title: EGFR mutation detection on routine cytological smears of non-small cell lung cancer by digital PCR: a validation study.
abstract: Highly sensitive genotyping techniques are useful to detect epidermal growth factor receptor (EGFR) mutations on lung cancer cytological samples, when these specimens feature only few neoplastic cells. This study aimed to validate digital PCR (dPCR) methodology on cytological material. In plasmid model system, dPCR allowed for the detection of a minimal percentage (1%) of EGFR mutant alleles. Cytological samples (n = 30), with neoplastic cell percentage ranging from 10% to 80% and yielding a quantity of extracted DNA ranging from 1.75 to 60 ng/µL were selected. Results previously generated by fragment length and TaqMan assays (n = 8 exon 19 deletions, n = 2 L858R mutations and n = 20 wild-type DNA) were compared with those obtained by dPCR. Data were highly concordant (96.6%). However, dPCR detected an additional L858R mutation that had been missed by TaqMan assay on a paucicellular smear. This mutation was confirmed by cloning PCR products and sequencing. Thus, dPCR can reliably be used to increase EGFR mutation detection rate on scarcely cellular lung cancer smears.
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Umberto
authors.initials: U
authors.lastname: Malapelle
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: de Luca
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Vigliar
authors.affiliation: Azienda Ospedaliera di Rilievo Nazionale, Antonio Cardarelli, Naples, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Ambrosio
authors.affiliation: Azienda Ospedaliera di Rilievo Nazionale, Ospedale dei Colli, Naples, Italy.
authors.forename: Danilo
authors.initials: D
authors.lastname: Rocco
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Pasquale
authors.initials: P
authors.lastname: Pisapia
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Claudio
authors.identifier: http://orcid.org/0000-0002-7479-6457
authors.initials: C
authors.lastname: Bellevicine
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Troncone
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies
chemical_list: C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: CYTOPATHOLOGY; EGFR; LUNG CANCER; PCR