AG

AA

bst

ract

sSa1771

Role of DNA Methylation on Epithelial Mesenchymal Transition (EMT)-LikePhenotype of Colon Cancer CellsJumpei Kondo, Takuya Inoue, Tomofumi Akasaka, Yoshito Hayashi, Shunsuke Yamamoto,Motohiko Kato, Tsutomu Nishida, Kenji Watabe, Masahiko Tsujii, Tetsuo Takehara

Background: Epigenetic regulation of gene expression plays a key role in cancer biology. Itis well known that gene-specific hypermethylation and genomic global hypomethylation isrelated to progression of cancer, and the methylation status is regulated by DNA methyltrans-ferase (DNMT). DNMT1 is not only responsible for the maintenance of global DNA methyl-ation pattern during cell proliferation, DNMT1 is also necessary for aberrant CpG islandmethylation in human cancer cells. Several researches demonstrate the therapeutic potentialsof genetic or pharmacologic inhibition of DNMT1 by reactivating silenced tumor-suppressorgenes. On the other hand, some literatures show that decreasing DNMT1 levels or activitycan potentially enhance invasiveness of cancer cells, indicating that the effect of DNMT1inhibition is cell context-specific. Aim: We evaluated the impact of DNMT1 inhibition ordemethylation drug on epithelial mesenchymal transition (EMT)-like phenotype, includingthe determination of key molecule on the induction of EMT-like phenotype. Materials andMethods: Wild type and DNMT1 knock-out (KO) colon cancer HCT-116 cells were usedin this study. Alternatively, small interfering RNA (siRNA), and 5-aza-2'-deoxycytidine (5-aza) was used to silence DNMT1 expression in HCT116 cells. The resulting cell lineswere validated by reverse transcriptase-polymerase chain reaction and Western blotting.Proliferation, migration and invasion assays were done in engineered cells to evaluate theeffect of DNMT1 silencing on cellular phenotype. Result: Our data shows that DNMT1 KOand 5-aza treatment on HCT-116 cells resulted in EMT-like gene expression change. Genesspecific to epithelial cells, such as E-cadherin, were down regulated, and genes related tomesenchymal markers, such as vimentin, were up regulated. Those engineered cells alsoexhibit higher cellular motility and acquired anoikis resistance. Those EMT-like phenotypewere not correlated to the expression of Snail or Twist, but had propensity for the involvementof Zeb1 expression. Conclusion: Inhibiting DNMT1 on cancer cells, at least in some particularcontext, result in acquisition of EMT-like property. Our data indicates the necessity ofselecting situation or patient when applying DNMT inhibition as treatment sensitizing agents.

Sa1772

Intestinal Isthmus Keratin-19 Positive Stem Cells Contribute to IntestinalTumorsSamuel Asfaha, Christoph B. Westphalen, Russell Ericksen, Johannes von Burstin, SarahC. Stokes, Teresa L. Mastracci, Michael Quante, Wataru Shibata, Daniel L. Worthley, AnilK. Rustgi, Timothy C. Wang

Tumors are postulated to arise from tissue stem or progenitor cells. Previous studies havesuggested a polyclonal origin for small intestinal tumors. In the intestine, keratin 19 (K19)is thought to mark a potential progenitor cell within the intestinal isthmus. We generateda new K19-BAC-CreER transgenic line to study the lineage of K19+ cells and determinewhether K19+ might mark both normal and cancer initiating intestinal progenitors. K19-BAC-CreER mice were crossed to a ROSA26r(LacZ or GFP) reporter line. The offspring wereinduced with tamoxifen and studied in a AOM/DSS model of inflammatory colorectalcarcinogenesis. In separate experiments, K19CreER/ROSA26r mice were crossed to ApcMinmice to examine the contribution of the K19 lineage to a genetic model of intestinalcarcinogenesis. Tamoxifen-induced X-gal labeling occurred in ~20-50% of colonic andintestinal glands. The labeling persisted for greater than 52 weeks, consistent with K19marking long-lived intestinal stem cells. In K19CreER/ROSA26r mice treated with AOM orDSS alone there was a dramatic increase in the labeling of contiguous X-gal positive glands,consistent with K19+ stem cell expansion and crypt fission. To test whether expansion ofcolonic progenitors represents a critical event in the initiation of tumors, we compared thepattern of lineage tracing in tumors relative to the timing of recombinase induction. WhenK19+ progenitor cells were labeled by tamoxifen prior to AOM and DSS, the majority oflineage traced tumors were entirely X-gal positive, suggesting they were each derived froma single recombined K19+ cell. Interestingly, when K19+ cells were labeled by tamoxifenafter tumor initiation with AOM, tumors originated from both K19-Cre recombined andnon-recombined cells, suggesting that AOM stimulates the K19+ progenitor cell to give riseto multiple K19+ daughter cells that contribute to the tumor. In contrast, in ApcMin mice,all X-gal marked small intestinal adenomas were derived from both K19 recombined andnon-recombined cells. In summary, K19 marks intestinal and colonic stem cells located inthe isthmus (above the crypt base), and these K19+ cells serve as cancer initiating cells inboth carcinogen and genetic models of intestinal tumorigenesis. AOM-induced division ofa K19+ cell, evidenced by contiguous crypt labeling and suggestive of symmetric cell division,appears to be an initiating event. Colonic tumors are polyclonal, but the multiple cancerinitiating cells appear to arise from a single K19+ progenitor. The polyclonal adenomas fromK19+ cells in APCmin mice, however, suggests much earlier cancer initiation, consistentwith an inherited abnormality in stem cell division. We identify a key role for early stemcell amplification in cancer initiation, and a potentially important distinction between inher-ited and sporadic forms of intestinal cancer.

