AIM: Chemotherapeutic treatment of pancreatic carcinomas is often impeded by intrinsic multidrug resistance (MDR). This MDR phenotype can be caused by transporters of the MDR P-glycoprotein (ABCB) or multidrug resistance related protein (MRP) family(ABCC). To elucidate the role of ABCB and ABCC family members in pancreatic carcinomas, we analyzed mRNA expression of MDR-1, MDR-3, MRP-1, MRP-3 and MRP-5, which have been shown to confer resistance to chemotherapeutic drugs. METHODS: mRNA expression was quantified in 10 different human pancreatic carcinoma cell lines before and during continuous in vitro chemotherapy including gemcitabine (0.5 μg/mL), 5-fluorouracil (5-FU; 0.5μg/mL), cisplatin (0.05 μg/mL) or the combination of 5-FU and cisplatin. RESULTS: Quantitative RT-PCR demonstrated high base line expression levels of MRP-3> MRP-1> MRP-5 in the pancreatic cell lines Panc-1, KCI-MOH1, MIAPaCa-2, PK-1, PK-8, PK-9 and AsPC-1, whereas PaTu8902, HuP-T4 und FAMPAC revealed low base line mRNA transcript levels. 5-FU and cisplatin caused a significant elevation of MRP-3> MRP-1> MRP-5, while gemcitabine affected the mRNA expression less. CONCLUSION: MRP-1, MRP-3 and MRP-5 are likely to be involved in the MDR phenotype. The majority of pancreatic carcinoma cells express high levels of MRPs even without prior chemotherapeutic treatment. Quantification of MRP expression may predict individual tumor responses and guide chemotherapeutic treatment.

Background. Discussion of the volume-outcome relationship in pancreatic surgery has gained increasing interest. Currently, no data describe the situation in Germany. Pursuant to a recent legislative reform, a threshold of 10 operations per year was introduced for pancreatic surgery in 2006. This study describes the situation in Germany and the effect of the legislative reform between 2006 and 2009.Methods. In 2007 and 2010, anonymous questionnaires were sent to leading surgeons in the German Society of General and Gastrointestinal Surgery asking for the numbers of pancreatic operations, methods of operation, and mortality for the years 2006, 2008, and 2009. Volume categories were defined by dividing hospitals into quartiles according to their annual volume of operations.Results. The return rate was about 48%. In the years 2006, 2008, and 2009, overall mortality in all hospitals was 2.85%, 3.98%, and 2.58%. High volume was defined as >= 32 pancreatic operations (2006) and >= 34 pancreatic operations (2008, 2009). Although mortality decreased with increasing volume, mortality between each volume category was not statistically different in any year. In the years 2006, 2008, and 2009, the number of operations increased in university hospitals (38.4%, 51.2%, and 50.4%, P < .001) and decreased in teaching hospitals (51.8%, 41.3%, and 41.2%, P < .001). The number of hospitals that did not perform pancreatic operations increased from 15.6% to 32.5% and 31% (P < .001).Conclusion. In pancreatic surgery, a centralization effect occurred after a legislative reform in Germany. Overall mortality after pancreatic resection in German hospitals is good. Although mortality decreases with greater volume, there were no differences compared to other volume categories. Also, low-volume hospitals can produce good results; however, the difference in quality is considerable among these institutions. Our data suggest that the German threshold agreement in pancreatic surgery might have a positive effect with regard to reproducible quality and outcome. (Surgery 2012;152:S50-5.)

Background Multidrug resistance (MDR) is a clinically, highly relevant phenomenon. Under chemotherapy many tumors show an increasing resistance towards the applied substance(s) and to a certain extent also towards other agents. An important molecular cause of this phenomenon is an increased expression of transporter proteins. The functional relationship between high expression levels and chemotherapy resistance makes these MDR and MRP (MDR related protein) proteins to interesting therapeutic targets. We here wanted to systematically analyze, whether these proteins are tumor specific antigens which could be targeted immunologically. Results Using the reverse immunology approach, 30 HLA-A2.1 restricted MDR and MRP derived peptides (MDP) were selected. Stimulated T cell lines grew well and mainly contained activated CD8 + cells. Peptide specificity and HLA-A2.1 restriction were proven in IFN-γ-ELISpot analyses and in cytotoxicity tests against MDP loaded target cells for a total of twelve peptides derived from MDR-1, MDR-3, MRP-1, MRP-2, MRP-3 and MRP-5. Of note, two of these epitopes are shared between MDR-1 and MDR-3 as well as MRP-2 and MRP-3. However, comparably weak cytotoxic activities were additionally observed against HLA-A2.1 + tumor cells even after upregulation of MDR protein expression by in vitro chemotherapy. Conclusions Taken together, these data demonstrate that human T cells can be sensitised towards MDPs and hence, there is no absolute immunological tolerance. However, our data also hint towards rather low endogenous tumor cell processing and presentation of MDPs in the context of HLA-A2.1 molecules. Consequently, we conclude that MDR and MRP proteins must be considered as weak tumor specific antigens-at least for colorectal carcinoma. Their direct contribution to therapy-failure implies however, that it is worth to further pursue this approach.

