Langenbeck’s Arch Surg (1999) 384: 90–129 © Springer-Verlag 1999

ABSTRACTS

2nd International Congress on Gastroenterological Carcinogenesis

Date: Location:

March 25–27, 1999 Hotel Maritim / Congress Center; CCU Ulm / Germany,

A Biannual Meeting of

The International Society of Gastroenterological Carcinogenesis with Scientific Support of

– Deutsches Krebsforschungszentrum – DKFZ, Heidelberg – German Cancer Society – Arbeitsgemeinschaft Experimentelle Krebsforschung – Arbeitsgemeinschaft Internistische Onkologie – European Association for Cancer Research – Deutsche Gesellschaft für Chirurgie – Chirurgische Arbeitsgemeinschaft für Onkologie – Chirurgische Arbeitsgemeinschaft Molekulare Diagnostik und Therapie – Deutsche Gesellschaft für Verdauungs– und Stoffwechselkrankheiten – Arbeitsgemeinschaft Gastroenterologische Onkologie – University of Ulm Congress-Organization Congress President: H. G. Beger Head and Chairman of Department of General and Visceral Surgery University of Ulm

Local Organization Committee: K. H. Link F. Gansauge M. Kornmann Department of General and Visceral Surgery University of Ulm

International and National Faculty H. Bartsch (D) H. Biesalski (D) P. Brennan (F) M. Büchler (D) W. Caselmann (D) K. Danenberg (USA) P. Danenberg (USA) K. Debatin (D) I. J. Fidler (USA) P. Galle (D) Th. Gress (D) P. Hainaut (F) C. C. Harris (USA) P. Hermanek (D) O. Hino (J)

K. Höffken (D) H. Höfler (D) P. Jones (USA) H. Kalthoff (D) M. Kaminishi (J) G. Klöppel (D) M. v. Knebel-Doeberitz (D) M. Korc (USA) N. Lemoine (UK) B. Levin (USA) H. Maier (D) M. Manns (D) C. J. Marshall (UK) J. O’D McGee (UK) F. Momburg (D)

R. Montesano (F) J. Neoptolemos (U.K.) T. Oohara (J) K. Pantel (D) P. Pour (USA) E. O. Riecken (D) V. Rotter (Israel) T. Sauerbruch (D) H. Schackert (D) R. Schmid (D) H. J. Schmoll (D) J. Schölmerich (D) E. Tahara (J) A. Ullrich (D) H. zur Hausen (D)

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Contents Thursday, March 25, 1999 Session 1: Session 2: Session 3: Session 4: Session 5: Workshop:

Esophageal carcinogenesis From reflux esophagitis to Barrett carcinoma S1.01-S1.05 / P1.01 Gastric carcinogenesis Helicobacter and gastric malignancy S2.01-S2.07 / P2.01-P2.13 Liver Carcinogenesis Hepatitis B and C and hepatocellular cancer S3.01-S3.07 Pancreatic carcinogenesis From chronic pancreatitis to ductal pancreatic cancer? S4.01-S4.11 / P4.01-P4.04 Colorectal carcinogenesis Hereditary cancer syndromes S5.01-S5.07 / P5.01-P5.05 Molecular factors of prognosis in GI-tract cancer WS.01-WS.05

Friday, March 26, 1999 Session 6: Session 7: Session 8: Session 9: SatelliteSymposium:

Virus infection and carcinogenesis/Tumor immunology S6.01-S6.05 / P6.01-P6.02 Genetic changes during carcinogenesis S7.01-S7.06 / P7.01-P7.03 Molecular biological approaches for early diagnosis and treatment S8.01-S8.11 / P8.01-P8.08 Extracellular matrix and micro-environment S9.01-S9.04 Selenium and Carcinogenesis SS.01-SS.04

Saturday, March 27, 1999 Session 10: Session 11: Symposium:

Author index

Clinical and pathological studies in gastrointestinal carcinogenesis S10.01-S10.06 / P10.01-P10.04 Frontiers between dysplasia and early cancer S11.01-S11.04 Prevention of Cancer Nutrition and GI-cancer Alcohol and GI-cancer Tobacco and cancer Occupational exposition and cancer Chemoprevention of GI-cancer SP.01-SP.10

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Session 1: Esophagal carcinogenesis From reflux esophagitis to Barrett carcinoma

suggest that potentially new tumor suppressor gene(s) may locate at 4q.

S1.01

S1.03

Recent advances in molecular mechanisms of human stomach carcinogenesis E.Tahara, Y.Yasui, H.Yokozaki. First Department of Pathology, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan

Barrett’s Esophagus: Genetic alterations in the Metaplasia-Dysplasia-Carcinoma Sequence detected by CGH A. Walch1, H. Zitzelsberger2,3, J. Bruch3, J. Mueller4,5, G. Keller4, H. Höfler1,4, and M. Werner4 1 GSF-Institut für Pathologie, 2GSF-Institut für Strahlenbiologie, 3Institut für Strahlenbiologie, LMU München,4Institut für Pathologie, TU München, 5Chirurgische Klinik, TU München

Recent advances in molecular dissection of preneoplastic and neoplastic lesions of the stomach are uncovering a large number of epigenetic and genetic alterations in oncogenes, tumor suppressor genes, telomere and telomerase as well as genetic instability at several microsatellite loci, which are responsible for multistep of stomach carcinogenesis. The scenario of these changes found in gastric carcinoma differs on the two histological types, strongly indicating that well differentiated or intestinal type and poorly differentiated or diffuse type carcinomas have different genetic pathways. At least 30% of gastric intestinal metaplasia and adenoma, which are candidate precancerous lesions of well differentiated adenocarcinoma, share the same genetic changes including telomere shortening, telomerase activity, microsatellite instability at the locus D1S191, aberrant CD44 transcript, APC and p53 mutations as those found in well differentiated adenocarcinoma. Interestingly, LOH of 1p36 including the p73 gene locus is frequently associated with pS2 expressed foveolar cell type gastric cancer.

S1.02 Molecular pathogenesis of esophageal cancers R. Montesano and P. Hainaut Unit of Mechanisms of Carcinogenesis, International Agency for Research on Cancer, Lyon, France Cancers of the esophagus (squamous cell- and adenocarcinoma) usually arise from well-defined, precancerous dysplatic lesions and show multiple, sequential alterations in well-defined oncogenes and tumors suppressor genes. Point mutation of p53 is an early event that occurs in at least half of the tumors. SCC differs from ADC by the type and distribution of mutations, and, within SCC, the p53 mutation spectra significantly differ from one geographic area to the other. These observations suggest that mutations in p53 may be informative of the nature of the mutagenic events that might have caused these cancers. Other frequently altered genes (in at least 20% of the cases) include c-myc, CyclinD1 and HER-2 (amplifications), and RB1, APC, MLH1 and DCC (loss of alleles). P16CDKN2A is frequently inactivated by multiple mechanisms including mutation, deletion, hypermethylation and altered expression. Compared to p53 mutations, alterations in other genes appear to be late events. We have recently analysed alterations at multiple loci in SCC and in ADC using Comparative Genomic Hybridization and allelotyping. We found that the number of chromosomes altered is correlated with tumor stage and grade, and that chromosome 4q is a frequent site of LOH, in particular in ADC. These data

Aims: The molecular genetic mechanisms underlying the development of adenocarcinoma in Barrett’s esophagus are poorly understood. In order to characterize the genetic alterations in this cancer and especially its premalignant lesions we studied chromosomal imbalances by comparative genomic hybridization (CGH) and allelic instabilities by microsatellite analysis in the proposed metaplasia-dysplasia-carcinoma-sequence. Methods: CGH was performed on laser-microdissected lesions of Barrett adenocarcinomas (n=30), high-grade dysplasias (n=11), low-grade dysplasias (n=8), metaplasias (n=6) and metastasis (n=8) as well as normal squamous epithelium (n=10). Additionally the results were validated by microsatellite analysis. Results: In Barrett’s adenocarcinoma the most common regions of DNA loss were detected on 4q (50%), 5q (43%), 9p (43%), 18q (40%), and a loss of Y-chromosome (77%). Recurrent DNA-gains were found on 2p (47%), 7p (47%), 7q (27%), 8q (80%), 10q (47%), 17q (27%), and on chromosome 20 (60%). High-level ampifications were observed on 7p, 7q, 8q, 10q and 17q. There was an increase in the frequency of chromosomal imbalances in the metaplasia-dysplasia-carcinoma-sequence. The pattern of DNA gains and losses in high-grade dysplasias were similar to aberrations detected in adenocarcinoma, while chromosomal imbalances are less frequent in low-grade dyplasias and metaplasias. In low-grade dysplasia and even in metaplasia we detected chromosomal imbalances. The DNA copy number changes identified in more than one low-grade dysplasia or meta-plasia were losses on 9p, 13q and Y-loss, as well as gains on 8q. Conclusions: Our results demonstrate recurrent chromosomal imbalances and their increase during tumor progression of Barrett adenocarcinoma. Chromosomal gains indicate a possible involvement of well-characterized oncogenes like c-MYC (8q24), MET (7q31), ERBB2 (17q21) and AIB (20q12-q13). Tumor suppressor genes associated with tumorigenesis might be located on deleted regions on 4q, 5q (e.g., APC), 9p (CDKN2A, CDKN2B) and 18q (e.g., DCC). We show that recurrent chromosomal changes are detectable in low-grade dysplasia and even in metaplasia, which may be crucial events in the progression to Barrett’s adenocarcinoma. Microsatellite analysis of selected loci like APC and DCC confirmed our CGH results. The detection of shared chromosomal imbalances in the proposed metaplasia-dysplasia-carcinoma sequence in Barrett’s esophagus supports the hypothesis of a process of clonal expansion underlying this progression

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S1.04 The importance of Trp53 and Apc in esophageal carcinogenesis in mice with duodenoesophageal reflux M Fein, KH Fuchs, PW Laird*, P Chandrasoma*, KA Skinner* Dept. Surgery, Univ Würzburg, Germany and *USC, Los Angeles Aim: There is a well established cancer model for esophageal adenocarcinoma in rats, which is based on the surgical induction of duodeno-esophageal reflux. To determine the role of tumor suppressor genes for the carcinogenesis of esophageal adenocarcinoma, we transferred this surgical cancer model into transgenic mice gene-targeted for either Trp53 or Apc. Methods: Wild-type (WT), p53-knockout (Trp53-/-), or Apcmutated (ApcMin/+) mice were operated using microsurgical techniques with total gastrectomy and esophagojejunostomy at 6 weeks of age, creating duodenoesophageal reflux. Mice were sacrificed at 30 weeks of age. H&E stains were reviewed by a single pathologist blinded to the genotype of the animal. Results: All mice (25 WT, 4 Trp53-/-, and 6 ApcMin/+) developed esophagitis with squamous hyperplasia and focal ulceration. The extent of esophagitis was less severe compared to the rat model. No carcinoma were identified in any of the WT or ApcMin/+ mice. All 4 Trp53-/- mice developed dysplasia of the squamous esophageal epithelium and 2 had adenocarcinoma on histology. Conclusion: The combination of gene-targeting technology with experimental surgical models provided a powerful tool for studies on the gene envolvement in carcinogenesis. S1.05 Expression and up-regulation of RAR-b is associated with RA sensitivity and colony formation in esophageal cancer cell lines X-C Xu, X Liu, E Tahara, S M Lippman, and R Lotan UT M. D. Anderson Cancer Center, Houston, USA and Hiroshima University School of Medicine, Hiroshima, Japan Aim Retinoic acids (RA) exhibit therapeutic and preventive activities possibly due to their ability to modulate cell growth, differentiation, and apoptosis. These effects are mediated mainly by RA receptors, RARs and RXRs. Thus, this study examined RA action on human esophageal cancer (HEC) and RAR expression, and modulation to determine whether RA and RARs play a role in HEC carcinogenesis. Material/Methods 7 HEC cell lines and 157 surgical specimens were analyzed by growth inhibition assay, Northern blots, and in situ hybridization. Results RA inhibited the growth of 5 of 7 cell lines and also the constitutive expression of the squamous differentiation markers cytokeratin 1 and transgluta-minase I in all cell lines. All 7 cell lines expressed RAR-a and RAR-g at levels which were not changed by RA, whereas RAR-b was expressed in 5 out of 7 cell lines and up-regulated by RA in these 5 cell lines, which was associated with growth inhibition. Two cell lines that failed to express RAR-b showed no growth inhibition and no RAR-b inducibility. Interestingly, only these two cell lines were able to form colonies in soft agar. RAR-b mRNA was expressed in all adjacent and distant normal tissues, whereas its

expression was decreased to 54% (84/157) of HEC. Conclusion RAR-b expression is associated with the response of HEC cells to RA and that the loss of RAR-b expression may be associated with HEC development.

Session 2: Gastric carcinogenesis Helicobacter and gastric malignancy S2.01 Hereditary Aspects of Gastric Cancer Heinz Höfler2, Holger Vogelsang3 and Gisela Keller1 1lnstitute of Pathology of the Technical University Munich; 2lnstitute of Pathology, GSF-Forschungszentrum Neuherberg; 3Department of Surgery, Technical University Munich. Germline mutations in the cell adhesion molecule E-Cadherin have recently been described as the molecular basis of a familial gastric cancer syndrome. In addition, gastric cancer is observed significantly more frequently in some hereditary tumor syndromes, among which the HNPCC snydrome is the most important. An anaylsis of seven diffuse type gastric cancer patients with a variably strong family history of gastric cancer in our group, revealed a new germlime mutation in the E-cadherin gene in a patient with a strong familial clustering of gastric cancer. This underlines the importance of the E-cadherin gene for a genetic predisposition. In respect to HNPCC we identified microsatellite instability (MSI) at multiple loci in approximately 20% of gastric cancer patients with a positive family history of gastric or colorectal cancer. In the early onset group (diagnosis < 45 years) and an affected first degree relative, 2/6 patients showed MSI at multiple loci in the tumor, for which in one patient a germline mutation could be identified in the hMLH1gene. In contrast, we did not find MSI in the tumors of 12 patients under the age of 45 with a negative family history or having only a second degree relative, indicating that an early onset of the disease alone is rarely associated with an HNPCC syndrome. In summary, a genetic predisposition to gastric cancer may be associated with defects in various genes and it is likely that new genes will be identified responsible for the hereditary component in the pathogenesis of this disease. S2.02 HELICOBACTER PYLORI INFECTION ENHANCES GLANDULAR STOMACH CARCINOGENESIS IN MONGOLIAN GERBILS TREATED WITH MNNG. N. Shimizu, T. Oohara, M. Kaminishi, M. Tatematsu, Lab. of Pathol., Aichi Cancer Center Res. Inst., Aichi & Dept. of Gastrointestinal Surg., Univ. of Tokyo, Tokyo, Japan Aims: Helicobacter pylori (Hp) is thought to be a stomach carcinogen from epidemiological findings. To determine the effects of infection with the bacteria on experimental carcinogenesis, a study of the glandular stomach of Mongolian gerbils (MGs) was performed. Methods: Male MGs were treated with

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N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) followed by inoculation with Hp, or infected with Hp followed by MNNG administration. Animals were killed at week 50 and their excised stomachs underwent microbiological and histopathological examinations. Results: The incidences of adenocarcinomas were significantly higher in animals treated with 300 or 60 ppm MNNG for 10 weeks followed by Hp inoculation, or Hp followed by 20 ppm MNNG for 30 weeks than in the respective controls. Of interest was the finding that a dose of 100 ppm MNNG given to infected gerbils eradicated the Hp in about half the animals, with a concomitant reduction in the enhancing effect. No tumors were found in animals infected with Hp without MNNG or non-treated gerbils. Conclusions: Hp infection enhances glandular stomach carcinogenesis in MGs treated with MNNG. S2.03 The pathogenic marker inducible NO-synthase (iNOS) in Helicobacter pylori-associated gastric cancer G. Rieder1,2, J. A. Hofmann1,2, R. A. Hatz1, M. Stolte3, G. Enders2, and F. W. Schildberg1 1 Dept. of Surgery and 2Institute for Surgical Research, Klinikum Grosshadern, Munich, and 3Institute of Pathology, Klinikum Bayreuth, Bayreuth, Germany. Nitric oxide (NO) generated by inducible NO-synthase is part of the host defence mechanism against microbial infection. At high concentrations NO reacts cytotoxic and causes DNA damage. Therefore, the aim of our study was to investigate the iNOS expression in gastric biopsies of patients with H. pylori-associated gastritis and gastric carcinoma by RT-PCR. The results were correlated with pathogenic and histological markers of the biopsies analysed. iNOS-producing cells were detected immunohistochemically in gastric mucosa. Only one third of the gastritis biopsies of patients with H. pylori-associated gastritis showed an increased level of iNOS mRNA. There was no correlation with any of the analysed markers except intestinal metaplasia which revealed a 100% correlation with an increased iNOS transcription. Furthermore, the majority (80%) of gastritis samples taken from patients with gastric cancer of the intestinal type and less than 20% of the diffuse type showed an increased iNOS expression. We conclude that during chronic H. pylori infection constant NO production may enhance or even initiate together with other radicals the development of stomach cancer of the intestinal type by formation of the genotoxic molecule peroxynitrite. The expression of the enzyme iNOS may be an additional marker besides intestinal metaplasia suitable to assess the risk of developing stomach cancer of the intestinal type.

ogy, Klinikum Grosshadern, Marchioninistrasse 15, 81377 Munich, Germany, Phone xx49-89-7095-1, FAX -89-7004418 The uPA (urokinase-type plasminogen activator)-system (uPA, uPA-receptor, PAI-1) has been recently described as a new biological risk factor in gastric cancer. The uPA-receptor at tumor cell membranes, is the functional center of this highly efficient and dynamic protease system, which is cascadically modulated by other tumor-associated proteases and inhibitors. However, these interactions have never been considered in previous studies. In a consecutive prospective series of 203 gastric cancer patients, expression of uPA-activators (cathepsin D, antithrombin III), uPA-substrates (MMP-2, plasminogen), uPA-inhibitors (α-1-antitrypsin, α-2-antiplasmin, PAI-2) and factors protective to uPA-receptor (PAI-1, α-2-macroglobulin, α-1-antichymotrypsin) were studied immunohistochemically (semiquantitative score 0-3). Kaplan-Meier analysis (median follow-up 31 months) revealed univariate prognostic impact on survival of antitrypsin (p=0.0012), antichymotrypsin (p=0.0002), α-2-macroglobulin (p=0.0281) and cathepsin D (p=0.0042) besides the uPAsystem. In multivariate analysis considering all conventional risk factors in gastric cancer, cathepsin D was a new independent prognostic parameter for disease-free survival (p=0.020, rel. risk 2.98) besides PAI-1 for disease-free and overall survival (p=0.002, rel. risk 1.86; p=0.005, rel. risk 1.39). From these data stepwise survival analyses were performed in subgroups of high uPA, uPA-receptor, PAI-1 and cathepsin D expression according to high and low expression of two additional proteases each. The results revealed a combination of high levels of uPA-receptor, PAI-1, antichymotrypsin and α-2-macroglobulin as high risk protease pattern (23% survival, mean survival time 25.65 months), parameters known to be essential for uPA-receptor recirculation and -protection. These data indicate additional biologic prognostic parameters in gastric cancer (antichymotrypsin, antitrypsin, α-2-macroglobulin), with cathepsin D and PAI-1as independent factors. A protease pattern promoting uPA-receptor has been identified as a first high risk protease pattern, leading to individualized characterization of risk in single tumors. S2.05

S2.04

Improvement of gastric atrophy after eradication of Helicobacter Pylori (Hp) is associated with decreased expression for interleukin-1b converting enzyme (ICE). P. C. Konturek*, P. Pierzchalski*, A. Duda*, S. J. Konturek**, A. Rembiasz**, H. Steininger***, T. Kirchner***, E. G. Hahn*. *Dept. Med. I, ***Inst. Pathol., Univ. Erlangen-Nuremberg, Germany and **Inst. Physiol., Univ. Med. Sch. Cracow, Poland.

IDENTIFICATION OF HIGH RISK PATIENTS IN GASTRIC CANCER BY SPECIFIC TUMOR – ASSOCIATED PROTEASE PATTERNS Heiss Markus Maria MD, Allgayer Heike MD, Babic Rudolf MD, Gruetzner Klaus Uwe, Schildberg Friedrich Wilhelm MD. Ludwig Maximilians University. Dept. of Surgery and Pathol-

Chronic infection with Hp has been linked to gastric atrophy and intestinal-type gastric cancer. No information is available regarding the implication of apoptosis-related protein such as ICE in the development of this condition. The aim of the present study was to determine the effect of Hp eradication therapy combined with prolong administration of vitamin C on gastric

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mucosal atrophy and on mRNA and protein expression for ICE in the gastric mucosa of patients with atrophic gastritis. 20 patients with atrophic gastritis were enrolled in this study. Antral and corpus biopsies were taken for assessment of gastritis according to updated Sydney system and for analysis of expression for ICE and GAPDH by RT-PCR. The quantity of PCR products was evaluated by Southern blot using specific probes. Each RNA sample was assessed for cagA mRNA by RT-PCR. In addition, protein expression for ICE was analyzed by Western blot. All these parameters were determined before and 4 weeks and 3 months after eradication therapy combined with oral supplementation with vitamin C (1 g tid) for 3 months. The eradication rate was 95% and the cagA positivity reached 50%. The Hp therapy combined with vitamin C led to significant improvement of gastric atrophy (atrophy score before eradication: 2.12±0.15 vs. 1.0±0.23 after eradication) and other features of gastritis such as activity and grade. Before eradication both mRNA and protein expressions for ICE were about 3 folds stronger in antrum than in corpus. Following eradication therapy a significant decrease in ICE expression on mRNA and protein level was observed both in antrum and corpus. We conclude that the eradication therapy combined with vitamin C for 3 months results in an improvement of atrophic gastritis and is associated with decreased expression of apoptosis related proteins such as ICE. S2.06 Oxidative stress and expression of interleukin-8 and inducible synthase in the human stomach infected with H. pylori B. Pignatelli, C.-Q. Li, B. Bancel&, S. Toyokuni* and H. Ohshima IARC, &C. R. Hospital, Lyon, France; *Kyoto University, Japan H. pylori infection has been associated with stomach cancer but the mechanism by which H. pylori contributes to gastric carcinogenesis is largely unknown. NO and its derivatives produced in inflamed tissue couLd contribute to the neoplastic transformation. H. pylori strains possessing the cagA gene are thought to induce interleukin-8 (IL-8) and to be associated with gastroduodenal diseases. We studied the levels of expression of IL-8 and inducible NO synthase (iNOS) mRNAs in infected human gastric mucosa of 108 Chinese patients using RT-PCR in relation to the presence of the cagA gene and stomach pathology. H. pylori strains positive for cagA had a strong capacity to induce severe inflammation and co-expression of IL-8 and iNOS in gastritis patients. We also investigated the effects of H. pylori eradication on oxidative stress in 31 French patients by comparing stomach pathology and biomarkers of oxidative stress before and after treatment. Expression of iNOS and the presence of 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine were analysed immunohistochemically as markers for oxidative/ nitrosative DNA/tissue damage. Successful bacterial eradication resulted in: a) reduction in the inflammatory infiltrate, b) strong reduction in gastritis activity, c) disappearance or decreased expression of iNOS in inflammatory cells of antral biopsies, d) decrease in levels of 8-OHdG in deep foveolar cells of antral biopsies. Both studies suggest that oxidative

stress induced by certain H. pylori strains may play an important role in stomach pathology. S2.07 Lineage and clonal development of gastric glands. + S. Nomura,*,† M. Kaminishi,† H. Esumi,* C. Job, + and + S-S. Tan+ *Investivative Treatment Division, National Cancer Center Research Institute East, Kashiwa, Japan, † Gastrointestinal Depatment of Surgery, Faculty of Medicine, University of Tokyo, To+ kyo, Japan, + Developmental Biology Group, Howard Florey Institute, The University of Melbourne, Australia Aim: Individual gastric glands of the stomach are composed of cells of different phenotypes. These are derived from multipotent progenitor stem cells located at the isthmus region of the gland. Previous cell lineage analyses suggest that gastric glands, as in the colon and small intestine, are invariably monoclonal by adult stages. However, little is known about the ontogenetic progression of glandular clonality in the stomach. The aim of this study is to examine this issue. Material/Method: We employed an in situ cell lineage marker in female mice heterozygous for an X-linked transgene. Results: We found that stomach glands commence development as polyclonal units, but by adulthood (6 weeks), the majority progressed to monoclonal units. Hence, unlike the colon and small intestine, stomachs showed a significant fraction (Å10%) of polyclonal glands at adult stages. Conclusion: We suggest that these glands persist from polyclonal glands present in the embryonic stomach and hypothesize that they represent a subpopulation of glands with larger numbers of self-renewing stem cells.

