title: Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR.
abstract: EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)-polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18-21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.
authors.affiliation: Division of Asthma, Allergy & Lung Biology, King's College London, London, United Kingdom. george.santis@kcl.ac.uk
authors.forename: George
authors.initials: G
authors.lastname: Santis
authors.forename: Roger
authors.initials: R
authors.lastname: Angell
authors.forename: Guillermina
authors.initials: G
authors.lastname: Nickless
authors.forename: Alison
authors.initials: A
authors.lastname: Quinn
authors.forename: Amanda
authors.initials: A
authors.lastname: Herbert
authors.forename: Paul
authors.initials: P
authors.lastname: Cane
authors.forename: James
authors.initials: J
authors.lastname: Spicer
authors.forename: Ronan
authors.initials: R
authors.lastname: Breen
authors.forename: Emma
authors.initials: E
authors.lastname: McLean
authors.forename: Khalid
authors.initials: K
authors.lastname: Tobal
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics; D000368:Aged; D000369:Aged, 80 and over; D044963:Biopsy, Fine-Needle; D001980:Bronchi / Q000000981:diagnostic imaging*; D018287:Carcinoma, Large Cell / Q000175:diagnosis / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D019160:Endosonography; D066246:ErbB Receptors / Q000235:genetics*; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics; D008207:Lymphatic Metastasis; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000706:statistics & numerical data*; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins