AGA Abstracts NS-398 has a reverse effect on the expression of genes with altered expression in the colorectal adenoma-carcinoma sequence. NS-398 more efficiently inverted the expression changes at the adenoma than in carcinoma stage suggesting it is an effective drug in CRC chemoprevention. S1979 Routine MSI-Analysis in Colorectal Cancer Patients ≤ 70 Years Leads to the Identification of More Patients At High Risk for Lynch Syndrome Margot G. van Lier, A. Wagner, Ernst J. Kuipers, W. Dinjens, M. E. Leerdam van, Ewout W Steyerberg Background: Lynch Syndrome (LS), an inherited disorder caused by a germline mutation in one of the mismatch repair (MMR) genes, is responsible for approximately 3% of all colorectal carcinomas (CRC). The molecular hallmark of these tumors is microsatellite instability (MSI). Early detection of LS is of great importance, since surveillance colonoscopies reduce CRC morbidity and mortality. However, a considerable proportion of patients at high risk for LS are not recognized. Aim: In the search for a new diagnostic strategy, we prospectively studied the yield of routine molecular analysis in CRC patients ≤ 70 years. Methods: In six hospitals, all consecutive patients ≤ 70 years diagnosed with invasive CRC were included from May 2007 - September 2008. Tumorspecimens were analyzed for MSI, immunohistochemistry (IHC) of the MMR proteins, MLH1 promoter hypermethylation and BRAF mutation. Tumors were classified as; 1) suspect for LS if MSI-H and simultaneously showing absent MMR protein expression with exclusion of epigenetic silencing if absent MLH1 expression, 2) sporadic MSI-H tumors displaying absent MLH1 expression with MLH1 promoter hypermethylation and/or BRAF-mutation, 3) sporadic, microsatellite stable (MSS) tumors. Clinical data including age at diagnosis, sex, tumor localization, the presence of MSI-related histology features and synchronous CRCs were recorded. Results: Tumors of 365 patients (60% males) with a mean age of 60 years (SD ±8) were analyzed. CRCs were left-sided in 75%, right-sided in 22% and in 3% the location was unknown. MSI- related pathology features were seen in 43 tumors (12%) and synchronous CRCs were seen in 11 cases (3%). The molecular analyses revealed 9 sporadic MSI-H tumors (2.5%) and 13 CRCs suspect for LS (3.6%; 95% CI 1.7-5.5). Among the 13 patients suspect for LS, 8 (62%) were > 50 years. Based on IHC, 3 patients were suspect for a MLH1 gene defect, 3 for MSH2, 4 for MSH6 and 3 for a PMS2 defect. In logistic regression analyses patients suspect for LS were significantly younger (OR 1.1; 95% CI 1.03-1.17) and cancers were located more often right-sided (OR 27; 95% CI 6-117) than in patients with MSS tumors. Sporadic MSI-H tumors were significantly more often located right-sided than in the MSS group (OR 27; 95% CI 3-228). Conclusion: Molecular screening for LS in patients presenting with CRC ≤ 70 years leads to identification of a profile pathognomic for LS in 3.6% of patients. More than half of those do not meet the age-criterium routinely used for LS assessment. The routine use of molecular screening thus helps to identify more LS patients. The cost-effectiveness of such an approach has to be determined. S1980 Two Potential Biomarkers Identified By Whole Genome Microarrays from Laser Microdissected Colonic Cells, Downregulated Prostaglandin D2 Receptor and Upregulated C-SRC Tyrosine Kinase Sándor Spisák, Orsolya Galamb, Norbert Solymosi, Ferenc Sipos, Kinga Tóth, Ottó Jász, Béla Molnár, Zsolt Tulassay Background: Detection of histological region specific biomarkers, which continuously altering along the adenoma - carcinoma sequence is very important task in the recent molecular biology. The best approach for solving of this problem the whole genome microarray analysis combined with laser captured microdissection (LCM). Aims: Our aims were to identify mRNA expression patterns using LCM samples and to compare different histological regions of colorectal tumor and adenoma. Methods: From 6 colorectal cancers and from 6 adenomas, altered and normal specimens were collected and frozen immediately after surgery. Six um thick tissue sections were cut, fixed and stained. 