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sTu1908

DNA Methylation Level of Mir-124a Could Be Individual Risk Marker ofColitis-Associated Cancer in Ulcerative ColitisTakayuki Ando, Yuko Ueda, Sohachi Nanjo, Hiroshi Mihara, Haruka Fujinami, JunNishikawa, Ayumu Hosokawa, Toshiro Sugiyama

OBJECTIVE: Colitis-associated cancer (CAC) is the most serious complication of ulcerativecolitis (UC). Recent studies have recommended that surveillance intervals should be tailoredwith individual risk, and useful molecular markers focusing on high-risk patients for CACare needed. This study evaluated the usefulness of quantification of miR-124a methylationas a risk marker of CAC development. METHODS: Twelve healthy volunteers (HV), 40 UCpatients without CAC, and 4 patients with CAC or dysplasia were recruited. DNAmethylationlevels of miR-124a, a tumor-suppressive microRNA, were quantified in the colorectal mucosaof UC patients, and analyzed the association with major risk factors, disease extent andduration. The methylation statuses of miR-124a genes (miR-124a-1, -2, and -3) in colorectalmucosa and tumor tissue were analyzed by methylation-specific polymerase chain reaction(MSP), and their methylation levels were quantified by real-time MSP. Expression of cyclin-dependent kinase 6 (CDK6), a target of miR-124a, was analyzed by immunohistochemistry.RESULTS: All three miR-124a genes were methylated, and CDK6 was expressed in all CACand dysplasia. Mean methylation levels of miR-124a-3 in HV, non-pancolitis, and pancolitispatients were 2.0%, 5.3%, and 12.3%, respectively, and were significantly higher in pancolitispatients than in HV (p,0.01). As for disease duration, mean methylation levels in patientswith short-term and longstanding duration were 2.5%, and 13.2%, respectively. Patientswith longstanding duration had significantly higher methylation levels than HV (p ,0.01).Moreover, UC patients with both pancolitis and longstanding duration had 7.4-fold highermethylation levels than those without two risk factors. CONCLUSIONS: MiR-124a genesare methylated during carcinogenesis in UC patients, and their methylation levels correlatewith risk factors for CAC development. Therefore they could be novel markers for estimatingindividual risk of CAC.

Tu1909

APC I1307k Is a Predictive Factor for Adenoma RecurrenceMenachem Moshkowitz, Ben Boursi, Tal Sella, Eliezer Liberman, Ravit Geva, EinatShacham-Shmueli, Diana Kazanov, Sarah Kraus, Nadir Arber

Background: The use of surveillance colonoscopy to detect colorectal neoplasia recurrencefollowing initial resection has increased significantly in the past decade. Currently, predictivefactors at index colonoscopy include only histopathologic characteristics such as adenomanumber, size, type and dysplasia. The aim of the current study was to identify additionalfactors (i.e., genetic markers) that increase the risk of subsequent lesions and may optimizethe use of surveillance colonoscopies. Patients and Methods: A prospective analysis, over aperiod of 10 years, of 383 consecutive Israeli subjects that underwent up to five surveillancecolonoscopies after an index neoplastic finding in screening colonoscopy. Clinical dataregarding potential risk factors for colorectal cancer as well as blood samples were collected.Genetic polymorphisms were detected using real-time PCR from DNA extracted from periph-eral mononuclear cells. Results: The overall prevalence of recurrent colorectal carcinomaand adenoma was 9.4% (36/383) and 69% (268/383) respectively, with a median of 4.8years from index colonoscopy. In a univariate analysis, subjects with recurrent lesions hadsignificantly higher number of adenomas at index colonoscopy (3.9 Vs 1.1, p=0.001),increased rate of high grade dysplasia (65.8% Vs 50%, p=0.018), and a non-significant trendfor larger adenomas and villous histology. The APC I1307K gene variant was detected in11.8% of subjects with recurrent lesions compared to 3.8% of subjects with normal colonos-copies (p=0.03). In a multivariate logistic regression (adjusted to age, sex, family history ofCR neoplasia and time to recurrence), the I1307K variant and the presence of dysplasiawere the only significant predictive factors for recurrent neoplasia with an OR of 3.27 (1-11.02, p-0.05) and 1.72 (1.02-2.89, p=0.04), respectively. Conclusions: The APC I1307Kgene variant is an important predictive factor for recurrent colorectal neoplasia after a positiveindex colonoscopy and should be considered as part of the criteria for high-risk subjects.

Tu1910

Methylated Cell-Free DNA of Reprimo in Plasma for Non-Invasive Diagnosisof Gastric Cancer and DysplasiaTeresa Martinez, Maria J. Maturana, Jacqueline Fry, Maria Mercedes Bravo, GustavoHernandez, DunFa Peng, Barbara G. Schneider, Wael El-Rifai, Alejandro H. Corvalan

