T2014 Analysis of K-RAS Mutations in Laterally Spreading Tumors of the Colorectum

female: 64/34, mean age 68.9±9.8 years) were as follows: non-granular type (NG type)/ granular type (G-type): 33/68, right-sided/left-sided (including rectum): 58/43, and median size: 25 mm (range 10-105) in diameter, respectively. Carcinoma histology was found in the 43 LSTs (43%) and invasion into submucosal layer was found in 9 LSTs (9%). As the controls, we also examined the K-ras gene mutations in 66 protruded colon adenomas (pedunculated or semi-pedunculated type) larger than 10 mm and the 41 advanced colon cancers which were excised surgically. Results: Of 101 examined LSTs, K-ras mutations were observed in 59 (58%). We found the K-ras mutations more frequently in the LSTs than the protruded adenomas (p<0.001) and even than the advanced colon cancers (p= 0.03). Frequency of the K-ras mutations in the LSTs was particularly dominant in the leftsided colorectum (LST vs. protruded adenoma; p<0.001, LST vs. advanced cancer; p=0.004). The comparisons among LSTs revealed that K-ras mutations were more frequently observed in the G-type LSTs or in those larger than 20 mm than each counterpart (p<0.01 and p= 0.015, respectively). In the right-sided LSTs, G-type and carcinoma histology were significant factors correlated with existence of K-ras mutations (OR, 11.64; 95% CI: 3.1-55.2 and OR, 3.8; 95% CI: 1.17-14.2, respectively), whereas in the left-sided LSTs, the size larger than 20 mm was the only predictor of K-ras mutations (OR, 9.5; 95% CI: 1.7-80.1). Conclusion: K-ras mutations were frequently observed in LSTs, particularly in the G-type or larger LSTs. Carcinoma histology was a predictor of K-ras mutations in the right-sided LSTs, while larger size was correlated with K-ras mutations in the left-sided LSTs. These suggest that K-ras mutation is correlated with histological progression in the right-sided LSTs, in contrast, that it is correlated with lateral expansion in the left-sided LSTs.

Cytokine Gene Polymorphisms, Cytokine Levels and Risk of Colorectal Neoplasia in the Screened Population of Northeast Scotland Umesh Basavaraju, Fatma M. Shebl, Andrew J. Palmer, Georgina L. Hold, Charles S. Rabkin, Emad El-Omar Introduction: Chronic inflammation plays a key role in the pathogenesis of many cancers but its role in colorectal neoplasia (CRN) is not clear. Our aim was to investigate the effect of cytokine gene polymorphisms and circulating cytokine levels on risk of CRN in North East Scotland, which has a high incidence of colon cancer. Methods: Subjects underwent a complete bowel cancer screening colonoscopy and were free of IBD. Blood-derived DNA was used to genotype polymorphisms in the IL1B, IL1-RN, IL6, IL8, IL10, PTGS2 and TNFA genes using 5'nuclease taqman assays. Serum high-sensitivity C-reactive protein (Hs-CRP) and plasma levels of 6 cytokines (IL-1beta, IL-4, IL-6, IL-8, IL-10 and TNF-alpha) were measured. Subjects with adenomas with or without cancer (cases) were compared against subjects with no evidence of adenoma or cancer (controls). For Hs-CRP and plasma cytokines, levels were divided into tertiles and compared against each other. Chi-square test was used for comparing cases and controls. Logistic regression analysis adjusting for gender was used and odds ratios (OR) with 95% confidence intervals (CI) were calculated. Results: For the genetic study, a total of 295 cases were compared with 398 controls. Mean age of the cases was 64.4 yrs compared to 62.3 yrs in controls (p<0.01) and males were overrepresented in the cases (66.1% vs. 53.3% in controls, p<0.001). Regular use of NSAID was more common among controls compared to cases (p<0.0001). The pro-inflammatory IL-1B-31 C*C genotype increased the risk of having an adenoma by 1.9-fold compared to the IL-1B31 T*T genotype (95% CI: 1.2-3.0). Among subjects with adenoma, the pro-inflammatory IL8-251-A*A genotype increased the risk of having a high risk lesion (cancer, large adenoma, or multiple adenomas) compared to the IL-8-251 T*T + T*A genotypes (OR 3.4, 95% CI 1.7-7.0). None of the other polymorphisms showed any significant associations. Having a Hs-CRP level in the highest tertile compared to the lowest two tertiles increased the risk of having an adenoma by 1.6-fold (95% CI 1.0-2.6) and cancer by 2.1-fold (95% CI 0.9- 5.0). Among subjects with adenomas, having an IL-8 plasma level in the highest tertile compared to the lowest two tertiles doubled the risk of having a high risk lesion. Conclusions: Proinflammatory cytokine gene polymorphisms in IL-1B increase the risk of developing adenomas while those in the IL-8 increase the risk of progression to advanced lesions. HsCRP and IL-8 levels predict presence and severity of CRN. Along with the NSAID findings, our data point to inflammation as an underlying pathogenetic mechanism in this cancer.

