Experimental Pancreatic Cancer

1  ABERRANT EXPRESSION AND TGF-BETA-DEPENDENT REGULATION OF ADAM23 mRNA IN HUMAN PANCREATIC CANCER CELLS

Ringel J¹, Jesenowski R¹, Batra S², Fleig W³, Löhr M⁴, (1) Section Molecular Gastroenterology, German Cancer Center, Heidelberg, Germany; (2) Department of Biochemistry, UNMC, Omaha, NE, USA; (3) Department of Medicine I, University of Halle, Halle, Germany; (4) Department of Medicine II, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany

INTRODUCTION AND AIM: Recently we have shown the aberrant expression of ADAM17 in pancreatic cancer. ADAM23, another member of this family, was found to be expressed during brain development and in brain neoplasia. It plays an important role in cell–cell and cell–matrix interactions. Here we investigated ADAM23 mRNA expression in pancreatic cells and tissues. PATIENTS AND METHODS: The expression of ADAM23 mRNA in 8 normal pancreas tissues (NP), 8 pancreatic cancer tissues and 9 pancreatic cancer cell lines was analyzed by RT-PCR. The regulation of ADAM23 mRNA was investigated in vivo and in vitro. Therefore, pancreatic cancer cells were injected orthotopically and subcutaneously using an established mouse model. RESULTS: ADAM23 mRNA was detectable in 6/8 pancreatic cancer tissues and 7/9 pancreatic cancer cell lines but in none of the normal pancreas tissues. ADAM23 mRNA was down-regulated in subcutaneous pancreatic tumors, whereas it was unaffected in tumors of orthotopically implanted pancreatic cancer cells. First experiments showed an ADAM23 mRNA induction in TGF-beta1 transfected Panc1 cells. CONCLUSION: Our results demonstrate for the first time an aberrant mRNA expression of ADAM23 in a non-brain malignancy. Published data showed the involvement of ADAM23 in adhesion processes by the interaction of ADAM23 with integrins. Our mouse model data suggest an environment-dependent regulation mechanism for the ADAM23 expression. TGF-beta1, which is known to play an important role in pancreatic cancer, might be a regulation factor. This might be critical for invasion and metastasis.

2  LOSS OF BNIP3 EXPRESSION IS A LATE EVENT IN PANCREATIC CARCINOGENESIS CONTRIBUTING TO CHEMORESISTANCE AND WORSE PROGNOSIS

Erkan M¹, Kleeff J¹, Esposito I², Giese T³, Ketterer K¹, Büchler M¹, Giese N¹, Friess H¹, (1) Department of General Surgery; (2) Institute of Pathology; (3) Institute of Immunology, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Altered expression of apoptosis-regulating genes plays an important role in the aggressive growth behavior and chemoresistance of pancreatic ductal adenocarcinoma. In the present study the hypoxia-inducible pro-apoptotic gene BNIP3 was analyzed in terms of expression, effect on patient survival, and chemo-responsiveness in pancreatic cancer cell lines. PATIENTS AND METHODS: cDNA microarray, QRT-PCR, laser-capture microdissection and immunohistochemistry analyses were used to evaluate BNIP3 expression in normal and diseased pancreatic specimens. Modulation ofBNIP3 expression was achieved using specific siRNA molecules. The effect of chemotherapeutic agents on pancreatic cancer cells were assessed utilizing MTT assays. RESULTS: BNIP3 mRNA levels were 3.0-fold and 6.3-fold lower in chronic pancreatitis and pancreatic cancer compared to the normal pancreas, respectively. Microdissection analysis confirmed the reduction of BNIP3 expression from normal ducts to pancreatic cancer cells. By immunohistochemistry, BNIP3 was predominantly expressed in the acinar cells of the normal and diseased pancreas. Interestingly, while BNIP3 was undetectable in the cancer cells of 59% of the cases, 75–100% of PanIN2/3 lesions displayed BNIP3 immunoreactivity. Loss of BNIP3 expression correlated with poorer survival of patients (8 vs 14 months for BNIP3 negative vs positive patients). Hypoxia induced BNIP3 expression in 4 of 8 pancreatic cancer cell lines, while in the remaining 4 it was absent under normoxic and hypoxic conditions. Down-regulation of BNIP3 resulted in increased resistance to 5-FU and gemcitabine.

CONCLUSION: Loss of BNIP3 expression occurs late in pancreatic carcinogenesis, contributes to the resistance to chemotherapy, and correlates with a worsened prognosis in this disease.

3  EXPRESSION OF TISSUE FACTOR IN PANCREATIC ADENOCARCINOMA IS ASSOCIATED WITH ACTIVATION OF COAGULATION

Haas SL², SteinerM¹, Hummel F², Ringel J³, Burstein C¹, Horst N⁴, Liebe S⁵, Löhr M², (1) Department of Clinical Chemistry and Pathobiochemistry, University of Rostock, Rostock, Germany; (2) II. Medical Department, University of Heidelberg at Mannheim, Mannheim, Germany; (3) I. Department of Internal Medicine, Martin Luther University at Halle-Wittenberg, Halle-Wittenberg, Germany; (4) Department of Pathology, University of Rostock; (5) Department of Internal Medicine, University of Rostock, Rostock, Germany

INTRODUCTION AND AIM: Pancreatic cancer is the tumor entity with the highest risk for thromboembolic complications. Tissue factor represents the principal initiator of coagulation. Aim: To study expression of tissue factor in pancreatic cancer. PATIENTS AND METHODS: TF expression was studied in eight human pancreatic tumor cell lines by Northern blot and indirect immunofluorescence. In addition TF expression was determined by immunofluorescence in pancreatic tissues of 19 patients with pancreatic adenocarcinoma (PC), 9 patients with chronic pancreatitis (CP) and in 20 normal controls (C). Plasma samples (30 PC patients, 13 CP patients and 20 normal controls) were investigated for soluble TF levels and coagulation activation markers (thrombin-antithrombin complex [TAT], prothrombin fragment 1.2). RESULTS: All pancreatic carcinoma cell lines expressed TF. Expression was not correlated with differentiation of the carcinoma cell line. In pancreatic tissue, TF expression was negative in all normal controls and weakly positive in chronic pancreatitis. 17/19 pancreatic carcinomas stained positive for TF. TF (pg/ml), TAT (ug/L) and PT1.2 (nM) levels in PC patients were significantly elevated compared to normal controls (288 vs 139, p=0.005, 89 vs 4, p=0.002, 1.2 vs 0.75, p=0.003, respectively). CONCLUSION: We conclude that in addition to the up-regulated expresssion of TF on the cell membrane soluble TF might be pivotal for activation of the coagulation system in pancreatic cancer.

4  PKC 412 – A NOVEL COMPOUND FOR THERAPY OF PANCREA TIC CANCER

Büchler P, Elfitori J, Chirurgische Universitätsklinik, Abteilung für Allgemein-, Viszeral- und Unfallchirurgie, Heidelberg, Germany

INTRODUCTION AND AIM: Pancreatic cancer is an aggressive malignancy with a poor prognosis. Currently there is no effective therapy including surgery since most patients will experience tumor recurrence shortly thereafter. The increasing awareness of the role of tumor neo-angiogenesis in growth of solid tumors resulted in a consensual opinion that abrogated neo-angiogenesis may cause tumor growth arrest. Angiogenesis is also necessary for local and systemic tumor progression – both hallmarks of pancreatic cancer. PKC412 is a novel inhibitor of mutated FLT3, PKC, KDR, c-KIT, PDGFRá, and PDGFRâ.The aims of this study were to analyse the therapeutic efficiency of this novel compound in vitro and in vivo using a novel orthotopic model for experimental pancreatic cancer. PATIENTS AND METHODS: Five human pancreatic cancer cell lines AsPc-1 (A1), Capan-1 (C1), HPAF-2 (HP2), PANC-1 (P1) and MIA PaCa-2 (MP2) were analyzed. RT-PCR was used for FLT3 mutation in pancreatic cancer cells. Anchorage-dependent cell growth was quantified with the MTT assay. Soft agar assays were used to study anchorage-independent growth. Cell cycle progression was analyzed by flow cytometry. In vivo HP2 and AsPC-1 cells were used to induce orthotopic tumors in a novel murine model for pancreatic cancer (n=12 each). Animals received 10 mg/ kg PKC412 i.p. every day for 8 weeks. Immunohistochemistry (anti-CD31) was used to quantify microvessel density. RESULTS: No FLT3 mutation could be detected in any of the pancreatic cancer cell lines. Cell growth was inhibited in all cell lines tested; PKC412 resulted in a dose- and time-dependent growth suppression. In flow cytometric analysis a strong G2/M-phase arrest was detectable. Anchorage-independent growth was also significantly reduced in a dose-dependent and time-dependent manner. The in vivo correlate was a significant growth reduction of orthotopic tumors growth in both cell lines tested. Growth suppression in vivo was mediated not only by antimitogenic activity but also by suppression of tumor neo-angiogenesis, as the number of blood vessels in treated animals was significantly lower. CONCLUSION: PKC412 is a promising new compound with strong anti-mitogenic and in vivo anti-angiogenic activity. This dual efficiency will likely be of therapeutic value for patients with pancreatic cancer.

5  RELEASE OF iC3b FROM APOPTOTIC TUMOR CELLS INDUCES TOLERANCE BY BINDING TO IMMA TURE DENDRITIC CELLS IN VITRO AND IN VIVO

Schmidt J, Märten A, Büchler MW, Department of Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Chemotherapeutic and immuno-therapeutic approaches induce apoptosis in tumor cells. Apoptotic cells are known to activate homologous complement and to be opsonized with iC3b. As maturation of dendritic cells (DCs) can be inhibited by binding of iC3b to the complement receptor 3 (CR3, CD1 1b/CD18) and because immature DCs induce tolerance, we investigated the induction of tolerance after pulsing DCs with apoptotic cells in the presence or absence of native serum. PATIENTS AND METHODS: Apoptosis in pancreatic carcinoma cells was induced either by heat-stress, chemotherapy or anti-Her2 antibody. Monocyte-derived DCs were pulsed with apoptotic cells with or without native serum. DCs were analyzed for the maturation state by flow cytometry and the cytotoxic activity was determined. Tolerance was prevented by addition of substances such as anti-CD11b or N-acetyl-D-glucosamine (NADG) which block iC3b binding to CR3. Furthermore, binding of iC3b from apoptotic cells to DCs was blocked in a syngeneic pancreatic carcinoma mouse model. RESULTS: All of the above strategies for apoptosis induction resulted in iC3b release. Pulsing DCs with apoptotic cells in the presence of serum prevents maturation of DCs and finally induces tolerance. This tolerance could be prevented almost completely by blocking the interaction of iC3b with the CR3 receptor. This could be shown as well in an immunocompetent mouse model. CONCLUSION: Chemotherapeutic and immunotherapeutic approaches induce apoptosis in tumor cells. Release of iC3b from apoptotic tumor cells prevents full maturation of DCs and immature DCs induce antigen-specific silencing or tolerance. Blocking of iC3b-binding could mostly prevent this effect.

6  FXYD3 IS OVER-EXPRESSED IN PANCREATIC DUCTAL ADENOCARCINOMA AND INFLUENCES PANCREATIC CANCER CELL GROWTH

Kayed H⁶, Kleeff J¹, Kolb A¹, Keleg S¹, Felix K¹, Giese T², Penzel R³, Zentgraf H⁴, Buechler MW¹, Korc M⁵, Friess H¹, (1) General Surgery, University of Heidelberg; (2) University of Heidelberg, Institute of Immunology; (3) University of Heidelberg, Institute of Pathology; (4) Applied Tumor Virology, German Cancer Research Center, General Surgery, University of Heidelberg, Heidelberg, Germany; (5) Medicine and Pharmacology and Toxicology, Dartmouth Medical School, Dartmouth, Dartmouth, USA; (6) General Surgery – European Pancreas Research Center, Heidelberg, Germany

INTRODUCTION AND AIM: Deregulation of FXYD3, a trans-membrane protein acting as a chloride channel or chloride channel regulator, has been found in some malignancies. In the present study, the expression and localization of FXYD3 were analyzed in pancreatic tissues derived from donors and patients suffering from chronic pancreatitis (CP) or pancreatic ductal adenocarcinoma (PDAC) as well as in pancreatic cancer cells. PATIENTS AND METHODS: QRT-PCR, laser-capture microdissection and micro-array analysis, in situ hybridization and immunohistochemistry were used to determine the expression and localization of FXYD3. FXYD3 antisense expressing T3M4 pancreatic cancer cells were generated and compared with control T3M4 cells in terms of growth and chemoresistance using anchorage-dependent and -independent growth assays, and xenotransplantation into nude mice. RESULTS: FXYD3 mRNA levels were 3.4-fold increased in PDAC tissues compared to donor specimens (p=0.006), and 3.9-fold increased in microdissected cancer cells compared to normal ductal cells (p=0.02). By in situ hybridization and immunohistochemistry, FXYD3 was localized in the tubular complexes and PanIN lesions of both CP and PDAC, as well as in pancreatic cancer cells. Down-regulation of FXYD3 by stable anti-sense transfection significantly increased the doubling time of T3M4 cells in vitro from 44 h ±2 to 55 h ±12 (p=0.02), and in vivo from 3.32 days ±1.02 to 4.30 days ±0.43 (p=0.058). Anchorage-independent growth and sensitivity of T3M4 cells to chemotherapeutic drugs and MgCl₂ were not dependent on the level of FXYD3 expression. CONCLUSION: Over-expression of FXYD3 in pancreatic cancer may contribute to the proliferation of this malignancy.

