Promoter hypermethylation (PM) of the tumor-suppressor genes p16INK4A and p14ARF during neoplastic progression in long-standing ulcerative colitis (UC)

immnunoreactive cells were observed within the glands, concentrated in the middle of the mucosal region. Bcl-2 expression was unreactive in all tumor lesions. The mean follow-up period was 36.8 months (SD= 45.5), varying from 3 to 144 months. Three patients were lost of follow-up. Survival of patients with p53 expression and high proliferative rate (Ki67) was significantly lower than that of negative patients (overall survival of 10.4 months vs. 111.06 months, p = 0.01). Conclusions: 1. p53 expressionassociatedwith high proliferative rate help to distinguish between type I and type III gastric carcinoid tumors, suggesting a biologically aggressive growth pattern in the sporadic carcinoid tumors; 2. These markers can be used as prognostic factors for ECL cell tumors.

1543 Expression of Homeobox Gene CDX2 precedes CDXl During the Transition from Atrophic Gastritis to intestinal Metaptasia Akashi Eda, Hiroyuki Muto, Ichiro Yanaka, Hiroyuki Osawa, Kentaro Sugano, Jichi Medical Sch, Yakushiji, Tochigi Japan Background- CDX1 and CDX2 are homeodomaintranscription factors that regulate intestinespecific geneexpression.CDX1/2are tumor-sapprassor genes involved in colon carcinogenesis, but their roles in atrophic gastritis and gastric intestinal metaplasiaare unknown. There have been no detailed report that directly compared CDX1 expression with that of COX2 during atrophy/metaplasia transition in human stomach. Aims-To examine expression of COX1/2 and their association with the expression of other intestinal metaplasia-associeted genes during the development process of intestinal metaplasia. Methods-This study was approved by the ethical committee of the Jichi Medical School, Japan. Informed consent was obtained from all patients. The expression of CDX1/2 gone was analyzed using reverse transeriptase-polymerasechain reaction in 44 human gastric tissues obtained endoscopically and comparedthe results with the expressionsof mucin markers(MUC2, MUC5AC), intestinespecific genes (sucrase-isomaltase, human defensin-5, alkaline phosphatase) and gastric phenotype genes (H+/K +ATPase- psubunit and pepsinogen C). Results-The expression of CDX1/2 were absent in the normal epithelium of the stomach without Helicobecterpylori infection. The prevalenceof CDX1/2 mRNA expressionwas significantly higher in the tissue with intestinal metaplasiathan in the tissue without intestinal metaplasia. Coexprassionof COX1 and CDX2 was observed in 86% of intestinal metaplasia. Especially,the expression of COX1was more closely associatedwith intestinal metaplasiathan that of CDX2.It is noteworthy that the expression of COX2 was already detected in the tissue with atrophic gastritis in the antrum and endoscopically normal epithelium in middle corpus greater curvature (fundic gland area) without expression of gene markers for intestinal mstaplasia. Conclusion-The expression of CDX2 precedes those of CDX1, sucrase-isomaltase, other intestine-specific genes (human detensin-5, alkaline phosphatase) and MUC2 in the sequence from atrophic gastritis to intestinal metaplasia.Thesefindings implies that the expressionof CDX2 may not be the effect, but the trigger of the development of intestinal metaplasia.

