1. AB Financial Support: CNPq, Fundação do Câncer, - Fiocruz 30/2006, Proteomic core facility from Fiocruz – PR, PDTIS. Epigenetic and proteomic analysis of gastric tumor and its histologically free, proximal and distal margins Paulo C. Carvalho 1 ; Guilherme P. Bravo Neto 2 ; Carlos Eduardo Carvalho 3 ; Priscila Aquino 1 ; Marcelo Mota 3 ; Thais Mac-Cormikc 3 ; Juliana S G Fischer 1 and, Maria da Gloria da C. Carvalho 3 1- Laboratory for proteomics and protein engineering, Carlos Chagas Instituto, Fiocruz Curitiba – Brazil 2.-Department of Surgery of Federal University of Rio de Janeiro-Brazil 3. Department of Pathology of Federal University of Rio de Janeiro- Brazil 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 : Eighteen 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. pylori and EBV negative patient was performed using an Orbitrap Velos mass spectrometry. TABLE 1: PATIENTS AND TUMOR CHARACTERISTICS, SURGICAL AND PATHOLOGICAL FINDINGS AND STAGING Patient / EBV / H.Pylori E-Cadherin PM T DM P16 PM T DM DAPK PM T DM RB PM T DM 1 + NDU U U U U MU U U 2 _ _ M U UM M M U U M 3 + _U M MM U UM M MU U U 4 _ NDU U U U U M 5 + +M M M U U U 6 _ NDM M MM U MM M MU NA M TABLE 2: DNA-EBV DETECTION AND PROMOTER METHYLATION STATUS OF E-CADHERIN, p16, DAPK, Rb GENES IN PROXIMAL MARGIN (PM), TUMOR AREA (T) AND DISTAL MARGIN (DM) OF GASTRIC CANCER PATIENTS. ND: Note done; NA: not amplified; M: Methylated, U: Unmethylated.Proximal Margin( PM), gastric adenocarcinomas (T) and Distal Margin (DM) Figure 2: VenDiagram of patient 2- 4B Tumor (786 proteins); 5B Proximal Margin (777 proteins); 6B Distal Margin(750 proteínas ) PATIENTAGEGENDERTUMOR LOCATION HISTOLOGIC TYPESURGERYMARGIN RESECTION STAGING 151MCardia III Poorly differentiated / difuse type Total gastrectomyNegativeT3N3aM1 275MCorpus Moderately differentiated/ intestinal type Subtotal gastrectomyNegativeT2N0M0 371MAntrum Moderately differentiated/ intestinal type Subtotal gastrectomyNegativeT3N1M0 453MCardia III Poorly differentiated / difuse type Total gastrectomyPositiveT3N2M0 556FAntrum Moderately differentiated/ intestinal type Subtotal gastrectomyNegativeT3N3aM0 653FFundus Poorly differentiated / difuse type Total gastrectomy Positive T3N1M0 Results: Fig 1 shown the surgical specimes from gastrectomy of the 6 patients and table 1 the tumor characteristcs, surgical, pathological finding and stagin. Table 2 shown the DNA-EBV status and promoter methylation of genes investigated. 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 shown in figure 3, 4 and 5 respectively: 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.The HDGF identified in tumor sample, is described to be involved in cell growth, cell invasion, and apoptosis. The up-regulated AnxA1 expression detected in proximal margin, is described to be associated with serosal invasion, peritoneal metastasis, and poorer overall survival and overexpression of MUC1-C oncoprotein, in distal margin, is related in Warburg effect of aerobic glycolysis. The molecular analysis of these genetically transformed but histologically normal tissues may be very important tool to predict tumor metastasis and personalized patient treatment. Figure 4: Annexin A1 is a calcium / phospholipid-binding protein which promotes membrane fusion and is involved in exocytosis. High AnxA1 expression was associated with more serosal invasion, more peritoneal metastasis, and poorer overall survival in GC patients. (Cancer 2012 Dec 1;118(23):5757-67). Figure 3: Hepatoma-derived growth factor (HDGF) is a unique nuclear/growth factor. Expression is correlated with lymph node metastasis and prognosis of gastric carcinoma. (Clin Cancer Res 2006;12:117-122). Figure 1: Tissues specimes from gastrectomy of patients 1 to 6. Tumor (T), proximal area (PM) and distal margins (DM) are indicated in the figure. Figure 5: Overexpression of MUC1 is reported in malignant neoplasms including breast, pancreatic, colorectal or non-small-cell carcinoma of the lung and is associated with a poor prognosis (Exp Ther Med. 2012 August; 4(2): 311–316).