title: Methylation analysis in spontaneous sputum for lung cancer diagnosis.
abstract: OBJECTIVE
  Lung cancer is the most fatal cancer in the developed world due to presence of metastases at time of diagnosis. The aim of this study is to examine DNA hypermethylation in sputum compared to sputum cytology for the diagnosis of lung cancer. A novel risk analysis is introduced, using the distinction between diagnostic and risk markers.
  
  
  METHODS
  Two independent sets were randomly composed from a prospectively collected sputum bank (Set 1: n = 98 lung cancer patients, n = 90 controls; Set 2: n = 60 lung cancer patients, n = 445 controls). Sputum cytology was performed for all samples. The following DNA hypermethylation markers were tested in both sets: RASSF1A, APC and cytoglobin (CYGB). Two statistical analyses were conducted: multivariate logistic regression and a risk classification model based on post-test probabilities.
  
  
  RESULTS
  In multivariate analysis, RASSF1A was the best of the three markers in discriminating lung cancer cases from controls in both sets (sensitivity 41-52%, specificity 94-96%). The risk model showed that 36% of lung cancer patients were defined as "high risk" (≥ 60% chance on lung cancer) based on RASSF1A hypermethylation in Set 1. The model was reproducible in Set 2. Risk markers (APC, CYGB) have less diagnostic value. Sensitivity of cytology for lung cancer diagnosis was 22%. RASSF1A hypermethylation yielded a sensitivity of 45%. The combined sensitivity for RASSF1A with cytological diagnosis increased to 52% with similar specificity (94%).
  
  
  CONCLUSIONS
  In a diagnostic setting, hypermethylation analysis in sputum is possible when a diagnostic marker is used. However, risk markers are insufficient for this purpose.
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: A Jasmijn
authors.initials: AJ
authors.lastname: Hubers
authors.affiliation: Department of Pulmonology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Miep A
authors.initials: MA
authors.lastname: van der Drift
authors.affiliation: Department of Pathology, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands.
authors.forename: Clemens F M
authors.initials: CF
authors.lastname: Prinsen
authors.affiliation: Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Birgit I
authors.initials: BI
authors.lastname: Witte
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, USA.
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas, USA.
authors.forename: Narayan
authors.initials: N
authors.lastname: Shivapurkar
authors.affiliation: Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Stastny
authors.affiliation: Department of Pathology, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Bolijn
authors.affiliation: Department of Pulmonology, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.
authors.forename: Bernard E A
authors.initials: BE
authors.lastname: Hol
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, USA.
authors.forename: Ziding
authors.initials: Z
authors.lastname: Feng
authors.affiliation: Department of Pulmonology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: P N Richard
authors.initials: PN
authors.lastname: Dekhuijzen
authors.affiliation: Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas, USA.
authors.forename: Adi F
authors.initials: AF
authors.lastname: Gazdar
authors.affiliation: Department of Pathology, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands. Electronic address: e.thunnissen@vumc.nl.
authors.forename: Erik
authors.initials: E
authors.lastname: Thunnissen
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D000078842:Cytoglobin; D019175:DNA Methylation; D055088:Early Detection of Cancer; D005260:Female; D005914:Globins / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D015999:Multivariate Analysis; D012372:ROC Curve; D012306:Risk; D013183:Sputum / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; D014408:Biomarkers, Tumor; C000627265:CYGB protein, human; D000078842:Cytoglobin; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D005914:Globins
keywords: Biomarkers; Diagnosis; Early detection; Epigenetics; Methylation; Non-small cell lung cancer; Sputum