title: Viability of lymph node samples obtained by echobronchoscopy in the study of epigenetic alterations in patients with lung cancer.
abstract: BACKGROUND
  The diagnosis of microscopic lymph node metastasis in lung cancer is challenging despite the constant advances in tumor staging. The analysis of the methylation status of certain genes in lymph node samples could improve the diagnostic capability of conventional cyto-histological methods. The aim of this study was to demonstrate the feasibility of methylation studies using cytological lymph node samples.
  
  
  METHODS
  Prospective study including 88 patients with a diagnosis or strong suspicion of non-small cell lung cancer, in which an echobronchoscopy was performed on mediastinal or hilar lymph nodes for diagnostic and/or staging. DNA was extracted from cytological lymph node samples and sodium bisulfite modification was performed. Methylation studies for p16/INK4a and SHOX2 were accomplished by MS-qPCR and pyrosequencing.
  
  
  RESULTS
  The methodology used in our study yielded optimal/good DNA quality in 90% of the cases. No differences in DNA concentration were observed with respect to the lymph node biopsied and final diagnosis. Methylation analyses using MS-qPCR and pyrosequencing were not possible in a small number of samples mainly due to low DNA concentration, inadequate purity, fragmentation and/or degradation as a consequence of bisulfite conversion.
  
  
  CONCLUSIONS
  Methylation quantification using MS-qPCR and pyrosequencing of cytological lymph node samples obtained using echobronchoscopy is feasible if an appropriate DNA concentration is obtained, notably contributing to the identification of epigenetic biomarkers capable of improving decision-making for the benefit of potentially curable lung cancer patients.
authors.affiliation: Servicio de Neumología, Complexo Hospitalario Universitario de Vigo (CHUVI), Vigo, Pontevedra, España.
authors.forename: Virginia
authors.initials: V
authors.lastname: Leiro-Fernández
authors.affiliation: Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Vigo, Pontevedra, España.
authors.forename: Loretta
authors.initials: L
authors.lastname: De Chiara
authors.affiliation: Servicio de Neumología, Complexo Hospitalario Universitario de Vigo (CHUVI), Vigo, Pontevedra, España.
authors.forename: Maribel
authors.initials: M
authors.lastname: Botana-Rial
authors.affiliation: Servicio de Anatomía Patológica, Complexo Hospitalario Universitario de Vigo (CHUVI), Vigo, Pontevedra, España.
authors.forename: Ana
authors.initials: A
authors.lastname: González-Piñeiro
authors.affiliation: Servicio de Anatomía Patológica, Complexo Hospitalario Universitario de Vigo (CHUVI), Vigo, Pontevedra, España.
authors.forename: Antoni
authors.initials: A
authors.lastname: Tardio-Baiges
authors.affiliation: Servicio de Neumología, Complexo Hospitalario Universitario de Vigo (CHUVI), Vigo, Pontevedra, España.
authors.forename: Manuel
authors.initials: M
authors.lastname: Núñez-Delgado
authors.affiliation: Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Vigo, Pontevedra, España.
authors.forename: Diana
authors.initials: D
authors.lastname: Valverde Pérez
authors.affiliation: Servicio de Neumología, Complexo Hospitalario Universitario de Vigo (CHUVI), Vigo, Pontevedra, España. Electronic address: alberto.fernandez.villar@sergas.es.
authors.forename: Alberto
authors.initials: A
authors.lastname: Fernández-Villar
mesh_terms: D000368:Aged; D001707:Biopsy, Needle / Q000379:methods*; D001999:Bronchoscopy / Q000379:methods*; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry / Q000000981:diagnostic imaging / Q000235:genetics / Q000556:secondary*; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000032:analysis* / Q000302:isolation & purification; D019160:Endosonography; D005240:Feasibility Studies; D005260:Female; D019942:Genes, p16; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000000981:diagnostic imaging / Q000235:genetics / Q000473:pathology*; D008198:Lymph Nodes / Q000473:pathology*; D008207:Lymphatic Metastasis / Q000000981:diagnostic imaging / Q000473:pathology*; D008297:Male; D008482:Mediastinum; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics*; D011446:Prospective Studies; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000494:pharmacology; D018084:Ultrasonography, Interventional
chemical_list: D004273:DNA, Neoplasm; D018398:Homeodomain Proteins; D009363:Neoplasm Proteins; C484664:SHOX2 protein, human; D013447:Sulfites; C009279:sodium bisulfite
keywords: Adenopatías; Carcinoma pulmonar; Deoxyribonucleic acid; Echobronchoscopy; Ecobroncoscopia; Lung cancer; Lymph node; Methylation; Metilación; Punción transbronquial; Transbronchial needle aspiration; Ácido desoxirribonucleico