T2015 Routine MSI-Analysis in Endometrial Cancer ≤ 70 Years Increases Identification of Patients at Risk for Lynch Syndrome Celine H. Leenen, Margot G. van Lier, Anja Wagner, W. Dinjens, Erik Jan Dubbink, Monique E. van Leerdam, Helena C. van Doorn, Ernst J. Kuipers, Ewout W Steyerberg Background: Lynch syndrome (LS) is an autosomal dominant syndrome that predisposes to various malignancies, including colorectal cancer (CRC) and endometrial cancer (EC). The risk of EC may exceed the risk of CRC in LS. Diagnostic strategies for LS include assessment for microsatellite instability (MSI) and immunohistochemical analysis (IHC) of tumor tissue. Identification of LS patients is important, because of the risk for synchronous and metachronous tumors. However, detection of LS is far from optimal. The aim of this study therefore was to evaluate a new diagnostic strategy for LS by performing routine molecular analysis of EC in patients ≤ 70 years. Methods: In eight hospitals, all consecutive patients ≤ 70 years with EC were included from May 2007-November 2009 as part of a large population-based study. Endometrial tumor specimens were analyzed for MSI and IHC of the mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2 and MLH1 promoter hypermethylation. Tumors were classified as: 1) suspect for LS if MSI-high (MSIH) and simultaneously showing absent MMR protein expression with exclusion of absent MLH1 expression in combination with MLH1 promoter hypermethylation, 2) sporadic MSIH tumors displaying absent MLH1 expression with MLH1 promoter hypermethylation, 3) sporadic, microsatellite stable (MSS/ MSI-L) tumors. Clinical data were recorded. Results: Tumorspecimens of 96 patients with a mean age of 62 years (range 26-70) were analyzed. 5% of patients were ≤ 50 years of age. EC were classified histologically as: endometrioid adenocarcinoma in 83 patients (87%), serous adenocarcinoma in four (4%), mixed adenocarcinoma in five (5%) and clear cell carcinoma in two patients (2%). 44% of the carcinomas were grade 1. Molecular analyses revealed 3 EC suspect for LS (3.1%; 95% CI 2.1-4.1). All three patients were > 50 years (range 53-70). 2/3 families did not fulfill Amsterdam II criteria. Family history of the third family is not available. Histology showed endometroid grade 1 cancer in 2 cases and adenocarcinoma grade 3 in 1. Based on IHC, 1 patient was suspect for a MLH1 gene defect, 1 for MSH6 and 1 for a PMS2 defect. In addition, 9 sporadic MSI-H tumors with MLH1 promoter hypermethylation (9.4%; 95% CI 8.4-10.3) were identified. Conclusion: Molecular screening for LS in patients ≤ 70 years with EC leads to identification of a profile pathognomic for LS in 3.1%. All three cases were ≥ age 50 and would without MSI and IHC have been diagnosed as sporadic EC. Molecular screening by a combination of MSI and IHC analysis contributes to the identification of LS patients, although a larger sample size is needed to confirm these results.

