.forename: Yoonjung
authors.initials: Y
authors.lastname: Kim
authors.affiliation: .
authors.forename: Sun-Mi
authors.initials: SM
authors.lastname: Cho
authors.affiliation: .
authors.forename: Kyung-A
authors.initials: KA
authors.lastname: Lee
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016066:Pleural Effusion, Malignant / Q000235:genetics* / Q000473:pathology; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: EGFR; cobas; malignant pleural effusion; non-small-cell lung cancer; real-time PCR