BACKGROUND:Our aim was to compare cardiovascular and stress response to robotic technology during thoracoscopic mobilization and anastomosis of the esophagus vs the conventional open approach.DESIGN:Randomized experimental study.SETTING:Department of Experimental Surgery, University of Heidelberg.SUBJECTS:Twelve pigs randomized to undergo robotic or conventional surgery (6 animals each).INTERVENTIONS:Fundus rotation gastroplasty followed by esophageal mobilization and intrathoracic anastomosis by conventional or robotic surgery.MAIN OUTCOME MEASURES:Mean arterial pressure, central venous pressure, mean pulmonary arterial pressure, pulmonary capillary wedge pressure, cardiac output, pulmonary vascular resistance, partial oxygen pressure, alveolar-arterial difference in partial pressure of oxygen, and arteriovenous oxygen content difference measured preoperatively, during esophageal manipulation, and 30 minutes after operation. Operative stress was assessed by plasma levels of cortisol and substance P.RESULTS:Hemodynamic measures showed higher intraoperative central venous pressure and pulmonary vascular resistance in the open surgery group, whereas cardiac output was significantly decreased compared with the robotic group. Blood gas values showed significant deterioration during esophageal manipulation with open surgery in contrast to the robotic group. Substance P and cortisol levels were significantly higher with the open approach.CONCLUSIONS:The robot-assisted approach is associated with improved intraoperative cardiopulmonary function and seems to be a less stressful technique.

and with an Institutional questionnaire on pancreatic exocrine-endocrine function.Mortality after surgical resection was zero, and there were no deaths related to the long-term complications.Overall QoL after TP was acceptable (median:5, scale from 1 to 7), despite a slightly decrease compared to the preoperative period (median:6).A significant impairment in QoL was found only in patients with malignancies (P<0.05).Twenty-nine patients (91%) complained of hypoglycemia, 72% of them at least once a week.Steatorrhea and abdominal pain were found in 66% and 44% of patients.Major impairments of leisure and work activities were reported in 56% and 31% of cases.In experienced centers TP is a viable and safe procedure.However TP substantially affects health status, and the risk for long-term complications should be well evaluated; therefore careful patient-selection and long-term follow-up are of paramount importance in this setting.

We analyzed the immunologic impact of adeno-associated virus type 2 (AAV-2), a small single-stranded parvovirus with tumorsuppressive properties, on DSL6A pancreatic carcinoma in syngeneic rats. Established tumors of animals treated with AAV-2 or mock infected were resected (Ro), and DSL6A cells were rechallenged on the different site. Eleven (92%) of 12 mock-infected animals but only 3 (25%) of 12 AAV-2-treated animals redeveloped tumors. Adeno-associated virus type 2 infection provoked systemic raises in monocytes and neutrophils numbers and in levels of the proinflammatory monocyte chemoattractant protein 1 and interleukin 10. Adeno-associated virus type 2-treated tumors were infiltrated with monocytes, macrophages, natural killer cells, CD4+ T cells, and especially CD8+ T cells. In cytotoxicity assays, AAV-2-infected DSL6A tumor cells were recognized by lymphocytes from AAV-2-treated animals and from controls. Yet, uninfected DSL6A cells were exclusively killed by lymphocytes from AAV-2-treated animals. Additionally, those lymphocytes displayed high natural killer cell activity but failed to attack unrelated tumor targets. Taken together, these results suggest that the antiviral response toward AAV-2 cross-activates the immune system toward simultaneously present tumor disease. This and the known potential to significantly reduce toxic side effects of chemotherapy make nonpathogenic viruses such as AAV-2 as "1-agent combination therapy" to an interesting treatment option of residual tumor disease. Abbreviations: AAV-2 - Adeno-Associated Virus type 2, Ad-2 - Adenovirus type 2, CCR-2 - monocyte chemoattractant protein-1 receptor, IL - interleukin, INF - interferon, MCP-1 - monocyte chemoattractant protein-1, MOI - multiplicity of infection, NK-cells - natural killer cells, TNF - tumor necrosis factor

The aim of this study was to investigate the effects of an activating anti-CD40 antibody (aCD40Ab) on leukocyte adhesion to tumour vessels, leukocyte migration and tumour growth in experimental liver cancer. Morris-Hepatoma was induced by subcapsular inoculation of tumour cells in the liver of ACI-rats. On day 7 and 8 after tumour cell injection, one group of the animals received aCD40Ab. On day 13 the tumour volume was measured and intravital microscopy was performed quantifying leukocyte adherence in the liver. Furthermore, immunohistological analyses were performed. aCD40Ab-Treated animals showed increased leukocyte–endothelium interaction, demonstrated substantially more T- and natural killer (NK) cells in the tumour and had a distinctly decreased tumour volume. Our results show that treatment with aCD40Ab stimulates endothelial leukocyte adhesion in tumour vessels and migration of CD4 cells/CD8 T-cells and NK cells into the tumour and inhibits tumour growth. Thus, the CD40/CD154 pathway is a worthwhile target for adjuvant immunotherapy.