Session 3: Liver carcinogenesis Hepatitis B and C hepatocellular cancer S3.01 Aetiology and pathogenesis for HCC Manns M.P. and Kubicka S. Department of Gastroenterology and Hepatology, Medical School Hannover. Germany Hepatocellular carcinoma is one of the main complications of liver cirrhosis with an annual incidence in liver cirrhosis of approximately 5%. Inherited metabolic diseases which result in hepatocyte injury as haemochromatosis, α-l-antitrypsin deficiency, glycogen storage disease, and tyrosinaemia are well known risk factors for the development of HCC Secondary risk factors for HCC among patients with chronic liver diseases are viral hepatitis B and C, male sex, age and cigarette smoking. Chronic viral hepatitis B and C accounts for the large majority of HCC around the world. Alcoholic cirrhosis may result in HCC, but it is not certain whether alcohol is an intrinsic carcinogen in hepatocarcinogenesis. In contrast, there are evidences that hepatitis B and C viruses are directly involved in the

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transformation of hepatocytes. The hepatitis B X gene transactivates myc, fos and TGFβ. HBX-transgenic mice frequently develop HCC. Besides the promiscuous gene transactivation, other X-mediated mechanisms have been described which may also contribute to hepatocyte transformation as kinase activity and inhibition of p53 nuclear translocation. The mechanism by which chronic HCV infection results in hepatocarcinogenesis remains unknown. Recently it has been shown that the hepatitis C core protein activates myc and induces HCC in transgenic mice. But also the non-structural protein NS3 is capable to transform cells in vitro. However in contrast to hepatitis B, which can induce HCC without liver cirrhosis in humans, the development ot HCV associated HCC is closely related to high graded liver cirrhosis. The molecular events leading to non viral associated hepatocellular carcinomas remains unknown. Several studies on human HCC suggest that overexpression of myc, fos, cyclin Dl, met, TGFα, IGFII and retinoid acid receptor and mutations in the tumour suppressor genes p53 and RB may play a role in hepatocarcinogenesis. Epidemiology and in vitro data revealed that the p53 mutation 249ser is specifically induced by aflatoxin B 1 in human HCC. However the stepwise molecular process of hepatocarcinogenesis remains to be established by histologic characterisation and molecular analysis of the nontransformed HCC-precursor lesions in the liver. S3.02 Apoptosis and liver cancer PR Galle First Department Internal Medicine, University of Mainz, Mainz / Germany The CD95 system is an important mediator of T-celI cytotoxicity and involved in hepatocytic injury. We investigated this system in 22 hepatocellular carcinomas (HCC) from patients. All HCCs had partially or completely lost the expression of the CD95 receptor constitutively expressed by normal liver cells and might thus evade CD95 mediated killing. Loss of CD95 correlated positively with mutation of p53. We also considered a new mechanism of immune evasion, namely, the active destruction of T-lymphocytes by tumor cells expressing CD95 ligand (CD95L). CD95L mRNA and protein could be detected in the HCCs. In coculture experiments, HepG2 hepatoblastoma cells, expressing CD95L mRNA after treatment with cytostatic drugs, killed CD95+ Jurkat lymphocytes. Our data suggest that tumor cells can evade immune attack by downregulation of the CD95 receptor and killing of lymphocytes through expression of CD95L. S3.03 Inflammation mediated carcinogenesis. Okio Hino Dept. of Experimental Pathology, Cancer Institute, Tokyo 1708455, Japan Persistent hepatitis B virus (HBV)/hepatitis C virus (HCV) infection is epidemiologically closely associated with the development of human hepatocellular carcinoma (HCC). Among

HBV/HCV carriers, HCC usually develops in patients with chronic liver disease, such as chronic active hepatitis or cirrhosis. The accumulation of mutations, which are likely to occur during continuous cycles of cell division, may eventually transform some hepatocytes through a multistage process. Hepatitis viral “inflammation mediated” hepatocarcinogenesis greatly influences the incidence of somatic genetic events in hepatocytes, by increasing the number of target cells, or the proliferation of once-hit hepatocytes, eventually leading to hepatocellular carcinomas (HCCs). These conditions may be designated as the “hypercarcinogenic state”. Our goal is to lead the “hypercarcinogenic state” to the “normo- or hypocarcinogenic” state and to prevent HCC development. S3.04 Activation of cellular gene expression by hepatitis B surface and X transactivators: impact on hepatocarcinogenesis Wolfgang H. Caselmann Department of General Internal Medicine, University of Bonn, Bonn, Germany Epidemiological and molecular data strongly suggest that the hepatitis B virus (HBV) carries considerable carcinogenic potential to induce hepatocellular carcinoma (HCC). In this regard integration of HBV DNA into the hepatocyte genome is considered a crucial event. Insertional mutagenesis can result in rearrangements of viral and cellular DNA and lead to altered gene expression by cis-mechanisms. More frequently in humans, viral X-protein (RBX) or large hepatitis B surface (LHBS) transactivators as well as the carboxyterminal truncated middle hepatitis B surface protein (MHBSt) are expressed and imbalance cellular gene expression in trans. Some truncted MHBSt proteins are retained in the endoplasmic reticulum and do not enter the secretory pathway anymore. Via different signalling cascades involving radical oxygen intermediates-, protein kinase C- dependent or mitogen-activated signal transduction pathways these transactivators induce expression of cellular genes through transcription factors such as NF-κB, API or SP l. Altered expression of genes relevant for proliferation, inflammation or malignant hepatocyte transformation may thereby contribute to the development of HBV-related HCC. HBV transactivator sequences are frequently (>80 %) detected in HCC tissue and in hepatoma cell lines, which emphasizes their pathogenic role. S3.05 Molecular mechanism of tumor promotion by testosterone during N-diethylnitros-amine/Nodularin induced hepatocarcino-genesis in Sprague-Dawley male rat. In Kyoung Lim1, Tae Jun Park1, In Hae Kwak1, Woon Ki Paik1, Kwang Wook Suh2 and Myung Wook Kim2. Department of Biochemistry & Molecular Biology1 and Department of Surgery2, Ajou University School of Medicine, Suwon, 442-749, Korea Purpose: Mechanism of impaired metabolism of testosterone (T) during carcinogenesis and changes of serum T were inves-

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tigated. Materials and Methods: A single i.p. injection of Ndiethylnitrosamine (DEN, 200 mg/kg) was employed in Sprague Dawley male rats as an initiator and nodularin (25 mg/kg, twice/wk) as a promoter for 2 wks from the 3rd wk after the initiation. Rats were sacrificed periodically and liver and blood were collected. Result: DEN treatment with or without nodularin significantly decreased the level of serum T, whereas the levels of lutenizing hormone and LDL-cholesterol markedly increased in serum. The DEN treatment significantly depressed T metabolism in liver microsome. Decreases of T content and the activities of side chain cleavage enzyme and 17ahydroxylase were also found in the testes of the animals treated with DEN with or without nodularin. However, daily supplement of T (5 mg/kg body weight) to the orchiectomized rats for 2 wks restored the serum T and lutenizing hormone levels to the control level even with DEN/nodularin injections. The above results led us to conclude that the reduction of serum T was not due to an increased metabolism of T in liver but rather due to inhibition of T biosynthesis in testes. Employing intact or orchiectomized male rats, effect of T on the expression of Bcl2 family genes were investigated. Nodularin injections after the initiation significantly induced Bax expression in both animals. Expressions of Bcl2 and Bcl-xL were reciprocally varied in both rats; Bcl2 decreased significantly by the treatment of DEN/nodularin, whereas expression of Bcl-xL did not show any change in the intact rats. In contrast, orchiectomy alone reduced Bcl-xL expression and the DEN/nodular in treatment accelerated reduction of Bcl-xL, as opposed to no significant change of Bcl2. Conclusion: These results indicate that decrease of T during carcinogenes is increase apoptotic reactions through the acceleration of BclxL inactivation in the DEN/nodularin treated male rats. S3.06 Genome Analysis of Hepatocellular Carcinoma with Expressed Sequence Tags and GenChip Analysis Stephan Kaiser1 , A. M. Keck1 , M. Gregor1 and Jung-Joo Hwang2 Dept. of Medicine, University of Tübingen, Germany1 and Gene Therapy Laboratories, University of Southern California School of Medicine, Los Angeles, USA2 Modern tools of genome analysis such as Expressed Sequence Tags (EST) analysis and High Throughput Microarray GenChip analysis provide the basis of a computerized genomic screening of biological samples within extremely short time frames. We used these two novel methods to identify over- and underexpressed genes in samples of human hepatocellular carcinoma (HCC). An EST analysis (HumanGenomeSciences, Inc.) was perfomed with RNA pooled from 5 different HCC samples. A total of 19.000 EST clones were generated. The analysis showed that a total of 609 sequences were differentially expressed in HCC tissue. 22 of exclusively in HCC expressed sequences were corresponding to sequences identified by DD-RT-PCR analysis in different HCC samples. To prove that these data were representative we conducted a GenChip analysis (Affymetrix, Inc.) on 12 HCC samples using Microarrays with oligomers from 8.600 cDNA sequences based on data from GenBank and TIGR data

bases. The data show that specific gene families and groups of genes are either under- or overexpressed in HCC. Furthermore, specific genes could be identified, which are expressed exclusively in HCC or normal liver tissue. Interestingly, the two different methods ( EST and GenChip analysis) led to the identification of the same over- or exclusively expressed genes in tumor tissue. The identified sequences will be used to conjunction with a proteome analysis to create novel HCC – specific markers. S3.07 Expression of endothelial adhesion molecules on primary liver tumors M. K. Frerker1, P. Flemming2, H. Aselmann1, K. J. Oldhafer1 1 Abdom.- and Transplantation Surgery, 2Institute of Pathology, Hannover Medical School, Germany Aim: Immune adhesion molecules play important roles for various cell interactions. We investigated expression patterns of vascular adhesion molecules on primary liver tumors with regards to their importance for pathogenesis and progression of those tumors as well as anti-tumor defense mechanisms. Patients/ Methods: Specimens were intraoperatively obtained from 44 patients with hepato- (HCC) and 19 patients with cholangio-cellular carcinomas (CCC) and immunohistochemically stained with monoclonal antibodies against ICAM2, PECAM1, CD34 and VCAM1. Results: The vascular adhesion molecules ICAM2, PECAM1 and CD34 showed constitutive endothelial expression and universal non-expression on tumor cells. VCAM1, however, was also expressed on tumor cells in 12/44 HCCs (3 G3, 6 G2, 1 G1, 2 Gx) and 2/19 CCCs (G2). All 3 patients with simultaneous hepatitis C induced liver cirrhosis expressed VCAM1 on tumor cells. Tumor-free specimens from patients with serologically/ histologically confirmed hepatitis, however, showed no VCAM-1 expression on hepatocytes. Conclusion: Expression patterns of adhesion molecules are related to concomitant diseases. Cytokines and gene transfer may represent potential tools for new immune therapies, e.g. using anti-vascular antibodies such as anti-CD31 and antiCD34 to prevent metastasis, inhibit angiogenesis and vanquish chemotherapy resistance.

Session 4: Pancreatic carcinogenesis From chronic pancreatitis to ductal pancreatic cancer S4.01 PERSISTENT OXIDATIVE DNA DAMAGE AS A DRIVING FORCE IN PANCREAS AND COLON CARCINOGENESIS H. Bartsch and J. Nair German Cancer Research Center (DKFZ), Heidelberg, Germany Lipid peroxidation (LPO) is thought to be involved in promotion and progression stages of carcinogenesis. We determined

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exocyclic etheno-DNA adducts (1,N6-ethenodeoxyadenosine: edA and 3,N4-ethenodeoxycytidine: edC) in human chronic pancreatitis (CP) and familial adenomatous polyposis (FAP) (in collaboration with H. G. Beger, University of Ulm, and G. Winde, University of Münster). These adducts are formed as a consequence of oxidative stress and LPO. Using an immunoaffinity-32P-postlabelling method, edA was found to be ~ 3.2 and edC ~ 28 times higher in DNA of CP than in normal pancreas DNA. The mean edA and edC levels in FAP versus normal colon mucosa were ~ 2- to 3-fold elevated. Our results show for the first time that etheno-DNA adducts are greatly increased in pancreatic cells and colonic epithelium of CP and FAP patients, suggesting that these adducts are formed in the target cells that may develop to tumors, as a consequence of oxidative stress and LPO. Etheno-DNA adducts are promutagenic, and their formation will thus contribute to increased genetic instability in the target cells in CP and FAP. This hypothesis is being investigated in other human tissues and experimental models. S4.03 INHIBITION OF THE GROWIH OF HUMAN PANCREATIC CANCER CELLS BY TRAIL P. M. Pour, H. Matsuzaki, B. Schmied The Eppley Institute for Research in Cancer Univ of Nebraska Med Center, Omaha, NE Introduction: Members of the TNF family mediate cell apoptosis. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a new member of the TNF family. TRAIL has been known to be most effective in lymphoma cells with variable effects on solid tumors. We tested the effect of TRIAL in human pancreatic cancer cells in vitro. Methods: Eight human pancreatic cancer cell lines (HPAF, Panc1, ASPC1, Miapaca2, Capan2, BXPC3, Panc89, SW979) were cultured and treated with TRAIL (8 ng/ml to 300 ng/ml). We examined cell cycle and the rate of apoptosis by FACS (TUNEL method) and internucleosomal DNA fragmentation. Results: All of the eight pancreatic cancer cell lines showed retarded cell growth and a significant dose dependent increase in apoptosis. The apoptotic rate was as follows. Capan2 (12.2%), HPAF (39.9%), Miapaca2 (21.8%), Panc1 (14.0%), ASPC1 (6.8%), SW979 (53.9%), BXPC3 (73.1%) and Panc89 (11.6%). Interestingly, the aneuploid cells were most affected by apoptosis. Conclusion: TRIAL caused apoptosis and reduced cell growth in eight human pancreatic cancer cell lines. However, the rate of apoptosis differed among the cell lines. BXPC3 was the most responsive and ASPC I was least responsive. S4.04 Inhibition of cyclin D1 in pancreatic cancer cells increases chemosensitivity and decreases expression of multiple chemoresistance genes M. Kornmann, K. D. Danenberg1, H. G. Beger2, P. V. Danenberg1 and M. Korc. Dept. of Medicine, University of California, Irvine, CA 92697; 1 Dept. of Biochemistry, University of Southern California

School of Medicine, Los Angeles, CA 90033; and 2Dept. of General Surgery, University of Ulm, 89075 Ulm, Germany Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. We have previously demonstrated that increased cyclin D1 levels correlate with decreased survival in human pancreatic cancer, whereas inhibition of cyclin D1 expression suppresses basal and mitogenstimulated pancreatic cancer cell growth. Moreover, cisplatin toxicity was increased by cyclin D1 antisense expression. The aim of the present study was to determine whether suppression of cyclin D1 modulates the effects of other antineoplastic drugs, and whether it is associated with alterations in the expression levels of drug resistance genes. Suppression of cyclin D1 expression following stable transfection of a cyclin D1 antisense construct in PANC-1 and COLO-357 human pancreatic cancer cells resulted in a significant increase in sensitivity to 5-fluorouracil (5-FU), 5-fluoro-2’-deoxyuridine (5-FUdR), mitoxantrone, and carboplatin. This increased sensitivity in all the antisense expressing clones was associated with a decrease in thymidylate synthase (TS) and an increase in thymidine phosphorylase (TP) expression as determined by a real-time fluorescence PCR technique. High TS and low TP levels have been shown to correlate with resistance to the antimetabolites 5-FU and 5FUdR. Cyclin D1 suppression also decreased the mRNA expression of the multi-drug resistance 1 (MDR1) P-glycoprotein and the multi-drug resistance protein (MRP) as determined by Northern blotting. High expression of MDR1 has been associated with resistance to the anthracenedione mitoxantrone and also to cross-linking agents like cisplatin and carboplatin. These findings demonstrate that inhibition of cyclin D1, besides suppressing pancreatic cancer cell growth, enhances chemosensitivity to several agents and suggest that cyclin D1 may regulate the expression of multiple genes that have been implicated in chemoresistance. S4.05 Cooperative effects of TGF-a and loss of p53 in pancreatic carcinoma susceptibility in vivo M. Wagner#, W. Deppert§, V. Stagge#, H. Kern$, G. Adler#, R. M. Schmid# # Dept. of Internal Medicine I, University of Ulm, §Heinrich Pette Institution for Experimental Virology and Immunology, University of Hamburg, $Dept. for Cytobiology and Cytopathology, University of Marburg Introduction: We have previously shown an acinar-ductal-carcinoma-sequence in the pancreas of TGF-a transgenic mice (Wagner et al., Gastroenterology, 115, 1998). Nuclear staining of p53 is markly increased in tubular complexes as well as in the papillary to cystic tumors in these animals suggesting a role of p53 in the tumor progression. Methods: TGF-aTG+xp53-/-+ and TGF-aTG+xp53-/- mice were generated by crossbreeding. Results: While TGF-a transgenic mice developed tumors only at the age of more than 365 days (n=19), the tumor free interval was reduced to 211±80 days in TGF-aTG+xp53-/+ (n=35) and to 80±20 days in TGF-aTG+xp53-/-+ (n=10) mice. Necropsy of TGF-aTG+xp53-/- and TGF-aTG+xp53-/+mice revealed ascitic fluid and large pancreatic mass indicative of advanced lo-

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cal disease. In the morphological examination papillary to cystic tumors originating from tubular complexes as well as diffuse infiltrating, anaplastic tumors were detected. Some tumors showed an infiltration and destruction of the duodenal wall as well as liver metastasis. Discussion: In the present study we report pancreatic tumor development in TGF-aTG+xp53-/- and TGF-aTG+xp53+/- mice. S4.06 RelA is constitutively activated in pancreatic adenocarcinomas PJ Chiao, JL Abbruzzese, K Cleary, L Larry and DB Evan The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 USA Aims: Recent studies have revealed that oncogenesis mediated by Ras may also involve activation of Rel/NF-kB. The c-rel member of Rel/NF-kB transcription factor family was first identified as a cellular homologue of the v-rel oncogene, suggesting that other members of the Rel/NF-kB family are potentially oncogenes. We therefore investigated the possibility that Rel/NF-kB is activated in pancreatic cancer. Material/Methods: Immunohistochemical analysis, western blot and northern blot analysis, electrophoretic mobility shift assays, and reporter gene assays were performed using normal and tumor pancreatic tissues and cell lines. Results: We show that RelA, the p65 subunit of NF-kB, was constitutively activated in ~67% (16 of 24) of pancreatic adenocarcinomas, but not in normal pancreatic tissues. Constitutive RelA activity was also detected in 9 of 11 human pancreatic tumor cell lines, but not in nontumorigenic Syrian golden hamster cell lines. IkBa, a previously identified NF-kB-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines, and RelA activation could be inhibited by curcumin and dominant negative mutants of IkBa, raf, and MEKK1. Conclusion: These data are consistent with the possibility that RelA is constitutively activated by the upstream signaling pathways involving Ras and MAP kinases. This is the first report demonstrating constitutive activation of RelA may play a key role in pancreatic tumorigenesis through induction of its target genes. S4.07 Expression or VEGF and its receptor in pancreatic cancer J. Itakura1, Y. Matsumoto1 and M. Korc2 1 Dept. of Surg., Yamanashi Medical University, Yamanashi, Japan; 2 Dept. of Medicine, University of California, Irvine, USA Vascular endothelial growth factor (VEGF) is an angiogenic polypeptide that has been implicated in cancer growth. In the present study we characterized the expression of VEGF and its receptor (KDR and flt-1) including co-receptor neuropilin-1 in pancreatic cancer tissues and the possibility of autocrine interaction of these ligand and receptor in pancreatic cancer cell line. Northern blot analysis of total RNA isolated from normal and cancerous pancreatic tissues revealed that VEGF and its receptor KDR, flt-1 and neuropilin-1 were overexpressed in pan-

creatic cancer tissues. Immunohistochemical study indicated the co-localization of VEGF, KDR and flt-1 in pancreatic cancer cells in human tissue. Analysis of 75 pancreatic cancer tissues revealed the presence of VEGF immunoreactivity in the cancer cells in the 64% of the cancer tissues. The presence of VEGF in the cancer cells correlated with increased tumor size and extension to the adjacent tissues. In Capan-1 human pancreatic cancer cells VEGF stimulated the growth of this cells in a dose dependent manner, and enhanced tyrosin phosphorylation of KDR, mitogen activated protein kinase family (MAPK, p38 and SAPK/c-JNK) and c-fos mRNA levels. PD98059, a MAPK inhibitor, blocked VEGF mediated MAPK activation and growth stimulation. These data suggest that in addition to enhancing pancreatic cancer growth by promoting angiogenesis VEGF may act directly on the cancer cells through autocrine and paracrine mechanisms and that anti-VEGF therapy may have a dual benefit in the therapy of this disorder. S4.08 Expressional and functional loss of Fas/CD95 in pancreatic cancer: tumour escape and immuno-protective approaches Wv Bernstorff, RA Spanjaard, AK Chan, JN Glickman, RD Odze, H Kalthoff, PS Goedegebuure, TJ Eberlein Harvard Medical School, Boston and Washington University, St. Louis, USA; Christian-Albrechts-Universität, Kiel, Germany Aim: 1. To investigate the possibility of a Fas tumour escape mechanism in pancreatic cancer cells. 2. To test immuno-protective/-therapeutic approaches against Fas ligand (FasL) expressing pancreatic cancer cells. Methods and Results: Immunohistochemically, 8/12 pancreatic cancer specimens showed a lower Fas staining intensity in invasive tumour than in benign pancreas. 6/12 specimens expressed Fas heterogeneously, particularly with loss of Fas staining in less differentiated areas. Fas was expressed by all investigated 14 pancreatic cancer cell lines. Fas apoptosis could be induced in only 1/14 cell lines when cells were cultured as monolayer, whereas 4/14 cell lines underwent Fas induced apoptosis when grown in suspension, thereby mimicking disseminating cells. Cytotoxic T lymphocytes (CTL) cultured in IL-2 were resistant to Fas apoptosis and could effectively kill the FasL expressing pancreatic cancer cell line CFPAC-1. Conclusions: Pancreatic cancer cells can evade immune surveillance by downregulation/loss of Fas expression or by blocking Fas-mediated apoptosis. IL-2 effectively protects activated CTL from an attack of FasL expressing pancreatic cancer cells. Future pancreatic cancer therapies should therefore aim at the restoration of Fas function, in particular with regard to disseminating cells, supported by immuno-protective substances such as IL-2.

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S4.09

S4.11

TGFa: and GSH in the pancreatic carcinoma cell line AsPC-l A. Nussler, T. Schnelldorfer, S. Gansauge, F. Gansauge, H. Beger Dept. of General Surgery, University of Ulm, Ulm,Germany

Malignant transformation of immortal pancreatic duct cells by transfection of mutated ki-ras. R. Jesnowski, S. Liebe, M. Löhr Dept. of Medicine, Univ. of Rostock, Rostock, Germany

Aim: Recent evidence suggest that mitogen such as TGF-α and glutathione (GSH) are crucial for cell proliferation of various tumors and may play an certain role against oxidative injury as demonstrated by an enhanced GSH synthesis of EGF-treated human breast cancer cells. Therefore,we investigated the link between mitogens (TGF-α) and GSH via the cell cycle in the human adenocarcinoma cell line AsPC-1. Methods: Cells were incubated in the presence or absence of the GSH depleting agent BSO and exposed either to a single or a continuous TGF-α: (25 ng/ml) dose. Then, cell cycle analysis and proliferation tests were performed, followed by GSH determination during G1/G0, G1/GO to S-phase, and G2/M-phase of cell cycle. In addition, we measured GSH levels in human pancreatic adenocarcinomas and normal pancreatic tissue. Results: We found that basic GSH levels were high in AsPC-l cells (median: 50.7 nmol/mg prot.) and were not further significantly increased in the presence of TGF-α-mediated cell proliferation. In the absence of TGF-α: or GSH cell proliferation was unchanged. A crucial role for GSH was confirmed by elevated levels in pancreatic carcinoma as compaired with normal pancreatic tissue (16.1 ± 8.5 vs. 8.5 ± 4.2 nmol/mg prot.; p<0.007). Our results show that TGF-α and GSH are necessary for the proliferation of AsPC-l carcinoma cells. Conclusion: Beside the role of GSH in the cell cycle, it may protect tumors against cytotoxicity, radiotherapy or chemotherapy-mediated radical injury that may explain resistance against adjuvant therapies of pancreatic cancers.

Introduction Although pancreatic adenocarcinoma meanwhile has become one of the best characterized malignant diseases, still severe diagnostic and therapeutic problems are associated with this disease. The establishment of a molecular model of pancreatic carcinogenesis may improve the prognosis of the malignancy. Material and Methods In order to mimic one of the first steps in pancreatic carcinogenesis SV40 immortalized bovine pancreatic duct cells established previously by our group additionally were transfected with a plasmid coding for a mutated ki-ras gene. Resulting clones were injected orthotopically into athymic nude mice to assay the ability of tumor induction. Results We succeeded in the establishment of an additionally ki-ras transfected bovine clone. The stable uptake of the transfected DNA was verified by restriction enzyme mismatch PCR. In contrast to the only SV40 large T immortalized PDEC the additional ki-ras transfected cells did induce tumor growth when injected orthotopically into athymic nude mice. Conclusion The additional transfection of a mutated ki-ras into the SV40 immortalized cells resulted in an induction of tumor growth in nude mice. Thus the combined effects of SV40 large T (funct. inactivation of p53) and of mutated ki-ras (const. activation of map kinase pathway) results in the transformation to a malignant cell, an effect yet described for other cells.

