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Background  This study was aimed at investigating if the effect of  RARβ2  hypermethylation on recurrence-free survival (RFS) in non-small cell lung cancer (NSCLC) depends on one’s smoking status and specific interacting proteins.    Results  We retrospectively analyzed the expressions of five proteins using immunohistochemistry in archival formalin-fixed and paraffin-embedded tissues from 578 NSCLC patients who had undergone surgical resection from 1994 through 2004. Promoter methylation of  RARβ2  was assessed by bisulfite pyrosequencing. Recurrence was found in 268 (46%) of 578 NSCLCs with a median follow-up period of 4.8 years. Overexpression of β-catenin, c-MET, cyclin D1, and EGFR occurred in 55%, 72%, 51%, and 41% of the patients, respectively. E-cadherin expression was negative in 62% of the patients, and  RARβ2  hypermethylation was found in 37%. The abnormal expression of  c-MET  ( P  = 0.002) and  EGFR  ( P  = 0.001) was found to be highly prevalent in never-smokers.  RARβ2  hypermethylation was significantly associated with poor recurrence-free survival (RFS) in 128 never-smokers with adenocarcinoma ( P  = 0.01) For parsimonious model building, the five proteins were clustered into three groups (β-catenin and E-cadherin; c-MET; cyclin D1 and EGFR) by an unsupervised hierarchical clustering and were included in a multivariate analysis. Cox proportional hazard analysis showed that  RARβ2  hypermethylation was significantly associated with poor RFS in 128 never-smokers with adenocarcinoma (adjusted hazard ratio [HR] = 2.19, 95% confidence interval [CI] = 1.28 to 3.47;  P  = 0.009), after adjusting for interacting proteins.    Conclusions  The present study suggests that  RARβ2  hypermethylation may be an independent prognostic factor of RFS in never-smokers with adenocarcinoma of the lung.

RARβ2 hypermethylation is associated with poor recurrence-free survival in never-smokers with adenocarcinoma of the lung