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Introduction: Gastric cancer (GC) corresponds to the fourth most common malignancy among men and sixth among women in Brazil. GC is a multifactorial disease that results from individual genetic predisposition and exposure to ambient factors such as diet, alcohol consumption, smoking, chronic Helicobacter pylori infection or Epstein-Barr virus (EBV). H. pylori and EBV are known to up-regulate DNA methyltransferases. Aim: Compare the promoter methylation profiles of E-cadherin, p16, DAPK, and Rb genes of histologically tumor free proximal and distal margins from fresh tissues and perform a quantitative proteomic comparison of the tumor, distal and proximal resection margins profiles from the same patient. Methods: 18 samples consisting of six gastric carcinomas, their corresponding proximal margins (PM), and distal margins (DM) were obtained from six patients subjected to gastric resection at the Federal University of Rio de Janeiro, Brazil. DNA was extracted from the fresh tissues by using proteinase K digestion and Phenol-chloroform isoamilic alcohol followed by ethanol precipitation. Methylation-specific PCR analysis was used to determine the methylation status of E-cadherin, p16, DAPK, and Rb genes promoter by bisulfite modification. The presence of EBV was investigated by PCR and a shotgun proteomic analysis of all tissues from the H pilory and EBV negative patient was performed using an Orbitrap Velos. Results: 3 of 6 patients were positive for EBV viz: 3 tumors plus 6 margins. The total methylation for the 4 genes in all 9 samples (p16, E-cadherin, DAPK, and Rb genes) were: 5/36 in PM (esophagus); 5/36 in tumor, and 9/36 for DM (intestinal); in the negative EBV samples: 6/36 in PM; 4/36 in tumor and 8/36 in the DM. The proteomic analysis disclosed 786 proteins identified in the tumor fragment (58 proteins uniquely identified in this tissue), 777 to histologically normal proximal margin (87 unique proteins) and 750 to histologically normal distal margin (132 unique proteins). In all three fragments analyzed, unique proteins related to tumor progression or metastasis were identified; examples are: hepatoma- derived growth (HDGF) in the tumor, Annexin 1A in the PM and Mucin 1 in the DM. Conclusion: Our results show that histologically free tumor margins are molecularly compromised by methylation and by up regulation of proteins correlated to tumor progression and metastasis even in samples not infected with EBV or H.pylori Financial support CNPq Citation Format: Paulo Costa Carvalho, Carlos Eduardo Carvalho, Guilherme Pinto Bravo Neto, Juliana Fischer Carvalho, Thais Mac Cormick, Priscila F. Aquino, Marcelo Mota Silva, Maria da Gloria da Costa Carvalho. Epigenetic and proteomic analysis of gastric tumor and its histologically free proximal and distal margins. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 389. doi:10.1158/1538-7445.AM2014-389