Session 5: Colorectal carcinogenesis Hereditary cancer syndromes

S4.10 S5.01 Dietary Modulation of TSNA-Induced Panercas Carcinogenesis in Rats. B. Prokopzcyk, E. L. Wynder, A Rivenson, S. Amin, and D.Hoffmann. American Health Foundation. Valhalla, N.Y. 10595. U.S.A Tobacco-specific N-nitrosamines are formed from nicotine and minor alkaloids during the processing and smoking of tobacco products. Four of seven TSNA have been identified as carcinogens in mice, rats, and hamsters. The smoke of one cigarette contains 1.3 mg and that of a cigar between 0.5 and 7 mg TSNA; levels in indoor air have measured as much as 0.05 mg/m3 The nicotine-derived NNK and NNAL are the only known environmental pancreas carcinogens per se. Fifty-two percent of F-344 rats who were given 2 mg NNK/L drinking water and maintained on a high-fat diet (23.5% corn oil) developed endocrine and exocrine pancreas tumors; among the rats receiving the same dose of NNK but a low-fat diet (5% corn oil), only 34 percent were found to have pancreas tumors. Tumor histology and and history of COX-2 expression at advanced stages of tumorigenesis will be presented. In addition, the finding of TSNA in pancreatic fluid of smokers will be discussed. Supported by grant No. ROl CA 76328-01A1 from the U.S. National Cancer Institute, Bethesda, MD.

DE NOVO METHYLATION OF CpG ISLANDS IN HUMAN CANCER P. A. Jones, M. Gonzalgo, C. Bender, C. Salem, M. Tsutsumi, G. Liang USC/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Los Angeles, California 90033 Mammalian genomes are strongly suppressed for the methyl acceptor site CpG, however, CpG islands have been protected from this loss, presumably because they are not methylated in the germline. CpG islands are protected from methylation in somatic cells with the exceptions of genes located on the inactive X chromosome in female mammals, imprinted genes and parasitic DNA sequences where methylation in the relevant promoters is thought to be part of a mechanism for promoter suppression. CpG islands can occur both upstream and downstream of the transcriptional initiation site yet methylation of downstream islands does not block transcription elongation and is often associated with expression rather than repression in both normal cells and cancer. We have developed two new techniques to assess the methylation changes which are associated with carcinogenesis. The first is a genome scanning technique called methylation sensitive arbitrarily primed PCR (AP-PCR)

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which has allowed us to isolate 20 CpG islands commonly methylated in cancer. Interestingly, these islands are located both in the promoters and coding regions of genes including the endothelin B receptor gene and the exon 5 region of PAX6. We have also developed a quantitative bisulfite-based method (MsSNuPE) to assess the level of methylation at CpG sites identified by the AP-PCR approach. The coupling of these two techniques has allowed us to investigate the kinetics of de novo methylation of CpG sites relevant to carcinogenesis. S5.02 Clinical consequences of predictive molecular testing for hereditary colorectal cancer syndromes Hans K. Schackert, Matthias Hahn, Steffen Pistorius1, H. D. Saeger1 Department of Surgical Research and 1 Department of Surgery, University of Technology Dresden, Germany Aim: Hereditary nonpolyposis colorectal cancer (HNPCC) and adenomatous polyposis coli (APC) are autosomal dominantly inherited cancer susceptibility syndromes with high penetrance. Cloning of genes responsible for the syndromes allows for identification of mutation carriers within affected families. Predictive molecular diagnostics has considerable impact on clinical decision making concerning clinical surveillance and preventive surgery. Material/Methods: Registration of families with hereditary colorectal cancer was performed since 1995 at the Department of Surgical Research and the Department of Surgery, University of Technology Dresden. Molecular analysis of mismatch repair genes was performed in index patients of families fulfilling specific criteria for microsatellite instability and Amsterdam and Bethesda citeria. Results: We report results of sequence analysis of hMSH2, hMLH1 and hMSH6 mismatch repair genes in HNPCC and HNPCC-like families. In addition, microsatellite analysis data are presented. All patients undergoing predictive genetic testing were counseled by geneticists and surgeons. Colorectal cancer risk in mutation carriers is high, wheras the risk of non-mutation carriers is comparable to the risk of the general population. Therefore, non-carriers of the individual mutation of the family were excluded from the specific HNPCC surveillance program. Mutation carriers were encouraged to adhere to the specific and strict HNPCC surveillance program. We report the case of one patient who fulfilled the prerequisites for preventive surgery and underwent preventive subtotal colectomy, hysterectomy and oophorectomy. Conclusions: Clinical implications of predictive molecular testing should be discussed on an individual basis, with special consideration of family history, compliance with the surveillance program and the risk for cancer. Evaluation of the value of prophylactic surgery versus surveillance-based procedures must be the aim of future clinical studies.

S5.03 Diagnostic strategies in patients with hereditary colorectal cancer T. Sauerbruch Department of Internal Medicine, University of Bonn, Germany Less than 5 % of colorectal cancers (CRC) are hereditary, namely familiar adenomatosis polyposis (FAP) caused by a germline mutation of the adenomatous polyposis coli (APC) gene or hereditary nonpolyposis colorectal cancer (HNPCC) caused by mutations in at least five missmatch repair genes. Patients with CRC diagnosed before age 40 are suspicious of having HNPCC. A good family history is decisive. Tumors of these patients should be tested for microsatellite instability (MSI) which is always positive in case of HNPCC. MSI positive patients should undergo genetic testing. Mutations of typical genes, however, are only detected in about 30–40 %. In case of a known mutation in the index person further screening is only indicated in gene cariers. In case of unknown mutation colonoscopic screening should be performed from 25 yrs onwards in the descendants. In clinical manifestation of FAP search for APC mutation should be performed to define gene cariers in the familiy. These have to be screened from 10 yrs onwards. S5.04 Microsatellite instability in colorectal and gastric cancer F. Kullmann°, W. Dietmaier*, J. Rüschoff*, J. Schölmerich° Depts. of °Internal Medicine I and *Pathology, Univ. Regensburg Aim: To determine the incidence of microsatellite instability (MSI) in colorectal and gastric cancer and to relate these results to tumor type, especially growth pattern, stage and site. Methods: MSI-status was determined in 210 primary colorectal (CRC) and 41 primary gastric adenocarcinoma (GC) using 10 microsatellite markers that comprised the best rate of definition and reliability in diagnosing MSI, as we have recently shown. Tumors were defined as highly unstable if at least 40% of the analyzed loci showed instability, whereas <20% instability was classified as „low MSI“ and 0% as stable (MSS). Results: Twenty five patients (12%) with CRC showed high MSI, 28 (13%) had low MSI only and 157 CRC (75%) were stable. The clinicopathological variables right-sided location, tumor type, presence of Crohn’s like lesions and better prognosis were significantly (p<0.001, p<0.025 for prognosis) different between high MSI and MSS tumors. No relevant differences were found between low MSI and MSS tumors. Five patients (12%) with GC showed high MSI, 4 (10%) had low MSI and 32 GCs (78%) were stable. The tumors with solid growth pattern showed a higher frequency of MSI (7/21=33% vs. 2/20 =10%, p=0.13). We observed no strong association between MSI and histology (Laurén), TNM-classification, differentiation or mucin production of the tumor. Conclusion: The high MSI-CRC is characterized by a distinct clinicopathological phenotype. Low MSI and MSS-CRC are phenotypic identical. The relevance of the low MSI-status as a molecular and clinical entity is low. The incidence of MSI in gastric carcinoma is comparable with that of colorectal cancer, especially consider-

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ing the high MSI-status. High MSI was associated with younger age of the patients, early tumor stage and occurrence at the proximal third of the stomach. Most interestingly, MSI tends to occur preferably in gastric tumors of solid/medullary type, as already observed in primary colonic adenocarcinoma with solid growth pattern. S5.05 De novo Colorectal Carcinoma and the Adenoma- Carcinoma Carcinogenetic Sequence J. Mueller 1, N. Haegele 2, G. Keller 2, M. Stolte 3, H. Höfler 2 1 Department of Surgery, Technische Universität München; 2 Institute of Pathology, Technische Universität München; 3 Institute of Pathology, Klinikum Bayreuth Aim: The term “de novo” carcinoma has been given to small colorectal carcinomas without evidence of an adenoma in their vicinity. This name implies an alternate carcinogenesis. We tested this concept using comparative immunohistochemistry (IHC) and loss of heterozygosity (LOH) analysis. Material and Methods: Two groups of early (UICC pT1) de novo (n = 35) and ex-adenoma (n = 36) colorectal carcinomas were compared for their rates of overexpression of p53 by IHC and for their rates of LOH at eight different loci (including the APC, DCC and p53 loci) important in the adenoma-carcinoma sequence. Results: By immunohistochemistry, 23 of 35 de novo and 15 of 36 ex adenoma overexpressed p53, a significantly higher rate in the de novo type (p = 0.04). Except for the 17p (p53) locus (de novo - 73% vs. ex-adenoma - 37%, p= 0.004), LOH analysis showed similar rates of LOH for the two groups at all loci including the 5q (APC) and 18q (DCC) loci. Conclusion: The similar rate of LOH at APC and DCC implies that so-called colorectal “de novo” carcinoma develops via the adenoma-carcinoma sequence, but that accelerated p53 abnormalities may contribute to their distinctive morphology by promoting early destruction of precursor adenoma elements. S5.06 Overexpression of cyclin E is an early event in Human colon carcinogenesis T. Sutter, T. Dansranjavin, K. Besler, H. Dralle Dept. of Surgery, Martin-Luther-University, Halle, Germany Aim: Dysregulation of the cell cycle machinery is a critical event in human carcinogenesis. Studying various cyclins, that promote G1-phase transition in colon cancer cell lines, we observed markedly high expression of cyclin E protein in a subset of these cells. In order to investigate whether cyclin E expression could be correlated to tumor progression, we further examined cyclin E in colon tumors of various stages. Methods: Thirty-eight primary colorectal tumors of stage Dukes A (n=8), B (n=8), C (n=13) and D (n=7), adjacent normal colon mucosa and recurrent rectal tumors (n=2) were examined for cyclin E protein expression in 37 patients (m/f=20/17, age: 44-83 yrs., mean: 66 yrs.). Data obtained from

western blot analysis were quantitated by 2-D laser densitometry. Results: All samples of normal colon mucosa displayed low cyclin E protein expression. Classified into stages, an 3- to 10fold cyclin E expression was detected in 7 out of 8 Dukes A tumors. In contrast, 8 of 21 Dukes B and C and only one out of 7 Dukes D carcinomas displayed an increase of cyclin E protein. Recurrent rectal cancer did not show altered cyclin E expression when compared to normal colon mucosa. Conclusion: Increased cyclin E expression is one of the early steps in colon carcinogenesis and cyclin E expression is inversely related to progression of colorectal tumors. S5.07 TUMOR ANGIOGENESIS IN PRIMARY COLON ADENOCARCINOMAS S SÖKMEN *S SARIOGLU M FÜZÜN C TERZY´ *AA KÜPELY´OGLU ´zmir, Dokuz Eylül Univ. Depts. of * Surgery & **Pathology, Y Turkey In order to evaluate the role of angiogenesis as a prognostic factor in a series of 64 colon adenocarcinoma cases, four different angiogenic, morphometric values, other well-known clinicopathologic variables and prognosis were estimated. Tumoural tissue sections were stained with monoclonal antibody against Factor VIII by immunohistochemistry. Vascular surface density (VSD), number of vessels per square mm (nVES) and mean number of nVES according to 3 maximum values (nVESmax) and number of vessels in unit area (N) were determined. Paired samples t-test and Kruskal-Wallis one way ANOVA were used for suitable variables. There was a significant correlation between colonic tumour size and angiogenesis for VSD (p=.002), nVES (p=.001) and nVESmax (p=.001) except N(p=.063). However, no significant correlation was detected between angiogenic parameters and tumour stage, metastasis, recurrence, survival, mode of surgical technique, adjuvant treatment and other variables. Our results suggest that angiogenesis, which is evaluated by different morphometric methods cannot predict the important clinical outcomes in this relatively large colon carcinoma series.

Workshop: Molecular factors of prognosis in GI-tract cancer W5 Molecular prognostic factors in pancreatic cancer JP Neoptolemos, P. Ghaneh, J. Evans, A Kawesha, C. Foster Departments of Surgery and Pathology, University of Liverpool, 5th Floor UCD Block, Duncan Building, Liverpool L69 3GA, U. K. Molecular studies have identified geneic alterations which may be important in the prognosis of pancreatic cancer. Overexpres-

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sion of growth factors (EGF, TGFα, TGFβ 1–3, FGFs) and receptors (ERBB2-4, TGFβ 1-3) is common. High levels of mutations are found in genes which control the cell cycle such as p53, p16, p21, DPC4, cyclin D1, and of apoptosis (bcl-2, bclXL and bax). Studies including our own have shown no significant correlation between overexpression of growth factors and their receptors and survival with the exception of TGFβ. p16, p53 and p21 havc not been found to be of any prognostic significance. In one of two studies, overexpression of cyclin D1 was associated with shorter patient survival. Bax expression has been shown to prcdict survival in one study, but was not confirmed in our own. There is no relationship between bcl-2 expression and survival. Two studies (including our own) have confirmed bcl-XL expression to be significantly associated with poor outcome. Different types of KRAS mutation are associated with survival. These results confirm the potential use of molecular markers to predict the outcome of patients with pancreatic cancer.

Session 6: Virus infection and carcinogenesis/ Tumor immunology S6.01 Immune response to gastrointestinal cancer F. Momburg Division of Molecular Immunology, Tumor Immunology Program, Deutsches Krebsforschungszentrum, Heidelberg, Germany Positive responses of even advanced gastrointestinal carcinomas to adjuvant locoregional therapy with immunostimulating lymphokines, IL-2 and IFN-γ, suggest a role for immune mechanisms to control tumor growth in at least a subset of patients. Antigen-specific immunotherapy of gastric, pancreatic or colorectal malignancies is still in the early experimental stage. Although gastrointestinal tumors are notorious for poor immunogenicity, a number of fairly tumor-specific antigens are potential targets for an antigen-specific cellular or humoral immune response. Induction of cytotoxic T cells recognizing the frequently found tumor antigens of the MAGE family, carcinoembryonic antigen (CEA), MUC-1 mucus protein, or overexpressed or mutant oncogene products such as p53, Ki-ras, or HER2/neu seem to be feasible goals. Peptide- or protein-pulsed dendritic cells appear to be the most efficient inducers of MHC class I-restricted CTL. Tumor-derived lymphokines such as IL10 or TGF-β may, however, limit the success of tumor-reactive CTL in situ. Eliciting antibodies in patients to CEA, MUC-1, etc. may have additional beneficial effects. Adoptive immunotherapy strategies include the expansion of tumor-infiltrating CTL, or the infusion of in vitro expanded natural killer or lymphokine-activated T cells. Antibody-based passive approaches involve bispecific monoclonal antibodies to facilitate T cell targeting to the tumor site, radiolabeled antibodies, antibody-cytokine fusion products or toxin immunoconjugates. Xenotransplant mouse models of gastrointestinal tumors will be reviewed.

S6.04 Induction of a cellular immune response against autologous tumor cells of gastrointestinal carcinoma by Interleukin 12: “Intrinsic immunological potential” in curatively resected tumor patients M. A. Ströhlein, K. U. Grützner, F. W. Schildberg, M. M. Heiss Dept. of Surgery, Klinikum Grosshadern, LMU Munich, Germany Immunological mechanisms against disseminated tumor cells have been shown to be critical for tumor recurrence and prognosis. IL-12 is a central cytokine between immunological recognition and effector phase by activating tumor-primed T cells. We tried to induce an immune response against autologous tumor cells (auTu) by IL-12 in order to examine “intrinsic potential” in curatively resected tumor patients. From 14 patients with gastrointesinal carcinoma PBMC were incubated without stimulation, with 100 I. U. IL-12/ml, and c) with 1000 I. U. IL-2/ml without contact to tumor cells for 36 h. Specific cytotoxic activity of PBMC against auTu, NK-sensible cells (K 562) and allogeneic tumor cells (RF 48/HT 29) was determined by a fluroescence assay. The auTu of 5 patients were incubated with mAb FMC 16 and W6/32 in order to inhibit specific lysis by CTL via the MHC I system. Specific cytotoxic activity of PBMC without stimulation was not different among the three targets. IL-12 caused a 3.9 fold elevation of cytotoxic activity against auTu (p<0.01). In contrast, after stimulation with IL-2, only a slight increase was seen. After identical IL-12 stimulation, cytotoxic activity against auTu was 2.5 / 4.2 fold higher than corresponding activity against NK sensible K 562 / allogeneic GIT tumor cells (p<0.01). After blocking of the MHC I complex on auTu by FMC 16 or W6/32 mAb, an 75% reduction of specific cytotoxic activity of IL-12 stimulated PBMC was seen indicating the participation of specific cytotoxic T8 lymphocytes. A de novo generation of the observed activity in 36 h without antigen contact is unlikely. Therefore, IL-12 triggered activation of a specific preexisting immune response from the interaction tumor – immune system may be supposed. These findings indicate “intrinsic immunological potential” against auTu in curatively resected patients, which might be used for adjuvant therapy strategies following curative surgery. S6.05 PREOPERATIVE IL-2-INDUCED LYMPHOCYTE AND EOSINOPHIL TUMOR INFILTRATION Brivio F, Lissoni P, Mandala M, Nespoli A Surgery Division, Monza Hospital, Italy It is known that lymphocyte and eosinophil tumor infiltration is associated with a better prognosis.Unfortunately, the evidence of immune tumor infiltration is very rare. Since IL-2 plays a main role in antitumor immunity, we evaluated the influence of presurgical IL-2 in patients (pts) with gastrointestinal tumors, who were randomized to be treated with (n=28)or without (n=32) preoperative IL-2 (9 million IU/day S. C. for 3 days prior to surgery). The percent of lymphocyte and/or eosinophil tumor infiltration was significantly higher in pts pre-

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treated with IL-2 than in controls (25/28 vs 2/32,P < O.OO1).This study shows that preoperative IL-2 may modify tumor histological feature, by inducing immune cell infiltration.

Session 7: Genetic changes during carcinogenesis S7.01 SMALL GTPASES AND CELL PROLIFERATION C. J. Marshall, M. F. Olson, H. F. Paterson, O. Rausch, M. Rosario, I. Treinies. CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, England Small GTPases of the Ras and Rho families are involved in transmitting signals to intracellular signalling pathways Activation of Ras has been shown to be essential for normal growth factor signalling and in a sizable fraction of some human tumours, particularly colon cancer and pancreatic cancer, Ras proteins are mutated to constitutively active forms. In many tumour cell lines which lack mutated oncogenic Ras, activation of normal Ras is required for proliferation. It is now clear that active Ras is capable of activating multiple signalling pathways These pathways include the ERK MAPkinase cascade via Ras dependent activation of the Raf protein, P13 kinase and guanine nucleotide exchange factors for the Ral GTPases. In some contexts we have also shown that Ras activation plays a key role in activation of the p38 and JNK SAPK pathways. Inhibition of each of these Ras dependent signalling pathways, either by chemical inhibitors or through expression of interfering mutants, suppresses cell proliferation. Cell proliferation is controlled through the regulation of cyclin dependent kinase activity. The activity of these kinases is regulated via multiple routes which include the control of phosphorylation of the kinases and the expression levels of cyclins and cyclin dependent kinase inhibitors. Signal transduction events must interface with the cell cycle machinery but much still needs to be learnt of how this takes place. We know that Ras signalling is required for growth factors to stimulate cell cycle entry but we still need to learn which cell cycle control points are regulated by Ras dependent signals? As well as Ras, activation of Rho family GTPases has been shown to be required for growth factor induced cell proliferation. A key issue is to discover how Ras dependent signalling pathways and Rho family GTPases interact to control proliferation. Some growth factors require both a Ras and a Rho signal to activate ERKs. Furthermore in the case of the Ras dependent ERK cascade it is now clear that activation of this signalling pathway can either promote cell proliferation or suppress it through induction of the cyclin dependent kinase inhibitor p21Waf-1. We have shown that when Rho signalling is blocked, Ras induces p21Waf-1 and cells do not go into DNA synthesis. Thus signalling through Rho creates permissive conditions for Ras to induce DNA synthesis. This regulation of p21Waf-1 levels appears to be the major function of Rho in Ras driven DNA synthesis since Ras can induce DNA synthesis in p21Waf-1 cells when Rho is blocked.

S7.02 Chromosome 11q in Solid Cancers James O’D McGee, MD, PhD University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, Oxford OX3 9DU, UK Allelic deletion at chromosome 11q22-123.1 occurs frequently in breast, gastric and other malignancies, suggesting putative tumour suppressor gene(s) within the ~8Mb deleted region. Recently we identified another locus at 11q25-qter which is also frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this ~2Mb deleted region. We explored the functional significance of these observations. Briefly, an 11q YAC contig was accessed and three YACs, one containing the ATM gene, at 11q23.1; and two contiguous YACs (overlapping for ~400-600kb) overlying most of the 11q25 deleted region, were retrofitted with a G418 resistance marker and transfected into murine A9 fibrosarcoma cells. Selected A9 transfectant clones (and control untransfected and “irrelevant” alphoid YAC transfectant A9 clones) were assayed for in vivo tumorigenicity in athymic female Balb c-nu/nu mice. All the 11q YAC transfectant clones demonstrated significant tumour suppression compared to the control untransfected and “irrelevant”, YAC transfected A9 cells. These results define two putative discrete tumour suppressor loci on chromosome 11q by functional complementation, one to a ~1.2Mb region on 11q23.1 (containing the ATM and other genes) and another to a ~400–600kb subterminal region on 11q25-qter. The significance of these findings for GI cancer will be discussed S7.03 P53 and other cell cycle regulators of cell-cycle and apoptosis in esophageal cancer lines P. Hainaut, C. Barnas, K. Castren, O. Pluquet, C. Méplan, and R. Montesano MCA, International Agency for Research on Cancer, Lyon, France Similar to primary cancers, esophageal squamous cell carcinoma cell lines of the TE series show diverse and frequent alterations in genes regulating cell-cycle and apoptosis, including p53 (point mutations, loss of alleles), p16CDKN2A (deletions), CyclinD1 (amplifications) and RB1 (loss of expression). These lines are good models for studying functional consequences of such alterations. TE-1 cells express a temperature-sensitive p53 mutant (p53met272) which determines reversible cell-cycle arrest in G1 at the restrictive temperature. These cells retain p16CDKN2A and express p14arf, the product of the alternative CDKN2A open reading frame. Thus, TE-1 are well-suited to analyse interactions between CDKN2A and p53 in the regulation of G/S transition. We have used several TE lines to analyse the effects of aspirin and cholesterol sulfate, two agents with potential chemo-protective or preventive effects, on cell cycle and survival. Aspirin induces a delay in G1 that does not require CDKN2A nor p53, but involves p27Kip1. Cholesterol sulfate, an inducer of keratinocyte differentiation, uses p53-independent pathways to

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sensitize cells to killing by cytotoxic drugs and by agonists of the APO1/Fas receptor. Together, our data suggest that several classes of pharmaceutical agents can be used to modulate cellcycle and survival in esophageal cancer cells having lost p53 function. S7.06 Allelic loss of the Retinoblastoma (RB) tumor suppressor gene and the p16INK4A tumor suppressor gene in the development of dysplasia and carcinoma in Barrett’s esophagus M. Sarbia, U. Tekin, F. Borchard1, H. E. Gabbert Institute of Pathology, University of Düsseldorf and 1Institute of Pathology, Klinikum Aschaffenburg, Germany Aims: Barrett’s esophagus (BE) is a condition in which the normal squamous epithelium of the esophagus is replaced by a metaplastic columnar epithelium. BE is a premalignant lesion because it is the initiating factor of a metaplasia - dysplasia - carcinoma sequence. In the present study, the role of RB and p16INK4A in this malignant transformation was investigated. Materials/Methods: DNA was extracted from paraffin-embedded tissue samples including normal esophageal tissue (n=36), metaplastic specialized epithelium (SE; n=29), low grade dysplasia (LGD; n=21), high grade dysplasia (HGD; n=19), and invasive adenocarcinoma (CA; n=34) derived from 36 esophagectomy specimens. Following PCR amplification, the samples were checked for loss of heterozygosity (LOH) at polymorphic sites of the RB gene and the p16INK4A gene using silver-stained polyacrylamid gels. Results: Of the 36 cases in this study, 27 (75.0%) were heterozygous for the RB gene and 23 (63.9%) were heterozygous for the p16INK4A gene. With regard to the 27 cases infomative for the RB gene, LOH was found in 0 out of 22 SEs (0%), in 0 out of 14 LGDs (0%), in 1 out of 12 HGDs (8.3%), and in 5 out of 27 CAs (18.5%). Of the 23 cases infomative for the p16INK4A gene, LOH was found in 1 out of 18 SEs (5.6%), in 1 out of 9 LGDs (11.1%), in 4 out of 13 HGDs (30.8%), and in 5 out of 20 CAs (25.0%). Conclusion: Our results suggest that LOH of the tumor suppressor genes RB and p16INK4A are frequent events in Barrett’s carcinomas. Allelic loss of p16INK4A occurs earlier in the metaplasia - dysplasia - carcinoma sequence than LOH of the RB gene. The determination of LOH of p16INK4A may therefore provide important information concerning the development of carcinoma and support the surveillance of patients with BE.