7  HEME OXYGENASE-1 (HO-1) MEDIATED IMPAIRED FUNCTIONALITY OF PERIPHERAL MONONUCLEAR CELLS IN PANCREATIC CANCER PA TIENTS

Dambrauskas Z², Berberat P¹, Gulbinas A², Mitkus T¹, Giese T³, Giese N¹, Meuer S³, Friess H¹, Buechler M¹, (1) Department of Surgery, Heidelberg University, Heidelberg, Germany; (2) Department of Surgery, Kaunas University of Medicine, Kaunas, Lithuania; (3) Immunology Institute, Heidelberg University, Heidelberg, Germany

INTRODUCTION AND AIM: Tumor-induced impairment of immune function may be the reason for fast progression of pancreatic cancer. This study determines if the function of peripheral mononuclear cells (PMNCs) of pancreatic cancer patients shows changed functionality in comparison to PMNCs of healthy controls. Furthermore, the role of HO-1 in the function PMNCs of pancreatic cancer patients is analyzed. PATIENTS AND METHODS: The expression of HO-1 was analyzed in PMNCs of pancreatic cancer patients and age-matched healthy controls. The functionality of PMNCs was assessed by measuring H₂O₂ and NO production. IL-1 and IL-10 secretion was determined in both groups. Alteration of PMNC function from healthy controls was analyzed after induction of HO-1. RESULTS: PMNCs from cancer patients showed marked overexpression of HO-1 in comparison to healthy controls (p<0.05). HO-1 mRNA was mainly found in CD11- and CD56-positive cells. HO-1 overexpression correlated with decreased production of H₂O₂ and NO in cancer patients. After LPS stimulation the PMNCs also showed decreased IL-1 production and marked increase of IL-10 secretion in comparison to healthy controls. Induction of HO-1 in PMNCs of healthy controls led to similar alteration of the function as seen in cancer patients. CONCLUSION: PMNCs of pancreatic cancer patients show impaired effector functions, including reduced oxidative burst and anti-inflammatory cytokine secretion pattern. This phenomenon is at least partly mediated by overexpression of HO-1 in PMNCs of pancreatic cancer patients. Tumor-mediated up-regulation of HO-1 in PMNCs may lead to a systemic immuno-suppressive state in pancreatic cancer patients.

8  THE NEUTROPHIC FACTOR ARTEMIN INCREASES CANCER CELL INVASION BUT NOT CELL PROLIFERA TION IN PANCREA TIC CANCER

Ceyhan GO, Müller M, Giese N, Mert E, Wente M, Büchler M, Friess H, Department of General Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Invasion of pancreatic nerves by pancreatic cancer and its extension to the extrapancreatic nerve plexus are still poorly understood events. It leads to retropancreatic tumor extension, precludes curative resection, promotes local recurrence and has a negative impact on survival. To understand the process of neural invasion, Artemin – a neurotrophic factor – and its receptors (RET-GFRa3) were studied in pancreatic cancer. PATIENTS AND METHODS: Tissues from 31 patients undergoing resection for pancreatic cancer, 10 liver metastases and 19 healthy organ donors were investigated by Western blot analysis, immunohistochemistry and QRT-PCR. Artemin was also determined in the pancreatic cancer cell lines Colo-357, Mia-PaCa-2, BxPc-3, SU-8686, Panc-1, Capan-1, Aspc-1 and T3M4. The influence of Artemin on proliferation was analyzed via a MTT test and its effects on invasion were investigated by using Biocoat-Matrigel invasion chambers. RESULTS: Artemin mRNA expression was increased in pancreatic cancer compared to normal pancreas. The highest and lowest levels of Artemin mRNA in pancreatic cancer cell lines were found in T3M4 and Panc-1, respectively. Western blot analysis revealed strong Artemin and RET-GFRa3 levels in pancreatic cancer, compared to normal pancreas. By immuno-histochemistry, pancreatic cancer and liver metastasis samples exhibited strong Artemin and RET-GFRa3 immunoreactivity in arteries, nerves, tubular complexes and pancreatic cancer cells. In normal pancreas, however, Artemin immunostaining was only present faintly in arteries. Artemin caused no striking effect on cell proliferation in none of the pancreatic cancer cell lines. However, Artemin increased the invasion capacity of pancreatic cancer cell lines up to three to five times. CONCLUSION: Artemin promotes pancreatic cancer cell invasion, but not proliferation. Its presence in metastatic cancer cells suggests that Artemin might also have an influence on the formation of metastases.

9  OSTEOPONTIN MODIFIES THE INVASIVENESS OF PANCREATIC CANCER CELLS: POTENTIAL THERAPEUTIC AND DIAGNOSTIC MARKER

Kolb A, Kleeff J, Guweidhi A, Büchler MW, Friess H, Department of General Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Osteopontin (OPN) is a secretory protein with a variety of functions, in for example, cell adhesion and migration, inflammatory reaction and apoptosis. OPN can increase tumor growth, invasiveness and metastases, and it has also been suggested to serve as a biological marker in certain tumors. Aim: The functional role of OPN in human pancreatic cancer and its potential use as a new disease marker were analyzed. PATIENTS AND METHODS: OPN expression in normal pancreas and pancreatic cancer were examined by RT-QPCR and immuno-histochemistry. Serum levels of donors and patients with pancreatic diseases were analyzed by ELISA. Growth experiments, RT-QPCR and ELISA were used to analyze cultures of pancreatic cancer cells. Invasion experiments were performed with siRNA transfected pancreatic cancer cells and specific OPN receptor blockade. RESULTS: In RT-QPCR, there was a 2.5-fold increase of OPN mRNA in pancreatic cancers compared to normal pancreatic tissues. Immunohistochemical analysis demonstrated OPN staining in 60% of the primary pancreatic tumors and in 72% of the metastases. ELISA analysis of serum samples showed a 2.1-fold increase in OPN serum levels in tumor patients. Recombinant human OPN significantly increased the invasiveness of pancreatic cancer cells, without having any impact on cell proliferation. In addition, down-regulation of OPN by specific siRNA molecules decreased pancreatic cancer cell invasion. CONCLUSION: OPN serum levels in pancreatic cancer patients derive most probably from the increased OPN production by the tumor cells, thus suggesting a possible role of OPN as a prognostic or follow-up marker. Our results also suggest that blockade of OPN might be useful as a therapeutic approach to inhibit invasion and metastasis of pancreatic cancer cells.

10  IN VITRO ANALYSIS OF THE ROLE OF INTERFERON-ALPHA IN COMBINED CHEMORADIOIMMUNOTHERAPY IN THE ADJUVANT TREATMENT OF PANCREA TIC ADENOCARCINOMA (CapRI)

Patrut E, Märten A, Schmidt J, Knaebel HP, Büchler M, Department of Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Adjuvant treatment of pancreatic adenocarcinoma has failed so far to produce long-lasting benefits. Results from a phase II trial, where chemoradiotherapy was combined with IFN-alpha (CapRI scheme), are very encouraging. We hypothesize that IFN-alpha is the agent that turns a slightly effective treatment (radiochemotherapy) into a potent therapy. PATIENTS AND METHODS: Eight pancreatic carcinoma cell lines were treated with the single agents and combinations of these. The role of IFN-alpha regarding a) direct inhibitory effects; b) radio- and chemo-sensitizing effects; c) anti-angiogenic properties and d) enhancement of immunogenicity was investigated. RESULTS: Our results show that IFN-alpha has direct inhibitory properties and some synergistic influence as determined by Annexin V/PI stain and cell count. IFN-alpha is also able to prevent the increase in proliferation rate and VEGF secretion of CDDP resistant cells. Taking the results from immunogenicity experiments together, we found cells that can be influenced by IFN-alpha but that are less susceptible against T cells. Furthermore, high expression of MHC molecules, CD118, EGF-R and Fas was predictive of a good response. CONCLUSION: In conclusion, IFN-alpha has direct cytotoxic effects, acts as a radiosensitizer and circumvents tumor cell regrowth after CDDP treatment. These mechanisms may be responsible for the good clinical outcome of CapRI.

11  IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN THE STROMAL TISSUE OF PANCREA TIC DUCTAL ADENOCARCINOMA AND CHRONIC PANCREATITIS

Grützmann R³, Lüttges J¹, Klüppel G¹, Ammerpohl O², Kalthoff H², Saeger HD³, (1) Pathology, University Kiel; (2) Surgery, University Kiel, Kiel, Germany; (3) Department of Visceral-, Thoracic and Vascular Surgery, University Hospital Dresden, Dresden, Germany

INTRODUCTION AND AIM: Prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite increasing knowledge about the molecular basis of PDAC no specific marker for early diagnosis nor a target protein for a new therapeutic approach have been identified so far. Moreover, PDACs display large desmoplasia, indicating that the activation of tumor surrounding stroma plays a key role during PDAC development. We were therefore interested in the analysis of differential gene expression between tumor surrounding stroma and the stroma compartment of chronic pancreatitis. PATIENTS AND METHODS: We micro-dissected the stromal tissue compartment of six pancreatic cancer and six chronic pancreatitis tissues. Moreover 20 microdissected samples of PDAC and 15 of normal ductal cells have been used. Manual microdissection has been applied to get pure cell compartments. The tissues were obtained during surgery and freshly frozen. The mRNA was extracted, amplified using repetitive in vitro transcription and hybridized to the Affymetrix U133 A + B Gene-Chip set. The data obtained from the microarray were normalized and signal intensities were calculated using dCHIP. Differentially expressed genes were identified using SAM (cut-off: fold change >3, q-value 55%). RESULTS: We identified 195 differentially expressed genes of which 39 were over-expressed and 157 were under-expressed in pancreatic cancer stroma. Hierarchical clustering using the 195 genes and the gene expression profiles of microdissected pancreatic tumor epithelia identified a subset of pancreatic cancer epithelia displaying gene expression similar to the cancer stroma. Annotation of the 195 genes resulted in the identification of soluble factors from the Wnt and the Notch signalling pathways over-expressed in the stroma from cancer tissue. Several genes are now subjected to further validation. CONCLUSION: In conclusion, gene expression analysis of the stromal compartment could identify new markers and therapeutic targets for the tumor-associated stroma of pancreatic cancer.

12  MOESIN: EXPRESSION AND FUNCTIONAL STUDIES IN PANCREA TIC CANCER

Abiatari I², Kleeff J¹, Giese NA¹, Buchler MW¹, Friess H¹, (1) Department of General Surgery, University of Heidelberg; (2) University of Heidelberg, Department of General Surgery, European Pancreas Research Center, Heidelberg, Germany

INTRODUCTION AND AIM: Moesin (membrane organizing extension spike protein) is a member of the ERM (Ezrin, Radixin, Moesin) protein subfamily, which has been proposed to link integral proteins to the actin component of the cytoskeleton. Having structural functions, it has been implicated in the regulation of signaling events in epithelial cells. Recent studies have shown a correlation between the deregulation of Moesin expression and the aggressiveness of tumor cells in different types of cancer. This study aimed to investigate the expression and localization of Moesin in pancreatic cancer tissues and pancreatic cancer cell lines, and to evaluate its importance in adhesion and invasion of pancreatic cancer cells. PATIENTS AND METHODS: Quantitative RT-PCR was used to determine Moesin mRNA levels. Immunohistochemistry and immunofluorescence were carried out to localize Moesin in pancreatic tissues and in pancreatic cancer cell lines. Adhesion and invasion assays were performed in control and following Moesin-specific siRNA transfection. Immunoblot analysis was used to monitor protein levels. RESULTS: QRT-PCR revealed a 2.4-fold increase of Moesin mRNA in pancreatic cancer tissue in comparison to normal pancreatic tissues. Moesin was expressed in the proximity of the plasma membrane in pancreatic cancer cell lines. A significant down-regulation of Moesin was observed 72 h after siRNA transfection in pancreatic cancer cell lines, which was accompanied by an increase of the invasion capacity and a reduction of adhesion in pancreatic cancer cell lines. CONCLUSION: Moesin displays a membranous pattern of expression in pancreatic cancer cell lines and suppression of Moesin expression decreases adhesion and increases invasion in pancreatic cancer cells.