Sa1773

Clinicopathological and Molecular Correlates of PIK3CA Mutations in GastricCancerYasutaka Sukawa, Hiroyuki Yamamoto, Katsuhiko Nosho, Yasushi Adachi, HiroakiKunimoto, Hisayoshi Igarashi, Mayumi Nakazawa, Hiromu Suzuki, Shigeru Sasaki,Yasuhisa Shinomura

Background: We have characterized the molecular alterations in gastric cancers, especiallythose with microsatellite instability (MSI) (Nature, Science, Nature Genetics 2006, 2009,Cancer Cell 2010). PIK3CA is mutated in a wide variety of human tumor types, includinggastric cancers. Activating mutations in this gene up-regulates the PI3K-AKT signalingpathway, making it a potentially useful therapeutic target. However, little work has been

S-322AGA Abstracts

performed on clinicopathological and molecular correlates of PIK3CA in gastric cancer. Theaim of this study was to characterize the mutational status of PIK3CA in a large cohort ofgastric cancers (n = 380) to determine its prevalence with a high precision and to correlateit with clinicopathological and molecular features, such as MSI, HER2 expression, and EBVinfection.Materials and Methods: Pyrosequencing-based methods facilitate the identificationof low-frequency tumor mutations and allow more accurate assessment of tumor mutationburden. We analyzed 380 gastric cancers, including 145 T1 cancers, for PIK3CA mutationsin exons 9 (helical domain) and 20 (kinase domain) as mutational hotspot regions bypyrosequencing of DNA obtained from formalin-fixed, paraffin-embedded tissues. Expressionof HER2 was analyzed by immunohistochemistry (IHC). Results: PIK3CA mutations werepresent in 19 cases (5%). The mutation frequency was high (27.3%) in T4 cancers, but itwas very low (1.4%) in T1 cancers. Ex20/Ex9 prevalence ratio was 1.38 (11/8). All mutationswere heterozygous missense single base substitutions and the most common mutation wasH1047R (7/19; 37%) in exon 20. PIK3CA mutations were significantly correlated with depthof invasion (p = 0.0088), advanced stage (p = 0.0499), invasive type of Bormann's classifica-tion (p = 0.0111). Two-year survival rates were 67% in patients with PIK3CA mutationsand 79% in those without, respectively (p = 0.6334). HER2 overexpression (IHC 3+) waspresent in 32 samples (8%), being significantly correlated with intestinal histological type.PIK3CA mutations were not present in cancers with HER2 overexpression, but were signific-antly associated with MSI-positive (p < 0.0001). Twenty-four cancers (6.7%) were EBV-positive, being significantly correlated with diffuse histological type. PIK3CA mutations andHER2 overexpression were present in 2 (8.3%) EBV-positive cancers. Conclusions: PIK3CAmutations appear to be late events in gastric carcinogenesis, leading to tumor progression.PIK3CA mutations are one of the most important oncogenes activated by missense mutationsin MSI-positive gastric cancer. Analyses of additional gastric cancers and other genetic andepigenetic alterations are underway and we will present comprehensive data in the meeting.