Session 8: Molecular biological approaches for early diagnosis and treatment S8.01 GENETIC INTERVENTION FOR PANCREATIC AND LIVER CANCER. Professor Nicholas R Lemoine, ICRF Molecular Oncology Unit, ICSM at Hammersmith, London UK Advances in our understanding of the molecular genetics of pancreatic and liver cancer have given us new targets for therapy using molecular and genetic approaches. Replacement of tumour suppressor gene function using adenoviruses to transfer wild type p53 and p16 genes can produce dramatic anti-tumour effects both in vitro and in vivo. Blockade of dominant oncogene function using dominant negative technology may have particular application for mutated K-Ras which occurs ubiquitously in pancreatic carcinoma. Genetic prodrug activation therapy using tumour selective and tissue selective gene promoters to drive expression of suicide genes is showing remarkable promise in both pancreatic and hepatic malignancies. Targeted delivery of such therapeutic constructs may also be possible through knowledge of the expression of surface receptors by particular tumour cell types. Genetic imunomodulation using cytokine genes as well as specific vaccines against tumour associated antigens are now being brought into clinical trial for both tumour types. S8.02 Apoptosis resistance mechanisms in pancreatic cancer H. Ungefroren, A. Trauzold, M. Jansen, D. Henne-Bruns, B. Kremer, H. Kalthoff. Clinic for General Surgery,-University of Kiel, Kiel, Germany Aim: Previously we have demonstrated that, despite expression of Fas (CD95, APO-1), the majority of pancreatic adenocarcinoma cell lines was resistant to Fas-induced apoptosis. As resistance correlated with expression of FAP-1 (Fas-associated phosphatase-1) in pancreatic cancer cells, we investigated the functional role of FAP-1 as a potential inhibitor of Fas-mediated apoptosis. Material and Methods: In the Fas-resistant, FAP-1-positive cell line Panc89 Fas/FAP-1 interactions were blocked by cytoplasmic microinjection of a synthetic tripeptide (Ac-Ser-Leu-Val) mimicking the C-terminus of Fas. The Fassensitive, FAP-1-negative Capan1 cell line was stably transfected with a FAP-1 cDNA and its sensitivity to Fas-mediated apoptosis was measured by the JAM DNA-fragmentation assay. Results: Inhibition of the Fas/FAP-1 interaction in Panc89 cells by cytoplasmic microinjection of a specific blocking peptide resulted in a 4-fold increase in apoptosis (after Fas stimulation) compared to cells that received a negative control peptide (Ac-Ser-Leu-Tyr). Furthermore, whereas wild type and vector-transfected Capan1 cells showed apoptotic rates of 70%, those in FAP-1-expressing Capan1 clones were reduced down to 30% after 24 hours of Fas stimulation. Conclusion: These results show that FAP-1 can protect pancreatic carcinoma cells from Fas-mediated apoptosis.

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S8.03 Genetic determinants of tumor response to drugs. K. Danenberg, J. M. Park and P. V. Danenberg USC/Norris Cancer Center, USC, Los Angeles, CA 90033 Because of low tumor response rates to most anti-cancer drugs, the majority of patients not do not derive any benefit from chemotherapy. Aim: To show that analysis of biochemical response determinants in tumor biopsy specimens can identify patients that are not likely to respond to a particular drug, thus allowing them the option to bypass fruitless treatment. Methodology: Quantitative RT-PCR was used to measure expressions of the genes of candidate drug response determinants in biopsies of colorectal, gastric and esophageal cancers. Results: Colorectal tumors with high gene expression levels of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydro-pyrimidine dehydrogenase (DPD) were unresponsive to 5-fluorouracil (5-FU)-based protocols. However, tumors with sufficiently high TS expression to be unresponsive to 5-FU did respond to CPT-11. Also, colorectal tumors that would be unresponsive to 5-FU because of high DPD levels responded to Tomudex. The sensitivity of gastric tumors to 5-FU-cisplatin was associated with gene expression of TS as well as ERCC1. Preliminary data indicate that one of the determinants for response of esophageal tumors to taxol-cisplatin-radiation may be low expression of cyclooxygenase (COX)-2. Conclusions: Measurement of molecular determinants can determine whether a tumor will or will not respond to specific drugs, thus permitting chemotherapy treatment to be optimized for individual patients. S8.04 Dominant negative effects of APC mutants - Molecular basis for genotype-phenotype correlations in FAP-patients M. v.Knebel Doeberitz1, S. Dihlmann1, J. Gebert1, A. Siermann1, Ch. Herfarth2, 1 Division for Molecular Diagnostics and Therapy, Department of Surgery, University of Heidelberg, D-69120 Heidelberg, Germany - 2 Department of Surgery, University of Heidelberg, D-69120 Heidelberg, Germany Inactivation of the adenomatous polyposis coli (APC) gene product initiates colorectal tumorigenesis (1,2). Patients with familial adenomatous polyposis coli (FAP) carry germline mutations in the APC-gene and develop multiple colorectal adenomas and subsequent carcinomas early in life (2). The severity of the disease correlates with the position of the inherited APC mutation (genotype-phenotype correlation) (3-10). Together with the fact that both, germline and sporadic APC mutations cluster in the central region of the APC gene (11), this points to a dominant negative effect of certain APC mutants. Loss of APC function was recently shown to result in enhanced β-catenin mediated transcription (CMT) in colon epithelial cells (12,13). Here, we provide experimental evidence for a dominant negative effect of APC gene products associated with severe polyposis. Wild-type APC activity on CMT is strongly inhibited by mutant APC, truncated at codon 1309. In contrast, mutant APC gene products associated with attenuated polyposis (codon 386 or 1465) interfere only weakly with wild type

APC activity. These results suggest a molecular explanation for the genotype-phenotype correlation in FAP patients and support the idea that colorectal tumor growth might be, in part, driven by selection for a mutation in the mutation cluster region (MCR) (11). S8.05 Epidermal growth factor (EGF) receptor-mediated molecular treatment of metastasized colorectal cancer. F. Ciardiello1, S. Agrawal2, J. Mendelsohn3, A. R. Bianco1, G. Tortora1. 1 Division of Medical Oncology, Univ. of Naples, Italy; 2Hybridon Inc., Cambridge, MA, USA; 3UT-MDACC, Houston, TX, USA Aim: EGF-related proteins such as transforming growth factor a (TGFa) control colorectal cancer growth through autocrine mechanisms. Overexpression of TGFα and ist receptor (EGFR) is associated with aggressive disease and poor prognosis. Therefore, blockade of EGFR has been proposed as target for anticancer therapy. Materials and Methods: The antitumor activity of selective inhibitors of EGFR activation, including a chimeric human-mouse anti-EGFR blocking monoclonal antibody (MAb C225), various anti-EGFR antisense oligonucleotides, an inhibitor of type I protein kinase A (PKAI), that is a EGFR-downstream signaling enzyme (8-Cl-cAMP), was evaluated in vitro and in vivo in human GEO colon cancer cells. Results: MAb C225 determined a dose-dependent cytostatic growth inhibition of GEO cells in vitro and in nude mice. A similar effect was observed with anti-EGFR antisense oligonucleotides or with 8-Cl-cAMP. A cooperative antitumor activity in vitro and in vivo was obtained by the combined treatment with MAb C225 and 8-Cl-cAMP or with MAb C225 and the topoisomerase I-inhibitor topotecan with also a significant improvement in mice survival. Tumor eradication of established GEO tumors was observed in 5-20% of mice. Conclusion: Since MAb C225 and 8-Cl-cAMP are in phase II studies, these results provide a rationale for the evaluation of the antitumor activity of inhibition EGFR by selective drugs alone or in combination with cytotoxic drugs in colorectal cancer. S8.06 IN VIVO Gene therapy Monitoring by Positron-emitting Radiodiagnostica U. Lauer, P. Staehler, R. M. Lambrecht, F. Oberdorfer1, M. Spiegel, W. A. Wybranietz, C. D. Groß, M. Gregor. Abt. Innere Medizin I, Universitätsklinikum Tübingen, and 1DKFZ, Heidelberg, Germany Aim: Dramatic expansion of clinical cancer gene therapy trials requires development of non-invasive clinical monitoring procedures, which provide information about expression levels/kinetics and spatial distribution of transduced therapeutic genes. We investigated the feasibility of the ∆LNGFR/NGF transduction tag system for a future PET-based gene therapy monitoring, which employs a mutated version of the low affinity nerve

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growth factor receptor (∆LNGFR) and PET-labeled nerve growth factor (NGF). Material/Methods: NIH 3T3 and Morris hepatoma cells were stably transduced by ∆LNGFR encoding retroviral vector pLDNSN. NGF was radioiodinated via the N-bromosuccinimid method. Results: Specific binding of 125I-NGF was found only for stably ∆LNGFR transduced cells, but not for parental ∆LNGFR-negative cells. Employing a MicroImager™ detector specific binding of 125I-NGF to ∆LNGFR-positive target cells was made directly visible. 125I-labeled monoclonal anti-LNGFR antibodies also showed a specific binding to ∆LNGFR-positive target cells. Binding studies employing PET-tracer labeled 124I-NGF demonstrated similar results. Conclusion: In this study, we have developed an experimental platform for the rational design and in vitro-testing of suitable receptor-ligand couples as components of future transduction tag systems for non-invasive gene therapy monitoring applications. S8.07 Molecular detection of liver micro-metastases by a PCRRFLP assay specific for K-ras mutations. C. Schimanski, U. Linnemann, M. R. Berger Unit of Toxicology and Chemotherapy, DKFZ, 69120 Heidelberg, Surgical Department, Municipal Clinic, Nürnberg, Germany Aim: Postoperative survival of colorectal cancer patients is determined mainly by the presence of liver metastases. We developed a diagnostic tool for detecting micro-metastases of the liver at the time of surgery. Material and Methods: A PCR-RFLP assay was set up tracking point mutations of the K-ras oncogene at codons 12 and 13 with restriction enzymes BstXI and XcmI. The detection limit of this assay was one mutant in a million wild type cells in mixtures of K-ras wild-type (colon cancer cell line HDC63) and mutant cells (colon cancer cell lines HDC8 [codon12] and HDC101 [codon13]). Using this assay 142 patients with colorectal carcinoma were screened for mutations in tissue samples of their tumor, adjacent mucosa, and liver. Results: Of 142 patients 60 patients (42%) were positive for a K-ras mutation, of which 53 had a codon 12 and 9 a codon 13 mutation, with two patients having both mutations. No patient without a K-ras positive tumor showed a mutation in mucosa or liver tissue, but 11 patients with a K-ras positive tumor (18%) were found to bear a K-ras mutation in their mucosa and for 21 patients (35%) a K-ras mutation was detected in liver tissue. Conclusion: The assay is a useful tool for detecting micro- metastases of the liver which are positive for K-ras codon 12 and 13 mutations. The detection of metastasis as early as at the time of surgery might well have implications for the patients’ further medical treatment.

S8.08 Adenovirus-mediated transfer of wild-Type p53 gene results in apoptosis or growth arrest in human cultured gastric and colorectal carcinoma cells H Ito, S Tatebe, R Doi, K Endo, I Kuratate, and K Teramachi Dep. of Pathology, Tottori Univ. Yonago/Tottori, Japan Aim: This study examines the susceptibility of gastric and colorectal carcinoma cell lines to infection with recombinant p53 adenovirus vector (AxCA-p53). Materials/Methods: The human cultured gastric carcinoma cell lines include MKN-1 (carrying mt p53 gene) , MKN-45(wt), MKN-74 (wt), OCUM2M (wt) and KATO-III (deletion), and the colorectal carcinomas include LoVo(wt), DLD-1(mt), COLO320(mt), COLO201(mt), SW83(mt), and WiDr(mt). Expression of p53 generelated molecules and bcl-2 family was examined by Western blot analysis. Results: A rapid loss of cell viability due to cell death was observed in MKN-1 and DLD-1 culture 72h after infection with AxCA-p53 at a multiplicity of infection (MOI) of 50. Flow cytometric analysis demonstrated that AxCA-p53 infection resulted in apoptotic cell death (MKN-1, DLD-1, COLO320, and LoVo; death (D)-line), growth arrest (MKN-45, MKN-74 and KATO-III, COLO201, and WiDr; arrest(A)lines), or non-effectiveness (TMK-1, OCUM-2M, and SW837). Western blotting analysis revealed increasing expression levels of p21/WAF1 protein after infection with AxCA-p53 both in D- and A-lines. After infection with AxCA-p53, the expression levels of bax or bcl-XL protein changed in D-lines, but was stable in all A-lines. Conclusion: The apoptotic pathway dominates the growth arrest pathway (dependence on the expressions of p21/WAF1 protein) after infection with AxCAp53. Thus, the bcl-2 family might play a crucial role in p53mediated growth arrest and apoptosis in human gastric and colorectal carcinoma cells. S8.09 Detection of Tpr-Met Rearrangement in Patients with Gastric Cancer (GC) and First-Degree Relatives J. Yu, M. Ebert, S. Miehlke, M. Breidert, U. Pich, P. Malfertheiner, M. Stolte, E. Bayerdörffer. Dept. of Gastroenterology, Univ. of Magdeburg, Med. Dept. I, Techn. Univ. Dresden, Inst. f. Pathology, Klinikum Bayreuth, Germany Aim: The presence of tpr-met mRNA in GC and precancerous lesons has been reported. We aimed to analyse tpr-met rearrangement in GC patients and in first-degree relatives of GC patients. Material/Methods: The presence of tpr-met mRNA was examined in tumour tissue and in normal mucosa of 8 GC patients, and in gastric mucosa of 8 first-degree relatives of GC patients. A 205 bp fragment and 70 bp fragment spanning the breakpoint were amplified by nested PCR. The PCR products were examined by hybridization with a Dig 3′-end labelled oligonucleotide spanning the breakpoint using Southern blot analysis. The MNNG-HOS cell line was used as positive control. Results: Four of 8 GC patients and 1 first-degree relative exhibited the tpr-met rearrangement. In the four GC patients with tpr-met activation the rearrangement was also detectable

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in the normal mucosa. Conclusion: The tpr-met rearrangement can be found in GC and in normal mucosa of these patients. It may be an early event in gastric carcinogenesis, and thus useful to identify individuals with an increased risk for gastric cancer. S8.10 Regression of human pancreatic carcinoma growing orthotopically in athymic nude mice by blockade of epidermal growth factor receptor (EGF-R) signaling in combination with Gemcitabine. C. J.Bruns, C. A.Portera, M. T.Harbison, R.Tsan, D. J.Hicklin, and R.Radinsky. Dept. of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas; ImClone Systems, Inc., New York, NY (DJH) Overexpression of the EGF-R and its ligands contribute to the malignant phenotype of pancreatic carcinoma. Neither surgical nor chemo-radiation therapies significantly improve overall survival, and agents such as Gemcitabine show a response rate of only 27% with a 3.85 months median survival. This study evaluated whether EGF-R blockade using anti-EGF-R antibody C225 has therapeutic benefits alone or in combination with Gemcitabine. In vitro C225 treatment of high metastatic L3.6pl human pancreatic carcinoma cells resulted in a dose-dependent inhibition of EGF-R-specific autophosphorylation in response to ligand and a maximal 30% cytostasis; whereas, Gemcitabine treatment (<0.04mg/ml) resulted >90% cytotoxicity. C225 treatment (i.p.,1 mg biweekly for 3 wks) of L3.6pl tumors growing orthotopically in nude mice resulted in tumor regression and abrogation of liver metastases (p<0.0001) vs. treatment with Gemcitabine (i.p.,125mg/kg biweekly for 3 weeks) or saline alone. Combination treatment with Gemcitabine+C225 resulted in no macroscopic tumors and only 1/10 animals with microscopically visible lesions. The expression of PCNA, VEGF, and IL-8 (protein and mRNA) were significantly reduced in C225 vs. Gemcitabine-treated or control tumors. These data indicate that C225 EGF-R blockade therapy (alone and with Gemcitabine) has a significant anti-tumor effect on human pancreatic carcinoma growth and metastasis in nude mice. S8.11 CHEMOTHERAPY-SENSITIVITY OF HUMAN LIVER TUMOR CELLS IS ENHANCED BY INTERFERING WITH THE IGF-II/IGF-I RECEPTOR SIGNALLING PATHWAY P. Lund, F. Niketeghad, HP. Dienes, P. Schirmacher Institute of Pathology, University Hospital, Cologne, Germany Aim: Insulinlikc Growth Factor II (IGP-II) is overexpressed in 40 to 60% of human hepatocellular carcinomas and exerts autocrine stimulatory effects via the IGF-I receptor. Since IGFIR mediated signalling may have antiapoptotic effects, we investigated whether neutralization of IGF-II or the IGF-IR increases the chemotherapy-sensitivity of human liver tumor cell lines.

Material/Methods: HEPG1 and HEP3B coexpressing IGF-II and IGF-IR were incubated with representative chemotherapeutic agents (etoposide, cisplatin, doxorubicin) to determine the dose response curves using a modified MTT-assay (EZ4U, BioZol). Neutralization was performed with aIGF-II Abs (Upstate Biotechnology) or αIGF-IR-Abs (R&D), stimulation with recombinant human IGF-II (R&D). Results: Cell growth was significanty reduced by aIGF-II Abs and αIGF-IR-Abs, while recombinant IGF-II only slightly enhanced cell growth. The cytostatic effect of etoposide, cisplatin and doxorubicin in HEPG2 and of etoposide in HEP3B cells was augmented when either IGF-II or the IGF-IR were neutralized. Conclusion: Autocrine growth-factor-mediated signalling, like demonstrated for IGF-II/IGF-IR, may increase chemotherapyresistance of HCC cells, thus inhibition of this pathway might increase the efficiency of chemotherapeutic treatment

Session 9: Extracellular matrix and microenvironment S9.02 Modulation of growth factor action in pancreatic cancer by components of the extracellular matrix M. Korc, J. Kleeff, H. Friess, A. D. Lander, and M. W. Buechler. Univ. of California, Irvine, USA and Univ. of Bern, Bern, Switzerland The mitogenic actions of many growth factors are dependent on interactions with heparan sulfate proteoglycans (HSPG) that facilitate binding to specific high affinity receptors. Proteoglycans consist of a core protein that is covalently attached to 1 or more polysaccharides called glycosaminoglycans. They are found in the extracellular matrix and on the surfaces of many different types of cells. Although the cell surface HSPG modulate growth factor signaling, cell-cell and cell-extracellular matrix adhesion and, their potential role in pancreatic cancer has not been adequately assessed. Therefore, we sought to characterize the expression of the glypican family of polypeptides in this disease. We determined that glypican-1 expression, both at the mRNA and protein levels, is dramatically up-regulated in human pancreatic cancers. In contrast, we found that expression of glypican-1 is low in the normal pancreas and in chronic pancreatitis, and that glypicans 2-5 are not overexpressed in the cancer samples. We also demonstrated that glypican-1 is highly expressed by human pancreatic carcinoma cell lines in vitro, and is readily released by the cancer cells into the medium. We took advantage of the fact that glypican-1 is a glycosylphosphatidylinositol- (GPI-) anchored HSPG. Therefore, the enzyme phosphoinositide-specific phospholipase-C (PIPLC) cleaves glypican-1 from the cell surface. We showed that treatment with PI-PLC of two pancreatic cancer cell lines that express glypican-1 abrogated their mitogenic responses to two factors that are commonly overexpressed in pancreatic cancer, FGF-2 and heparin binding EGF-like growth factor (HB-EGF). We also showed that stable expression of a form of glypican-1 engineered to possess a transmembrane domain instead of a

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GPI-anchor conferred resistance to the inhibitory effects of PIPLC on growth factor responsiveness. Furthermore, transient transfection of a glypican-1 antisense construct attenuated the mitogenic response to FGF-2 and HB-EGF. In contrast, neither PI-PLC treatment nor glypican-1 antisense expression, altered the response of the cancer cells to EGF or insulin-like growth factor -1 (IGF-1). Our findings suggest, therefore, that glypican-1 may play a crucial role in enhancing the actions of certain growth factors in human pancreatic cancer, that its expression by pancreatic cancer cells may be important in the pathobiology of this disorder, and that glypican1 contributes to aberrant epithelial-mesenchymal interactions in pancreatic cancer. S9.03 Strategies for the identification of genetic alterations in pancreatic cancer. C.Wallrapp1, F.Müller-Pillasch1, M.Geng1, JD.Hoheisel2 M..Frohme2 S.Solinas-Toldo2, P.Lichter2 ,G.Adler1, T. M. Gress1 1 Department of Internal Medicine I, Ulm, Germany, 2 DKFZ Heidelberg, Germany Aim: In our presentation we will summarize our strategies aimed at identifying and characterizing genetic alterations occuring at the transcriptional and chromosomal level in pancreatic cancer. Methods: To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA-RDA (representational difference analysis). Comparative genomic hybridization (CGH) was used to study chromosomal aberrations occuring in pancreatic cancer tissues. Results: The study of transcriptional alterations has led to the identification of more than 500 genes with differential expression in pancreatic cancer. The sum of these alterations represented the first expression profile characteristic for pancreatic tumors. The CGH analysis allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. These regions are presently being characterized at the molecular level. In a first approach the myb-oncogene was identified as the relvevant oncogene of an amplification on 6q ocurring in up to 10% of pancreatic cancer patients. Conclusions: Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis. S9.04 Cell-Cell Contact Regulation by Protein-Tyrosin-Phosphatases M. M. Lerch, J. Schnekenburger. Department of Medicine B, Westfälische Wilhelms-Universität Münster, Germany Malignant tumours metastasise only when individual cancer cells dissolve their cell-cell contacts and thus escape the confines or the original tumour. Classical cell adhesion proteins of

the cadherin family are therefore regarded as tumour suppressors. We have identified four protein tyrosine phosphatases (PTPs) that are differentially expressed in the pancreas and have studied their involvement in the regulation of cadherin-controlled cell-cell adhesions. PTPm, PTPk, SHP1 and SHP2 were all detected in pancreatic acinar and duct cells. PTP-Inhibition with orthovanadate in vitro was sufficient to dissociate the adherens junctions of isolated pancreatic acini and induced an association of SHP1 with cell-adhesion proteins of the cadherin/catenin complex. In in vivo experiments in which we dissociated adherens junctions by supramaximal hormone stimulation PTPk was found to associate with the cadherin/catenin complex constitutively and at intervals when adherens junctions were found intact. Conversely, at intervals when the junctions and the cell adhesion complex were dissociated PTPk dissociated from this complex and, instead, the highly homologous PTPm was found to associate with cell adhesion proteins. These data indicate that PTPm, PTPk and SHP1 play a critical and complementary role in the maintenance of pancreatic adherens junctions and that this role could involve the regulation of cadherins and catenins - a known class of tumour suppressors. (supported by the Deutsche Forschungsgemeinschaft: Le 625/4)

Satellite Symposium: Selenium and carcinogenesis SS01 Hepatitis C virus encodes a selenium-dependent glutathione peroxidase gene: implications for oxidative stress as a risk factor in progression to hepatocelluar carcinoma E. W. Taylor, W. Zhang, A. G. Cox College of Pharmacy, University of Georgia, Athens, GA 30602 USA Aim: To assess the hypothesis that hepatitis C virus (HCV) encodes a glutathione peroxidase (GPx) gene in an overlapping reading frame, linking HCV expression and pathogenesis to the Se status and dietary oxidant/antioxidant balance of the host. Methods: The putative HCV GPx gene was identified by searching viral sequence databases, using conserved GPx active site sequences as probes, giving particular weight to the UGA (selenocysteine) codon. Multiple sequence alignments were generated and analyzed to validate the sequence similarity, and to establish the degree of conservation of the identified genomic features in HCV. Molecular modeling was used to assess the structural feasibility of the proposed homology. Results: The GPx homology region overlaps the NS4 gene, and is well conserved in HCV. The sequence similarity of the conserved active site regions to a set of known GPx is high (4 to 6 S. D. greater than expected for similar random sequences). The computed strain energy of a molecular model of the HCV GPx is energetically favorable, comparable to the bovine GPx structure.