13  INCREASED EXPRESSION OF TUMOR-ASSOCIATED ANTIGEN RCAS1 ALONG WITH PANIN PROGRESSION TO INVASIVE PANCREATIC ADENOCARCINOMA

Ito D, Fujimoto K, Kami K, Doi R, Surgery and Surgical Basic Science, Kyoto University, Kyoto, Japan

INTRODUCTION AND AIM: RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) has been reported as a tumor-associated antigen, which contributes to immunologic tolerance of cancer through induction of apoptosis of T, B or NK cells. Over-expression of this molecule has been reported in many types of cancers. We hypothesized that RCAS1 might also play a certain role in pancreatic cancer development. To assess this hypothesis, we immunohistochemically examined the expression of RCAS1 in invasive pancreatic ductal adenocarcinomas (IPDAs) and pancreatic intraepithelial neoplasia (PanINs), which are now proposed as precancerous lesions of pancreatic ducts. PATIENTS AND METHODS: Immunohistochemical analysis was performed in 24 IPDAs, 1 IPDA with PanINs and 3 carcinoma in situ with PanINs using anti-RCAS 1 antibody. Correlation between the expression of RCAS1 and clinocopathological features was evaluated. RESULTS: RCAS1 was highly expressed in strong cytosolic and membranous staining of RCAS 1 was seen in 19 cases of 25 IPDAs (76%), whereas no staining was detected in normal pancreatic epithelia. In PanINs, the proportion of the specimens regarded as positive was as follows: 0/2 lesions in 0%, 1/4 lesions in 25%, 1/2 lesions in 50% and 4/4 lesions in 100%, respectively. There was no statistical correlation with survival and clinocopathological features in these series of immunohistochemstry. CONCLUSION: RCAS1 was highly expressed in IPDAs and the proportion of the specimens regarded as positive increased along with PanIN progression. These results suggest that RCAS1 might play a certain role in pancreatic cancer development.

14  HISTONE DEACETYLASE (HDAC) INHIBITORS ENHANCE THE APOPTOTIC EFFECTS AND THE GROWTH REDUCTION ACTIVITY OF GEMCITABINE IN PANCREATIC CARCINOMA CELL LINES

Bonora A³, Beghelli S¹, Donadelli M², Cavallini A³, Bassi C³, Palmieri M², Pederzoli P³, (1) Department of Pathology, University of Verona; (2) Department of Neurological Sciences, Biochemistry Unit, University of Verona; (3) Department of Surgical Sciences, University of Verona, Verona, Italy

INTRODUCTION AND AIM: Gemcitabine, though considered the most active agent in pancreatic cancer chemotherapy, does not exceed a 20% rate in clinical response, this resistance being probably related to the alteration of apoptosis-regulating genes, mainly p53. HDAC inhibitors promote hyperacetylation and highly induce apoptosis in both p53-positive and -negative tumour cells. Therefore we evaluated the effects in nude mice of the association of a HDAC inhibitor, like TSA, and gemcitabine. PATIENTS AND METHODS: A p53-defective pancreatic cancer cell line (T3M4) was subcutaneously implanted in four groups of five nude mice. Each group was then randomly treated with a twice-weekly administration of either DMSO (as control), or TSA (0.25 mg/kg), or gemcitabine (2.5 mg/kg), or TSA plus gemcitabine during a 4-week period. Tumour growth and body weight were monitored and the animals were sacrificed at the end of the scheduled treatment. RESULTS: Neither toxicity nor mortality was observed in any group. The combined treatment significantly enhanced cell apoptosis in tumour section compared with the control group in a 1.43:1 rate (p<0.02). Histone acetylation levels showed a three-fold increase in TSA treated versus untreated groups. We reported a tumour weight significant reduction (p<0.01) in the combined treatment group (2.98 + 1.5) if compared both with control (5.74 + 1.7) and single treatment groups (6.34 + 1 and 5.98 + 1.7, respectively). CONCLUSION: The association of TSA and gemcitabine seems able to reduce pancreatic cancer tumour growth in vivo. These data need to be confirmed in larger studies to provide a rational basis for further clinical trials.

15  STANNIOCALCIN-1 IS UP-REGULA TED IN PANCREATIC CANCER

Salabat MR¹, Adrian T², Ding XZ¹, Wagner GF¹, DiMattia GE¹, Talamonti MS¹, Bell RH¹, (1) Department of Surgery and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, Department of Physiology and the London Regional Cancer Center, University of Western Ontario, USA; (2) Northwestern University Feinberg School of Medicine, Surgical Research Departments of Surgery and Pathology, Chicago, USA

INTRODUCTION AND AIM: Stanniocalcin-1 (STC-1) is a polypeptide hormone originally found in bony fish, involved in calcium and phosphate homeostasis. STC-1 is widely expressed in different tissues in mammals, including the pancreas. STC-1 has diverse roles in mammals, including metabolic effects on target cell mitochondria. Transgenic mice overexpressing STC1 exhibit metabolic wasting, hyperphagia and a dwarf phenotype. STC-1 may be a marker for breast cancer and to detect minimal residual disease in acute myelogenous leukemia. PATIENTS AND METHODS: The present study investigated the expression of STC-1 in pancreatic cancer, using expression microarray, real-time RT-PCR, Western blotting and immunocytochemistry. RESULTS: Microarray analysis of CD 18 pancreatic cancer cells, revealed STC-1 among up-regulated genes in response to the phorbol ester, TPA. Time- and concentration-dependent increased expression of STC-1 was confirmed by real-time RT-PCR. Western blotting showed that STC-1 protein was markedly increased in media, but not lysates of TPA-treated CD18 cells. TPA also increased STC-1 promoter activity. Immunocytochemistry showed intense STC-1 staining in pancreatic cancer tissue. Investigation of the putative receptor using an STC-1/ alkaline phosphatase fusion protein, showed increased ligand binding in cancer tissues. CONCLUSION: STC-1 and its putative receptor are up-regulated in pancreatic cancer tissue and cells. TPA induces time- and concentration-dependent expression of STC-1 mRNA and protein in pancreatic cancer cells. Further investigation will reveal the role of stanniocalcin-1 in pancreatic cancer and whether this polypeptide may be valuable as a marker or therapeutic target in this disease.

16  p-21waf1 EXPRESSION IS UP-REGULATED THROUGH A P38 KINASE-DEPENDENTMECHANISM INASSOCIATION WITH FRONDANOL®-A5P-INDUCED CELL CYCLE ARREST AND APOPTOSIS IN PANCREATIC CANCER

Roginsky AB¹, Adrian T², Singh B¹, Ujiki MB¹, Ding XZ¹, Collin P¹, Woodward C¹, Talamonti MS¹, Bell RH¹, (1) Department of Surgery and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL and Coastside Research, Stonington, ME, USA, (2) Northwestern University Feinberg School of Medicine, Surgical Research Departments of Surgery and Pathology, Chicago, USA

INTRODUCTION AND AIM: Pancreatic cancer has a grave prognosis due to late diagnosis and lack of effective therapy. Frondanol® A5, produced from an edible sea cucumber, inhibits growth of pancreatic cancer cells. In the present study, we examined the effects of a polar sub-fraction, Frondanol®-A5P on cell cycle and apoptosis. PATIENTS AND METHODS: Cells were cultured in serum-free conditions 24 h prior to treatment. Cells were stained with propidium iodide for cell cycle analysis. Cell cycle and apoptosis-related proteins were evaluated by Western blotting. mRNA was quantified by real-time RT-PCR. RESULTS: G2/M arrest was seen by flow cytometry in both AsPC-1 and S2-O13 cells after treatment with 15.5 µg/ml for 24 h. Treatment decreased expression of cyclins A, B, D and E. Phosphorylation of p-38 and SAPK/JNK were seen at 5 min. Treatment also increased p21waf1 protein and mRNA (8–32 fold at 3 h). Pre-treatment of the cells with the phospho-p38 inhibitor, SB203580, reduced the amount of p21waf1 mRNA. Caspase-3 activation provided evidence of apoptosis. CONCLUSION: Apoptosis induced by Frondanol®-A5P is associated with a marked increase in expression of the p-21waf1 mRNA and protein. It is likely that p-21waf1 mediates the apoptotic and cell cycle effects of the sea cucumber-derived agent. The increased expression of this pro-apoptotic and cell cycle arrest protein is mediated by a p38 kinase-dependent mechanism and is likely stabilized by phospho-JNK. Since Frondanol®-A5P comes from an edible, nontoxic source, it may be valuable for therapy or prevention of pancreatic cancer.

17  EXPRESSION OF STEROID HORMONE RECEPTORS AND OTHER BREAST CANCER-ASSOCIATED GROWTH FACTORS IN PANCREATIC ADENOCARCINOMA

Spinelli A¹, Raab C¹, Heinz T², Benckert C¹, Langrehr JM¹, Rudolph B³, Neuhaus P¹, Jonas S¹, (1) Allgemein-, Visceral- und Transplantationschirurgie, Charité Campus Virchow Klinikum; (2) Universitaetsfrauenklinik, Hormonlabor, Charité Campus Virchow-Klinikum; (3) Institut f. Pathologie, Charité Campus Mitte, Berlin, Germany

INTRODUCTION AND AIM: The role of hormonal regulation in pancreatic adenocarcinoma growth is still unclear. Our aim is to dose the expression of progesterone receptors (PRs), estrogens receptors (ERs), cathepsin D, pS2, p53 and c-erbB2 in specimens of pancreas adenocarcinoma and to correlate these data with the clinical parameters and outcome of patients. PATIENTS AND METHODS: Hormone receptors and growth factors were quantitatively determined by enzymatic immunoassay in specimens from 56 pancreas carcinomas and from 11 chronic pancreatitis. RESULTS: Nine pancreatic carcinomas (16%) were ER-positive (cut-off: 1.5 fmol/mg), 16 pancreatic carcinomas (28.5%) PR-positive (cut-off: 1.7 fmol/mg). PR and ER were common in poorly differentiated carcinomas (G3) (p=0.15 and 0.12, respectively). There was a trend towards an increased rate of detection of PRs in advanced tumor stages (p=0.07). Two specimens obtained from patients suffering from pancreatitis expressed hormonal receptors (1 ERs, 1 PRs). A trend towards an accumulation of PRs in pancreatic carcinomas compared with pancreatitis was identified (p = 0.17). pS2 was found to be expressed in 10.7% of the carcinomas (all of them hormone receptor positive) and in none of the pancreatitic specimens. The mediane cathepsin D concentration in cancers was significantly higher than in the control group (p=0.02; Mann-Whitney test). Additionally, 13 (23%) carcinomas were p53-positive and 45 (80%) carcinomas were c-erbB2-positive. None of the considered factors, except radical resection, revealed a prognostic influence on the survival (univariate analysis, log-rank-test). CONCLUSION: The significantly different concentration of cathepsin D in pancreatitis and tumor could suggest a role of this protein as a parameter for tumor progression.

18  NEUROMEDIN U IS A SPECIFIC MARKER FOR PANCREATIC DUCTAL ADENOCARCINOMA AND ENHANCES INVASIVENESS

Ketterer KM, Frank D, Giese N, Büchler MW, Friess H, Department of General Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Microarray analysis revealed a specific overexpression of the neuropeptide Neuromedin U (NmU) in pancreatic cancer samples compared with chronic pancreatitis and normal pancreas. Therefore the potential of NmU as a diagnostic marker and the putative role of NmU in pancreatic cancer carcino-genesis was further investigated. PATIENTS AND METHODS: Expression of NmU and its receptors NmUR1 and 2 was analysed in pancreatic cancer cell lines and human pancreatic specimens by QT-PCR, immunohistochemistry, Western blot and by ELISA of serum samples. NmU-stimulated cells were analysed by cDNA microarrays. In addition the role of NmU in cell proliferation and migration was tested. RESULTS: A significant overexpression of NmU (p=0.0022) and NmUR2 (p=0.0016) mRNA in cancer and metastatic tissues was found by QT-PCR. According to IHC, protein expression was localized predominantly in cancer ducts but also hypertrophic nerves and tubular complexes in primary tumours and in cancer cells of metastasic lesions. Serum levels of NmU did not differ significantly between the various groups; however, individual NmU-serum levels dropped considerably after tumour resection, indicating NmU as a possible serum marker for tumour recurrence. NmU treatment induced c-met up-regulation and accordingly NmU treatment promoted rHGF-induced scattering of tumour cells. CONCLUSION: We report that NmU and its receptor NmUR2 are over-expressed in pancreatic cancer on mRNA and on protein level. Besides its role as a clinical marker, we suggest an involvement of NmU in the HGF-Met paracrine loop regulating cell migration and thus invasive tumour growth and dissemination of cancer cells in PDAC.