BACKGROUND:Experimental gene transfer can make tumors more immunogenic, leading to local regression and inducing immunological memory sufficient to permit resistance to a tumor rechallenge. However, this rarely had any significant impact on large established tumors.METHODS:To analyze potential immunological effects, we used weakly immunogenic pancreatic carcinomas in syngeneic, immunocompetent Lewis rats and performed in situ adenoviral mediated cytosine deaminase (CD) gene transfer followed by administration of the prodrug, 5-fluorocytosine (5FC). In order to reflect the clinical situation, such treated tumors were surgically resected and animals were rechallenged with parental DSL6A pancreatic tumor cells. Tumor growth and cytotoxic activity of immune cells were determined.RESULTS:CD/5FC treatment of the DSL6A cells revealed significant induction of apoptosis in vitro and slowed down tumor progression in syngeneic hosts. Furthermore, we observed neither significant change in tumor growth nor protective immunity in the rechallenged animals. Analysis of T lymphocytes showed no specific cytotoxic activity against DSL6A cells. There was only a trend towards a minor NK cell activation.CONCLUSIONS:Albeit the present study failed to induce protective antitumor immunity, the initial finding of reduced tumor growth argues for the development of multimodal therapeutic options to overcome negative impacts of advanced malignant disease or chemotherapy-related anergy and immunosuppression.

AIM AND BACKGROUND: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. Therefore, we determined the percentage of regulatory T cells in cytokine-induced killer (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 interleukin 10 (IL-10) after unspecific stimulation of the TCR complex and stimulation with interleukin 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 coculture of CIK cells with dendritic cells (DCs). After coculture 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:Our established effector cells possess some regulatory features induced by unspecific TCR-activation that could be prevented by coculture with DCs. CIK cells have desirable properties for immunotherapeutical approaches, especially after coculture with DCs, which could be used additionally for induction of a specific immune response.

AIM: There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status.METHODS: Human pancreatic cancer cell lines Capan-1(p53mut), Capan-2(p53wt), FAMPAC(p53mut), PANC1(p53mut), and rat pancreatic cancer cell lines AS(p53wt) and DSL6A(p53null) were used for in vitro studies. Following infection with different ratios of Ad-p53-particles (MOI) in combination with 5-FU, proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining). In addition, DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size, apoptosis (TUNEL) and survival were determined.RESULTS: Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53. In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU. Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU.CONCLUSION: Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function. These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.

IN VIVO ADENOVIRUS MEDIATED GENE TRANSFER OF THE ESCHERICHA COLI CYTOSINE DEAMINASE GENE TO PANCREATIC TUMOURS INDUCES CHEMOSENSITIVITY TO 5-FLUOROCYTOSINE. Dalibor Antolovic, Sven C. Eisold, Michael Linnebacher, Geeske C. Meyer, Susanne Dihlmann, Jan Schmidt, Ernst KIar, Christian Herfarth, Magnus von Knebel-Doeberitz, Univ of Heidelberg, Dept Surg, Heidelberg, Germany; Univ of Heidelberg, Heidelberg, Germany.

The use of the cytosine deaminase (CD)/5-fluorocytosine suicide system as a cancer gene therapy approach enables selective killing of CD-modified cells as well as the ablation of non-modified tumor cells due to a bystander effect that has been suggested to involve the immune system in vivo. Using a stable CD transfectant of the tumorigenic rat adenocarcinoma cell line AS (AS/CD), an antitumoral response against the CD expressing cell line as well as the parental cell line could be induced by stepwise vaccinations in syngeneic animals. AS/CD tumor regression occurred independently of 5-fluorocytosine treatment and was sufficient to protect 37% of the animals against subsequent challenge with tumorigenic doses of the parental AS cell line. Immune rats contained lymphocytes able to specifically lyse CD modified as well as unmodified AS tumor cells in vitro, most likely contributing to the in vivo antitumoral reaction. Thus, the CD suicide system seems to be suitable not only for a local tumor gene therapy but also for the application as therapy of metastatic tumors and minimal residual disease.

Backgrounds: Nitric oxide (NO) is a well-known inducer of apoptosis, but there have been many unknown issues in its apoptotic signal pathway.Recent studies have clarified that the release of cytochrome C (cyt-C) from mitochondria triggers the subsequent activation of caspase-cascade and leads to apoptosis.Bax, BcI-2 and others are considered to regulate this process.However, the mechanisms of cyt-C releasing have not been elucidated yet.The present study was aimed to clarify the possible implication of mitochondrial functional molecules such as FOFI-ATPase and PT pore in cyt-C releasing process of NO induced apoptosis, using the rat normal gastric mucosal cell line, RGMI.Materials and Methods: RGMI cells were cultured in a high glucose medium for 24hrs to maintain intracellular ATP level.Thereafter, the cells were exposed to pure NO donor (NOCI8) under the presence or absence of various inhibitors of mitochondrial function, oligomycin as a FOFI-ATPase inhibitor, antimy-cinA as a respiratory chain complex III inhibitor and cyclosporineA as a PT pore inhibitor.The followings were studied, cell viability by MTT assay,