1545 Promoter HypermethylaUon (PM) of the Tumor-Suppressor Genes p161NK4Aand p14ARF During Neoplasti© Progression in Long-Standing Ulcerative Colitis (UC) Bodo Klump, Chih-Jen Hsieh, Kariheinz Hotzmann, Oliver Nehls, Veronika Gaco, Michael Gragor, Univ Hosp Tuebingen, Tuebingen Germany Background:UC is considered as premalignant condition predisposing for the development of colorectal carcinoma (CRC). Own results have demonstrated that the TSG p16tNK4A is involved at an early stage in the multistep carcinogenesis of UC associated CRC. PM was suggestedas major inactivating mechanism, p14ARFrepresentsanother TSG also located in the genetic locus INK4a. Both genes share two out of three exons and are suspectedto be involved in different tumor suppressor pathways, respectively. This study should further characterizethe locus' INK4a significance in the developmentof UC associatedCRC. Material and Methods:83 samples were retrieved from three colectomy specimens of pts with UC. In 2 pts surgery was recommendeddue to the previous detection of dysplasia, in one pt due to clinical deterioration under medical treatment. A methylation specific PCR protocol was applied to determine the p14ARF PM status. The PM status was compared to the exact histopathologicai diagnosis of each sample. Resu/ts: p14ARF PM was detected in merely 14.5%(8/55) of samples without dysplastic alteration (NEG). All NEG samples with p14ARF PM were retrieved from pts who underwent surgery due to the finding of dysplssia. 16.7% of sampleswhich were classified as "indefinite for dysplasia" (INDEF)revealedto havep14ARF PM. However, in 41.7% (5/12) of samples with low grade dysplasia (LGD), 57.1% (4/7) of samples with high grade dysplasia (HGD) and 33.3% (1/3) of carcinomatous samples (CA) was detected p14ARF PM. In comparison, PM of p161NK4Awas found in 12.7% of "NEG," 50% of "INOEF," 75% of "LGD," 43% of "pHGD" and all "CA" samples (Cancer Res. 1998). In all dysplastic categoriesa high rate of coincidentalp161NK4Aand pl 4ARFPM was observed. Conc/usion:Theseresults indicatethat the TSG p14ARFis involved in the processof neoplastic transformation in UC and suggest PM as major inactivating mechanism. A high rate of coincidental p161NK4Aand p14ARFPM in dysplastic specimensfurther supports the hypothesis that the two TSGsact in different tumor suppressive pathways.The predominanceof one pathway could not be demonstrated. This study has been supported by a grant of the Interdisciplinary ResearchCenterof the University of Tuehingen(iZKF, illB2). The first author is supported by the Deutsche Krabshilfe (70-2601) and the German CompetenceCenter for Inflammatory Bowel Disease (Kompetenznet2CEO). 1546 CENP-C: An Oncogene or Tumor Suppressor Gene? A Possible New Tumor Marker Diana Kazanov,Baruch S. Stem, TeI-Aviv Medical Ctr, TeI-Aviv Israel; Walter Pyerin, Oliver Boecher, DKFZ, HeidelbergGermany; Hana Strui, Menahem Moskowifz, Ludmila Strier, TelAviv Medical Ctr, TeI-Aviv Israel; Raanan Sbemir, Rambem Medical Ctr, Haita israel; Nadir Arber, TeI-Aviv Medical Ctr, TeI-Aviv Israel Background: Centromera protein-C (CENP-C) is one of three centromere proteins (A,B,C) presentthroughout the cell cycle. Theyform a kinetochoraprecursor that provides scaffolding for additional transiently expressed proteins that result in a funetionafly active kinstochora. Mutations causethe centromerefunction to become progressivelydeficient leadingto increasing mitotic disarray,embryonic lethality,aneuploidy,cancerand cell death.Hypothesis:Upregulafion of CENP-Cin a normal enterocytecell line IEC18)will result in malignanttransformation. Material and Msthods:The human cDNA CENP-~jenewas cloned into the expression vector pCDNA3.1Zeo using PCR.IEC18cells weretransfested with the construct, which also encodes for the Zeocin drug resistanceselectablemarker. Resistant clones were isolated and further characterized.Results: Clonesexpressingincreasedlevels of CENP-Cwere chosen for further evaluation. The integration of CENP-Cinto the genuine was confirmed by Southern blot, and expressionwas determinedusing RT-PCR.The biological activity was assessed using various proliferation assays and we established that these clones exhibited an increased doubling time. Like the parental cells, their plating efficiency was zero, they did not grow in soft agar, and they were not tumorigenic in nudes mice. We also tested the transformed cells for an altered sensitivity to the induction of apoptosis using various NSAIDs compounds. CENP-C clones are more resistant to celecoxib (Pharmacia), sulindac sulfide (Cell Pathways) and sulindac sulfone (Cell Pathways). They were sensitive to the newer cGMP phosphodiesterase CP-248 and CP-461(Cell Pathway). Western blot displayed higher amounts of cyclin O1, Rb and Fas-ligand and lower amount of COX 1 in the CENP-Ccells. There was no change in other protein of the bcl2, cyclins and cyclin inhibitors families. Expressionarray analysis of 1156 genes revealed upregulation of 11 genes and downregulation of 16 among them cell adhesion molecules, receptors and other functionally unclassified genes. Conclusions: 1. CENP-C up-regulation is not sufficient to cause a malignant transformation in IEC-18 cells. 2. CENP-Cmight serve as a tumor supressor gene as it inhibits cell growth. 3. Increased resistance of the clones (compared with IEC18) to apoptosis induced by the different drugs may be explainedbythe up regulationof Rb and down regulation of FAS ligand. 4. The analysis of differentially expressedgenes may yield novel targets for cancer prevention and therapy.