19  EXPRESSION OF CYTOKERA TIN-20: AN INDICATOR OF POOR OUTCOME IN RESECTED PANCREATIC CANCER

Schmitz-Winnenthal FH¹, Z'graggen K³, Berger S¹, Volk C¹, Kleeff J¹, Friess H¹, Weitz J¹, Helmke BC², Hinz U¹, Büchler MW¹, (1) Department of Surgery, University of Heidelberg; (2) Department of Pathology, University of Heidelberg, Heidelberg, Germany; (3) Klinik Beau-Site Hirslanden, Department of Surgery, Bern, Switzerland

INTRODUCTION AND AIM: Even after radical surgery (R0) ∼ 80% of pancreatic cancer patients will die of the disease within 5 years. At present no reliable indicators of prognosis after radical resection are available. The expression panel of cytokeratins (CK) is closely linked with specific programs of cell differentiation. The aim of the study was to investigate the expression of CK-20 in pancreatic cancer tissue and to correlate CK-20 expression with tumor recurrence and survival in radically resected pancreatic cancer patients. PATIENTS AND METHODS: Tissue samples of 63 patients with pancreatic cancer were obtained intraoperatively and subjected to CK-20 RT-PCR. 34/63 patients underwent potentially curative resection and were followed for disease recurrence and survival. From the 34 R0 patients, 26 (76.5%) tumors were CK-20-positive and 8 (23.5%) tumors were CK-20-negative. The mean follow-up period for the entire group was 12.7 months (range 4–24.3), follow-up in censored patients was 14.7 months (range 6.3–24.3). RESULTS: Considering the R0 resected group, none of the patients with CK-20-negative tumors, but 12 of 26 (46%) patients with CK-20-positive tumors (p=0.02) died of recurrent disease. The median survival time of patients with CK-20-positive tumors was 9 months (4–11.6), median survival was not reached in R0 resected patients with CK-20-negative tumors (follow-up period of 16.7 months). The survival advantage observed in patients with CK-20-negative tumors could not be attributed to intergroup variations in tumor stage or tumor grade. CONCLUSION: The data suggest that in patients undergoing radical resection of pancreatic cancer, CK-20 expression is associated with increased aggressiveness and a poorer survival. Accordingly, negativity for CK-20 defines a subgroup of patients exhibiting a more favorable disease outcome. The expression of CK-20 in resected pancreatic cancer may be of interest as a prognostic test.

20  IMMUNOMODULATORY IMPACT OF INTERFERON-ALPHA IN COMBINATION WITH CHEMORADIATION OF PANCREATIC ADENOCARCINOMA (CapRI)

Ma J, Schmidt J, Märten A, Patrut E, Knaebel HP, Büchler M, Department of Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Adjuvant treatment of pancreatic adenocarcinoma has so far shown only moderate results. Data from a phase II trial, where chemoradiotherapy was combined with IFN-alpha (CapRI scheme), are very encouraging. We hypothesize that IFN-alpha is the agent that improves radiochemotherapy significantly. Here, we focused on the immunomodulatory effect of IFN-alpha. PATIENTS AND METHODS: Eight pancreatic carcinoma cell lines were treated with the single agents 5-FU, CDDP, IFN-alpha, irradiation and/or combinations of these. The immunomodulatory capacity of IFN-alpha was investigated in cytotoxicity assays against these cell lines. Immune cells were preincubated with 1000 U/ml IFN-alpha over 24 h. RESULTS: Our results show an increase in cytotoxic activity of peripheral blood mononuclear cells after IFN-alpha treatment from 12.5% to 34.3% (p<0.05). This cytotoxicity was NK cell-mediated as it was shown after depletion of T cells (T cells 4% lysis, NK cells 42.7% lysis). The T cell depleted fraction was to 60% CD16 +, CD56 + and expressed to 50% the IFN-a receptors CD118 and CD21. Monocytes were not involved either in killing or in activation (IFN-alpha stimulation in the absence of monocytes 29.4% killing vs 34.3% after stimulation of all mononuclear cells). Pretreatment of tumor cells with chemoradiation showed a significant increase in susceptibility of tumor cells against NK cells after treatment with 5-FU and combinations (5-FU alone 66.9% lysis, CapRI scheme 69.1%, untreated tumor cells 34.3%). CONCLUSION: IFN-alpha activates NK cells against pancreatic carcinoma cells and 5-FU treatment makes tumor cells more susceptible. These mechanisms may be responsible for the good clinical outcome of CapRI.

21  TREA TMENT OF PANCREA TIC CANCER WITH ANTISENSE THERAPY SPECIFIC TO MUTATED K-RAS GENE IN VITRO AND IN VIVO IN SYRIAN GOLDEN HAMSTERS – IS IT SUITABLE?

Morioka CY¹, Matheus AS¹, Saito S², Machado MCC¹, Cunha JEM¹, Yamago GI³, Watanabe A², (1) Department of Surgery, University of São Paulo, Brazil; (2) 3rd Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan; (3) Department of Cell Biology, Okayama University Medical School, Okayama, Japan

INTRODUCTION AND AIM: Pancreas cancer prognosis is still reserved constituting a medical challenge. About >90% of this type of cancer carry the K-ras point mutation, which may play an important role in tumoral progression. Antisense oligonucleotides (ASO) targeting this gene may be a therapeutic choice. Thus, our aim was to elucidate the effectiveness of this gene therapy in a hamster experimental pancreatic cancer model. PATIENTS AND METHODS: HaP-T1, a cell culture derived from nitrosamine-induced hamster pancreatic cancer was used. MTT, MTT-agarose, Western blotting, and in vitro chemoinvasion assays were performed using ASO specific to K-ras gene. In exponential phase of growth, a tissue derived from subcutaneously implanted cancer cells was implanted into the pancreas. Animals were divided in 3 groups: 1. Positive control (PC), 2. Sense-treated hamsters (STH), and 3. Antisense-treated hamsters (ATH). Oligonucleotides were administered for 2 weeks. Follow-up was done by evaluation of the tumor growth by palpation,'general state', weight, and side effects. Five animals of each group were sacrificed at days 10, 17, 24, 31, 38, to study the local response and metastatic sites. Five animals of each group were left to study the survival time. Necropsy was performed and specimens were studied histopathologically. RESULTS: ASO inhibited the tumoral growth by suppression of K-ras p21 protein synthesis. It could also inhibit the invasiveness. All tumors were palpable. Positive controls, STH, and ATH survived on average 72.7, 73.8, and 79.6 days, respectively. Side effects were observed in both oligonucleotide-injected groups. Tumor sizes were on average smaller in the ATH group throughout the study. Spontaneous lymph node metastases were found from 31 days in the ATH group, while PC and STH groups showed metastases and direct invasion to adjacent organs from 17 days. After death, metastatic sites were similar in the 3 groups. Liver metastasis has a higher incidence in PC; moreover, only the PC group showed ascites. CONCLUSION: Antisense oligonucleotides inhibited tumor growth and invasiveness, and improved liver metastatic rate and ascites. Therefore, it may be a good approach in the management of pancreatic cancer. Moreover, it may contribute as neoadjuvant and/or adjuvant therapy.

22  METHYLENE BLUE AND THE REDUCTION OF PERITONEAL CARCINOMA TOSIS IN HAMSTER EXPERIMENTAL PANCREATIC CANCER MODEL – IS IT A PROMISING THERAPY?

Matheus AS¹, Morioka CY¹, Machado MCC¹, Saito S², Jukemura J¹, Cunha JEM¹, Watanabe A³, (1) Department of Surgery, University of São Paulo, Brazil; (2) 3rd Department of Internal Medicine, Toyama Medical and Pharmaceutical University; (3) 3rd Department of Internal Medicine, University of São Paulo, Brazil

INTRODUCTION AND AIM: Methylene blue, hematoporphyrin derivative Halle and chlorophyllin produce a marked reduction of tumor weight. In addition, it is known to inhibit the generation of oxygen radicals. This dye has been tried experimentally to prevent adhesion formation. However, it has never been reported in the prevention of tumoral adhesion. The aim of the present study was to elucidate the effectiveness of methylene blue to prevent peritoneal tumoral implantation in a hamster experimental pancreatic cancer model. PATIENTS AND METHODS: Tumor cell suspensions were injected intraperitoneally. Animals were divided in two groups: A. only injection (positive control, n=10) and B. injection and administration of methylene blue after the injection (n=10). The animals were observed and weighed until 14 days, when they were sacrificed. After necropsy, ascites volume was quantified and numbers of implantations were measured RESULTS: Hamsters of Group A were shown to be heavier throughout the experiments. After necropsy, Group A had an average 7.4 ml of ascites and generalized peritoneal carcinomatosis including diaphragm and Group B showed an average 2.6 ml of ascites and an average 9.4 implants located mainly in the pelvic region. CONCLUSION: The present study showed that methylene blue decreased the number of pancreatic cancer implants and the volume of ascites. This substance may be used as an adjuvant therapy to decrease or even prevent the adhesion of possible metastatic cells in the peritoneal wall.

23  PANCREATIC CANCER CELLS EXPRESS A UNIQUE NEUROGENIC FACTOR, BETA-TUBULIN

Standop J², Yalniz M¹, Saruc M¹, Pour PM¹, (1) Eppley Research Institute, UNMC, University of Nebraska Medical Center (UNMC), Omaha, USA; (2) University of Bonn, Department of Surgery, Bonn, Germany

INTRODUCTION AND AIM: It has been known that islets of Langerhans share similarities in the expression of neuroendocrine markers, including N-C AM, NSE, synaptophysin and chromogranin A. These markers are also expressed in some pancreatic carcinomas of ductal phenotype, suggestive of a neuroendocrine differentiation. One of the most specialized tubulins specific for neurons is class III beta-tubulin, which is essential throughout neuronal differentiation. Because we believe that pancreatic ductal cell carcinomas derive from transdifferentiated islet cells, we investigated the expression of beta-tubulin in pancreatic tissue. PATIENTS AND METHODS: Three normal pancreata (NP) and 15 ductal type adenocarcinomata (PC) with different differentiation grade as well as cultured human islet cells were examined by immunohistochemistry using a monoclonal and a polyclonal antibody against (class III) beta-tubulin. RESULTS: In the normal pancreatic tissue, the immunoreactivity of the polyclonal antibody was restricted to the islet cells, whereas 14 of 15 PC showed the expression of beta-tubulin. Within the islets, alpha-cells showed the strongest immunoreactivity. The reactivity of the monoclonal antibody with cancer cells, however, differed greatly. Only in three cases all pancreatic cancer cells were stained, three cases showed no staining and in the remaining cases the immunoreactivity varied between 20% and 70%. The immunoreactivity of islets within and outside of cancer were diminished or lost. Cultured human islet cells were immunoreactive with both antibodies. CONCLUSION: The results, thus, lend further support for the common origin of pancreatic ductal carcinomas and pancreatic islets from neuroendocrine cells.

24  PRIMARY AND METASTASTIC PANCREATIC ACINAR CELL CARCINOMA: A MOLECULAR ANALYSIS

Bergmann F¹, Aulmann S¹, Esposito I¹, Kleeff J², Friess H², Schirmacher P¹, (1) Institute of Pathology; (2) Department of General Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Pancreatic acinar cell carcinomas (PAC) are associated with a poor prognosis. At the time of diagnosis, approximately half of the patients display metastatic spread, usually affecting regional lymph nodes and the liver. To date, cytogenetic and molecular analyses have been limited to a few cases of primary PAC; however, no data have been obtained for their metastases. Therefore, the present study was designed to detect possible differences between primary and metastatic PAC. PATIENTS AND METHODS: Three primary PAC, one corresponding regional lymph node metastasis and two corresponding hepatic metastases were characterised by means of comparative genomic hybridisation, fluorescence in situ hybridisation, and immunohistochemistry. RESULTS: Chromosomal imbalances were detected in all tumors, most consistently affecting gains of 1 q, 8q, 12, 17, 20q and 22q, as well as losses of 1 p, 4q, 11 q and 18q. In general, similar chromosomal imbalances were seen in primary and metastatic PAC. However, some changes, including gains of 3p, 8q, and 17q, were more frequent or limited to metastatic PAC. Low level amplifications of c-MYC (8q24) were detected in the primary tumor and in one hepatic metastasis of one patient. Neither primary nor metastatic PAC revealed expression of Her2/Neu (17q21). CONCLUSION: PAC display a consistent chromosomal profile, which shows a large overlap between primary and metastatic PAC. Chromosomal imbalances, more frequently or exclusively occurring in metastatic PAC, indicate chromosomal loci possibly harboring candidate genes that are involved in tumor progression and spread.

25  REDUCED EXPRESSION OF TUBER IN ISASSOCIATED WITH PANCREATIC CANCER DEVELOPMENT

Kataoka K, Ito D, Fujimoto K, Doi R, Surgery and Surgical Basic Science, Kyoto University, Kyoto, Japan

INTRODUCTION AND AIM: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutation in either the TSC1 or TSC2 tumor suppressor genes. Recent studies have revealed that TSC1/TSC2 proteins (hamartin/tuberin, respectively) function downstream of Akt and upstream of mTOR, and are one of the regulators of cell growth and proliferation. It has been reported that amplification of AKT2 is observed in pancreatic cancer and mTOR/p70S6K signaling pathway is required for pancreatic cancer cell proliferation. But the precise role of tuberin in cancer development remains unclear. The purpose of this study is to examine whether tuberin expression is expressed or lost in pancreatic cancer tissues and cell lines, and to evaluate the correlation between tuberin expression and clinocopathological features. PATIENTS AND METHODS: First, immunohistochemical analysis was performed in 42 pancreatic cancer tissues and 10 noncancerous pancreatic tissues with an anti-tuberin antibody. Next, we examined mRNA expression of tuberin in pancreatic cancer tissues by RT-PCR analysis. Correlation between tuberin expression and clinocopathological features was evaluated. We also examined tuberin expression in 6 pancreatic cancer cell lines by Western blot and RT-PCR analysis. RESULTS: All the noncancerous tissues showed strong expression, whereas 57% of pancreatic cancer tissues showed reduced expression of tuberin. Especially, 11 specimens of pancreatic cancer were stained negligibly negative for tuberin. RT-PCR also showed weak to strong expresson in cancer tissues. The reduced tuberin expression was significantly correlated with patients with advanced pT factor (3 and 4) of UICC classification (p = 0.024) and male patients (p=0.014). No statistical correlation with survival was found in these series of immunohistochemstry. In pancreatic cancer cell lines, the expression of tuberin was observed at almost equal level. CONCLUSION: We demonstrated that tuberin expression was reduced and associated with pT factor in pancreatic cancer. These results suggest that tuberin plays a certain role in pancreatic cancer development.

26  MODULATION OF PANCREATIC ABLA TION BY SIMULATED PORTAL CIRCULATION IN AN EX VIVO PORCINE MODEL OF RADIOFREQUENCY ABLATION

Date RS², McMahon R¹, Siriwardena A², (1) University Department of Histopathology, Manchester Royal Infirmary; (2) HPB Unit, Department of Surgery, Manchester Royal Infirmary, Manchester, UK

INTRODUCTION AND AIM: We have reported the development of an ex vivo porcine model for the study of radiofrequency ablation of pancreatic parenchyma. This study evaluates the effects of variation of target temperature, duration of ablation and heat-sink modulation of portal circulation on thermal injury as a precursor to human trial. PATIENTS AND METHODS: The starburst probe (RITA, Mountainview CA) was used. Radiofrequency was applied to a pre-marked area in the centre of the pancreatic head. The effect of variations in temperature, duration and simulated portal circulation were studied. Portal circulation was simulated by the use of a recir-culating haemofiltration pump which perfused warm saline at a rate of 100 ml/h. Post-ablation pancreatic biopsies incorporated the duodenum, portal vein and bile duct. Control non-ablated biopsies were from the tail of the pancreas. Temperatures between 70 and 100°C were evaluated in 10° increments keeping duration constant at 10 min. All experiments were repeated 5 times in separate pancreata at each setting. RESULTS: The minimum temperature needed to produce complete pancreatic ablation was 90°C. There was no additional benefit from ablation at higher temperatures or greater duration. Simulated portal circulation had no effect on ablation. Histologic analysis showed no evidence of thermal injury to the duodenum. CONCLUSION: This study has demonstrated that pancreatic parenchymal ablation is consistently achieved at 90° C for 10 min. At this temperature there is no thermal injury to the duodenum and this effect of radiofrequency was not affected by simulated portal circulation. These findings are an important step in the validation process towards radiofrequency ablation of non-resectable pancreatic tumours in man.

27  EFFECT OF RETINOIC ACID ON P21WAF1 TURNOVER IN HUMAN PANCREATIC CANCER CELLS

Singh B¹, Adrian T², Roginsky AB¹, Ding XZ¹, Murphy RF¹, Talamonti MS¹, Bell RH¹, (1) Department of Surgery, Northwestern University, Chicago, IL, Department of Biomedical Sciences, Creighton University, Omaha, USA; (2) Northwestern University Feinberg School of Medicine, Surgical Research Departments of Surgery and Pathology, Chicago, USA

INTRODUCTION AND AIM: Retinoic acid (RA) inhibits growth by modulating cell cycle proteins, including p21, in various types of cancer cells. The effects of RA on pancreatic cancer cell growth are controversial. We investigated whether p21 was involved in RA-induced growth inhibition, using conditions optimized for RA response. RESULTS: In CD-18 cells, RA caused a massive but transient 60-fold increase in p21 mRNA levels at 18 h, accompanied by a three-fold increase in p21 protein levels. Levels of p21 mRNA had normalized by 48 h, while p21 protein declined to levels 80% lower than control. In HS766T cells the transient 8-fold increase inp21 mRNA came later (24–48 h). No early increase in p21 protein was seen in HS766T, but levels declined at later times. The decrease of p21 protein levels following an increase in the mRNA suggested that degradation of the protein was induced. The RA-induced decrease in p21 was blocked by the proteosome inhibitor, MG132, but not by the caspase-3 inhibitor III. These findings suggest that the RA-induced p21 degradation is mediated by the proteosome but not by executioner caspases. However, we also detected ∼ 32-fold increase in the mRNA expression of the recently discovered p53 inducible ring finger protein (p53RFP), which is an E3 ligase. This suggests that some form of p21 is targeted for ubiquitination. CONCLUSION: These findings demonstrate RA-induced p21 proteosomal degradation which may be ubiquitin-independent. This decrease in p21 is unexpected in the face of RA-induced growth arrest and may represent a cell-survival mechanism.

28  TARGETED AND INDUCIBLE GENE/IMMUNOTHERAPY OF PANCREATIC CARCINOMA

Wenger TU¹, Ucur E¹, Moldenhauer G², Friess H³, Herr I¹, (1) CCU Pediatric Oncology, DKFZ, Heidelberg; (2) Moecular Immunology, DKFZ, Heidelberg; (3) General Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Pancreatic carcinoma is a frequent malignancy of the gartrointestinal system and associated with poor prognosis. Due to the unsatisfactory response to conventional therapy and 5-year survival rates below 15%, it ranks fifth among death rates by cancer. Hence, new approaches for therapy of pancreatic carcinoma are needed. PATIENTS AND METHODS: Our combination gene/immunotherapy approach uses tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL/Apo2L) as therapeutic gene as TRAIL has been shown to induce apoptosis specifically in cancer cells but not in normal cells. For tumor-specific and inducible delivery patient-derived cytotoxic T cells (CTLs) are transduced with recombinant lentiviral vectors which express TRAIL under the control of a tetracycline-inducible promoter. The manipulated CTLs are armed with bispecific antibodies able to crosslink antigens commonly overexpressed in malignant cells (e.g. Her2, EGFR) and the CD3 T-cell surface marker. Thus, the nonspecific T cells are converted to tumor-specific TRAIL-KILLER cells, which can home to the site of the tumor and metastases. After switching on TRAIL expression by the antibiotic tetracycline, apoptosis is induced in tumors. Our inducible TRAIL-based immunotherapeutic approach may prove to be effective for targeting a variety of pancreatic tumors with or without chemotherapy. RESULTS: Current experiments focus on preclinical studies using established cell lines in vitro and in vivo studies using xenografts in nude mice. We have used stably transfected Jurkat T-cells for preliminary experiments, showing that tet-induced TRAIL surface overexpression on these cells efficiently induces apoptosis in target cells, and are currently optimizing the lentiviral inducible expression system.

29  PROTEIN EXTERNALIZATION DURING ANGIOGENESIS IN EXPERIMENTAL PANCREA TIC AND HEPATOCELLULAR CARCINOMA

Ryschich E, Knaebel HP, Büchler M, Schmidt J, Department of Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: The process of tumor angiogenesis comprises a strong re-organization of intracellular proteins in endo-thelial cells including protein externalization. Externalized proteins could distinguish tumor endothelial cells from normal endothelium and represent a possible vascular target for antitumoral therapy. The externalization of several proteins such as actin, tropomyosin and F0/F1-ATP-synthase has been found on proliferating endothelial cells in vitro. The aim of the study was to identify whether these proteins are externalized on tumor endothelium in experimental pancreatic and hepatocellular cancer in vivo. PATIENTS AND METHODS: Rat pancreatic (DSL6A) and hepatocellular cancer (MH3924) were induced by subcutaneous inoculation of tumor cells in male Lewis (DSL6A) and ACI (MH3924) rats. After establishment of a solid tumor, 0.25 mg of mouse antibodies against actin, tropomyosin, F0/F1-ATP-synthase, ICAM-1 (positive control) and mouse immunoglobulin G (negative control) were injected intravenously. Three tumor-bearing rats were used for every single antibody. Tumors were removed 10 min after injection of antibody. The tumors were cut and stained with anti-mouse secondary antibodies. Additionally, intracellular staining was performed by standard immunohistochemistry with identical antibodies. RESULTS: The endothelium of venular endothelium was strongly stained after injection of anti-ICAM-1 antibodies. No cell membrane binding of anti-actin, tropomyosin or F0/F1-ATP-synthase antibodies after intravenous injection was identified, although a strong intracellular staining was found by immunohistochemistry. CONCLUSION: In contrast to in vitro data, actin, tropomyosin and F0/F1-ATP-synthase are not externalized in vivo and thus are unlikely to represent a vascular target for anticancer therapy.

30  CT ANTIGEN EXPRESSION IN PANCREATIC ADENOCARCINOMA AND NORMAL PANCREAS

Schmitz-Winnenthal FH¹, Z^’graggen K⁵, Volk C¹, Galindo LV¹, Tempia A², Rimoldi D³, Romero P⁴, Büchler MW¹, (1) Department of Surgery, University of Heidelberg, Heidelberg, Germany; (2) Department of Surgery, University of Lausanne, Lausanne; (3) Molecular Tumor Immunology Group, Ludwig Institute for Cancer Research; Lausanne; (4) Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research; Lausanne; Switzerland; (5) Klinik Beau-Site Hirslanden, Department of Surgery, Bern, Switzerland

INTRODUCTION AND AIM: Cancer testis (CT) antigens are antigens shared by a variety of malignant tumors, but usually not by normal tissue. The exceptions are germ cells in testis. Restricted expression in neoplastic tissues and immunogenic features make CT antigens ideal for use in immunotherapy. Effective immuno-therapy requires antigenic peptides with binding motifs restricted to specific MHC alleles. Pancreatic cancer has been poorly studied with regard to CT antigen expression. The investigation of a selected panel of CT antigens and tumor-related antigens in pancreatic cancer that have been shown to elicit an efficient immunogenic response in other malignancies was the aim of this study. PATIENTS AND METHODS: Pancreatic adenocarcinoma samples (n=76) were obtained intraoperatively, control tissues (n=5) were collected from cadaveric donor and from patients with chronic pancreatitis. CT antigen expression of MAGE A1, MAGE A3, MAGE A4, MAGE A10, LAGE 1, NY-ESO-1, SSX-2, and SSX-4 were investigated. Tumor-related antigens PRAME, NA-17A, as well as HERV-K-Mel expression were also assessed by PCR. Western blot analysis and sequencing were performed to assess the expression of SSX-4 in neoplastic and normal pancreatic tissues. RESULTS: SSX-4 was detected in 10.5% of the tested pancreatic adenocarcinoma specimens, the rest of the explored CT antigens were expressed in <3% of the samples. HERV-K-Mel was found positive in 20% of the tested pancreatic adenocarcinoma samples. Surprisingly, SSX-4 (100%) and faintly SSX-2 expression (60%) was detected in normal pancreatic tissue; Western blot analysis and sequencing confirmed the expression of SSX-4 in both malignant and normal pancreatic samples. CONCLUSION: The explored panel of CT antigens showed an overall low expression rate (<5%). SSX-4 expression in both neoplastic and normal pancreatic tissue may indicate immunological relevance. However, HERV-K-Mel is expressed with a relatively high prevalence and may be a candidate for specific immunotherapy in a large subgroup of pancreatic cancer patients.