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Conclusions: Expression of a viral GPx gene could induce an antioxidant defect in the host, suggesting that Se deficiency may be a risk factor in HCV disease progression, which is accelerated by oxidant stressors like alcoholism and iron overload. SS02 Expression of Selenoproteins in the Human Gastrointestinal (GI) Tract – Implications for the Carcinogenesis. J. Köhrle, H. Mörk, I. Dreher, K. Hesse, O. Al-Taie, S. Karvar, M. Scheurlen, F. Jakob. Medizinische Poliklinik, Universität Würzburg, D-97070 Würzburg Epidemiological studies revealed a correlation between low selenium (Se) intake and occurrence of several cancers. In a recent intervention study supplementation with 200 mg Se reduced tumor incidence in lung, prostate and colon. Oxygen radicals and reactive oxygen intermediates cause DNA damage and contribute to carcinogenesis. Selenoproteins, especially glutathione peroxidases (GPx), thioredoxin reductase (TRR), and Selenoprotein P (SeP) are part of the human antioxidative defense system. The aim of this study is to analyse the role of selenoproteins in the antioxidative defense of the GI-tract. We therefore investigated expression and regulation of selenoproteins in human cell lines and biopsies of the human GI-mucosa by functional enzyme assays, Western and Northern blot analysis, metabolic labeling with 75-Se, and transfection experiments. Among several selenoproteins expressed in GI-cell lines and in biopsies of the human GI-mucosa we identified gastrointestinal GPx, plasma-GPx, TRR, and SeP by functional assay or in blots, other bands metabolically labeled by 75-Se remain to be identified. Expression of GI-GPx, pGPx, and TRR shows a distinct differential pattern along the human GI-tract. Se supplementation increased expression of GPx in GI cell lines. Proinflammatory cytokines inhibit activity of the human SeP promoter in transient transfection experiments in CaCo-2 cells. These data indicate that selenoproteins, part of the human antioxidative defense, are regulated targets for Se-supplementation in prevention and intervention in colorectal cancer. Supported by DFG and Wilhelm Sander-Stiftung. SS04 Strategies for genetic counselling and testing in families with hereditary cancer. Elisabeth Gödde Berghäuser Str. 295, 45659 Recklinghausen Although everybody is at some risk of developing cancer, individuals whose families show certain charactaristics might be at high risk. The existance of families with hereditary cancers have been known for a long time. Meanwhile several genes for these dispositions have been identified, which gives high-risk individuals the opportunity to choose presymptomatic testing. This leads to a new group of „patients„, the healthy gene carriers. Genetic counselling is a process which includes verification of diagnosis, pedigree analysis, individual risk assessment, expla-

nation of complex medical and genetic facts and the choice of possible options to deal with the genetic burden (e.g. coping strategies, prevention). Presently cancer gene testing has two clinical applications. In affected individuals it is part of the diagnostic procedure, in at risk individuals it segregates gene carriers from non-carriers. With regard to the healthy carriers of highly penetrant mutated genes and to some extend also with regard to the non-carriers this means new medical, psychological, social and ethical tasks. Genetic counselling should be offered to any family with a history of cancer or cancer-linked conditions. It has to be considered that not only patients and their families are laymen with regard to medicine, but that we all are laymen with regard to the leading a life with the knowledge of once own deleterious heritage.

Session 10: Clinical and pathological studies in gastrointestinal carcinogenesis S10.01 Genetic differences among normal esophageal tissue, Barrett’s esophagus and esophageal adenocarcinomas. P. Danenberg, R. V. Lord, K. Danenberg, Ji Min Park, J. H. Peters, T. R. DeMeester and M. Kiyabu. USC/Norris Cancer Center and Department of Surgery, USC, Los Angeles, CA 90033 Cyclooxygenase (COX)-2, often upregulated in tumors, is thought to promote tumorigenesis by stimulating angiogenic factors and other genes. Aim: To characterize changes in expressions of cyclooxygenase (COX)-2 and COX-2-associated genes occurring in the Barrett’s metaplasia-dysplasia-adenocarcinoma sequence. Methods: Relative mRNA levels of COX-1 and -2, inducible nitric oxide synthetase (iNOS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF) and telomerase (hTRT), were determined using a fluorogenic dye quantitative RT-PCR (Taqman™)method in specimens of Barrett’s adenocarcinoma (19 patients), Barrett’s intestinal metaplasia (33 patients), and matched normal esophagus samples. Results: COX-2, iNOS, VEGF, bFGF and hTRT expressions all increased significantly during progression from normal to tumor tissue. In contrast, PD-ECGF expression was not different, whereas COX-1 expression was progressively inhibited at each pathologic stage. Levels of COX-1 and -2 also differed significantly in histologically normal or non-malignant tissues in the presence of cancer. Conclusions: Expression levels of these genes change consistently during esophageal tumorigenesis and could be useful adjuncts to conventional histopathological examination for Barrett’s tissues.

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S10.02 Duct changes and K-ras mutation in the disease-free pancreas: an analysis of type, age relation and spatial distribution G Klöppel, A Reinecke-Lüthge, B Möllmann, M Menke, J Lüttges Department of Pathology, University of Kiel, Germany Aim: Duct lesions of the pancreas might be potential precursors of pancreatic ductal adenocarcinoma. K-ras mutations are known to occur in duct lesions as well as in most carcinomas. This study examines the type, prevalence, distribution and Kras codon 12 mutation rate of duct lesions within the normal pancreas. Material/Methods: Autopsy pancreases from 101 subjects (6-92 y) were screened for the presence of mucinous cell hypertrophy (MHT), ductal papillary hyperplasia (DPH), adenomatoid ductal hyperplasia (ADH), and squamous metaplasia (SQM). In 70 pancreases a K-ras analysis was performed. Results: The various types of duct lesions were evenly distributed in the pancreas. They were more common beyond the age of 40, and especially MHT and ADH increased in frequency. MHT was present in 61% of the cases, DPH in 30%, ADH in 33%, and SQM in 37%. K-ras mutations were found in a total of 16 samples from 12/70 specimens (10 MHT, 1 DPH, 2 ADH, 1 SQM). Normal duct cells also showed K-ras mutations, but not acinar cells. The most common K-ras mutations were GGT to GAT(6) or GTT (5), followed by TGT (2), GCT (2) and CGT (1). Conclusions: Hyperplastic/metaplastic changes of the pancreatic ducts are common beyond the age of 40 but do not share the preferred localization of pancreatic carcinomas. Since Kras mutations occur in all types of duct lesions and also in normal duct cells, their presence is not inevitably associated with carcinoma development.

1991; 4. Pantel et al., JNCI 85:1419, 1993; 5. Pantel et al., JNCI 87:1162, 1995 S10.04 DETECTION OF P53 MUTATIONS IN HUMAN TUMORS: A TOOL FOR EARLY DIAGNOSIS Varda Rotter: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 76100. There is ample evidence for participation of wild type p53 in the positive regulation of apoptosis. Much less is known about the effects of various p53 mutants on apoptosis. Conceivably, in cells which have endogenous wild type p53, the addition of mutant p53 could inhibit the apoptotic activity of this endogenous wild type p53 in a negative transdominant fashion. However, the more challenging issue is whether mutant p53, on its own, has any effects on the cellular response to p53-independent apoptotic signals. This question is highly important because most tumors express mutant p53 protein types. Hence, a negative dominant effect over endogenous wild type p53 is irrelevant there. Yet, it is now known that in order for cancer therapy to eradicate such tumors, the therapy must be able to induce effective apoptotic death of the tumor cells. We have found that introduction of the mutant p53 into p53null cells protects them from p53-independent apoptosis that is induced by survival factor deprivation. Wild type p53, however, seems to induce an apoptotic response that co-operated with the p53-independent one. This is direct evidence that at least certain types of mutant p53 can exert a direct oncogenic effect by interfering with general apoptotic pathways. S10.05

S10.03 Disseminated colorectal cancer cells in bone marrow Klaus Pantel Universitätsfrauenklinik, UKE, Hamburg, Germany Individual tumor cells in bone marrow can be frequently found in patients with colorectal cancer (UICC stage II: 26%, UICC stage III: 38% [1]) despite the fact that overt skeletal metastases detectable by clinical staging are rare. The prognostic significance of this surprising finding has been demonstated by two different groups using either immunocytochemistry with monoclonal antibodies against cytokeratin-18 [1] or RT-PCR specific for cytokeratin-20 to reveal ectopic epithelial tumor cells in bone marrow [2]. The malignant nature of these cells was supported by downregulation of MHC class I antigens [3] erbB2 oncogene overexpression [4], and their capacity to grow in cell culture [5]. Thus, cytokeratin-positive cells in bone marrow of colorectal cancer patients appear to be dormant tumor cells that contribute to metastatic relapse at other secondary sites such as liver or lung. The dormant nature of these cells may explain the failure of adjuvant chemotherapy in relapsed patients. 1. Lindemann et al., Lancet 340:685, 1992, 2. Soeth et al., Cancer Res. 57:3106, 1997; 3. Pantel et al., Cancer Res. 51:4712,

Intestinal Non Hodgkin’s Lymphoma - Results of a multicenter Study E. O. Riecken, B. Dederke, W Heise S. Daum and the German Non Hodgkins’s Lymphoma Study Group Objective: Primary small bowel lymphomas are a rare lymphoma entity and characterized by a high rate of complications, late diagnosis and short survival. Since standardized therapy concepts do not exist, a prospective German multicenter study was started to investigate epidemiology, diagnostic procedures, histopathology and a standardized therapy. Methods: In addition to standard diagnostic procedures, histopathologic, immunphenotypic and molecular differentiation, Tcell receptor rearrangement was analyzed to proof early diagnosis and etiopathology. Standard therapy (after postsurgical stratification) included chemotherapy (CHOP) in Ann Arbor stage I and II, CHOP plus radiotherapy in stage III and IV. Results: From April 1995 to December 1998, 84 patients with intestinal lymphoma were included. Preliminary data from 60 patients (37 T-cell and 23 B-cell type, 2/3 Ann Arbor stage I/II) show a high rate of initial life threatening complications (bleeding, perforation, obstruction) in 65%. Significant factors for remission and survival are lymphoma type, stage of disease and CHOP chemotherapy, but not lymphoma localization or initial complication.

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Conclusion: Intestinal lymphoma remain a clinical entity with late diagnosis of and a high rate of initial complications. The receptor rearrangement in T-cell lymphoma may be used for early diagnosis in patients with celiac disease. S10.06 EFFECT OF URSODEOXYCHOLIC ACID ON COLONIC MUCOSAL PROLIFERATION: A RANDOMIZED DOUBLE-BLIND TRIAL. T. Ochsenkühn, I. Marsteller, U. Hay, G. B. Baretton, A. Meining, G. Paumgartner, M. Sackmann. Dept. of Medicine II, and Dept. of Pathology, Klinikum Großhadern, University of Munich and Dept. of Medicine II, Technical University of Munich, Germany Aim: Ursodeoxycholic acid (UDCA) has a chemoprotective effect in animal models of colon cancer. One possible explanation is the lowering effect of UDCA on the proportion of the tumor-promoting fecal deoxycholic acid (DCA). In the animal model DCA leads to colonic mucosal hyperproliferation, which is considered as a first step in colorectal carcinogenesis. UDCA might exert a tumorsuppressive effect by lowering fecal DCA and thus reduce the colonic mucosal proliferation rate. We tested this hypothesis in a randomized, double-blind trial. Methods: 6 women and 12 men with colonic adenoma were randomized to receive either 750mg UDCA (n=10) or placebo (n=8) for 6 months. Biopsies from the transverse colon were sampled before and at the end of the medica-tion by colonoscopy. Colonic mucosal proliferation was measured by analysis of the cell proliferation-associated antigen, Ki-67. The proliferation rates in the UDCA and the placebo group were compared before and at the end of treatment. Results:

Session 11: Frontiers between dysplasia and early cancer S11.02 Dysplasia stomach E. Tahara First Department of Pathology, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan Gastric dysplasia composed of nuclear atypia and structural atypia or loss of polarity has some important inerpopulation differences in the types, frequency and severity of dysplasia. Among several types of gastric dysplasia, however, adenoma or adenomatous hyperplasia and incomplete intestinal metaplasia are implicated in the development of gastric cancer. Excitingly, over 30% of incomplete intestinal metaplasia, associated with hyperplasia of hTERT/hTRT positive cells, harbor reduced telomeres, p53 mutation, altered microsatellites at the D1S191 locus (1q), and DNA hypermethylation at the D17S5 locus, followed by abnormal gland formation with dysplasia. In addition, abnormal CD44 transcripts containing the intron 9 sequence, loss of pS2 and cripto overexpression are commont events in gastric metaplasia and adenoma. Cyclin E overexpression and p27 loss in these precancerous lesions evidently confer progression to develop into malignancy. These genetic and epigenetic alterations in these gastric dysplasias can serve as molecular biomarkers for differential diagnosis and understanding biological malignancy of gastric dysplasia. In fact, through a good combination of these biomarkers, we have routinely implemented molecular diagnosis of gastric dysplasia and cancer at Hiroshima City Medical Association Laboratory since 1993.

S11.05 Medication:

UDCA

Placebo

Proliferation before treatment: (% of Ki-67-positive cells, X ± SD) Proliferation at end of treatment:

40.4 ± 13.1

34.9 ± 11.1

40.6 ± 12.9

39.5 ± 10.5

All p values > 0.3 (Student’s T-Test)

Conclusions: UDCA treatment for 6 months does not lead to changes in the proliferation of the human colonic mucosa. Thus a suggested chemopreventive effect of UDCA is not exerted by a reduction of the colonic proliferation, as measured by Ki-67 analysis.

Significance of Adenomas in Ulcerative Colitis E. Mueller 1, J. Mueller 2, M. Vieth 3, H. Höfler 1, M. Stolte 3 1 Institute of Pathology, Technische Universität München; 2 Department of Surgery, Technische Universität München; 3 Institute of Pathology, Klinikum Bayreuth Aim: Since the diagnostic criteria and biologic significance of adenomas in areas of ulcerative colitis (UCA) are uncertain, we analyzed the p53 and bcl-2 phenotype and clinicopathologic data of a group of UCA lesions in comparison to ulcerative colitis associated dysplasia (UCD) and sporadic adenomas. Material and Methods: 24 UCA (22 patients) were compared to 66 UCD (40 patients) with respect to clinical features (age, duration, extent of the disease, associated neoplasms) and immunohistochemical p53 and bcl-2 expression Results: The UCA patients differed from the UCD group in age (66.4 vs. 44.0 yr.) and extent of UC (56% vs. 13% with left or distal disease). p53 and bcl-2 expression rates were similar in the UCA group and the sporadic adenoma (n=25) control group but were significantly different from the UCD group (p53 positive: 4% UCA vs. 30% UCD, p = 0.01; increased bcl-2: 96% UCA vs. 70% UCD, p = 0.01). p53 in UCA vs. low grade dysplasic polypoid UCD was just below significance (p= 0.07).

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Conclusion: The results show that UCA has clinical and phenotypic features similar to sporadic adenomas but different from UCD. These findings support the concept that the biologic and clinical significance of adenomas in UC is distinct from that of UC associated dysplasia.

Symposium: Prevention of Cancer

whereas for colorectal cancer a decreased intake of red meat and alcohol contributes to lower incidences. Interindividual differences in the metabolism of carcinogens and nutrients may contribute to the effect of nutrition on these cancer sites. About five years ago the study “European Prospective Investigation into Cancer and Nutrition (EPIC)” has started as a cohort study. In Germany there are two study centers, Potsdam and Heidelberg, with approximately 53.000 participants. Of particular interest is the later analysis of the stored blood samples. Finally, we will discuss about a recently published prospective study on the effect of fruits and vegetables on stomach cancer risk.

SP01 The impact of carotenoids on prevention of cancer R. K. Biesalski Dept. Biol. Chemistry and Nutrition, Univ. Hohenheim The results of several epidemiological studies indicate that an increased dietary intake of B-carotene correlates with a decrease of cancers of epithelial cell origin, including colonic neoplasia. The cancer chemopreventive effects of β-carotene are believed to be due to i) antioxidative activity and ii) provitamin A activity. The latter results at least in formation of retinoic acid which regulates cellular growth and differentiation. Retinoic acid transrepresses transcription factors (e.g. AP- 1) and consequently the expression of depending genes (e.g. MMP, ODC). Accumulation of β-carotene in colonic cells following supplementation results in a decrease of ODCactivity. Whether this is due to either the antioxidant capacity or retinoic acid formation is up to now unknown. Formation of reactive oxygen species (UVB-irradiation) in human skin fibroblasts leads to an upregulation of ODC expression via API dependent signal pathways. Retinoic acid slightly down-regulates the expression. Incubation with β-carotene completely abolishes the expression of QDC. Consequently β-carotene seems to influence the ODC expression via singulett oxygen quenching and retinoic acid formation. The accumulation of β-carotene via nutrition might be achieved with a diet rich in fibres and rich in carotenoids. Fibres will adsorb carotenoids including β-carotene and consequently the amount of carotenoids in the colon will be increased. SP02 Nutrition and Cancer – Facts and Hypotheses J. Wahrendorf German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany While the incident cases of stomach cancer have declined worldwide, the incident cases of colorectal cancer are more or less stable. The convincing epidemiological data about stomach cancer and colorectal cancer are rare. For stomach cancer it is regarded that vegetables and fruits have a protective effect as well as the use of refrigerators. For colorectal cancer physical activity and vegetables are convincing protective factors. Probably evidence is presented for the protective effect of high vitamin C intake and low salt intake on stomach cancer risk

SP04 POSSIBLE MECHANISMS OF THE COCARCINOGENIC EFFECT OF ETHANOL H. K. Seitz Dept. of Medicine, Salem Medical Ctr., Heidelberg A great number of epidemiologic studies have identified chronic alcohol consumption as a risk factor for upper alimentary tract cancer, cancer of the liver, rectum and breast. Although the exact mechanisms by which chronic alcohol ingestion stimulates carcinogenesis are not known, experimental studies support the concept that ethanol is not a carcinogen, but under certain experimental conditions a cocarcinogen and/or a tumor promoter. The metabolism of alcohol leads to acetaldehyde (AA), a highly toxic compound which binds to protein and DNA. AA decreases DNA repair mechanisms and the methylation of cytosine in DNA. It also traps glutathione, an important peptide in detoxification. Furthermore, it leads to chromosomal aberrations and seems to be associated with tissue damage and secondary compensatory hyperregeneration. More recently, the finding of considerable production of AA by gastrointestinal bacteria was reported. Other methanisms by which alcohol stimulates carcinogenesis include the induction of cytochrome P4502E1, associated with an enhanced activation of various procarcinogens present in alcoholic beverages, in association with tobacco smoke and in diets, a change in the metabolism and distribution of carcinogens, alterations in cell cycle behavior such as cell cycle duration leading to hyperregeneration, nutritional deficiencies such as methyl, vitamin A, folate, pyrridoxalphosphate, zinc and selenium deficiency, and alterations of the immune system, eventually resulting in an increased susceptibility to certain viral infections such as hepatitis B virus and hepatitis C virus. SP05 Intrinsic susceptibility to cancer, particularly head and neck squamous cell carcinoma J.Cloos, C. R. Leemans, G. B. Snow, B. J. M. Braakhuis University Hospital Vrije Universiteit, Amsterdam, The Netherlands It has long been acknowledged that exposure to carcinogens alone is not sufficient to explain all environmentally related cancers. Currently, the research field of molecular epidemiology is exploring possibilities to improve cancer risk assessment

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by the inclusion of individual intrinsic biomarkers of cancer susceptibility. One such biomarker, mutagen sensitivity, is measuring the mean number of bleomycin induced chromatid break per cell in cultured peripheral blood lymphocytes and is thought to represent the way a person deals with carcinogenic challenges. It has been found to be increased in patients with environmentally related cancers such as lung, colon and head and neck cancer. In a multicenter trial, it was found that mutagen sensitivity itself was not greatly enhancing cancer risk in non-smoking subjects (odd ratio: 2.6 (95% confidence interval (CI): 0.9-7.6). However, the odds ratio for developing head and neck cancer increased up to 44.6 (CI: 17.4-114.0) in heavy smokers (≥ 25 pack-years). Our results of a combined family and twin study showed a heritability estimate of 77% for the susceptibility to bleomycin induced chromatid breaks which indicates a clear genetic basis. The findings of this study support the notion that a common genetic susceptibility to DNA damage and thereby a susceptibility to cancer exists in the general population. This is particularly important for the identification of person at high risk for cancer, who may get an intense follow-up and be enrolled in (chemo)prevention trials. SP06 Reduction of Tobacco-Related Cancer. D. Hoffmann and E. L. Wynder. American Health Foundation. In 1991. cigarette smoking contributed to 77,2 % of the 9.800 deatlis from cancer of the esophagus and to 31.2 % of the 25.200 deaths from cancer of the pancreas in the USA. In addition, smokers of cigars and pipes, as well as snuff dippers faced an increased risk of cancer at these two sites. Approaches toward reducing these cancer risk factors include health education from kindergarten through high school as well as counseling and treatment for cessation of tobacco use for those already afflicted with these habits. Treatineni consists mainly of medications such as nicotine gum, patch, or nasal spray. Where these cessation methods fail, preventive aims include modification of tobacco products and the use of chemopreventive agents. Moreover, attempts are made to substitute nicotine by analogs that do not lead to addiction, nor to the formation of carcinogens during smoking or snuff dipping . Clearly, there will never be a safe cigarette: however the methods currently under investigation are urgently needed to reduce tobacco-related cancer risk. Supported by research grants #CA 17613 and CA 76328 from the U. S. National Cancer Institute. SP08 Occupational exposures and cancer – epidemiological aspects and/or facts for carcinogenesis Paul Brennan International Agency for Research on Cancer, Lyon There exist strong socioeconomic differences in incidence and mortality rates of gastrointestinal (GI) cancer which indicate that occupational exposures may be relevant. The only estab-

lished occupational carcinogen of GI-cancer (Group 1 in the IARC Monograph series) is vinyl chloride which is strongly associated with angiosarcoma of the liver. Extremely high levels of exposure appear to be necessary for tumour induction although, because of the rarity of the tumour, the increased risk among highly exposed workers is in the order of 400 fold. Aflatoxin and hepatitis B and C infection may also be considered occupational risk factors for hepatocellular cancer in certain situations. Probable human carcinogenesis of GI cancers (Group 2A of the monograph series) include solvents which have been reported for both oesophageal and liver cancer. Several studies have reported increased risks of gastric cancer for occupations associated with high levels of fibre and dust exposure. These occupations include coal mining, asbestos mining, leather workers, rubber workers, construction workers and workers exposed to metal compounds. It is possible that carcinogenic dusts reach the stomach by first being inhaled, and then cleared by the lung and swallowed. Occupations associated with low physical activity are associated with an increased risk of colon cancer, particulary in the descending colon and sigmoideum. Biological mechanisms for this relationship have been proposed including the stimulation of colon peristalsis and prostaglandin by exercise. Although the validity of these association are still unclear they have a strong public health relevance and require further attention. SP10 Sulindac Chemoprevention : Metabolites and Dose Relations in FAP – Influencing PGE2 and Leucotrienes G Winde, B Glodny, N Lügering2, W Schlegel3, H Bartsch4 Depts. of Surgery,2 Medicine,3Gynecology of the WWU Muenster and 4German Cancer Research Center Heidelberg Aim: The NSAID sulindac and its metabolites (sulfide and sulfone) are preventive against colorectal adenomas in FAP. We aim on 1.) the necessity of giving sulindac metabolites as single chemopre-ventive agents, 2.) the effective dose and 3.) the dose dependent influence of sulindac on prostaglandins and leucotrienes. Material/methods: Patients with FAP (33) of an chemopreventive trial, Crohn’s disease (21), colorectal cancer (20) and 20 healthy volunteers had regular follow-up. Sulindac given as coated tablets or as suppositories (300 to 50 mg/day). PGE2/ PGF2alpha determined by time resolved fluorescence immuno assay; plasma leucotrienes (LTB4) detected by ELISAs; sulindac and metabolites detected by HPLC. Results: In a seven year follow-up, 50 mg/d was the median effective maintenance dose of adenoma reversion in 85% of the FAP patients. In FAP plasma/tissue samples after rectal low dose sulindac, sulfone/sulfide metabolites were detected more often than after high dose use (t-test p < 0.05). Oral sulindac in coated tablets (150 mg) for local drug delivery, sulindac sulfide and sulfone appeared dose dependent in plasma. PGE2/PGF2alpha in plasma decreased (Wilcoxon test, p< 0.05). LTB4 differed in FAP with complete vs.partial adenoma reversion vs. controls. Conclusions: Sulindac (50 mg/d) mostly is the effective dose in adenoma preven-tion in FAP.Sulindac sulfide/sulfone are metabolized in the epithe-lium; coated oral or rectal sulindac are prodrugs for anti-COX II activities. LTB4 aims on influencing the 5-lipoxigenase pathways .