Sa1774

Prevalence of MLH1 Constitutional Epimutations as a Cause of LynchSyndrome in Unselected Consecutive Cases of Colorectal CancerMaria Rodriguez-Soler, Lucía Pérez-Carbonell, Carla Guarinos, Adela Castillejo, CeciliaEgoavil, Victor M. Barberá, Eduardo Martinez-Dueñas, Maria-Isabel Castillejo, AnaMartinez-Canto, Ana B. Sanchez-Heras, Clara Ruiz-Ponte, Alejandro Brea, CristinaAlenda, Artemio Payá, Cristina Sanchez-Fortun, Miriam Juarez-Quesada, Luis Bujanda,Juan Clofent, Xavier Llor, Montserrat Andreu, Antoni Castells, Angel Carracedo, José-LuisSoto, Rodrigo Jover

Colorectal cancer with loss of MLH1 expression and hypermethylation of its promoter areusually considered as sporadic. The epigenetic inactivation of MLH1 has been reported ina small number of cases of CRC with a phenotype similar to Lynch Syndrome. However,all these cases have been reported from selected population and the prevalence of MLH1constitutional epimutations in patients with CRC is unknown. AIM: To assess the prevalenceof MLH1 constitutional epimutations as a cause of Lynch Syndrome in unselected consecutivecases of CRC and to compare that with a selected population fulfilling revised Bethesdaguidelines. METHODS: Patients from a prospective, population-based, observational cohortincluding 871 CRC patients from the Epicolon II cohort and 1405 cases from HospitalGeneral Universitario de Alicante were included. From them, 118 patients showed loss ofexpression of MLH1. For comparisons, a group of 54 selected CRC patients fulfilling revisedBethesda guidelines with loss of MLH1 expression coming from the Valencian CommunityGenetic Counseling program was also included. Somatic MLH1 methylation and germlineMLH1 mutations were assessed. Constitutional MLH1 methylation was studied with Methyl-ation Specific-MLPA of MLH1 in patients with somatic MLH1 methylation. RESULTS: A totalof 72 (61%) patients with loss of expression of MLH1 showed somatic MLH1methylation, 16(22.2%) from selected population and 56 (77.8%) from unselected population. The frequencyof fulfilment of the revised Bethesda guidelines in the group of unselected consecutivepatients was 26%. In the group of unselected consecutive CRC cases none constitutionalepimutation was found. Whereas, in the group of selected patients we found 1 out of 16(6.25%) cases withMLH1 epimutation, withmethylation in peripheral blood, normal colorec-tal mucosa and epithelial oral mucosa. CONCLUSION: We did not find any case of MLH1constitutional epimutation in an unselected population of 2276 CRC cases. However, betweenselected patients fulfilling revised Bethesda guidelines there are cases of constitutional epimut-ations. These results suggest that the analysis of MLH1 constitutional epimutation shouldbe included in the screening for Lynch Syndrome only in selected cases with high risk ofthis syndrome.

Sa1775

Moderate Microsatellite Instability is Associated With Recurrent DistantMetastasis in Stage II and III Primary Colorectal CancersMelissa Garcia, Chan Choi, Hyeong-Rok Kim, Yuji Toiyama, Masanobu Takahashi, AjayGoel, Clement R. Boland, Minoru Koi

(Background & Aims) We previously reported that elevated microsatellite alterations atselected tetranucleotide repeats (EMAST) and low level microsatellite instability (MSI-L) arefrequently observed in colorectal cancers (CRCs). However, their significance for CRCdevelopment is not known. In this study, we aimed to determinewhether any clinicopatholog-ical variables associated with primary CRC are linked to EMAST and/or MSI-L phenotype.(Materials and Methods) Tissues: One hundred sixty-seven consecutive cases of primaryCRC and matching normal colonic tissues were collected at Chonnam National UniversityHospital, Gwangju and Chonnam National University Hwasun Hospital National Biobankof Korea. All samples were obtained with informed consent under institutional review board-approved protocols. For DNA extraction, tumor and normal tissues were micro-dissectedseparately fromparaffin-embedded sections (10μm). Genomic DNAwas isolated and purifiedfrom micro-dissected tissues. Microsatellite Assay: PCR amplifications were performed fromgenomic DNA using fluorescently labeled primers. Two markers with mononucleotiderepeats, five markers with dinucleotide repeats, and seven EMAST markers were used.Amplified PCR products were denatured and electrophoresed on an ABI PRISM 3100 AvantGenetic Analyzer and analyzed by GeneMapper fragment analysis software. A locus was