Introduction: Gastric cancer (GC), the second leading cause of cancer-related deaths world-wide, is associated with the H.pylori infection and multistep progression of GC (multifocalatrophic gastritis [MAG], intestinal metaplasia [IM] and dysplasia [DY]). Although biomarkersfor MAG are well defined, currently no such biomarkers are available for the direct detectionof DY or GC. Our previous finding of the detection of methylated cell-free DNA of Reprimo(RPRM), a p53-dependent G2 arrest mediator candidate in plasma from GC patients butnot in healthy donors prompted us to investigate the possibility that RPRM could be sucha biomarker. Materials and Methods: The study population was GC (n=55), DY (n=23) andIM (n=65). Characteristics of the study population and frequency of infection by H.pyloristrains are shown in Table 1. Methylated cell-free RPRM DNA was detected by TaqManReal-Time PCR (MethyLight). Results were reported as the mean and media number ofcopies per mL of plasma (cp/mL). Positive and negative MethyLight cases were confirmedby Pyrosequencing of matched tissues. Results: Methylated cell-free RPRM DNA was detectedin 31 (56.4%), 13 (56.5%) and 24 (37.5%) cases of GC, DY and IM, respectively. The meanand median cp/mL of methylated cell-free RPRM DNA were 5,431 and 24 (range: 0 - 243,847cp/mL), 280 and 11 (range: 0 - 2,024 cp/mL) and 173 and 0 (range: 0 - 2,859 cp/mL) inGC, DY and IM, respectively (p=0.05, Kruskal-Wallis Test). After adjusted by age ≥ 50 y.o.(Bonferroni=0.37), 23 DY and 16 IM cases were selected. The mean and median cp/mL ofmethylated cell-free RPRM DNA were 147 and 0 (range: 0 - 2,859 cp/mL) and 205 and 27(0 - 2,023 cp/mL) in DY and IM patients, respectively. These differences were highlysignificant (p= 0.01, Mann-Whitney Test) (Fig 1). Among GC cases, no specific associations

S-878AGA Abstracts

were found, except higher levels of methylated cell-free DNA in tumors located in antrumand body. Pyrosequencing of matched tissues samples confirmed positive and negativeMethyLight results in 100% (3/3), 66%, (2/3) and 57% (4/7) of GC, DY and IM, respectively.Discussion: Our data suggest that methylated cell-free RPRM DNA may be a non-invasivebiomarker for the diagnosis of GC. The finding of significant differences between IM vs.DY suggests that our biomarker may also detect the direct precursor lesion of GC, thegastric dysplasia. Pyrosequencing confirmed that negative cases were associated with lackof methylation of RPRM at the tissue level. Grant Support: Fondecyt #1111014 and Fondef#D09I1137 to AHC.Table 1. Study population and characteristics of infecting H. pylori strains

Fig 2. Methylated cell-free DNA in plasma of Reprimo in Dysplasia (DY) and IntestinalMetaplasia (IM) after adjustment by age of 50 y.o. (Bonferroni=0,37). Reprimo levels wereexpressed as Log 10 of number of copies per mL of plasma.

Tu1911

Novel DNA Methylation Targets Associated With Resistance to AdjuvantChemotherapy in Colon Cancer PatientsHamed Rahi, Mohammad Daremiporan, Hassan Brim, Ron Leavitt, Xueguang Sun, SudhirVarma, Hassan Ashktorab

Novel DNA methylation targets associated with Resistance to adjuvant chemotherapy incolon cancer patients Background: Drug resistance remains a major obstacle to successfulcolon cancer (CRC) treatment. Our previous DNA methylation array analysis indicated thatepigenetics affect colon cancer progress and response to 5-FU therapy. Therefore, we aimedto investigate paired responder (R) and non-responder (NR) patients' tumor DNA usingwhole epigenomic sequencing to elucidate the epigenetic basis of resistance to chemotherapy.Patients and Methods: Genomic DNA was isolated from fresh frozen tissues of three CRCresponder patients, an African American (AA) (stage IIIC), a Caucasian (stage III) and aHispanic ( stage IIIb) , who were free of disease after 5FU treatment, and two non-responder(stage III & Stage IV) AA patients whose disease progressed despite 5-FU treatment. AReduced Representation Bisulfite Sequencing (RRBS) at .20X depth was performed. Align-ment, mapping and CpG methylation analyses were done. For each sample (responder andnon-responder), the top 200 hyper- and hypo-methylated genes were selected by comparingthe confidence intervals for the methylation status to 0 (un-methylated) or 1 (methylated).From this comparison, 21 hypermethylated and 25 hypomethylated genes were found tobe specific of the non-responder tumors. The ingenuity Pathway Analysis software (IPA)was used for pathway analysis of the affected genes. Results:We identified 4 hyper-methylatedand 2 hypo-methylated novel genes in non-responder tumors. IPA showed that the hyper-methylated genes in the resistance to 5FU were involved in the suppression of apoptosisby blocking the activation of caspase-3 via TGF-beta signaling pathway (COMP). The otherhypermethylated genes in the non-responder were involved in DNA replication and repair(CSNK1D, WNT pathway), negative regulation of cell proliferation and survival by activin/TGF-beta-induced apoptosis through its Smad-dependent expression (INPP5D), LTBP3 islikely involved in the assembly, secretion and targeting of TGFB1 to sites at which it isstored and/or activated and induce apoptosis through its Smad-dependent expression. Thehypo-methylated genes in the non-responder involved GAS6 whose signaling is implicatedin cell growth and survival, cell adhesion and cell migration, and TLE2 that Inhibits thetranscriptional activation mediated by CTNNB1 and TCF family members in Wnt signaling.Conclusion: This work provides insight into differential methylation profiles in colon tumors