title: Validation of a locked nucleic acid based wild-type blocking PCR for the detection of EGFR exon 18/19 mutations.
abstract: BACKGROUND
  Treatment decisions in advanced non-small cell lung cancer rely on accurate analysis of the EGFR mutation status in small tissue samples. Sanger sequencing of PCR products is unbiased and cheap, but its detection threshold requiring 20 % infiltration by malignant cells is not optimal. Commercial kits, based on quantitative real-time PCR have better detection limits and can detect a wide spectrum of mutations but are considerably more expensive.
  
  
  METHODS
  We developed a wild-type blocking PCR for EGFR G719A/S/C (exon 18), exon 19 deletions, and exon 20 insertions using locked nucleic acid (LNA) probes. The amplification products of positive reactions were analyzed by Sanger sequencing. We retrospectively validated this assay by comparison of the EGFR mutation status as obtained with Fragment Length Analysis and the Therascreen EGFR RGQ PCR kit.
  
  
  RESULTS
  The EGFR mutation status for exon 18 and 19 as obtained with the LNA-PCR/sequencing assay correlated adequately with the results obtained by the other independent methods. Due to the lack of structural consistency among the insertions in exon 20, the latter are less amenable for a LNA-PCR design.
  
  
  CONCLUSIONS
  The LNA-PCR/sequencing assay presented here is specific, sensitive, and has a low detection threshold. In combination with allele-specific PCR reactions for T790M (exon 20) and L858R (exon 21), a wider scope of EGFR mutations can be assessed at a lower cost.
  The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1272520418142748.
authors.affiliation: Center for Human Genetics, University Hospitals Leuven, Herestraat 49,, Leuven, B-3000, Belgium. liesbet.vliegen@uzleuven.be.
authors.forename: Liesbet
authors.initials: L
authors.lastname: Vliegen
authors.affiliation: Respiratory Oncology Unit, University Hospitals Leuven, Herestraat 49, Leuven, B-3000, Belgium. christophe.dooms@uzleuven.be.
authors.forename: Christophe
authors.initials: C
authors.lastname: Dooms
authors.affiliation: Center for Human Genetics, University Hospitals Leuven, Herestraat 49,, Leuven, B-3000, Belgium. wim.dekelver@uzleuven.be.
authors.forename: Wim
authors.initials: W
authors.lastname: De Kelver
authors.affiliation: Department of Pathology, University Hospitals Leuven, Herestraat 49, B-3000, Leuven, Belgium. eric.verbeken@uzleuven.be.
authors.forename: Eric
authors.initials: E
authors.lastname: Verbeken
authors.affiliation: Respiratory Oncology Unit, University Hospitals Leuven, Herestraat 49, Leuven, B-3000, Belgium. johan.vansteenkiste@uzleuven.be.
authors.forename: Johan
authors.initials: J
authors.lastname: Vansteenkiste
authors.affiliation: Center for Human Genetics, University Hospitals Leuven, Herestraat 49,, Leuven, B-3000, Belgium. peter.vandenberghe@med.kuleuven.be.
authors.forename: Peter
authors.initials: P
authors.lastname: Vandenberghe
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D020022:Genetic Predisposition to Disease; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D010641:Phenotype; D011237:Predictive Value of Tests; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012189:Retrospective Studies
chemical_list: D011933:Reagent Kits, Diagnostic; C512478:EGFR protein, human; D066246:ErbB Receptors