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POSTER-PRESENTATION Session 1: Esophageal carcinogenesis From reflux esophagitis to Barrett carcinoma P1.01 Rat model for esophageal adenocarcinoma M Fein, KH Fuchs, JH Peters*, P Chandrasoma*, TR DeMeester* Dept. Surgery, Univ Würzburg, Germany and *USC, Los Angeles Aim: In the rat model, esophageal adenocarcinoma develops following administration of nitrosamine and surgically induced duodenal reflux into the esophagus. Duodenal reflux alone could induce adenocarcinoma in the glandular stomach as well as in the squamous forestomach. We questioned whether carcinogen was necessary for cancer development in the esophagus following surgically induced reflux of duodenal juice. Methods: Three groups of 26 rats each were randomized to a surgical procedure to produce one of the following reflux models: Esophagojejunostomy with gastroduodenal reflux, total gastrectomy and esophagojejunostomy with duodenal reflux, or Roux-en-Y reconstruction without reflux. The animals were killed at 24 weeks of age and the esophagus was histologically evaluated. Results: Severe esophagitis was found in all animals with reflux. Sixteen weeks postoperative and without carcinogen administration 87% of the rats developed columnar-lining of the distal esophagus and 48% adenocarcinoma at the anastomotic site. Cancer prevalence was similar in rats with gastroduodenal or duodenal reflux. Conclusions: Duodenoesophageal reflux is carcinogenic in the rat model. Exogenous carcinogen is not necessary for cancer development.

Session 2: Gastric carcinogenesis Helicobacter and gastric malignancy P2.01 BILE ACIDS CONTENT AND MUTAGENICITY OF GASTRIC JUICE OF GASTRIC CANCER PATIENTS K. Choi, S. Lee, Y. Moon, Department of Surgery, M. Chang, Department of Microbiology, Kosin Medical College, Pusan, Republic of Korea B. Chung, Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea Purpose: Since duodenogastric reflux gastritis is a precancerous condition and one of the most important factors inducing gastric stump cancer, we compared bile acid content and mutagenicity of gastric juice between gastric cancer patients and controls. Methods: The gastric juice was collected during operation in the gastric cancer patients by a needle puncture of

the fundus of the stomach and during endoscopic examination in the control subjects. The samples were analyzed for the contents of various bile acids (gas chromatography/mass spectrometry) and mutagenicity (Xenometric AMAX™ system). Results: The gastric juice of gastric cancer patients showed significantly high contents of cholic acid (CA, 25.49±24.76 24 SD mg/ml), chenodeoxycholic acid (CDCA, 13.73¡3⁄4 11.19) and deoxycholic acid (DCA, 13.04±13.30), a significantly low content of ursodeoxycholic acid (UDCA, 3.52±2.49), and a similar content of lithocholic acid (LCA, 1.08±1.19), compared to those in the gastric juice of control subjects (CA, 11.68±2.84; CDCA, 5.84±2.86; DCA, 6.33±3.32; UDCA, 4.96±0.95; LCA,1.07± 0.88). Eleven out of 36 gastric juice samples of gastric cancer patients showed positive mutagenicity, whereas only one out of 35 control samples showed positive mutagenicity. The mutagenicity of the gastric cancer patients was significantly correlated with the CA and CDCA contents. Conclusion: The gastric juice of gastric cancer patients contained high concentrations of bile acids, some of which may act as mutagen. Reference: 1. van Heerden JA, Priestly, Farrow GM, Philips SF. Postoper-ative alkaline reflux gastritis. Am J Surg 1969; 118:427-433 2. Ritchie Jr.WP. Reflux gastritis as a surgical problem. Difficult Problems in General Surgery, Year Book Publishers, Inc 1989 pp 79-86 3. Miwa K, Hattori Y, Miyazaki I. Duodenogastric reflux and fregut carcinogenesis. Cancer 1995; 75:1426-32 4. Langhans P, Heger RA, Hohenstein J, Schlake W, B nte H. Operation-sequelae carcinoma of the stomach:experimental studies with or without resection. World J Surg 1981; 5:595-605 5. Correa P. A human model of gastric carcinogenesis. Cancer Res 1988; 48:3554-3569 P2.02 IS INTESTINAL METAPLASIA A REAL RISK FACTOR FOR GASTRIC CARCINOMA? S. Miehlke, A. Meining, E. Bayerdörffer, M. Stolte. Med. Dept. I, Technical Univ. Hospital, Dresden, Med. Dept. II; Technical Univ., Munich, Inst. of Pathology, Klinikum Bayreuth, Germany Background: Intestinal metaplasia (IM) is considered as a condition in a sequence leading to gastric carcinoma of the intestinal type. A history of duodenal ulcer disease (DU) associated with H. pylon (Hp) infection appears to protect of gastric carcinogenesis. Aim: To investigate the occurrence of IM in patients with Hp-associated DU in comparison with Hp-infected patients suffering from early gastric cancer (EGC). Methods: In a total of 400 patients (200 Hp+ EGC, 200 Hp+ DU), 2 antrum and 2 corpus biopsy specimens each were examined for the presence of IM. Hp gastritis was graded in accordance with the updated Sydney System. Results: Intestinal metaplasia was detected in 151/200 (75.5%) EGC patients (100/116 in intestinale type cancer (86.2%), 47/79 in diffuse type cancer (49.5%)) compared with 44/200 (22%) DU patients (p<0.001). After adjusting the data for antrum-predominant gastritis, the frequency of intestinal metaplasia was 49/200 (18.5%) and 37/200 (24.5%) in EGC and DU patients, respectively (n.s.). Conclusions: The presence of IM is highly associated with EGC. Yet, after adjusting the data to an equal distribution of gastritis there was no significant differences in the

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frequency of IM between EGC and DU patients. Thus, the association of IM and gastric cancer may depend on the distribution of gastritis. IM per se may therefore not be regarded as an independent risk factor of gastric cancer. P2.03 Does Helicobacter pylori accelerate gastric carcinogenesis or not T Sakagami, N Yamamoto, Y Nakamura, N Tanida, T Shimoyama Internal Medicine 4, Hyogo College of Medicine, Hyogo, Japan Aim : The aim of this study is to test the susceptibility of gastric carcinogen in Hp infected gastric mucosa using mice model. Materials and methods :Six weeks old SPF female C57BL/6 mice were used as experimental animal and Helicobacter pylori SS1 strain (cag ve+, vac ve+) was used. Animals were divided into 2 groups, ie; Hp infected and Hp uninfected group. At 4 months after infection, at this point chronic active gastritis was already induced by Hp infection in this model, N-methylN-nitrosourea (MNU) was inoculated in both group animals for 18 weeks. At 50 weeks after MNU treatment all animals were sacrificed to assess pathological findings. Result : Only one tumor in stomach was detected glossily in MNU alone group(1/13). No tumor in stomach was detected in Hp+MNU group. Three peritoneal tumors (including lymphnode) were observed in each groups. Conclusion : Our findings suggest the susceptibility of gastric chemical carcinogen is not enhanced in Hp infected gastric mucosa. P2.04 Endogenous formation of nitroso bile acids and carcinogenesis following reflux of duodenal juice M Fein, KH Fuchs, S Diem*, M Herderich* Chirurgie, *Inst. f. Lebensmittelchemie, Univ Würzburg, Germany Aim: Esophageal adenocarcinoma is induced by duodenal reflux in the rat. A proposed mechanism of carcinogenesis relies on the reaction of physiologic bile acids with nitrite to produce carcinogenic N-nitroso amides (Miwa et al, Cancer 1995). To test this hypothesis, we analyzed duodenal juice for the presence of nitroso bile acids before and after reflux inducing surgery. Methods: Rats were operated with total gastrectomy and esophagojejunostomy to induce reflux of duodenal juice into the esophagus. Duodenal juice (5 ml) was aspirated before suturing the anastomosis and stored at -80°C. After 2 weeks (n=3) and 6 weeks (n=3) duodenal juice was aspirated and analyzed within two hours together with the preoperative samples. Combined chromatography and mass spectroscopy analysis (HPLCESI-MS/MS) was focused on the detection of the two major bile acids TCA (m/z 516) and GCA (m/z 466) and their nitroso derivates (m/z 545 and m/z 495). Results: The expected pattern of bile derivatives with TCA and GCA as predominating conjugates was detected in all samples,

preoperative and after 2 and 6 weeks. However, even selective reaction monitoring experiments failed to demonstrate the presence of any N-nitroso TCA or GCA. In addition, other nitroso derivatives could not be detected in any of the samples by neutral loss experiments monitoring the loss of NO (-30 u). Conclusion: Because of the very high sensitivity of the applied methods endogenous formation of N-nitroso TCA and GCA appears questionable. Therefore, other mechanisms for carcinogenesis following reflux of duodenal juice have to be discussed. P2.05 Laser-Capture Microdissection: Modern Analysis of Molecular-Genetic Alterations in Gastric Cancer M Breidert, S Miehlke, J Gillespie, E Bayerdörffer, G Ehninger Internal Medicine I Techn.University of Dresden, Gerrnany; NCI, Bethesda, USA Many investigators have postulated a sequence of histologic events in the progression to gastric adenocarcinoma (Correa, 1998), definitive experimental or clinical evidence confirming this progression does not exist. Another hypothesis claims that gastric cancer develops from a stem cell (Hattori, 1986). Molecular analysis of cell populations in their native environment will be an important feature of medical genetics in the next years. Therefore, the recently new developed method of laser capture microdissection (LCM) is a reliable method to procure pure populations of cells from specific microscopic regions of tissue sections under direct visualization. The cells of interest are transferred to a polymer film that is activated by laser pulses. The exact morphology of the procured cells with intact DNA or RNA is retained and held on the transfer film. The fluctuation of expressed genes or alterations in the cellular DNA can ultimately be compared within or between individual patients. In the same tissue section, normal epithelium, premalignant lesions and cancer foci can be sampled and compared. Here we demonstrate the first data with the new LCM-technique in microdissected human gastric tissue of first degree relatives of patients with gastric carcinoma. P2.06 Expression of cyclin E, p27 and E2F-1 in gastric carcinomas and their lymph-node metastases W. Yasui, T. Suzuki, J. Fujimoto, K. Naka, H. Yokozaki and E. Tahara Dept. Pathology, Hiroshima Univ. Sch. Med., Hiroshima, Japan Aim: The cell cycle is controlled by positive and negative regulators. Gene abnormalities and aberrant expressions of various cyclins/CDKs and CDK inhibitors may play a pivotal role in the stomach carcinogenesis. To clarify the role of cyclin E, CDK inhibitor p27 and their target molecule, E2F-1 in tumor metastasis, the expression of these molecules was examined in gastric carcinomas with lymph-node metastasis. Materials and Methods: Immunohistochemical studies using specific antibodies were performed on paraffin-embedded sections of primary tumors and their lymph-node metastasis of gastric carcinoma.

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Results: Reduced expression of p27 as well as increased expression of cyclin E correlated well with advanced tumor stage, depth of tumor invasion and presence of lymph-node metastasis. Most of gastric carcinomas with metastasis showed reduced p27 expression. p27 was negative in 39% of primary tumors, while it was so in 52% of lymph-node metastases. p27 expression was weaker in lymph-node metastasis than in primary tumor in 43% of cases. On the other hand, over 70% and 50% of the cases expressed cyclin E and E2F-1 at nearly the same levels in both primary tumor and lymph-node metastasis, respectively. Conclusion: These results suggest that tumor cells with reduced p27 expression selectively metastasize to lymphnode or distant organs. P2.07 Analysis of diamine oxidase expression in gastrointestinal tumors H. G. Schwelberger, P. J. Klingler, E. Bodner Department of Surgery, University of Innsbruck, Austria Aim: Besides histamine, diamine oxidase (DAO, EC 1.4.3.6) also degrades putrescine and other biogenic amines. These polyamines are involved in cell proliferation and differentiation and therefore a role for DAO in these processes has been proposed. Especially the expression of DAO in various human tumors has been of interest but systematic investigations of tumor associated DAO expression in a larger series of patients have not been performed. Employing a highly sensitive and specific assay for DAO activity we analyzed the presence of the enzyme in various gastrointestinal tumors. Methods: Duplicate biopsies of 10-100 mg were obtained both from the tumor and from the macroscopically healthy adjacent tissue, respectively, immediately after resection. The specific activities of DAO were determined in cleared homogenates of the bioptic samples by a radiometric micro-assay based on the conversion of 14C-putrescine. Results: DAO activity was found in all tumors of the colon, the sigma, and the rectum but the activity was reduced compared to the adjacent normal tissue. In neoplasms of the stomach, DAO activity was usually found to be higher in tumor tissue than in adjacent healthy tissue. Whereas DAO activity can not be detected in human liver tissue we found the enzyme to be present in all liver metastases derived from colorectal neoplasms, but not in metastases from a lung tumor or in hepatocellular carcinoma. DAO activity was also found in a peritoneal metastasis of a large bowel carcinoma although DAO is normally not expressed at this site. Conclusion: Our results indicate that cells constituting gastrointestinal neoplasms retain the ability to express diamine oxidase not only in situ but also when tumor cells metastasize to other organs. Expression of DAO in tumor cells could possibly provide a growth advantage related to histamine or polyamine metabolism and might be interesting for the detection of tumor cells in tissues that normally lack this enzyme.

P2.08 Vitamin A deficient diet in experimental gastric carcinogenesis J. Klocker1, M. Gadenstätter1, P. Klingler1, R. Schindler2, A. Kreczy 3, E. Bodner1, J. Sattler1, H. G. Schwelberger1 1 Univ.-Klinik für Chirurgie, University of Innsbruck, Austria; 2 Institut f. Humanernährung und Lebensmittelkunde, University of Kiel, Germany; 3 Institut f. Pathologische Anatomie, University of Innsbruck, Austria Aim: It has been demonstrated consistently that individuals with diets rich in fruit and vegetable tend to have a lower risk of several types of cancer. Studies on specific nutrients in these foodstuffs raised interest in vitamin A. In contrast to data on vitamin A supplementation, information concerning possible effects of vitamin A depletion on gastrointestinal carcinogenesis is scarce. Therefore, we used a rat model of gastric stump cancer to study the influence of a long-term vitamin A deficient diet on the incidence of gastric cancer. Methods: A gastrojejunostomy was performed in male Wistar rats. Postoperatively, the animals received the carcinogen MNNG in the drinking water and either a standard diet or a vitamin A depleted diet. Subpopulations of each group were sacrificed 30 and 40 weeks after the operation and the anastomotic site was analyzed histologically. To detect changes in intestinal proliferation, activities of diamine oxidase (DAO), one of the major enzymes catabolizing polyamines, were determined using a radiometric assay. Plasma and liver vitamin A concentrations were analyzed by HPLC. Results: Whereas the rates of cancer and premalignant lesions were not different for the control and vitamin A depleted groups, DAO activities in all intestinal sections were significantly higher in the vitamin A depleted animals (duodenum: 0.198 vs. 0.102; jejunum: 0.377 vs. 0.323; ileum: 0.876 vs. 0.502, colon 0.069 vs. 0.055; median values in mU per mg protein). Conclusion: We conclude that in our rat model of gastric cancer vitamin A depletion does not lead to an increased incidence of neoplastic lesions as would have been expected. Nevertheless, changes observed in DAO activities indicate that vitamin A depletion may significantly influence polyamine metabolism und intestinal proliferation. P2.09 Clonal Analysis of Poorly Differentiated Gastric Carcinomas with Signet Ring Cells Masamichi BAMBA, Hiroyuki SUGIHARA, Katsuji OKADA, Ryoji KUSHIMA, Takanori HATTORI First Dept. of Pathology, Shiga Univ. Med. Sci., Ohtsu, JAPAN (AIM) An unanswered question concerning the histogenesis of superficial-type gastric carcinoma is whether it is monoclonal or multiclonal in origin. Therefore, we analyzed multiple areas of each cancer with a clonality assay that was subject to random inactivation of X chromosomes. (MATERIAL/METHODS) The human androgen receptor gene (HUMARA) assay was applied to 15 gastric carcinomas manifested in superficial depressed lesions of various sizes and at least some signet ring cells. (RESULTS) In the 15 cases examined, 10 cancers were

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informative, and in these 10 cancers a total of 63 areas were analyzed. In 8 of the 10 cancers, the inactiveted allele was common to all the informative areas of each tumor (80%) regardless of the macroscopic shape or the degree of histologic heterogeneity. In one of the two remaining cancers, the inactivated allele of one of the areas was different from those in the other areas. In a remaining cancer, the inactivated allele couldn’t be decided in all 3 areas. (CONCLUSIONS) Most of the superficial depressed-type, poorly differentiated gastric carcinomas with signet ring cells were demonstrated to be of monoclonal origin in this study. P2.10 Fractional allelic loss in gastric carcinoma correlates with growth patterns S-W Choi, S-W Park, K-Y Lee, K-M Kim, Y-J Chung and M-G Rhyu College of Medicine, the Catholic University of Korea, Seoul 137-701, Korea AIM: To gain insight into genetic events underlying morphological phenotypes, we analyzed 58 gastric carcinoma tissues for the genome-wide allelotype study using microsatellite markers. MATERIAL/ METHOD: Based on a binomial distribution, loss of heterozygosity (LOH) that was significantly more frequent than expected (P<0.05) thus interpreted as nonrandom LOH selected during tumorigenesis. The overall extent of chromosomes undergoing LOH, fractional allelic loss (FAL, the ratio of LOH-positive markers divided by the total number of informative markers), was measured in each tumor patient. RESULTS: Nonrandom LOH was found on 17p (48.0%), 18q (38.4%), 13q (38.1%), and 9p (36.4%). Overall, there were no significant phenotypes correlated with allelic loss on specific chromosome regions. Because FAL values showed bimodal distribution with two peaks bordered by a mean of 0.233, tumors were classified into LOH-related (>0.233) and LOH-unrelated (<0.233) types. Twenty-four patients with high FAL value, demonstrating LOH-related tumors, evidenced a significant trend for FAL to increase with development of an infiltrative type of cellular growth pattern. CONCLUSION: This study demonstrates that the growth pattern of gastric carcinoma correlates with FAL, a malignant phenotype influenced by LOH event. P2.11 Histological heterogeneity and genetic abnormalities in gastric carcinoma with microsatellite instability Y-J Chung, K-M Kim, J-R Choi, and M-G Rhyu College of Medicine, the Catholic University of Korea AIM: Microsatellite instability (MSI)-mutator phenotype variably targets microsatellite-like sequences in coding regions of cancer-related genes. Gastric tumor tissues with MSI were evaluated for their microscopic features in relation to the distribution of MSI-associated mutations. MATERIAL/METHOD: One hundred and fifty tumor sites obtained from 51 gastric cancer patients were microdissected with respect to histological

and topographical clonality. RESULTS: We found 11 gastric carcinomas with MSI, showing by marked morphological heterogeneity. Most tumor sites obtained from MSI-phenotype tumors exhibited intestinal-type histology accompanying the TGF-RII mutation but three tumor sites with the diffuse type. Of the three diffuse-type tumor sites obtained from the MSIcases, two had neither MSI nor TGF-RII mutation. Five patients had E2F-4 mutations in MSI expressing tumor sites in which glandular structures of intestinal-type tumor were variably differentiated. In two of the five patients, the better differentiation grade was observed in individual tumor sites showing no or less E2F-4 frameshift mutations. CONCLUSION: Overall, gastric carcinoma with MSI manifested heterogeneous histological subtypes, which were, in part, associated with frameshift mutations in TGF-RII and E2F-4 genes. P2.12 METHYLATION OF THE PEPSINOGEN A GENES IN 19 STOMACH CANCER PATIENTS K. P.Hanson, A. R. Shumakov, V. P.Kalinovsky N. N.Petrov Research Institute of Oncology, St.Petersburg, Russia Aim: study the methylation status of human pepsinogen A (PGA) genes in stomach cancer patients. Material/Methods: Genomic DNA, isolated from normal and tumor stomach mucosa of 19 patients, was digested with HpaII restriction enzyme and hybridized with cDNA probe of the Rhesus monkey PGA (Evers M. P. J. et al., 1988). Results: Methylation level of the PGA genes in stomach tumors of 12 patients (63 %) was lower than in normal mucosa. This reduction was opened not only in pronounced tumors, but also in early stomach cancers. There is a strong correlation (P < 0,01) between reduced metylation in tumors and grade of cell differentiation. Conclusion: Obtained data suggest that methylation of PGA genes is a process, which start at the early stages of gastric carcinogenesis. In analysis of human pepsinogen genes, obtained by M. Ichinose et al. (1991), screened the same data (reduction of methylation level at different stages of stomach cancer). P2.13 Chromosomal Imbalances In Gastric Cancer. A Comparative Genomic Hybridization Analysis W. Müller, H.-C. Wirtz, T. Noguchi, S. Michaelis, H. E. Gabbert Institute of Pathology, University of Düsseldorf, Germany Aims: The purpose of this study was to identify altered chromosomal regions that might contain candidate genes of importance for the development and progress of gastric carcinomas. Methods: Genetic instability was analyzed in 38 gastric carcinomas by comparative genomic hybridization (CGH). The profiles of DNA copy number changes were correlated with the pT and pN category as well as tumor histology according to Lauren. Results: In all 38 gastric carcinomas investigated DNA copy number changes could be demonstrated by CGH analysis rang-

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ing from 1 up to 11 different aberrations (mean: 7). Whereas gains of chromosomal regions could be observed frequently at the long arm of chromosomes 20, 12, 17, 15, 11, and 2 with frequent hyperamplifications at 20q 12-13 and 17q21, loss of tumor DNA was a rare event and referred to chromosomes 4q, 13q and 5q. Neither gains nor losses were correlated with the pT or pN category. Only gain of 17q was significantly more often found in intestinal type carcinomas than in the diffuse type. Widespread chromosomal aberrations could already be found in all early gastric carcinomas investigated. Conclusions: According to our results, genetic instability including multiple chromosomal aberrations is a common feature in gastric cancer occuring early during tumor progress. The present CGH analysis provides a new set of candidate regions of interest at 12q, 15q and 11q that have not yet been associated with gastric cancer

Session 4: Pancreatic carcinogenesis From chronic pancreatitis to ductal pancreatic cancer P4.01 Development of a Highly Metastatic Human Pancreatic Cancer Cell Line in a Murine Model T. Breslin, P. Hallin, J. Abbruzzese, L. Larry, P. Chiao, D. Evans The University of Texas M. D. Anderson Cancer Center, Houston, Texas USA Aim: To develop an experimental model of pancreatic carcinoma metastasis using direct injection of pancreatic carcinoma cell lines into the portal vein of nude mice. Material/Methods: ATCC cell lines AsPc1, MiaPaca, CFPac, CaPan1, and Panc1 were grown in tissue culture, and 1 × 107 cells were injected into the portal vein of nude mice. Animals were surgically explored to detect the presence of hepatic metastases. Metastatic lesions were harvested, cultured in vitro, and re-injected into the portal vein. Results: Hepatic metastases were established in AsPc1, MiaPaca, Panc1, and Capan1. Metastatic potential correlates with the alterations in the TGF-β / DPC4 signal transduction pathway. AsPc1 has been through 5 successive passages in the nude mouse. Time to metastasis has decreased with increasing passage number. See Table.(LM=Liver Metastasis) Cell Line

#w/ met

Days to met

AsPc1 LM-1 LM-2 LM-3 LM-4

2/3 2/5 2/5 1/6 3/9

59 26 35 15 16

Conclusions: A highly metastatic clone has been selected through successive injections into the nude mouse. The rapid of development of metastatic lesions may decrease the time required for evaluation of novel therapeutic agents aimed at eradication or inhibition of hepatic metastases. Further study of these metastatic cell lines may provide further insight into the

predilection of pancreatic carcinoma for development of hepatic metastases. P4.02 Angiogenesis, p53 and H-ras mutations in pancreatic ductal adenocarcinomas Erdener Özer, Sermin Özkal, Sedat Karademir, Özgül Sadol, Selman Sökmen, Ahmet Çoker, Ali Küpeliodlu, Y´brahim Astarcy´odlu Dokuz Eylül University Hospital, Izmir, TURKEY To evaluate the correlation of angiogenesis, p53 and H-ras mutations with prognostic factors and proliferative activity assessed with Ki-67 protein expression in primary pancreatic ductal adenocarcinomas, the archival tissues of twenty four patients were studied. Vascular endothelium were labeled immunohistochemically using factor-VIII-related antigen. The vascular surface density (VSD) and the microvessel number (NVES) were assessed by means of stereology. The expression of p53, H-ras and Ki-67 proteins were analyzed by immunohistochemical method. Both NVES and VSD showed a statistical significance with metastatic potential, Ki-67 expression, histologic differentiation and tumor size. p53 mutations were found in 11 cases (45.8%). However only 3 cases (12.5%), all negative for p53 mutations showed H-ras mutations. p53 mutations exhibited a statistical significance with metastatic behaviour and Ki67 expression, whereas H-ras mutations showed no significance with prognostic factors. We conclude that angiogenesis might have a role in predicting prognosis in pancreatic carcinomas and p53 mutations might be acquired in the later stage associated with metastatic progression and higher proliferative activity. Although H-ras mutations were rare in the present study, we think that they might play a role in a different carcinogenetic pathway excluding p53 mutations. P4.03 Cooperation between transcription factor AP-1 and NF-kB in the induction of interleukin-8 in human pancreatic adenocarcinoma cells by hypoxia Qian Shi, James L. Abbruzzese, Christiane Brun, Suyun Huang, Naofumi Mukaida, Kouji Matsushima, Isaiah J. Fidler, and Keping Xie Departments of Gastrointestinal Medical Oncology and Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030 The regulation of IL-8 gene expression in human pancreatic cancer by hypoxia was investigated. Exposure of FG human pancreatic cancer cells to hypoxia (< 1% O2 , 5% CO2 and > 94% N2) resulted in a time-dependent increase in steady-state levels of IL-8 mRNA and in increased amount of IL-8 protein secretion as compared to normoxia (5% CO2 and 95% air). Hypoxia-mediated transient induction of IL-8 expression involved the transcriptional activation of IL-8 gene as determined by nuclear run-on assay. Deletion mutation analysis of the IL-8 promotor revealed that the sequence between -133 and -85 bp relative to the transcription initiation site significantly contribut-

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ed to the IL-8 gene transactivation by hypoxia. Point-mutation analysis further indicated that AP-1 and NF-kB binding elements were responsible for the induction of the IL-8 gene by hypoxia and that IL-8 transcription activation by hypoxia required the cooperation of NF-kB and AP-1. In conclusion, hypoxia upregulates IL-8 gene and contribute to the progression and metastasis of human pancreatic cancer. P4.04 Overexpression of interleukin-8 correlates with increased malignancy of human pancreatic cancer cells Qian Shi, James I. Abbruzzese, Christian Brun, Suyun Huang, Isaiah J. Fidler, and Keping Xie Departments of Gastrointestinal Medical Oncology and Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030 The role of interleukin-8 in growth and metastasis of SG, FG and L3.3 variants derived from COLO 357 parental human pancreatic cancer cells was determined. After orthotopic implantation in pancreas of nude mouse, SG produced smallest tumors, whereas L3.3 produced largest tumors. SG did not produce any liver metastasis, whereas FG produced numerous liver metastases and L 3.3 produced more and larger liver metastases. In vitro analysis of IL-8 expression indicated that SG expressed lowest level of IL-8 gene as determined by northern blot analysis and ELISA, whereas L 3.3 expressed highest level of IL-8. To provide direct evidence for the role of IL-8 in aggressive biology of pancreatic cancer, FG cells were transfected with sence or antisence IL-8 expression vectors. The neomycin-transfected FG cells were used as controls. Overexpression of IL-8 accelerated growth of FG tumor cells in mice after intropancreatic implantation, whereas decreased IL-8 expression after IL-8 antisence transfection retarded the growth of FG in mice after intropancreatic implantation. Our data demonstrated that overexpression of IL-8 contribute to the aggressive biology of human pancreatic cancer.