T2013 Galectin-3 Sequence Polymorphisms Predict Response of Colon Cancer Cells to Chemotherapy Nachman Mazurek, Suguru Ueno, Yun Jie Sun, James C. Byrd, Robert S. Bresalier Galectin-3 is a beta-galactoside binding protein which has been implicated the pathogenesis of colon cancer. The gene for galectin-3, LGALS3, is polymorphic in human populations. SNP rs4644 encodes galectin-3 with either His64 or Pro64. The purpose of this study was to determine whether these common (heterozygosity >0.4) sequence variants of galectin-3 have different effects on sensitivity to chemotherapeutic agents. Methods: SNP analysis of eight human colon cancer cell lines was performed by mutation scanning of PCR-SCCP. Cell viability was assessed by the MTT assay and apoptosis was quantified with the annexin assay. A full-length galectin-3 cDNA encoding Pro64 galectin-3 was obtained by RT-PCR from LiM6 colon cancer cells and subcloned into a bicistronic lentiviral expression plasmid. A second cDNA encoding His64 galectin-3 was generated by site-directed mutagenesis, and the mutations were confirmed by DNA sequencing. Expression of the individual haplotypes of galectin-3 in low-galectin-3 expressing cell lines, RKO and LM12, was achieved by lentiviral based delivery, followed by sorting of the high-GFP-expressing cell population. Results: Eight colon cancer cell lines were examined for TRAIL sensitivity and for the rs4644 SNP. Six cell lines (Caco-2, HCT116, HT29, SW480, RKO & LM12) were associated with the Pro64 haplotype, and were resistant to TRAIL-mediated apoptosis. Two cell lines (RSB1 & KM12) expressed His64 galectin-3 and were sensitive to TRAIL (>95% cell death). The effects of forced expression of individual galectin-3 haplotypes in low-galectin-3-expressing, TRAIL-resistant RKO and LM12 cells were examined in detail. The His 64 haplotype partially sensitized RKO to TRAIL (<5%→25% apoptotic cell death) and augmented the TRAIL sensitivity of LM12 cells (35%→70%). However, the Pro64 haplotype had no effect on apoptosis in either cell line, in spite of equivalent levels of galectin-3 expression. Differential effects on genotoxic drug sensitivity were also observed depending on galectin-3 genotype. Pro64 galectin-3 conferred irinotecan resistance to RKO cells (80%→10% cell death), but the His64 haplotype did not. In contrast, overexpression of His64 galectin-3, but not Pro64, augmented the sensitivity of LM12 and RKO cells to oxaliplatin and 5-FU / leucovorin. Conclusions: These results indicate a functional link between the rs4644 sequence polymorphism of the LGALS3 gene and differential susceptibility of colon cancer cells to agents which promote apoptosis. This has implications for the individualized therapy of colon cancer.

T2016 Regulation of COX-2 Expression by MicroRNA-542-3p Ashleigh E. Moore, Dan A. Dixon The inducible cyclooxygenase-2 (COX-2) enzyme mediates inflammatory responses and plays a pivotal role in several cancer types. Overexpression of COX-2 has been linked with colon cancer and is an early event during tumorigenesis. In normal tissues, COX-2 levels are controlled through post-transcriptional regulation via regulatory elements present in the mRNA 3' untranslated region (3'UTR) that control the stability of the transcript. MicroRNAs (miRNAs) are small non-coding RNAs that act post-transcriptionally by binding mRNAs in the 3'UTR and function to regulate target gene expression by either degrading or translationally repressing their targets. We have identified a putative binding site for the microRNA miR542-3p within the COX-2 3'UTR and miR-542-3p specificity was demonstrated through efficient down regulation of a reporter construct bearing the COX-2 3'UTR. The ability of this miRNA to down regulate inducible COX-2 expression was observed in IL-1β-induced HeLa cells where miR-542-3p was able to attenuate COX-2 protein expression. Similarly, miR-542-3p inhibited COX-2-dependent prostaglandin synthesis as efficient as a COX-2selective NSAID. COX-2 mRNA levels were also decreased suggesting that miR-542-3p acts on the level of mRNA stability. Expression of COX-1 was unaffected by miR-542-3p, indicating specificity for COX-2. Furthermore, COX-2 overexpression occurring in colon cancer cell lines was efficiently downregulated with miR-542-3p expression. To determine the role of miR-542-3p in colon cancer, tissue samples were assayed for miR-542-3p levels. Matched tumor/normal tissue samples displayed similar expression levels of miR-542-3p indicating that loss of miRNA expression is not responsible for COX-2 overexpression observed in tumors. Interestingly, the target site for miR-542-3p contains a single nucleotide polymorphism T8473C SNP (rs5275) that is located in exon 10 of PTGS2 and the presence

T2014 Analysis of K-RAS Mutations in Laterally Spreading Tumors of the Colorectum Eisuke Kaji, Jun Kato, Shunsuke Saito, Hideyuki Suzuki, Reiji Higashi, Motoaki Kuriyama, Toshio Uraoka, Sakiko Hiraoka, Kazuhide Yamamoto Objective: Laterally spreading tumor (LST) is a new category of the colorectal neoplasia which is defined as a lesion larger than 10 mm in diameter and extending circumferentially rather than vertically. Although a lot of investigations regarding endoscopic and histopathological features of LSTs have been performed mainly in Japan, genetic features are not fully elucidated. The aim of this study is to examine the K-ras mutations in the LSTs, and evaluate the correlations between the K-ras status and the characteristics of LSTs, including endoscopic and histopathological features. Methods: We examined K-ras gene mutations of 101 LSTs by direct sequencing. Characteristics of the 101 LSTs (obtained from 98 patients: male/

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AGA Abstracts

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