title: Effective assessment of egfr mutation status in bronchoalveolar lavage and pleural fluids by next-generation sequencing.
abstract: OBJECTIVE
  The therapeutic choice for patients with lung adenocarcinoma depends on the presence of EGF receptor (EGFR) mutations. In many cases, only cytologic samples are available for molecular diagnosis. Bronchoalveolar lavage (BAL) and pleural fluid, which represent a considerable proportion of cytologic specimens, cannot always be used for molecular testing because of low rate of tumor cells.
  
  
  METHODS
  We tested the feasibility of EGFR mutation analysis on BAL and pleural fluid samples by next-generation sequencing (NGS), an innovative and extremely sensitive platform. The study was devised to extend the EGFR test to those patients who could not get it due to the paucity of biologic material. A series of 830 lung cytology specimens was used to select 48 samples (BAL and pleural fluid) from patients with EGFR mutations in resected tumors. These samples included 36 cases with 0.3% to 9% of neoplastic cells (series A) and 12 cases without evidence of tumor (series B). All samples were analyzed by Sanger sequencing and NGS on 454 Roche platform. A mean of 21,130 ± 2,370 sequences per sample were obtained by NGS.
  
  
  RESULTS
  In series A, EGFR mutations were detected in 16% of cases by Sanger sequencing and in 81% of cases by NGS. Seventy-seven percent of cases found to be negative by Sanger sequencing showed mutations by NGS. In series B, all samples were negative for EGFR mutation by Sanger sequencing whereas 42% of them were positive by NGS.
  
  
  CONCLUSIONS
  The very sensitive EGFR-NGS assay may open up to the possibility of specific treatments for patients otherwise doomed to re-biopsies or nontargeted therapies.
authors.affiliation: Center of Predictive Molecular Medicine, Center of Excellence on Aging, University G. D'Annunzio of Chieti, Chieti, Italy.
authors.forename: Fiamma
authors.initials: F
authors.lastname: Buttitta
authors.forename: Lara
authors.initials: L
authors.lastname: Felicioni
authors.forename: Maela
authors.initials: M
authors.lastname: Del Grammastro
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Filice
authors.forename: Alessia
authors.initials: A
authors.lastname: Di Lorito
authors.forename: Sara
authors.initials: S
authors.lastname: Malatesta
authors.forename: Patrizia
authors.initials: P
authors.lastname: Viola
authors.forename: Irene
authors.initials: I
authors.lastname: Centi
authors.forename: Tommaso
authors.initials: T
authors.lastname: D'Antuono
authors.forename: Roberta
authors.initials: R
authors.lastname: Zappacosta
authors.forename: Sandra
authors.initials: S
authors.lastname: Rosini
authors.forename: Franco
authors.initials: F
authors.lastname: Cuccurullo
authors.forename: Antonio
authors.initials: A
authors.lastname: Marchetti
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D000077192:Adenocarcinoma of Lung; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D009154:Mutation; D016066:Pleural Effusion, Malignant / Q000737:chemistry*
chemical_list: D066246:ErbB Receptors