5000 and 10000 epithelial and stromal cells were collected from the healthy and the pathological region by PALM LCM system. Total RNA was amplified and labelled using two-round In Vitro transcription and hybridized to HGU133Plus2.0 array. Significantly differentially expressed genes were identified by paired SAM test. Selected genes were verified by RT-PCR and tissue microarray. Results: The fixation and RNA isolation from LCM samples were successfully optimized. In tumor epithelial cells 28 genes (such as prostaglandin D2 receptor, forkhead box A1) were downreg- ulated, while 6 (like forkhead box Q1) were upregulated compared to the normal epithelium. The stromal samples required more cells and produced slighter results. Between tumor and normal stroma 12 differentially expressed genes were identified (including thrombospondin 2, collagen 8A1, collagen 11A1). In adenoma epithelial cells 100 genes showed upregulation (including c-src tyrosine kinase, high-mobility group box 3) and 1 was downregulated in proportion to normal epithelium. 14 overexpressed and 1 underexpressed genes were detected in stromal part of adenoma compared to normal. Tumor epithelial cells were characterized by 74 upregulated (like c-src tyrosine kinase, BCL2-like 1) and 26 downregul- ated (such as cysteine-rich protein 1) genes in proportion to adenoma epithelium. Between tumor and adenomatous stromal cells elevated mRNA level of 72 genes (including importin 7, collagen 8A1) and decreased expression of 27 genes (like LOC730091, F-box protein 10) were found. Conclusion: The microarray technology combined with LCM allows analyzing of microenvironment of tumor and examination of different rare cell histological region in order to understand the process of cancer development. A-306 AGA Abstracts S1981 Septin 9 mRNA, Protein Expression and Methylated DNA Level in Colorectal Adenoma-Dysplasia-Carcinoma Sequence Kinga Tóth, Sándor Spisák, Orsolya Galamb, Ferenc Sipos, Barnabas Wichmann, Katalin Leiszter, Gabor Valcz, Zsolt Tulassay, Béla Molnár Background: Free methylated septin 9 DNA was found to be a specific and sensitive peripheral blood marker of colorectal cancer (CRC) (Grützmann, Molnár et al. PLOS ONE 2008). The effect of increased methylation of this gene, that plays important role in the cell cycle related cytokinesis, on mRNA and protein expression was not analysed until now. Aim: Our aim was to detect the methylation related mRNA and protein expression changes of septin 9 in colorectal adenoma-dysplasia-carcinoma sequence and its reversibility by demethylation agent treatment. Material and methods: Septin 9 was detected in healthy, adenoma and colorectal cancer (CRC) samples by immunhistochemistry using septin 9 polyclonal antibody. Total RNA was extracted and amplified from colonic biopsies of 7 healthy, 6 low-grade adenoma, 9 high-grade adenoma and 15 colorectal cancer. Gene expression profile was evaluated using HGU133Plus2.0 microarrays. 6 normal, 6 adenoma and 6 colorectal cancer samples were collected and frozen immediately after surgery. 5000 and 10000 epithelial and stromal cells were collected from the samples by PALM LMD (laser microdissection) system. Total RNA was amplified and labeled using two-round In Vitro transcription and hybridized to HGU133Plus2.0 arrays. Further parallel DNA and mRNA analyses were made for the alternative transcripts of septin 9 in LMD samples. At last, HT29 cells were treated by 5-aza-2'deoxycitidine demethylation agent and mRNA, protein changes were identified. Results: Protein expression of septin 9 showed significant difference between normal and CRC (p<0,001) samples. According to biopsy microarray results, septin 9 mRNA expression decreased in the progression of colon neoplastic disease (p<0,001). In laser microdissected microarray results, especially in epithelial samples, septin 9 showed significant reduction between normal and cancer (p<0,001) samples. According to LMD splice variant analysis, expression of septin9_v2 region showed significant