PTEN, RASSF1 and DAPK site-specific hypermethylation and outcome in surgically treated stage I and II nonsmall cell lung cancer patients Lela Buckingham 1,4 , L. Penfield Faber 2 , Anthony Kim 2 , Michael Liptay 2 , Carter Barger 4 , Sanjib Basu 3 , Mary Fidler 3 , Kelly Walters 1 , Philip Bonomi 3 and John Coon 1 1 Departments of Pathology, Rush University Medical Center, Chicago, IL 60612 2 Thoracic Surgery, Rush University Medical Center, Chicago, IL 60612 3 Medical Oncology, Rush University Medical Center, Chicago, IL 60612 4 College of Health Sciences, Rush University Medical Center, Chicago, IL 60612 The primary objective of this study is to identify prognostic site-specific epigenetic changes in surgically treated Stage I and II nonsmall cell lung cancer (NSCLC) patients by quantifying methylation levels at multiple CpG sites within each gene promoter. Paraffin-embedded tumors from stage Ib, IIa and IIb in training and validation groups of 75 and 57 surgically treated NSCLC patients, respectively, were analyzed for p16, MGMT, RASSF1, RASSF5, CDH1, LET7, DAPK and PTEN promoter hypermethylation. Hypermethylation status was quantified individually at multiple CpG sites within each promoter by pyrosequencing. Molecular and clinical characteristics with time to recurrence (TTR) and overall survival (OS) were evaluated. Overall average promoter methylation levels of MGMT and RASSF1 were significantly higher in smokers than in nonsmokers (p 5 0.006 and p 5 0.029, respectively). Methylation levels of the p16 promoter were significantly higher in squamous cell carcinoma than in adenocarcinoma (p 5 0.020). In univariate analysis, hypermethylation of RASSF1 at CpG sites 253 and 248 and PTEN at CpG site 21310 were the significantly associated with shorter TTR (p 5 0.002 and p < 0.000, respectively). Hypermethylation of PTEN at 21310 and DAPK at 21482 were most significantly associated with outcome in multivariate analysis. These results show that methylation of specific promoter CpG sites in PTEN, RASSF1 and DAPK is associated with outcome in early stage surgically treated NSCLC. Genetic and epigenetic events largely determine tumor phe- notype and ultimately patient outcome. In carcinogenesis, tu- mor suppressor genes are frequently inactivated in two steps, starting with functional loss of one allele by a variety of mechanisms including mutations and methylation followed by deletion of the remaining allele. Methylation is the pre- dominant mechanism for promoter inactivation by postrepli- cative epigenetic modification. Normally, stable or transient methylation at CpG dinucleotide promoter sequences coordi- nates gene expression during cell cycling. 1,2 Aberrant methylation in tumor suppressor genes has been linked to disease course and outcome. 3–5 Gene expression profiling in primary tumors and metastases have revealed molecular signatures associated with a metastatic phenotype. For example, a 17-gene signature, characteristic of primary adenocarcinoma and associated with poor clinical outcome (p < 0.03), showed that cells preconfigured to metastasize are present in some primary tumors even at the time of diagnosis. 6–8 For this study, promoter methylation of genes involved in a variety of cellular functions including adhesion (CDH1), DNA repair (MGMT), gene expression (LET7), cell division (p16, RASSF1, RASSF5) and survival (DAPK, PTEN), was compared with time to recurrence and survival in surgically treated early stage lung cancer patients. The genes studied were selected based on reports of associations between loss of function and outcome in lung and other cancers. Hypermeth- ylation of p16, DAPK, MGMT and RASSF1 in bronchial epi- thelium and sputum have been linked to increased risk of lung cancer and hypermethylation of CDH1 and DAPK pro- moters were observed in lymphocytes from smokers. 9,10 Stud- ies on cell lines investigating the methylation status of the Ras association domain family proteins, RASSF1 and RASSF5, support the disruption of apoptotic pathways in the meta- static phenotype. 11–13 Aberrant methylation of a member of the Let-7 microRNA gene family, Let-7a-3 has been impli- cated in modification of expression of multiple genes that may affect outcome in ovarian 14 and lung cancer. 15 In previ- ously reported studies, we observed that PTEN expression is Key words: lung cancer, epigenetics, DNA methylation, biomarkers Additional supporting information may be found in the online version of this article DOI: 10.1002/ijc.24896 History: Received 1 Jul 2009; Accepted 27 Aug 2009; Online 30 Sep 2009 Correspondence to: Lela Buckingham, Department of Pathology, Rush Medical Center, 1653 W. Congress Pkwy, Chicago, IL 60612, Fax: (312)942-8142, E-mail: lela_buckingham@rush.edu Cancer Genetics Int. J. Cancer: 126, 1630–1639 (2010) V C 2009 UICC International Journal of Cancer IJC