31  DENDRITIC CELLS REDUCE NUMBER AND FUNCTION OF CD4 + CD25+ CELLS IN CYTOKINE-INDUCED KILLER CELLS DERIVED FROM PATIENTS WITH PANCREATIC CARCINOMA

Schmidt J, Märten A, Büchler M, Department of Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: CD4 + CD25 + cells are described as professional regulatory/suppressor T cells that are crucial for the prevention of spontaneous autoimmune diseases. They play an important role in maintaining a balanced peripheral immune system. On the other hand, it has been suggested that regulatory T cells (Treg) suppress antitumor immune responses after tumor-specific vaccinations. PATIENTS AND METHODS: Therefore, we determined the percentage of regulatory T cells in cytokine-induced killer cells (CIK cells), an effector cell population with high impact for adoptive immunotherapeutic strategies. RESULTS: CIK cells showed strong induction of CD4 + CD25 + cells with high secretion of IL-10 after nonspecific stimulation of the TCR complex and stimulation with IL-2. Depletion of CD25 + cells led to an increase in cytotoxic activity and a reduction of IL-10 release. A more pronounced reversal of suppression could be induced by co-culture of CIK cells with dendritic cells (DCs). After co-culture of CIK cells with DCs, the number of CD4 + CD25+ cells as well as the IL-10 concentration in the supernatant decreased and the cytotoxic activity against pancreatic carcinoma cells increased. This was shown for cells from healthy donors as well as for cells from patients with pancreatic carcinoma. CONCLUSION: In conclusion, our established effector cells possess some regulatory features induced by unspecific TCR activation that could be prevented by co-culture with DCs. CIK cells have desirable properties for immunotherapeutical approaches, especially after co-culture with DCs, which could be used additionally for induction of a specific immune response.

32  MOLECULAR CANCER DIAGNOSTICS OF PANCREATIC TUMOURS USINGMICROARRAY TECHNOLOGIESHoheisel J¹, Beier V¹, Kusnezow W¹, Kleeff J², Bauer A¹, Lyko F³, Friess H², (1) Funktionelle Genomanalyse, DKFZ, Heidelberg; (2) Department of General Surgery, University of Heidelberg; (3) Epigenetics, DKFZ, Heidelberg, Germany

INTRODUCTION AND AIM: The diagnosis of ductal adenocarcinoma of the pancreas is associated with a poor prognosis, an increasing incidence and no or only ineffective means of treatment. We are developing and evaluating chip-based diagnostic analyses at the level of epigenetic variations, changes in transcript levels and actual protein expression. PATIENTS AND METHODS: DNA-methylation patterns are analysed with in situ synthesised oligo-nucleotide arrays that query the promoter of currently some 250 genes. Transcript profiling is performed on a microarray that consists of PCR products, which represent about 3500 human genes that are known to be differentially transcribed in pancreatic cancer cells and thus expected to show a representative expression pattern in both ductal adenocarcinoma and cystic lesions. Based on earlier transcriptional analyses, we selected 900 genes, which exhibited a significant increase of RNA levels. From their sequences, peptides were selected and used for immunisation of rabbits. The resulting antibodies are also arrayed on microscopic slides for an investigation of variations in the protein expression. In addition, we isolate recombinant antibodies, which are specifically binding to either normal or tumour cells. RESULTS: Microarray experiments on various samples were performed and allowed a classification of different types of pancreas lesions. Genes or proteins were identified that are highly associated with the occurrence of tumours and provide reasonable chances of affecting pathways that are essential for pancreatic tumours. Analyses of serum samples and other body fluids for the establishment of an early, non-invasive diagnostic assay are ongoing and will be discussed at the meeting. CONCLUSION: The data resulting from our studies will allow a detailed analysis of regulative factors, which are critical to pancreatic tumours, as well as the identification of highly relevant molecule interactions. In addition, diagnostic assays are established, which might be of significant clinical utility to detect cancer cells in tissue from patients with different types of pancreatic carcinoma and to draw prognostic conclusions based on their molecular appearance.

33  PROFILING OF CYSTIC NEOPLASMS OF THE PANCREAS USINGMICROARRAY TECHNOLOGY

Bauer A¹, Fellenberg K¹, Friess H², Kleeff J², BierM¹, Hoheisel JD¹, (1) Funktionelle Genomanalyse, DKFZ Heidelberg; (2) Division of General Surgery, Universität Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Cystic pancreatic neoplasms account for approximately 5% of primary malignancies of the pancreas and may be benign, pre-malignant or malignant. These types of pancreatic tumours are characteristic for the better prognosis compared to the more aggressive ductal adenocarcinoma. In this study, we are developing and evaluating a DNA diagnostic chip with about 3500 human genes known to be differentially transcribed in pancreatic cancer cells and thus expected to show a representative expression pattern in both ductal adenocarcinoma and cystic lesions. PATIENTS AND METHODS: cDNAs representing different human genes were PCR-amplified, purified and robotically arrayed onto slides with an epoxy surface. Fluorescently labelled cDNA samples were prepared from total RNA isolated from cells of human pancreas tissue by incorporation of labelled dCTPs during reverse transcription. RESULTS: Currently, we can detect transcript variations with only 5 mg of total RNA as starting material with no need for amplification. Microarray experiments on various samples are being performed and analysed, allowing classification of different types of pancreas lesions and the identification of potential targets as a means of eventually developing new modes of treatment. CONCLUSION: The resulting diagnostic DNA chip will be of significant clinical utility to detect cancer cells in tissues from patients with different types of pancreatic carcinoma and to draw prognostic conclusions based on their molecular appearance. Furthermore, comparative studies on transcriptional profiling and actual protein expression by means of complex DNA and antibody microarrays are under way. Combining these data with clinical information permits the definition of subgroups within an analysed cohort and may eventually provide a robust means for diagnosis and prognosis as well as the identification of highly relevant molecular pathways.

34  INHIBITION OF CYTOTOXIC GAMMA/DELTA T CELLS BY PANCREATIC CARCINOMA PATIENTS’ DERIVED SOLUBLE MIC+ SERUM COULD BE RESTORED BY CAPTURING SOLUBLE MIC WITH ANTIBODIES

Salih H¹, Märten A², Steinle A¹, Büchler MW², Schmidt J², (1) Department of Internal Medicine, University of Tübingen, Tübingen, Germany; (2) Department of Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: NKG2D is known to stimulate tumor immunity through activation of T cells, NK cells and gamma/ delta T cells. Its ligands MICA/B and ULBPs have been shown to be broadly expressed on epithelial tumors. Shedding of MICA is suspected to modulate NKG2D-mediated tumor immune surveillance in a negative way. PATIENTS AND METHODS: Sera from 55 patients with pancreatic adenocarcinoma were tested for presence of soluble MICA/B (sMIC). NK cells or gamma/delta T cells were used as effector cells against pancreatic carcinoma cells in cytotoxicity assays in the presence of either MIC-positive or -negative serum. RESULTS: Patients showed elevated serum levels of sMICA/B. sMICA/B level correlated with tumor grade, grade of differentiation and tumor stage. NK92 cells were inhibited after addition of sMIC+ patients’ sera. Pancreatic tumor cells expressing MIC and or ULBPs were lysed by NK92 cells in an NKG2D-dependent fashion, this could be blocked by addition of sMIC+ sera. Gamma/delta T cells killed pancreatic tumor cells in a NKG2D-dependent way. The inhibition of cytolysis in the presence of sMIC+ sera was mediated by sMIC. Addition of anti-MIC to the patients' sera in cytotoxicity assays restored the cytolytic activity nearly completely. CONCLUSION: Pancreatic carcinomas express MIC and shed sMIC. This phenomenon is dependent on tumor grade, differentiation and stage. sMIC inhibits cytotoxic activity of NK cells as well as gamma/delta T cells. Gamma/delta T cells lyse pancreatic tumor cells after recognition of MIC or ULBPs via NKG2D receptor; this could be restored by capturing of sMIC by specific antibodies.

35  A NEW THERAPEUTIC STRATEGY FOR THE TREATMENT OF PANCREATIC CANCER BY A CONDITIONALLY REPLICATION-COMPETENT HSV-1 VECTOR

Kami K¹, Doi R¹, Ito D¹, Miyatake SI², Imamura M¹, (1) Department of Surgery and Surgical Basic Science, Kyoto University, Kyoto, Japan; (2) Department of Neurosurgery, Osaka Medical College, Osaka, Japan

INTRODUCTION AND AIM: In this study, we constructed an oncolytic herpes simplex virus 1 (HSV-1) vector (d120.survE) in which the survivin promoter drives expression of ICP4, a major transactivating factor for viral genes, so that replication of the vector is restricted to survivin-expressing cells. Survivin is not expressed in normal adult tissues, but it becomes highly expressed in transformed cell lines and in most of the common human cancers including pancreatic cancer. Then, we assessed the ability of d120.survE to inhibit the growth of pancreatic cancer cells in vitro and in vivo. PATIENTS AND METHODS: A 397-bp survivin promoter was characterized using luciferase reporter assays, and was cloned into the thymidine kinase (tk) gene of mutant HSV-1 d120, deleted for both copies of the ICP4 gene. This vector also contains the E. coli LacZ gene under control of the tk promoter. ICP4 and LacZ expression in the cells infected by d120.survE were examined by Western blot analysis and X-gal staining. The ability of d120.survE to replicate specifically in survivin-expressing cells was examined by a viral single-step growth experiment in three human pancreatic cancer cell lines (Panc-1, SUIT-2, and AsPC-1). The in vitro cytotoxic activity of d120.survE was examined by infecting the same cell lines with the vector at a low multiplicity of infection (0.001 to 10). For in vivo studies, nude mice harboring AsPC-1 tumors subcutaneously recieved twice on days 0 and 7 intra-neoplasmic injection of 10⁶ plaque-forming units of d120.survE. The tumor sizes were measured every 5 days. Expression of the lacZ gene was also examined by X-gal staining. RESULTS: ICP4 and LacZ were expressed in pancreatic cancer cells infected by d120.survE. The ability of d120.survE to replicate and the cytotoxic activity of d120.survE were correlated with survivin promoter activity of the host cells. There were 60% and 10% cells surviving respectively in Panc-1 (low expression of survivin) and AsPC-1 (high expression of survivin) cells infected by d120.survE at an MOI of 0.01 on day 7 post-infection compared to those of mock-infected cells. Subcutaneous tumors treated with d120.survE were significantly smaller than control tumors by day 30 post-infection. Positive X-gal staining was also observed in the tumor nodule which was challenged with this viral vector. CONCLUSION: A conditional replication-competent HSV-1 vector regulated by the survivin promoter may be a new therapeutic strategy for treatment of pancreatic cancer.

36  PLASMA LEVELS OF MATRIX METALLOPROTEINASE-7 CAN BE USED TO DIFFERENTIATE BETWEEN PERIAMPULLARY CARCINOMA AND CHRONIC PANCREATITIS

Kuhlmann KFD¹, van Till ORC¹, Boermeester M¹, Tsvetanova I³, Offerhaus J², ten Kate F², Busch O¹, van Gulik T¹, Gouma D¹, Crawford H³, (1) Department of Surgery, Academic Medical Center, Amsterdam; (2) Department of Pathology, Academic Medical Centre; Amsterdam, The Netherlands; (3) Department of Pharmaceutical Sciences, State University of New York at Stony Brook, New York, USA

INTRODUCTION AND AIM: The radiological differentiation between periampullary carcinoma and chronic pancreatitis (CP) with an inflammatory mass is difficult. Consequently, 5–10% of pancreatic resections are performed for CP. The aim of this study was to test whether the levels of matrix metalloproteinase-7 (MMP-7), a secreted protyolytic enzyme, can distinguish between these two disorders. PATIENTS AND METHODS: MMP-7 levels in plasma, pancreatic and duodenal juice were analyzed in 22 patients who underwent pancreaticojejunostomy for CP and 60 consecutive patients who underwent exploratory laparotomy for a suspected periampullary malignancy. RESULTS: MMP-7 plasma levels were significantly higher in patients with pancreatic carcinoma (1.99 ng/ml, range 0.50-10.18, n=28) and distal cholangiocarci-noma (1.83 ng/ml, range 0.66–6.44, n=10) compared to those with CP (0.83 ng/ml, range 0.25-3.53, n=27). MMP-7 levels in pancreatic juice were significantly elevated in pancreatic cancer (143 ng/mg protein, range 0.6–9789, n=17) compared to those in CP (15 ng/mg protein, range 0–2380, n=31). MMP-7 levels in duodenal juice did not significantly differ between groups. Plasma levels of 5 patients who underwent a resection for an inflammatory mass were comparable with those of the other patients with CP (1.16 ng/ml, range 0.79–2.16). When a plasma threshold of 1.5 ng/ml was used in patients who underwent exploration, the positive predictive value for the determination of periampullary (pancreatic and distal cholangio) carcinoma was 96% with a specificity of 80%. The sensitivity was 63%. CONCLUSION: Plasma MMP-7 levels can be used as a marker for pancreatic cancer and in particular to differentiate between periampullary cancer and CP in cases of diagnostic uncertainty by conventional diagnostics.