correlation with DNA methylation and RNA expression results in the epithelium, but not in stromal cells. The mRNA and protein levels of HT29 cells increased after demethylation agent treatment. Conclusions: The increas- ing level of methylation septin 9 gene in colorectal adenoma-dysplasia-carcinoma sequence is reflected in the decreasing mRNA and protein expression, especially in the epithelium. These changes can be reversed by demethylation agents converting this screening marker gene into therapeutic target. S1982 Identification of Methylation Related Biomarkers By Whole Genome Microarrays from Laser Microdissected Colonic Cells Sándor Spisák, Orsolya Galamb, Barnabas Wichmann, Norbert Solymosi, Ferenc Sipos, Kinga Tóth, Béla Molnár, Zsolt Tulassay Background: Detection of histological region specific biomarkers, which show continuously decreasing mRNA expression along the adenoma - carcinoma sequence is very important task in the recent molecular biology. One of the molecular mechanisms which are able to manifest this phenomenon are the epigenetic alterations. The best approach for analysis of these alterations is the whole genome microarray analysis combined with laser captured microdissection (LCM). Aims: Our aims were to identify mRNA expression patterns using LCM samples and to compare different histological regions of colorectal tumor and adenoma. Furthermore we aimed to determine the underexpressed genes and analyze the possible mechanisms of downregulation (like cancer related methylation) using a cell culture model. Methods: From 6 colorectal cancers and from 6 adenomas, altered and normal specimens were collected and frozen immediately after surgery. 5000 epithelial cells were collected from the healthy and the pathological region by PALM LCM system. Total RNA was amplified and labeled using two-round In Vitro transcription and hybridized to HGU133Plus2.0 array. Significantly differentially expressed genes were identified by paired SAM test. HT29 cell line was treated by 5-aza-2'deoxycytidine and the upregulated genes were identified compared to untreated group. Selected genes were verified by RT-PCR, bisulfite sequencing of the promoter regions and on tissue microarrays by imuunohistochemistry. Results: In adenoma epithelial cells 100 genes showed downregulation (including c-src tyrosine kinase, high- mobility group box 3) compared to normal epithelium, 22 of them showed reverse expression in 5-aza treated HT29 cells. In tumor epithelial cells 28 genes (such as prostaglandin D2 receptor, forkhead box A1) were downregulated, compared to the normal epithelium 16 of them changed reversely under the 5-aza treatment. Tumor epithelial cells were characterized by 27 downregulated (such as cysteine-rich protein 1) genes in proportion to adenoma epithelium, 12 of them expressed oppositely after the demethylation treatment. Conclusion: Considerable part of the downregulated alterations can be reversed by 5-aza treatment. The microarray technology combined with LCM allows analyzing of microenvironment of tumor and examination of different rare cell histological region in order to understand the process of cancer development including changing of the methylation status. S1983 Hypermethylation Attributes to PDX1 Expression Silencing in Human Gastric Cancer Juan Ma, Jide Wang, Bing Zou, Zesong Li, Wenjing Zhang, Qing Gu, Yun Dai, Liang Qiao, Hui Y. Lan, Benjamin C.Y. Wong Background and Aims: Pancreatic-duodenal homeobox 1(PDX1) is a homeobox gene that plays a role in differentiation and development of the pancreas, duodenum and antrum. We reported that PDX1 is aberrantly down-regulated and has tumor suppressor traits in gastric cancer. However, the relative molecular mechanism underlying the regulation of PDX1 gene expression in gastric cancer is unknown. So we aim to identify PDX1 putative promoter area and characterize CpG island methylation of the promoter resulting in PDX1 gene silencing. Methods: Four different fragments named F383, F314, F283 and F724 containing single CpG island were cloned and examined for putative promoter activities by