1544 Miorosatellite Instability Influences Histology And Prognosis In Mutinous Carcinoma Of The Coloreetum. Sanjay N/a Kakar, Lawrence J. Burgart, Stephen Thobodeau,Thomas C. Smyrk, Mayo Clin, Rochester, MN Background: Mucinous histology in colorectal cancer is an adverseprognostic factor in some studies, but not in others. Recentwork has indicatedthat largebowelcancerswith microsatellite instability (MSI-high) have better prognosis than those without microsatellite instability (MSIstable). We hypothesizedthat knowing MSI status would help clarify the question of prognosis in mucinous carcinoma. Methods: We studied 72 mucinous carcinomas of the colorectum resectedduring the years 1996-98. All tumors had mucin making up more than 50% of tumor volume. MSI status was determined by PCR using the BethesdaConsensusprimers. Tumors with 2 or more unstable microsatelliteswere designated MSI-high; all others were classified as MSI-stable. Without knowledge of MSI status, we recordedtumor size, site, grade, stage, growth pattern, Crohn s-like reaction, vascular invasion and numbers of tumor-infiltrating lymphocytes (TIL). Results:34 mucinous carcinomaswere MSI-high and 38 were MSI-stable. MSI-high cancers were more likely to be right-sided (30•34 vs 16/38, p
MSI-hioh Alive Dead

(Non-signet) 26 1

1547

MSl-stable (Signet) 4 3

(Non-signet) 27 9

p53 Mutations and Microsatellite Instabilities in the Subtype of Intestinal Metaplasia of the Stomach and Its Role as a Precancerous Lesion Sung Sou Kim, Catholic Univ of Korea, Seoul South Korea; Choon Sang Bhang, Catholic Univ of Korea, Suwan South Korea; Hak Sung Lee, Hiun Suk Chae, In Sik Chung, Dog Ho Park, Catholic Univ of Korea, Seoul South Korea BACKGROUND:Although there have been reports of the comparison of p53 mutation and microsatellite instability according to the subtype of gastric intestinal metaplasia,these were mostly studied with isolated lesions from different patients. The study was aimed to compare the prevalenceof p53alterafionsand microsatelliteinstability in the subtypeof gastric intestinal metaplasia.METHODS:We investigatedthese genetic abnormalitiesin the samples containing complete (type I) and sulphomucin-secreting incomplete type (type III) intestinal metaplasia from the same patient with gastric cancer. The DNA was extracted from each lesion with microdissection method. Exons5 to 8 of p53genewere examinedfor mutation by polymerase

(Signet) 1 1

A-299