Session 5: Colorectal carcinogenesis Hereditary cancer syndromes P5.01 Mutations of the mismatch repair gene hMSH6 in kindreds with familial colorectal cancer J. Plaschke, C. Kruppa, R. Tischler, S. Pistorius1, H. Dralle2, J. Rüschoff3, H. D. Saeger1, H. K. Schackert Surgical Research and 1 Surgery, Technical University Dresden, 2 General Surgery, University Halle, 3 Pathology, Klinikum Kassel, Germany Aim: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited cancer susceptibility syndrome with high penetrance. Germline mutations in the both mismatch repair genes hMSH2 and hMLH1 have been found as disease-causing predispositions for the majority of HNPCC

cases. Recently, two germline mutations of hMSH6 have been associated with HNPCC. In this study we analyzed the involvement of hMSH6 in kindreds without evidence of hMSH2 and hMLH1 mutations. Material/Methods: The coding sequence and flanking intron regions of hMSH6 were completely sequenced in eleven patients from HNPCC or HNPCC -like families. In addition, microsatellite analysis was performed using ten mono- and ten dinucleotide repeat markers. Results: One frameshift mutation was found in the germline of an HNPCClike kindred and was accompanied by a second, somatic frameshift mutation in the tumor. The tumor was unique among the analyzed cases for the combination of elevated mono- but no dinucleotide repeat instability and lack of LOH at loci frequently affected in colorectal carcinomas. A missense mutations with unknown biological consequence was detected in an HNPCC family and was accompanied by low MSI for mono- but no MSI for dinucleotide repeat markers in the tumor. Further we found 2 silent and 16 intronic variations previously not reported. Conclusions: This study presents further evidence for hMSH6 germline mutations to be predispositions for HNPCC-like phenotypes, although the frequency is low. Larger studies are needed to evaluate the benefit of predictive mono- and dinucleotide repeat instability testing for discrimination between individuals harboring an hMSH2 or hMLH1 germline mutation or a mutation of the hMSH6 gene. P5.02 Histological features of serrated adenocarcinoma(SACA)arising from serrated adenoma(SA) of the colorectum F.Shimamoto1, S.Tanaka2, T.Hiyama3, K.Hayashi3, T.Nishida4, K. Ono5, and E. Tahara3 1 Department of Pathology, Hiroshima University Hospital, 2 Department of Internal Medicine and 3 Department of Pathology, Hiroshima University School of Medicine, 4 Chugokurosai Hospital and 5 NTT Hiroshima Health Administration Center, Hiroshima, Japan We examined the histological features of SACA in order to make a precise diagnosis of colorectal carcinogenesis. Materials and Methods; Forty eight colorectal adenocarcinomas with / in SA were histologically examined with classification on the basis of tumor growth into superficial-type (S-SA) and polypoid-type serrated adenoma (P-SA). Result; 48 lesions were divided into 27 P-SAs and 21S-SAs. Various kinds of histological features found in SACA showed ordinary well differentiated adenocarcinomas with tubulovillous growth and / or serrated glands, ordinary moderately differentiated adenocarcinoma with pseudocribriform formation, mucinous carcinoma, and SACA with glands similar to gastric foveolar epithelium. In S-SA, early minute SACAs begin within the crypt base of SA, and then are prone to invade immediate submucosa beneath muscularis mucosa, whereas in P-SA they begin within the same upper crypt of SA as ordinary adenoma, but may not invade immediately muscularis mucosa. Conclusion; Although colorectal adenocarcinomas arising from S-SA and P-SA have histological and biological features, respectively, it may be difficult to make a precise diagnosis of adenocarcinomas asrising from SA, if they don’t contain component of SA within carcinoma.

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P5.03

P5.05

RELATIONSHIP OF ANGIOGENESIS AND p53 PROTEIN EXPRESSION IN COLORECTAL CARCINOMAS S SARIOGLU *S SÖKMEN *C TERZY´ *M FÜZÜN, AA KÜPELY´OGLU ´zmir, Dokuz Eylül Univ. Depts. of Pathology & *Surgery, Y TURKEY

GENETIC MARKERS OF COLORECTAL CANCERS P. Rahóty, O. Csuka, I. Péter, Sz. Ottó, I. Köves, I. Vámosi-Nagy, Z. Doleschall Nat. Inst. of Oncology, Dept. Surgery and Dept. Pathogenethics, Budapest, Hungary

Recently new functions have been attributed to the „policeman of the genome“, p53 protein. It is suggested that p53 protein plays a major role on the regulation of angiogenesis by downregulation of vascular endothelial growth factor (VEGF). In mutant forms of p53 protein increased angiogenesis is expected. In this study relationship between p53 protein expression and angiogenesis in 33 colorectal carcinomas have been searched. Mutant p53 protein expression and staining of the vascular structures with Factor VIII antibody were performed by immunohistochemistry. Vascular surface density (VSD), number of vessels per square mm (nVES) and mean number of nVES according to three maximum values (nVESmax) and number of vessels in unit area (N) were determined by morphometry. Statistical analysis by Spearman’s Correlation Test was not significant when VSD, nVES, nVESmax or N values were correlated with p53 protein expression. These results do not support the recent concept that, loss of wild type p53 expression gives the result of increased angiogenesis at the tissue level.

Aim: The aim of our study is to identify the expression levels of nm23, DCC, p53, CD44, Cadherin E and Ciklin D proteins in patients of colorectal cancer. Material / Methods: We have carried out immunochemistry, Western blot and PCR-SSCP analysis in 49 sporadic colorectal cancer – 18 Dukes B and 31 Dukes C tumours – and their normal mucosa of 23 female and 26 male patients. Results: In 25% of Dukes B tumors the quantity of DDC protein is decreasing or missing.The nm23 level in Dukes C tumors is lower than in Dukes B cases.The frequency of p53 mutation is the highest in Dukes C tumors,Cyclin D overexpression could be observed in 37% of colorectal cancers. Conclusion: The low level of nm23 and Cadherin E may caracterise the higher metastatisation ability of tumors.The overexpression of Cyclin D let us know about the frequent possiblity of the local recurrence of the tumor.

Session 6: Virus infection and Tumor immunology P6.01

P5.04 New hMLH1 and hMSH2 gene mutations in families with hereditary non-polyposis colorectal cancer (HNPCC) L.Papatheodorou, S.Glasl, Ch. Jung, M. Gross Medizinische Poliklinik, Klinikum Innenstadt, Ludwig-Maximilians- Universität München Aim: Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common genetic diseases. HNPCC results from germline mutations in the human mismatch-repair genes. The majority of the mutations are localised in hMLH1 and hMSH2. We screened for germline mutations in two families with HNPCC. Methods: Genomic DNA from two unrelated patients (patient 1, 37 years old; patient 2, 46 years old) that fullfil the “Amsterdam-Criteria” of HNPCC was subjected to mutation analysis by direct sequencing of hMLH1 and hMSH2. Results: In patient 1 we found a deletion of two nucleotides in exon 2 of hMLH1 (211-212, embl:hs07343) that results in a stop-codon at amino acid position 75. Patient 2 showed a deletion of six nucleotides in exon 2 of hMSH2 (deletion of Val100Glu101, nucleotide 300-305, embl:hs03911). Conclusion: The detection of two up to now unknown mutations in HNPCC shows the genetic heterogenicity of the disease. The pathogenetic role of the deletion of two amino acids has to be proved by functional assays. Interestingly the mutation in hMLH1 caused only carcinoma of the colon (Lynch I) whereas the hMSH2 deletion led to a wide spectrum of cancers (Lynch II).

Perianal skin lesions in transplant recipients M. Reh, N. Rellecke, E. Christophers, E. Stockfleth Universitäts-Hautklinik, Kiel Aim: Nearly 4000 organ transplantations are performed yearly. One of the greatest problems of the transplant recipients is the highly increased risk to develop HPV-associated warts and nonmelanoma skin cancer, predomiantly squamous cell carcinomas. The aim of this study was to investigate wether these patients have also an increased risk developing perianal HPV-associated skin tumors. Material/methods: We investigated 35 transplantated patients (kidney 17, liver 5, heart 12, lung 1) for HPV-associated skin tumors and perianal skin lesions (inspection of the anus and proctoscopy). Results: Nearly 70% of the patients showed skin tumors like warts>squamous cell carcinomas>basal cell carcinomas>Bowen‘s desease, etc. Most of these tumors had an untypical outcome and untypical location. Only one patient showed a lower rectum cancer. We found no HPV-associated tumors in the genital-/perianal region. 34 patients had typical anal diseases like anal fissure, haemorrhoidal prolapse, anal fistula, perianal abscess, etc. Conclusion: Organ transplant recipients had an highly increased risk to develop HPV-associated skin tumors. Interestingly, we didn‘t find such lesions in the anal/genital skin area. Nethertheless, the responsible Human Papillomvirus is harbored by these patients. One explanation could be that there must be a specific block of the HPV- receptor in the genital/anal area may be due to the immunosuppressive treatment. Further studies ars necessary to answer these questions.

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P6.02 CD44v6 expression as a predictive parameter of Neoplasms of the ampulla of vater M. Menges, C. Moser, M. Zeitz, A. Stallmach. Dep. Int. Medicine II, Saarland University, Homburg, Germany Accurate preoperative diagnosis of tumors of the ampulla of Vater is difficult because ampullary biopsies have a high falsenegative rate. Recently, it has been shown that expression of CD44 splicing variants including CD44v5 and v6 occur frequently in human cancers, including gallbladder carcinoma (1). Aims: In the present study, we determined whether CD44v2, -v5 and -v6 are expressed in endoscopically obtained biopsies of tumors of the ampulla of Vater. Methods: Expression of CD44v was analyzed in 14 patients with polypoid, tumor-suspect ampullae (TSA) in comparison to the pattern in adjacent duodenal normal mucosa (DM). CD44v were detected by RTPCR with primers hybridizing with CD44 sequences up- and downstream from variable exons. Subsequent southern blotting with specific probes identified CD44v2, -v5 and -v6. The quality of mRNA-preparation and cDNA synthesis of tissue samples was checked by RT-PCR using GAPDH-specific primers. Results: Histological examination revealed 6 adenomas, 2 adenocarcinomas and 6 ampullae without signs of malignancy but signs of unspecific inflammation. In 2 cases (1 carcinoma, 1inflamed papilla) quality of cDNA preparation was not sufficient. Expression of CD44-standard and CD44-epithelial was observed in all biopsies of TSA and DM. Expression of CD44v2 and -v5 was found in only 2 cases (1/ 6 adenomas, 1 carcinoma). In contrast CD44v6 was expressed in 5/6 adenomas, 1/5 normal ampullae, and in 1carcinoma. DM was negative for CD44v2 and -v5. Only 1/12 probes of DM was positive for CD44v6 (a patient with a tubulovillous adenoma of the papilla). Conclusion: These results suggest that 1) the molecular events leading to expression of CD44v6 in tumors of the ampulla of Vater occurs early in the neoplastic process and 2) the expression of CD44v6 in biopsies taken from the ampulla of Vater indicates the presence of a neoplasia. (1) Eur J Cancer. 34: 1607, 1998

Session 7: Genetic changes during carcinogenesis P7.01 Effect of tumor-associated E-cadherin mutations on cell motility and actinin-4 localization B. Luber1, G. Handschuh1, E. Mentele2, M. Sakamoto3, T. Genda3, Y. Ino3, T. Yamada3, P. Hutzler1, S. Hirohashi3, H. Höfler1,2, K.-F. Becker1,2. 1 GSF-Institute of Pathology, Neuherberg, Germany; 2 Institute of Pathology of the Technical University of Munich, Germany; 3 Pathology Division, National Cancer Center Research Institute, Tokyo, Japan We have shown that E-cadherin mutations are frequently observed in diffuse-type gastric carcinoma and influence cell adhesion, actin filament organization and cell motility in an in vitro model system. Aim: Our study was undertaken to investigate the influence of E-cadherin mutations on cell motility in living cells. We also studied the subcellular localization of the actin-bundling protein actinin-4 associated with cell motility and cancer invasion. Material/Methods: Motility of MDA-MB435S cells expressing intact or mutant E-cadherin cDNAs was investigated using a time-lapsed video system. Actinin-4 localization was studied by confocal fluorescence microscopy. Results: We found the lowest percentage of motile cells when wildtype E-cadherin was expressed. In contrast, mutant E-cadherin provoked higher motility and abundant formation of lamellipodia. The localization of actinin-4 depended on the functional status of E-cadherin with the highest concentration of actinin4 at cell protrusions involved in cell motility. In a wound healing assay, cells forced to be motile showed strong overexpression of actinin-4. Conclusion: Our results suggest that E-cadherin mutations provoke an increase in cell motility. Moreover, actinin-4 expression and localization vary according to the functional status of E-cadherin.

P7.02 HEPATOCYTE GROWTH FACTOR MODULATES INVASIVENESS OF GASTRIC CANCER VIA REDISTRIBUTION OF E-CADHERIN Sang-Uk Han, Yong-Kwan Cho, Myung-Wook Kim Departments of Surgery, School of Medicine, Ajou University, 5, Wonchon-Dong, Paldal-Gu, Suwon, Korea/442-749 Aim To study the role of hepatocyte growth factor (HGF) on the invasion of gastric cancer cells, we analyzed the expression of HGF and its receptor, Met in gastric cancer cells and studied its effect in vitro. Material/Methods Using immunohistochemistry and ELISA, we examined the expression of Met and HGF in gastric cancer. Scatter assay, and migration assay were used to study HGF-stimulated motility of gastric cancer cells. Ultrafiltration and immunocytochemistry were used to identify the localization of E-cadherin in the cells. Results In tumors, HGF was mainly detected in stromal cells, whereas Met was overexpressed in cancer cells. We observed 86% positive overexpression of Met in gastric cancer tissues. The level of HGF

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in the tumor tissue was increased according to the increase of the stage. 52% tumors revealed a presserved normal E-cadherin expression, but, abnormal E-cadherin expression was found in 48% tumors. There were significant correlations between the degrees of E-cadherin expression and the differentiation of the tumors. In vitro, 92% human gastric cancer cells overexpressed Met. HGF stimulated motogenesis and scattering of gastric cancer cells. The location of E-cadherin was shifted from membrane to cytoplasm after treatment with HGF. Conclusion HGFstimulated motogenesis of gastric cancer cells induced the redistribution of E-cadherin. P7.03 DECREASED EXPRESSION OF SHPS-1 IN TRANSFORMED CELLS S. Matsuda, K. Machida and M. Hamaguchi Department of Molecular Pathogenesis, Nagoya University School of Medicine, Nagoya 466-8550, Japan SHPS-1 is a novel transmembrane glycoprotein possessing three immunoglobulin-like domains in its putative extracellular region as well as four potential tyrosine phosphorylation sites and Src homology 2 (SH2) domain binding sites in its cytoplasmic region. It is indicated that SHPS-1 may be a direct substrate for activated protein tyrosine kinases and for nontransmembrane protein tyrosine phosphatases to regulate some physiological and pathological signals. In the process of characterizing the function of SHPS-1, we found unexpectedly that the expression of SHPS-1 was down regulated by activated tyrosine kinases such as v-Src in rat fibroblast. From the results of northern blot and RT-PCR analyses, SHPS-1 mRNA levels were specifically suppressed in the transformed cells, suggesting that SHPS-1 expression was transcriptionally down regulated. However, the protein half-life of SHPS-1 was not changed in the transformed cells compared to that in the non-transformed counterpart cells as it was examined by using a temperature sensitive mutant of v-Src. The expression of SHPS-1 was also transcriptionally silenced in the cells transformed with different type of oncogenes such as v-fps, v-crk, and H-ras, suggesting that a functional oncogenic cascade in malignant tumors can override the biological consequences of the expressed SHPS-1. This idea has been supported by our further findings from primary cancer specimens, demonstrating that tumors with poor prognosis predominantly lack SHPS-1 expression.

Session 8: Molecular biological approaches for early diagnosis and treatment P8.01 Detection of early cancer in Barrett’s esophagus. M. Vieth, M. Stolte. Institute of Pathology, Bayreuth, Germany Aim: In a recent analysis we showed that Barrett’s esophagus (BE) is correctly diagnosed at endoscopy in about 60%, BE-

carcinoma (BECA) in some 70%. BE-dysplasia in contrast was suspected in only 7.6% of the patients [1]. To investigate the question wether endoscopic evaluation of patients with BE is compatible with a high standard of qualitiy- that is how frequent early cancer in operation specimen of BECA-patients is detected. Material/methods: Medical reports on 100 consecutive patients with histologically confirmed BECA, seen between 1990 and 1996, were analysed (age: 66.8±11.3 years; m:f 3.5:1). 87 out of 100 patients with BECA were operated and complete histopathological diagnosis on the operation specimen was available. 8 patients died prior therapy and on 5 patients no sufficient medical reports were available. Results: The percentage of T4 cacinoma among the operated patients was 9.2 % (8 patients), T3 carcinoma was detected in 31.0 % (27 patients), T2 was detected in 23.0% (20 patients). T1 carcinoma in the operation specimen could be detected in 36.8 % (32 patients). Conclusion: The high percentage of detected T1 carcinoma can only be found due to sufficient endoscopy and biopsy-taking. The informative value of our study is somewhat restricted by the fact that it is a retrospective analysis. It can never be completely excluded that the endoscopist did in fact note a suspicious finding, without mentioning it. The high percentage of early cancer (36.8 %) might be an indicator for this fact. References 1. Grunewald M., Vieth M., Kreibich H., Bethke B., Stolte M. Current diagnosis of Barrett’s oesophagus: an analysis of 1000 histologically confirmed cases. Dtsch med Wschr 1997; 122: 427-431. P8.02 INTESTINAL METAPLASIA IN ROUTINE GASTRIC BIOPSY H. Steininger, G. Faller, A. Jung, R. Mäffert, Th. Kirchner Institute of Pathology, University of Erlangen-Nuremberg Aims: Intestinal metaplasia (IM) is considered to be a risk factor of intestinal type gastric cancer. The aim of this study was to evaluate the incidence of IM in a great number of gastric biopsies. Methods: 5415 routine gastric biopsies from 3011 patients were investigated. Gastritis was classified according to the UPDATED SYDNEY SYSTEM, typing of IM followed the proposal of JASS and FILIPE (IM I, II, III). Results: 25,5% normal mucosa, 64,5% different forms of gastritis, 7,2% ulcera ventriculi, 2,4% carcinomas (1,4% intestinal, 1% diffuse type), 0,2% adenomas, 0,1% carcinoid-tumors and malignant lymphomas. IM in general was observed in 568 patients (18,9%), IM III in 32 patients (1,1%). The three types of IM were found mixed in most cases. A significant correlation could be observed between the age of patients and IM and there was also a significant correlation between IM in general with all forms of inflammatory changes but not with premalignant or malignant changes of the gastric mucosa. Conclusion: 1) IM is related to age and to inflammatory changes of the gastric mucosa. 2) Different types of IM do not correlate with special forms of gastritis or special lesions of the gastric mucosa. 3) IM has no predictive value for the development of neoplastic lesions in gastric biopsy specimens.

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P8.03

P8.05

Thymidine phosphorylase (dThdPase) expression in human gastric and colorectal carcinoma: Role of P53 expression H Ito, K Shomori, T Sakatani, T Matsuura, E Okamoto, H Adachi Dep. of Pathology, Tottori Univ, Yonago/Tottori, Japan

Evidence of EBVand HSV(I+II) DNAin Ileum and Colon Mucosa in Colorectal Cancer,Crohn’s Disease orChronic Ulcerative Colitis U. Rüther1, C. Nunnensiek2, H. A. G. Müller3, A. Scheller1 1 Leonardis-Klinik, Kornwestheim, 2Ärztliche Praxis Reutlingen; 3Zentrum für Laboriumsmedizin, Klinik am Eichert, Göppingen

Aim: Clinical and biological relevance of thymidine phosphorylase (dThdPase) expression examines is in human gastric and colonic carcinomas, in terms of intratumoral microvessel density (IMVD) and P53 expression. Materials and Methods: A total of 123 gastric carcinomas and 97 colorectal carcinomas were examined for dThdPase and P53 expression. IMVD was visualized by CD34 immunoreaction. Western blot analysis was conducted in eight gastric and six colorectal carcinoma cell lines. Results: More than 10% of the carcinoma cells showed dThdPase expression in the 59 (48%) of gastric carcinomas and in the 61 (63%) of the colorectal carcinomas. Mean IMVD was significantly higher in dThdPase positive tumors than in the negative tumors in both the gastric and colonic carcinomas (P<0.05). The frequency of dThdPase expression was significantly lower in the P53-negative carcinomas than in the positive carcinomas (P<0.05). Western blot analysis showed the dThdPase expression in a variable degree, which was up-regulated by interferon-g. The expression level, however, was not correlated with p53 gene status. Conclusion: dThdPase plays a crucial role in the induction of intratumoral microvessels, regardless of tumor stage, and the expression might be modulated by not only P53 but also other unknown molecules.

Inview of the uncleared etiology of colorectal cancer,Crohn’s disease, and chronic ulcerative colitis, the question arose, whether or not Epstein-Barr virus (EBV) and herpes simplex virus (I+II) (HSV) are detectable in the invaded mucosa. Biopsies of five patients with adenocarcinoma of the colon or rectum,10patients with various benign diseases of the ileum and colon mucosa as well as 5 healthy controls were investigated for EBV and HSV DNA.The evidence was positive for EBV in 4/5 and for HSV in3/5 cases in adenocarcinomas, and 3/5 and 4/5 cases, respectively, in Crohn’s disease as well as 2/2 and 1/2 cases, respectively in chronic ulcerative colitis. Also a rectumadenom was EBV-positive and HSV-negative,whereas all controls were negative in both viruses.The evidence of EBV and HSV DNA in ileum and colon mucosa of patients with Crohn’s disease, ulcerative colitis, and colorectal cancer supports the idea of a virus etiology of these dieseases. Since EBV and HSV are possibly associated with malignant tranformation, the question arises with respect to the role of viruses in the etiopathogenity of colorectal cancer and in the malignant transformation of Crohn’s diesease or ulcerative colitis cells.

P8.04

P8.06

Colorectal cancer patients exhibits increased serum angiogenin concentration. S. Shimoyama, K. Yamasaki, M. Kawahara, M. Kaminishi Dep. of Gastroenterological Surgery,University of Tokyo Japan.