37  FUNCTIONAL TUMOR REACTIVE T CELLS IN BONE MARROW AND BLOOD OF PANCREATIC CANCER PATIENTS

Schmitz-Winnenthal FH¹, Beckhove P², Volk C¹, Weitz J¹, Nummer D², Galindo LV¹, Büchler MW¹, Schirrmacher V², Z'graggen K³, (1) Department of Surgery, University of Heidelberg, Heidelberg; (2) Division of Cellular Immunology, German Cancer Research Center, Heidelberg, Germany; (3) Department of Surgery, Klinik Beau-Site Hirslanden, Bern, Switzerland

INTRODUCTION AND AIM: Memory T cells specifically reactive to tumor antigens should be an ideal source to generate therapeutic effector cells against pancreatic cancer. To our knowledge functionally active, specific memory T cells have not been demonstrated in pancreatic cancer patients. To investigate if functional memory T cells are present in the bone marrow (BM) of pancreatic cancer patients, we performed in vitro stimulation with tumor-associated antigens presented by autologous dendritic cells (DCs). PATIENTS AND METHODS: DCs and T cells were generated in a 14-day culture with GM-CSF and IL-4 (DCs) and/or IL-2, IL-4, IL-7 (T cells) from BM-derived CD34+ progenitor cells of 35 patients with pancreatic carcinoma and chronic pancreatitis. DCs were pulsed with lysate from autologous tumor cells, Muc I peptide fragments and PBMCs (peripheral blood mononuclear cells as negative controls). Tumor specific T-cell response was evaluated in a single-cell interferon-a, IL-4, IL-10 and IL-12 release immunospot assay (Elispot). Cytotoxicity of CD8+ T cells against primary cultured tumor cells was measured by a functional DNA release, and trypan blue staining assay. RESULTS: In the individual patient, T cells stimulated by tumor lysate pulsed DCs as well as DCs pulsed with Muc I peptide showed a significantly higher response in the INF-α Elispot assay compared with T cells stimulated by PBMC-lysate pulsed DCs. The tumor specific T-cell frequency found in the bone marrow was consistently high (49/105 to 109/105) in all patients compared with the response found in the peripheral blood (9/105 to 53/105; p < 0.05 to p<0.001). In the cytotoxicity assay, the response was significantly higher when T cells were stimulated with tumor lysate pulsed DCs compared with PBMC-lysate pulsed DCs. CONCLUSION: Our data indicate for the first time a regular occurrence of tumor antigen specific T-cell responses during the course of pancreatic cancer that lead to the generation and enrichment of functional tumor cell-reactive memory T cells in the bone marrow. The bone marrow may therefore be considered an important lymphoid organ for the induction or maintenance of anti-tumor immune responses in pancreatic cancer.

38  DNA CONTENT AND SURVIVAL RATES OF PATIENTS WITH PANCREATIC CANCER: A PROSPECTIVE STUDY OF 65 CASES

Al-Abadi HPD², Abou-Rebyeh H¹, Schumacher G², (1) Gastroenterologie-Hepatology, Charite Campus Virchow-Klinikum; (2) Allgemein, Visceral, Transplantationschirurgie, Charite Campus Virchow-Klinikum, Berlin, Germany

INTRODUCTION AND AIM: Nuclear DNA content and pathology are considered to be prognostically relevant to several solid tumors, but controversial findings have emerged from pancreatic carcinoma (PC). PATIENTS AND METHODS: During the 3-year period between January 1999 and December 2001 we enrolled a total of 64 patients with histologically proven ductal adenocarcinoma of the pancreas in a prospective study. DNA ploidy was assessed by image cytometry in cytological specimens obtained from ERCP by aspiration and brush cytology. All 64 patients were followed up till death (n=48) or in case of survival (n=16) a mean follow-up period was 12±9 months. Survival was calculated by using the Kaplan-Meier method. Risk factors were identified with Cox regression analysis. RESULTS: DNA cytometry revealed aneuploid (n=47) as well as polyploid (n=15) and dilpoid (n=2) pancreatic cancer. Resection margins, lymph node status and tumor differentiation were independent prognostic factors. Patients with diploid and polyploid pancreatic adenocarcinoma had significantly longer survival (median survival was 35 months) compared to 15 months in patients suffering from aneuploid pancreatic cancer. This diploidy associated with survival benefit persisted after adjustment for prognostic factors including tumor size, margins, lymph node status and pathological stage. CONCLUSION: Patients with pancreatic adenocarcinoma will survive longer if their cancer is diploid or polyploid and will have a very poor prognosis if afflicted with DNA aneuploid cancer. Based on the analysis carried out the authors conclude that the main prognostic factor for long-term survival is DNA ploidy itself showing close association with natural development and tumor biology. The predictive value of DNA ploidy is present in patients with resectable or unresectable tumor stage. The results may be helpful for predicting life expectancy, determining treatment strategies and designing future clinical trials.

39  THE INHIBITION OF SRC TYROSINE KINASE ENHANCES THE EFFICACY OF CHEMOTHERAPEUTIC AGENTS AGAINST PANCREATIC CARCINOMA CELLS

Bruns CJ², Ischenko I¹, Papyan A¹, GubaM¹, Green T³, Jauch KW¹, (1) Surgery, University of Munich-Großhadern; (2) Department of Surgery; University of Munich-Großhadern, Munich, Germany; (3) AstraZeneca, Macclesfield, Alderley Park, UK

INTRODUCTION AND AIM: Pancreatic cancer is one of the most aggressive tumors and resistant to almost all cytotoxic agents. The majority of pancreatic cancer cell lines over-express Src tyrosine kinase (Src kinase). Here we explored whether inhibition of the Src can reduce resistance of the human pancreatic carcinoma cells to chemotherapy in vivo and in vitro. PATIENTS AND METHODS: We used an orthotopic animal model, immunohistochemical analyses of pancreatic tumor samples and MTT assays to evaluate in vivo and in vitro the effect of Src to reduce chemotherapy resistance in pancreatic tumor cells and human pancreatic tumors growing orthotopically in nude mice. RESULTS: The pre-existing resistance to chemotherapeutical agents such as gemcitabine and 5-FU in L3.6pl and AsPc human pancreatic cell lines was found to be associated with expression of the active Src kinase. IC50 doses detected by MTT cells viability assay of gemcitabine and 5-FU were 0.0487 µg/ml and 10.1 µg/ml for L3.6pl cells and 452.6 mg/ml and 112.9 µg/ml for AsPc, respectively. IC50 doses after combination of AZM with gemcitabine or 5-FU were significantly reduced in L3.6pl (0.0272 µg/ml and 0.532 µg/ml) and in AsPc (56.4 µg/ml and 24.3 µg/ml), respectively. The sensitizing effect of Src kinase inhibition towards cytotoxic agents such as gemcitabine was confirmed in vivo: the amount of TUNEL-positive apoptotic tumor cells was significantly higher in pancreatic tumors following orthotopic cell injection in nude mice when the animals were treated with AZM for 1 week at the end of a treatment period with increasing doses of gemcitabine as compared to those only treated with increasing doses of gemcitabine. Furthermore, treatment of the nude mice 7 days after orthotopic injection of L3.6pl human pancreatic cells with low dose gemcitabine (50 mg/kg, twice-weekly i.p.) in combination with AZM (50 mg/kg, daily oral) led to a 9 3.1% reduction of primary pancreatic tumor growth comparable with those treated with high dose gemcitabine (100 mg/kg, twice-weekly i.p.) in combination with AZM (50 mg/kg, daily oral) (92% reduction of primary pancreatic tumor weight). CONCLUSION: In summary, our data indicate that inhibition of Src significantly enhances the anti-tumor efficacy of standard chemotherapy in human pancreatic cancer. In consequence, the inhibition of Src can significantly reduce the dose of the cytotoxic agents such as gemcitabine without any deficit in their anti-tumor efficacy, but less side effects.

40  GAINOFCHROMOSOME 8Q DETECTED BY COMPARATIVE GENOMIC HYBRIDISATION (CGH) IS ASSOCIATED WITH POOR SURVIVAL IN PATIENTS WITH RESECTABLE PANCREATIC CANCER

Schleicher C², Poremba C¹, Wolters H², Boecker W³, SenningerN², Colombo-Benkmann M², (1) Institute of Pathology, University of Düsseldorf, Düsseldorf, Germany; (2) Department of General Surgery, University of Münster; (3) Institute of Pathology, University of Muenster, Muenster, Germany

INTRODUCTION AND AIM: The objective of this study was to detect specific genomic alterations involved in initiation and progression of pancreatic cancer (PCA). Chromosomal imbalances were correlated with histopathological and clinical data to verify the prognostic significance of identified cytogenetic changes. PATIENTS AND METHODS: Paraffin-embedded specimens from 33 patients with PC were investigated by comparative genomic hybridisation (CGH). Microdissection was used for separation of PC from normal cells before isolation of DNA, nick end labeling and hybridization were performed according to standard protocols. Aberrations were correlated with histopathological staging (UICC 2002) by univariate and multivariate anaylsis using log rank test and Cox regression, respectively. Survival rates were plotted using the Kaplan-Meier method. RESULTS: 28 (85%) PC showed chromosomal aberrations. Gains of chromosomal material were most frequently identified on 8q (42%), 13q (30%), 18p (21%), and 3q (18%). Genetic losses were frequently detected on 1p (45%), 22 (42%), 19 (36%), 17p (27%), 18q and 8p (15% each) and 3p (12%). Losses of 8p (n=5) and 3p (n=4) were only detected in stage III and IV PC (p<0.05). Median survival time of all patients was 13 months. Median survival time of patients with aberration of 8q (n=14) was 8.5 months compared to 16 months in patients without gain of 8q (n=19; p=0.029). CONCLUSION: The chromosomal regions containing genetic alterations represent potential loci for new target genes in PC. The significant correlation of gain of chromosome 8q with short survival time suggests that potential new prognostic markers could be located on this chromosomal region.

41  TRAF2 AND THE CD95 DEA TH RECEPTOR MEDIA TE INVASIVENESS OF PANCREATIC CANCER CELLS

Trauzold A¹, Röder C¹, Sipos B², Karsten K¹, Arlt A³, Kalthoff H¹, (1) Sektion Molekulare Onkologie, UKS-H, Klinik f. Allgemeine Chirurgie u. Thoraxchirurgie; (2) Institut f. Allgemeine Pathologie, UKS-H; (3) Labor f. Molekulare Gastroenterologie, UKS-H, 1. Med. Klinik, Kiel, Germany

INTRODUCTION AND AIM: Pancreatic ductal adenocarcinoma is characterized by poor prognosis due to apoptosis resistance and its highly metastatic potential. Anti-apoptotic changes in pancreatic tumor cells include high constitutive and death receptor-induced activity of the transcription factor NF-kappaB. TRAF2 is an adaptor molecule involved in death receptor-mediated NF-kappaB induction. The aim of this study was to investigate the role of TRAF2 in the pathophysiology of pancreatic adenocarcinoma. PATIENTS AND METHODS: Expression of TRAF2 was detected by immunohistochemistry and by Western blot analysis. Low TRAF2-expressing Colo357 cells were transfected with a TRAF2-expression plasmid and the effects were analyzed using an in vitro invasion assay and the ‘JAM’ -apoptosis assay. Activation of transcription factors was detected by EMSA. Secretion of IL-8, uPA, MMP-9, and MMP-2 was analyzed by ELISA or by zymography. RESULTS: TRAF2 was constitutively overexpressed in 34 of 36 pancreas adenocarcinomas and in pancreatic tumor cell lines. Transfection and overexpression of TRAF2 in apoptosis-sensitive Colo357 cells led to resistance against CD95-induced apoptosis, increased constitutive NF-kappaB and AP-1 activity, higher secretion of MMP-2, MMP-9, uPA, and IL-8, as well as enhanced invasive growth. Stimulation of Colo357/TRAF2 cells with CD95-ligand resulted in an additional NF-kappaB and AP-1 induction, secretion of IL-8 and uPA, and a further increased invasiveness. CONCLUSION: TRAF2 is involved in establishing the malignant phenotype of pancreatic carcinoma cells. Overexpression of TRAF2 switches CD95-induced apoptosis towards survival and enhanced invasiveness after stimulation of this death receptor. The high incidence of TRAF2 positivity in clinical tumor samples further underlines its pathophysiological importance.