Isolation of tumour cells from peripheral blood – comparison of PCR and immuno-chemistry B Illert, KH Horak, W Timmermann, C Otto, A Thiede Clinic of Surgery, University of Würzburg/Germany

Aim: We investigated the relationship between ANG expression and its clinical relevance in colorectal cancer. Methods: By enzyme-linked immunosorbent assay, the serum ANG concentrations (sANG) were determined in preoperative (n=34) and postoperative (n=25) colorectal cancer patients, in preoperative (n=9) and postoperative (n=4) hernia patients, and in 23 healthy volunteers. Results: Before surgery, the mean sANG of the cancer patients (411.8±106.3 ng/ml) was significantly higher than those of the hernia patients (344.0±60.7 ng/ml, p=0.04) and of the healthy volunteers (321.7±59.7 ng/ml, p=0.0001). The degree of elevation of mean sANG was more significant according to the cancer progression as compared with that in the healthy volunteers (vs.Tis+T1+T2; p=0.01, vs. T3+T4; p=0.002, vs. stage 0+I; p=0.02, vs. over stage III; p=0.001, vs. Dukes A; p=0.02, vs. Dukes C; p=0.006). After cancer resection, the mean sANG decreased to the same level as that of the healthy volunteers and the degree of reduction was significant in the more progressed patients. Conclusion: These results suggest that the increased sANG which may be derived from colorectal cancer correlates with and facilitates cancer progression.

Aim: Isolation of disseminated tumour cells is discussed as a prognostic marker in cancer disease. Most the isolation is performed from bone marrow or lymph nodes. For a better patient comfort and short intervall follow up of cancer patients, we started isolation of tumour cells from peripheral blood. In an in vitro-study we compared the sensitivity of tumour cell detection using polymerase-chain-reaction and immunochemistry. Material/methods: A cytokeratin antibody (CK 20) for detection of epithelial cells was used in both methods. Using a stomach cancer cell-line different dilutions of tumour cell containing blood samples were prepared with 1, 10, 100, 1000 and 10000 tumour cells /ml of whole blood. Afterwards both methods were performed for isolation and detection of tumour cells. Results: Sensitivity with PCR is 10-fold higher than immunochemistry. With immunochemistry tumour cells cold be detected constantly in dilutions of 100 cells/ml blood. Using PCR Detec-tion of tumour cells was possible constantly in a dilution of 10 cells /ml blood. Conclusion: Both techniques are usefull for detection of epithelial cells in peripheral blood. PCR has a higher sensitivity than immunochemistry but requires more expense of time and

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material. For optimal results use of both techniques enables a combination of high sensitivity and visual proof of tumour cells. P8.07 COMPARISON OF THREE ANTI-p53 ENZYME IMMUNOASSAYS IN THE DIAGNOSIS OF CANCER J. ROHAYEM, K. CONRAD, T. ZIMMERMANN, K-H. FRANK Institute for Immunology; Dresden, Germany AIM. We compared the clinical efficiency of three Enzyme Immunoassays (EIA) for the early diagnosis of cancer: the antip53 ELISATM ( Pharmacell, Paris; France), the p53-Autoantibodies ELISA™ (Dianova, Hamburg, Germany) and the p53Antibodies ELISA™ (Orga-med, Bad Heilbrunn, Germany). MATERIAL and METHODS: Sera from 72 patients admitted for suspicion of colorectal, lung cancer or other malignancies were tested, as well as sera from 72 apparently healthy individuals. Diagnostic accuracies were assessed using receiveroperating characteristic (ROC) curve analysis. RESULTS:

Test A Test B Test C

AUC

95% CI of AUC

0.535 0.902 0.614

0.440 – 0.631 0.846 – 0.958 0.522 – 0.706

The p53-Autoantibodies ELISA™ (Dianova, Hamburg, Germany) showed the highest diagnostic accuracy (area under curve = 0.902) with a significant difference to other tests (p<0.001). A possible explanation of the low clinical performance is to consider the analytical specificity. Indeed, one of the weakness of tests A and C is that negative values are obtained at various anti-p53 antibody concentrations. CONCLUSION: We conclude that for clinical efficiency, the p53-Autoantibodies ELISA™ (Dianova, Hamburg, Germany) is superior because of its higher analytical specificity. P8.08 A nested pcr strategy for diagnosis of hereditary pancreatitis Böttger Th, Hengstler J, Engelmann R Klinik und Poliklinik für Allgemein- und Abdominalchirurgie; Johannes Gutenberg Universitat Mainz Langenbeckstraße 1, 55101 Mainz Hereditary pancreatitis is an autosomal dominant disorder characterized by recurrent bouts of severe epigastric pain with onset usually but not always before 15 years age. Recently an ArgHis substitution at residue 117 of the cationic trypsinogen gene (third exon) was reported to be associated that the Arg 117 residue is required for inactivation of trypsine. Loss of this cleavage site was suggested to permit autodigestion resulting in pancreatitis (Whitecomb et al., Nature Genetics 14, 141-145, 1996).

In the present the study was established a technique that allows identification of the relevant point mutation. The third exon of the cationic trypsinogen gene was amplified by a nested PCR technique. The G:A mutation at Arg 117 creates a novel restriction hereditary pancreatitis mutation. DNA obtained from 34 patients with pancreatic cancer was analyzed by this technique. The G:A mutation was analyzed in one of the 34 patients.

Session 10: Clinical and pathological studies in gastrointestinal carcinogenesis P10.01 Detection of scattered tumor cells in central-venous blood during surgical manipulation in GI-tract cancer patients N. R. Frühauf, P. Kaudel and K. J. Oldhafer* Department of Abdominal- and Transplantsurgery, MHH, Hannover, *Department of General Surgery, University of Essen Subject: The reported incidence of perioperatively detected circulating tumor cells varies in a wide range, possibly due to the time of blood sampling. In this study we analyzed blood for tumor cells at the time of maximal surgical manipulation, during tumor dissection. Methods: 21 patients who underwent abdominal surgery for presumed GI-tract cancer were analyzed (colorectal liver metastasis, n=9; colorectal carcinoma, n=9; hepatocellular carcinoma, n=3). Central venous blood (30ml) was collected preoperative and at the end of the surgical dissection. Equal samples were collected from 10 patients undergoing abdominal surgery for benign diseases. Tumor cell density was determined after mechanical and enzymatic dissection of a tumor biopsy by a continuous percoll gradient. Blood samples were analyzed by individually adjusted density gradient centrifugation. Cytospin specimens were prepared and stained for cytokeratins using a pancytokeratin antibody, CEA and AFP. Results: In 17 out of the 21 patients circulating tumor cells could be detected perioperatively at the time of maximal surgical manipulation. In 7 out of these 17 patients circulating tumor cells could also be detected in preoperative blood samples. Circulating tumor cells were not detectable in the 10 control patients with benign diseases. Conclusions: Despite of notouch tumor surgery circulating tumor cells were found in perioperative blood samples in almost all cases. The meaning of this remained unknown. However, to prevent the emergence of metastatic disease, surgerons should minimize intraoperative manipulation and adjuvant treatment during surgery might be indicated.

126

P10.02

P10.04

Gastrointestinal Stromal Tumors - Problems in Diagnosis and Therapy Th. Günther1, M. Pross2, Th. Manger2, H.-U. Schulz2, H. Lippert2 ,A. Roessner1 Departments of Pathology1 and General Surgery2 Otto-vonGuericke-University Magdeburg, Germany

The distribution of two polymorphic enzymes in colon cancer cases in a highly industrialized area H. C. Römer1,2, C. Rötzel2, R. Thier2, U. Zorn1, T. Reckwitz2, K. Golka2, D. Löhlein1 1 Department of Surgery, Städtische Kliniken Dortmund; 2Institute of Occupational Physiology at the University of Dortmund, Germany

Aim: Gastrointestinal stromal tumors (GIST), which form a rare group of neoplasias of the gastrointestinal tract, have not yet been fully investigated. Although good progress has been made in the diagnosis, it is still a problem to assign these lesions concerning histogenesis and biological behavior. We investigated the clinical outcome and the follow up of all GISTs, operated in the Department of Surgery. Material: Between 1994 and 1998, 18 GISTs were diagnosed and treated. Twelve of these patients (67%) had stromal tumors in the stomach, and six (33%) had intestinal stromal tumors. Results: The primary tumor could be removed in all patients with R0-resection. Six patients developed hematogeneous liver metastasis, with the size of their primary tumor exceeding 10 cm. Extrahepatic distant metastases were not found in all cases investigated. Lymphadenectomy showed that lymph node metastases did not occur. Histological evaluation was made according to the guidelines of Lewin, Weinstein and Riddell. Conclusion: Currently, sure therapy is limited to complete surgical resection of the primary tumor and its metastases. To date, approaches in adjuvant or neoadjuvant chemotherapy have failed to achieve a success. P10.03 CLINICAL OBSERVATION OF APPLICATION OF PROGLUMIDE AFTER RESECTION OF CARDIAC CARCINOMA Tu Yuanrong , Guo Xiangyun , Lin Min Department of Thoracic Surgery, First Affiliated Hospital Medical College, Fuzhou, P. R.China Aim: To study actions of proglumide as an adjuvant treatment to raise the postoperative ling-term survival rate of patients with cardiac adenocarcinoma. Method: Proglumide was administered after proximal subtotal gastrectomy in 73 patients (proglumide group)with cardiac adenocarcinoma. The administration of proglumide 0.4g t.i.d, preprandially was maintained. In the control group, 62 patients received operation only. Followup lasted for more than 3 years. Result: In the proglumide group, the 1-,2- and 3 year survival rates were 87% (64/73),75.3% (55/73) and 43.8% (32/73) respectively,and were obviously higher than the control group (p<0.01 and p<0.001). Conclusion: the proglumide is convenient to use with no obvious toxic side effect, and prolonged postoperative administration of proglumide can raese the long-term survival rate of patients after resection of cardiac carcinoma.

Introduction: Colorectal cancer is associated with genetic, occupational, and environmental factors. The polymorphic enzymes N-acetyltransferase 2 (NAT2) and glutathiontransferase M1 (GSTM1) are predisposing factors for several human cancers. Material/Methods: Therefore, the distribution of NAT2 and GSTM1 was investigated in 28 colorectal cancer cases, 60 colon cancer cases and 127 controls (suffering from non-malignant diseases) by standard methods. Additionally, possible occupational and non-occupational risk factors were investigated using a questionnaire. Results: In the cancer cases 61% were „slow“ acetylators and 56% were GSTM1 negative. In the controls 62% were “slow” acetylators and 47% were GSTM1 negative. In contrast to most studies on colon cancer cases, an overrepresentation of the “rapid” acetylator status was not observed in our study group from an area with known elevated colon cancer mortality. Conclusion: The impact of occupational and/or environmental factors might be discussed as the cause for the shift observed.

127

Author index

The numbers are the abstract number

A Abbruzzese, J. L. Adachi, H. Adler, G. Agrawal, S. Al-Taie, O. Allgayer H. Amin, S. Aselmann, H. Astarcy´odlu, Y´.

P4.01, P4.04, S4.06, P4.03 P8.03 S4.05, S9.03 S8.05 SS02 S2.04 S4.10 S3.07 P4.02

B Babic, R. Bamba, M. Bancel, B. Baretton, G. B. Barnas, C. Bartsch, H. Bayerdörffer, E. Becker, K. F. Beger, H. G. Bender, C. Berger, M. R. Bernstorff, v. W. Besler, K. Bianco, A. R. Biesalski, R. K. Bodner, E. Bodner, E. Borchard, F. Böttger, Th. Braakhuis, B. J. M. Breidert, M. Brennan, P. Breslin, T. Brivio, F. Bruch, J. Brun, C. Bruns, C. J. Büchler, M. W.

S2.04 P2.09 S2.06 S10.06 S7.03 S4.01, SP10 S8.09, P2.02, P2.05 P7.01 S4.04, S4.09 S5.01 S8.07 S4.08 S5.06 S8.05 SP01 P2.07 P2.08 S7.06 P8.08 SP05 S8.09, P2.05 SP08 P4.01 S6.05 S1.03 P4.03, P4.04 S8.10 S9.02

C Caselmann, W. Castren, K. Chan, AK. Chandrasoma, P. Chang, M. Chiao, P. Cho, Y. K. Choi, J. R. Choi, K. Choi, S. W. Christophers, E. Chung, Y. J. Ciardiello, F. Cleary, K. Cloos, J. Coker, A.

S3.04 S7.03 S4.08 S1.04, P1.01 P2.01 P4.01, S4.06 P7.02 P2.11 P2.01 P2.10 P6.01 P2.10, P2.11 S8.05 S4.06 SP05 P4.02

Conrad, R. Cox, A. G. Csuka, O.

P8.07 SS01 P5.05

D Danenberg, K. D. Danenberg, P. V. Dansranjavin, T. Daum, S. Dederke, B. DeMeester, T. R. Deppert, W. Dien, S. Dienes, H. P. Dietmaier, W. Dihlmann, S. Doi, R. Doleschall, Z. Dralle, H. Dreher, I. Duda, A.

S10.01, S4.04, S8.03 S4.04, S8.03, S10.01 S5.06 S10.05 S10.05 S10.01, P1.01 S4.05 P2.04 S8.11 S5.04 S8.04 S8.08 P5.05 S5.06, P5.01 SS02 S2.05

E Eberlein, TJ. Ebert, M. Ehninger, G. Enders, G. Endo,. K. Engelmann, R. Esumi, H. Evan, DB. Evans, D. Evans, J.

S4.08 S8.09 P2.05 S2.03 S8.08 P8.08 S2.07 S4.06 P4.01 W5

F Faller, G. Fein, M. Fidler, I. J. Flemming, P. Foster, C. Frank, K. H. Frerker, M. K. Friess, H. Frohme, M. Frühauf, N. R. Fuchs, K. H. Fujimoto. J. Füzün, M.

P8.02 S1.04, P1.01, P2.04 P4.03, P4.04 S3.07 W5 P8.07 S3.07 S9.02 S9.03 P10.01 P1.01, P2.04, S1.04 P2.06 S5.07, P5.03

Gansauge, F. Gansauge, S. Gebert, J. Genda, T. Geng, M. Ghaneh, P. Gillespie, J. Glasl, S. Glickman, JN. Glodny, B. Gödde, E. Goedegebuure, P. S. Golka, K. Gonzalgo, M. Gregor, M. Gress, T. M. Gross, M. Gruetzner, K. Grützner, K. U. Günther, Th.

S4.09 S4.09 S8.04 P7.01 S9.03 W5 P2.05 P5.04 S4.08 SP10 SS04 S4.08 P10.04 S5.01 S3.06 S9.03 P5.04 S2.04 S6.04 P10.02

H Haegele, N. Hahn, E. G. Hahn, M. Hainaut, P. Hallin, P. Hamaguchi, M. Han, S. U. Handschuh, G. Hanson, K. P. Harbison, M. T. Hattori, T. Hatz, R. A. Hay, U. Hayashi, K. Heise, W. Heiss, M. M. Hengstler, J. Henne-Bruns, D. Herderich, M. Herfarth, Ch. Hesse, K. Hicklin, D. J. Hino, O. Hirohashi, S. Hiyama, T. Höfer, H. Hoffmann, D. Höfler, H. Hofmann, J. A. Hoheisel, J. D. Horak, K. H. Huang, S. Hutzler, P. Hwang, J. J.

S5.05 S2.05 S5.02 S1.02, S7.03 P4.01 P7.03 P7.02 P7.01 P2.12 S8.10 P2.09 S2.03 S10.06 P5.02 S10.05 S2.04, S6.04 P8.08 S8.02 P2.04 S8.04 SS02 S8.10 S3.03 P7.01 P5.02 S1.03 S4.10, SP06 S2.01, S5.05, S11.05, P7.01 S2.03 S9.03 P8.06 P4.03, P4.04 P7.01 S3.06

G I/J Gabbert, H. E. Gadenstätter, M. Galle, PR.

S7.06, P2.13 P2.08 S3.02

Illert, B. Ino, Y.

P8.06 P7.01

128

Itakura, J. Ito, H. Jakob, F. Jansen, M. Jesnowski, R. Job, C. Jones, P. A. Jung, A. Jung, Ch.

S4.07 S8.08, P8.03 SS02 S8.02 S4.11 S2.07 S5.01 P8.02 P5.04

K Kaiser, S. Kalinovsky, V. P. Kalthoff, H. Kaminishi, M. Karademir, S. Karvar, S. Kaudel, P. Kawahara, M. Kawesha, A. Keck, A. M. Keller, G. Kern, H. Kim, K. M. S3.05, P7.02 Kirchner, T. Kiyabu, M. Kleeff, J. Klingler, P. Klocker, J. Klöppel, G. Knebel-Doeberitz, v. M. Köhrle, J. Konturek, P. C. Konturek, S. J. Korc, M. Kornmann, M. Köves, I. Kreczy, A. Kremer, B. Kruppa, C. Kubicka, S. Kullmann, F. Küpely´oglu, A. Kuratate, I. Kushima, R. Kwak, I. H.

S3.06 P2.12 S4.08, S8.02 S2.02, S2.07, P8.04 P4.02 SS02 P10.01 P8.04 W5 S3.06 S1.03, S2.01, S5.05 S4.05 P2.10, P2.11, S2.05, P8.02 S10.01 S9.02 P2.08, P2.07 P2.08 S10.02 S8.04 SS02 S2.05 S2.05 S4.04, S4.07, S9.02 S4.04 P5.05 P2.08 S8.02 P5.01 S3.01 S5.04 S5.07, P4.02, P5.03 S8.08 P2.09 S3.05

S1.04 S8.06 S9.02 S4.06, P4.01 S8.06 P2.10 P2.01 SP05 S8.01 S9.04 S2.06 S5.01

S9.03 S4.11 S3.05 S8.07 P10.02 S1.05 S6.05 S1.05 P10.04 S4.11 S10.01 S1.05 P7.01 SP10 S8.11 S10.02

M Machida, K. Mäffert, R. Mandala, M. Manger, Th. Manns, M. P. Marfertheiner, P. Marshall, C. J. Marsteller, I. Matsuda, S. Matsumoto, Y. Matsushima, K. Matsuura, T. Matsuzaki, H. McGee, J. O’D. Meining, A. Mendelsohn, J. Menges, M. Menke, M. Mentele, E. Méplan, C. Michaelis, S. Miehlke, S. Min, L. Möllmann, B. Momburg, F. Montesano, R. Moon, Y. Mörk, H. Moser, C. Mueller, E. Mueller, J. Mukaida, N. Müller, H. A. G. Müller, W. Müller-Pillasch, F.

L Laird, P. W. Lambrecht, R. M. Lander, A. D. Larry L. Lauer, U. Lee, K. Y. Lee, S. Leemans, C. R. Lemoine, N. R. Lerch, M. M. Li, C.-Q. Liang, G.

Lichter, P. Liebe, S. Lim, K. Linnemann, U. Lippert, H. Lippman, SM. Lissoni, P. Liu, X. Löhlein, D. Löhr, M. Lord, R. V. Lotan, R. Luber, B. Lügering, N. Lund, P. Lüttges, J.

P7.03 P8.02 S6.05 P10.02 S3.01 S8.09 S7.01 S10.06 P7.03 S4.07 P4.03 P8.03 S4.03 S7.02 S10.06, P2.02 S8.05 P6.02 S10.02 P7.01 S7.03 P2.13 S8.09, P2.02, P2.05 P10.03 S10.02 S6.01 S1.02, S7.03 P2.01 SS02 P6.02 S11.05 S1.03, S5.05, S11.05 P4.03 P8.05 P2.13 S9.03

N Nair, J. Naka, K. Nakamura, Y. Neoptolemos, JP. Nespoli, A. Niketeghad, F. Nishida, T. Noguchi, T.

S4.01 P2.06 P2.03 W5 S6.05 S8.11 P5.02 P2.13

Nomura, S. Nunnensiek, C. Nussler, A.

S2.07 P8.05 S4.09

O Oberdorfer, F. Ochsenkühn, T. Odze, RD. Ohshima, H. Okada, K. Okamoto, E. Oldhafer, K. J. Olson, M. F. Ono, K. Oohara T. Otto, C. Ottó, Sz. Özer, E. Özkal, S.

S8.06 S10.06 S4.08 S2.06 P2.09 P8.03 S3.07, P10.01 S7.01 P5.02 S2.02 P8.06 P5.05 P4.02 P4.02

P Paik, W. K. Pantel, K. Papatheodorou, L. Park, J M. Park, S. W. Park, T. J. Paterson, H. F. Paumgartner, G. Péter, I. Peters, J. H. Pich, U. Pierzchalski, P. Pignatelli, B. Pistorius, S. Plaschke, J. Pluquet, O. Portera, C. A. Pour, P. M. Prokopzcyk, B. Pross, M.

S3.05 S10.03 P5.04 S10.01, S8.03 P2.10 S3.05 S7.01 S10.06 P5.05 S10.01, P1.01 S8.09 S2.05 S2.06 S5.02, P5.01 P5.01 S7.03 S8.10 S4.03 S4.10 P10.02

R Radinsky, R. Rahóty, P. Rausch, O. Reckwitz, T. Reh, N. Reinecke-Lüthge, A. Rellecke, N. Rembiasz, A. Rhyu, M. G. Riecken, E. O. Rieder, G. Rivenson, A. Roessner, A. Rohayem, J. Römer, H. C. Rosario, M. Rotter, V. Rötzel, C. Rüschoff, J. Rüther, U.

S8.10 P5.05 S7.01 P10.04 P6.01 S10.02 P6.01 S2.05 P2.10, P2.11 S10.05 S2.03 S4.10 P10.02 P8.07 P10.04 S7.01 S10.04 P10.04 S5.04, P5.01 P8.05

129

S Sackmann, M. Sadol, Özgül Saeger, H. D. Sakagami, T. Sakamoto, M. Sakatani, T. Salem, C. Sarbia, M. Sarioglu, S. Sattler, J. Sauerbruch, T. Schackert, H. K. Scheller, A. Scheurlen, M. Schildberg, F. W. Schimanski, C. Schindler, R. Schirmacher, P. Schlegel, W. Schmid, R. M. Schmied, R. Schnekenburger, J. Schnelldorfer, T. Schölmerich, J. Schulz, H. U. Schwelberger, H. G. Seitz, H. K. Shi, Q. Shimamoto, F. Shimizu, N. Shimoyama, S. Shimoyama, T. Shomori, K. Shumakov, A. R. Siermann, A. Skinner, KA Snow, G. B. Sökmen, S. Solinas-Toldo, S. Spanjaard, RA. Spiegel, M. Stachler, P. Stagge, V. Stallmach, A. Steininger, H. Stockfleth, E. Stolte, M. Ströhlein, M. A. Sugihara, H. Suh K. W. Sutter, T. Suzuki, T.

S10.06 P4.02 S5.02, P5.01 P2.03 P7.01 P8.03 S5.01 S7.06 S5.07, P5.03 P2.08 S5.03 S5.02, P5.01 P8.05 SS02 S2.03, S2.04, S6.04 S8.07 P2.08 S8.11 SP10 S4.05 S4.03 S9.04 S4.09 S5.04 P10.02 P2.07, P2.08 SP04 P4.03, P4.04 P5.02 S2.02 P8.04 P2.03 P8.03 P2.12 S8.04 S1.04 SP05 S5.07, P4.02, P5.03 S9.03 S4.08 S8.06 S8.06 S4.05 P6.02 S2.05, P8.02 P6.01 S2.03, S5.05, S8.09, S11.05, P2.02, P8.01 S6.04 P2.09 S3.05 S5.06 P2.06

T/U Tahara, E. Tan, S. S. Tanaka, S. Tanida, N.

S1.01, S1.05, S11.02, P2.06, P5.02 S2.07 P5.02 P2.03

Tatebe, S. Tatematsu, M. Taylor, E. W. Tekin, U. Teramachi, K. Terzy´, C. Thiede, A. Thier, R. Timmermann, W. Tischler, R. Tortoral, G. Toyokuni, S. Trauzold, A. Treinies, I. Tsan, R. Tsutsumi, M. Ungefroren, H.

S8.08 S2.02 SS01 S7.06 S8.08 S5.07, P5.03 P8.06 P10.04 P8.06 P5.01 S8.05 S2.06 S8.02 S7.01 S8.10 S5.01 S8.02

V Vámosi-Nagy, I. Vieth, M. Vogelsang, H.

P5.05 S11.05, P8.01 S2.01

W Wagner, M. Wahrendorf, J. Walch, A. Wallrapp, C. Werner, M. Winde, G. Wirtz, H. C. Wynder, E. L.

S4.05 SP02 S1.02 S9.03 S1.03 SP10 P2.13 SP06, S4.10

X/Y Xiangyun, G. Xie, K. Xu, X-C. Yamada, T. Yamamoto, N. Yamasaki, K. Yasui, W. Yasui, Y. Yokozaki, H. Yu, J. Yuanrong, T.

P10.03 P4.03, P4.04 S1.05 P7.01 P2.03 P8.04 P2.06 S1.01 S1.01, P2.06 S8.09 P10.03

Z Zeitz, M. Zhang, W. Zimmermann, T. Zitzelsberger, H. Zorn, U.

P6.02 SS01 P8.07 S1.03 P10.04