42  AN ANALOGUE OF SANSAL VAMIDE POTENTLY INHIBITS PANCREATIC CANCER CELL GROWTH THROUGH G0/G1 CELL CYCLE ARREST

Ujiki MB¹, Adrian T², Ding XZ¹, Liu S¹, Gu W¹, Roginsky A¹, Salabat MR¹, Silverman R¹, Talamonti MS¹, Bell RH¹, (1) Department of Surgery and Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL; (2) Northwestern University Feinberg School of Medicine, Surgical Research Departments of Surgical and Pathology, Chicago, USA

INTRODUCTION AND AIM: Patients with pancreatic cancer have little hope for cure because no effective therapies are available. Gemcitabine is the first-line chemotherapeutic agent used, but has little impact on survival. Sansalvamide A is a product of a marine fungus and has been used to develop a novel family of analogues for use as potential chemotherapeutic drugs. We hypothesized that these peptides would inhibit human pancreatic cancer cell growth in vitro. After screening for anti-cancer effects the present study focused on the most potent of these analogues. PATIENTS AND METHODS: Two human pancreatic cancer cell lines (AsPC-1 and S2-013) were treated with various concentrations (0.1–100 mM) of analogue. Proliferation was measured by 3H-thymidine incorporation and cell counting at different time points (24–72 h). Cell cycle analysis was determined by flow cytometry with propidium iodide DNA staining. Morphological changes were observed using light microscopy. RESULTS: The analogues caused both time- and concentration-dependent inhibition of DNA synthesis (85% decrease in AsPC-1, p<0.01 and 82% decrease in S2-013, p<0.01 at 24 h with 10 mM) and cell proliferation (82% decrease in AsPC-1, p50.001 and 69% decrease in S2-013, p<0.001 at 72 h with 10 mM). The peptides induced G0/G1 phase cell cycle arrest (AsPC-1 control 78.8% vs treated 86.7%, p<0.01; S2-013 control 55.8% vs treated 75.9%, p<0.01). At 24 h, obvious morphological changes could be seen through light microscopy at a concentration of 10 mM. CONCLUSION: These novel peptides inhibit growth of pancreatic cancer cells through G0/G1 arrest and may be valuable for the treatment of pancreatic cancer.

43  EPITHELIAL–STROMAL IN TERACTIONS IN PANCREA TIC CANCER: THE ROLE OF TENASCIN C ANDANNEXIN II

Esposito I¹, Penzel R¹, Bergmann F¹, Giese N², Kleeff J², Friess H², Schirmacher P¹, (1) Institute of Patholog; (2) Department of General Surgery, University of Heidelberg, Heidelberg, Germany

INTRODUCTION AND AIM: Epithelial-stromal interactions are relevant to the progression of pancreatic ductal adenocarcinoma (PDAC). Tenascin C (TNC) is an extracellular matrix protein with anti-adhesive and pro-invasive properties. The aim of this study was to elucidate the role of TNC and its cell surface receptor annexin II in the progression of PDAC. PATIENTS AND METHODS: Real-time quantitative PCR was used to evaluate the levels of TNC mRNA. The transcriptional levels of TNC splice variants were assessed by PCR. Immunohistochemistry was used to localize the expression of TNC and annexin II. RESULTS: TNC mRNA was overexpressed in pancreatic cancer tissues (4-fold) in comparison to the normal pancreas and it was demonstrated in 4 of 7 pancreatic cancer cell lines. TNC large splice variants were present in pancreatic cancer tissues and cell lines, but not in the normal pancreas. TNC mRNA was also detected in pancreatic stellate cells, where it increased under TNF-alpha stimulation. TNC expression was mostly stromal and increased in frequency in the progression from PanIN-1 lesions to PDAC. Annexin II was expressed in almost all the PanIN lesions and in all the cancer samples. A redistribution of annexin II from the cytoplasm to the cell membrane was observed in the progression from low-grade PanINs to PDAC. CONCLUSION: TNC is overexpressed in the stroma of PDAC, possibly under the influence of cytokines. The increase in TNC expression in the subsequent steps of pancreatic tumor progression and the parallel redistribution of annexin II to the cell membrane suggests an involvement of the two proteins in the establishment of a tumor-favorable microenvironment.

44  ANTISENSE OLIGONUCLEOTIDES TARGETED TO K-ras POINT MUTATION IN HAMSTER EXPERIMENTAL PANCREATIC CANCER MODEL – CAN IT INHIBIT THE GROWTH OF 5-FU- AND MMC-RESISTANTMETASTATIC AND REMETASTATIC CELL LINES?

Morioka CY¹, Machado MCC¹, Saito S², Ohzawa K², Matheus AS³, Jukemura J¹, Cunha JEM¹, Watanabe A¹, (1) Department of Surgery, University of São Paulo, São Paulo, Brazil; (2) 3rd Department of Internal Medicine, Toyama Medical and Pharmaceutical University; (3) Department of Surgery, Toyama Medical and Pharmaceutical University; Toyama, Japan

INTRODUCTION AND AIM: New genes have been discovered day after day and some of them have been related to disease states, including pancreatic cancer. K-ras point mutation at codon 12 has a relationship of >90% with pancreatic cancer. Cancer therapy should also include the treatment of metastatic disease because it is known that properties of metastatic cells may vary considerably from those of the primary tumor. The aim was to verify whether the same drugs, which can inhibit the tumor growth in the parental cell line, could inhibit the pancreatic metastatic and pancreatic remetastatic cell lines at the same concentrations and to compare the inhibition with antisense oligonucleotides mismatched to K-ras gene, in Syrian golden hamsters. PATIENTS AND METHODS: HaP-T1, a BHP-induced hamster pancreatic cancer cell line, MS-PaS-1 (a metastatic cell line established from ‘return trip’ metastases from the liver to the pancreas) and MS-PaS-2 named as ‘remetastatic cell line’ (i.e. metastases from MS-PaS-1) were used. MTT and MTT-agarose assays were performed, using 5-fluorouracil (5-FU), mitomycin C (MMC) and antisense oligonucleotide specific to K-ras oncogene. RESULTS: The inhibitory concentration (IC50) of 5-FU, which inhibited the HaP-T1, had to be increased by 50-fold to inhibit MS-PaS-1 and 100-fold to inhibit MS-PaS-2. MMC had to be increased by 10-fold to inhibit MS-PaS-1 and 50-fold to inhibit MS-PaS-2. However, ID50 was the same when antisense oligonucleotide was tried in these 3 cell lines. CONCLUSION: Antisense oligonucleotide targeted K-ras gene may be a good choice for therapy because it could inhibit the growth in metastatic and remetastatic pancreatic cells as well as in primary tumor cells.

45  ROLE OF IMMUNOHISTOCHEMICAL AND MOLECULAR IDENTIFICATION OF HER-2 GENE IN HUMAN PANCREATIC CANCER

Borka K², Szijjarto A¹, Kaliszky P¹, Kiss A², Schaff Z², Kupcsulik P¹, (1) 1st Department of Surgery; (2) 2nd Department of Pathology, Semmelweis University, Budapest, Hungary

INTRODUCTION AND AIM: The expression of epidermal growth factor receptor-2 (HER-2) has a role in malignant transformation and metastasis formation. Overexpression has also been demonstrated in adenocarcinomas of the pancreas but the significance is unclear. Following the success of HER-2 neutralizing antibody treatment in breast cancer, some authors reported results of the combinations of trastuzumab (Herceptin) and gemcitabine in HER2-overexpressing patients with pancreatic carcinoma. However, it is still controversial whether gene amplification or protein detection is required for the initiation of the treatment. The aim was to correlate HER-2 DNA amplification and protein overexpression in human pancreatic adenocarcinomas. PATIENTS AND METHODS: 27 pancreatic tumors were immunostained by the avidin-biotin peroxidase conjugate method. DNA was isolated from the same formalin-fixed, paraffin-embedded tissue sections. Real-time PCR was used to quantify gene amplification. Gene amplification was analyzed using internal control and classified as positive if the ratio was more than two. Immunostaining was classified as 0, 1+, 2 +, or 3 +. RESULTS: 12/27 tumors showed gene amplification. Only one tumor showed 3 + HER-2 positivity and another 1 + positivity as analysed by immunohistochemistry. The highest levels of DNA amplification corresponded to 3 + and 1+ positivity. CONCLUSION: HER-2 gene amplification data in pancreatic cancers did not correspond with the immunohisto-chemical detection. In contrast to the low number of HER-2 positive cases by immunohistochemistry, RT-PCR showed amplification of the gene in a high number of cases. Further analysis is required to explain the discrepancy between the results of the methods, especially since HER-2-overexpressing pancreatic cancers might be a target of Herceptin therapy.

46  RAPAMYCIN-INDUCED ENDOTHELIAL CELL DEATH AND TUMOR VESSEL THROMBOSIS OPTIMIZES GEMCITABINE'S CYTOTOXIC EFFECT AGAINST PANCREA TIC CANCER

Bruns CJ³, Guba M¹, Köhl G², Yezhelyev M¹, Jauch KWS¹, (1) Surgery, University of Munich-Großhadern, Munich, Germany; (2 )Surgery, University of Regensburg, Regensburg, Germany; (3) Department of Surgery, University of Munich-Großhadern, Munich Germany

INTRODUCTION AND AIM: Despite current chemotherapies pancreatic cancer steadfastly remains refractory to treatment. Here we tested a new approach of combining antiangiogenic and standard cytotoxic therapy in a metastatic human pancreatic cancer nude mouse model. PATIENTS AND METHODS: Nude athymic mice were injected orthotopically with metastatic human L3.6pl cancer cells. Pancreatic tumors were allowed to become established for 8 days before initiation of rapamycin or gemcitabine treatment. Standard doses of rapamycin (1.5 mg/kg/d) and gemcitabine (100 mg/kg, 2x/week) were used in the first group of experiments, and all animals were sacrificed 28 days after tumor cell injection. Immunohistochemical analysis was performed from primary pancreatic tumors for proliferation (Ki67), cell death (TUNEL), and apoptotic endothelial cells (CD31/TUNEL). To directly test the effect of rapamycin on tumor blood vessel flow dynamics, L3.6pl tumor cells were implanted into dorsal skin-fold chambers and vessels were examined by intravital microscopy on day 7. FACS analysis of HUVE cells was performed to detect annexin-V-positive cells. RESULTS: Following orthotopic tumor cell injection, rapamycin treatment alone reduced tumor volume 2-fold more than the standard gemcitabine therapy. Furthermore, when rapamycin and gemcitabine treatment were combined, tumors grew to only 19% of the size observed with gemcitabine treatment alone. Interestingly, histologic analysis revealed tumor vessel endothelium detachment and thrombosis with rapamycin treatment. Angiogenesis observation in dorsal skin-fold chambers after rapamycin treatment directly illustrated unusually dilated tumor vessels that were highly susceptible to thrombosis. Furthermore, when we photodynamically promoted vascular thrombosis in tumors, blood flow was very rapidly blocked by thrombosis in rapamycin-treated mice, compared to controls as monitored by intravital microscopy. Furthermore, CD31/TUNEL staining of orthotopic tumors demonstrated apoptotic endothelial cells with rapamycin treatment, which was substantiated in vitro by increased annexin-V staining of rapamycin-treated human endothelial cells. In contrast, gemcitabine showed no antiangiogenic effects, but induced extensive tumor cell apoptosis in vivo, albeit without concomitantly reducing cell proliferation. CONCLUSION: Our data suggest that rapamycin's antiangiogenic activity inhibits tumor expansion, thereby more positively balancing the potent cytotoxic effect of gemcitabine against tumor progression. Furthermore, our study provides the first evidence that tumor control achieved with rapamycin is related to tumor vessel thrombosis associated with the death of endothelial cells. Rapamycin promotion of thrombosis preferentially in new pancreatic tumor vessels introduces a novel mechanism potentially contributing to its anticancer action.