Multidrug-resistance proteins are weak tumor associated antigens for colorectal carcinoma.

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.

Activating anti-CD40 antibodies induce tumour invasion by cytotoxic T-lymphocytes and inhibition of tumour growth in experimental liver cancer.

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.

Sensitization of sarcoma cells to doxorubicin treatment by concomitant wild-type adeno-associated virus type 2 (AAV-2) infection.

Doxorubicin-based chemotherapy is used in the treatment of sarcomas. Toxic side effects and poor response rates underline the demand for an improvement in current chemotherapeutic protocols. Recently, it has been reported that parvoviruses confer various antineoplastic properties to infected cells, and that adeno-associated virus type 2 (AAV-2) infection sensitizes malignant epithelial cells to radiation- or chemotherapy-based genotoxic treatment. Thus, we analyzed whether AAV-2 infection leads to an improved efficacy of doxorubicin chemotherapy in malignant mesenchymal cells, using 13 human sarcoma cell lines. Therapeutic effects were analyzed by measuring cell viability and proliferation (WST-1, colony forming, and propidium iodide assays). Additionally, permissivity for AAV-2 infection was determined by Southern dot blot analysis. AAV-2 infection strongly increased the efficacy of doxorubicin treatment in rhabdomyo-, fibro-, osteo- and chondrosarcoma cells in a dose-dependent manner. This effect was not observed in liposarcoma and synovial sarcoma cells, although a susceptability to AAV-2 infection was documented. Our results indicate that the sensitization effects towards genotoxic treatment exerted by non-pathogenic AAV-2 infection are not restricted to epithelial malignancies but may also be exploited for the improvement of chemotherapy in patients suffering from rhabdomyo-, fibro-, osteo-, or chondrosarcomas.

The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines.

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.

Volume-outcome relationship in pancreatic surgery: the situation in Germany.

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.

Experimental study of cardiorespiratory and stress factors in esophageal surgery using robot-assisted thoracoscopic or open thoracic 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.

Induction of an antitumoral immune response by wild-type adeno-associated virus type 2 in an in vivo model of pancreatic carcinoma.

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.

Effective antitumoral immune responses are not induced by cytosine deaminase suicide gene transfer in a syngeneic rat pancreatic carcinoma model.

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.

Dendritic cells reduce number and function of CD4+CD25+ cells in cytokine-induced killer cells derived from patients with pancreatic carcinoma.

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.

Characterization of FAMPAC, a newly identified human pancreatic carcinoma cell line with a hereditary background.

BACKGROUND: A novel pancreatic carcinoma cell line, FAMPAC, was identified from investigation of poorly differentiated pancreatic adenocarcinoma cells found in a patient with a familial predisposition to pancreatic carcinoma. A gene responsible for familial pancreatic carcinoma has not been identified to date. METHODS: The FAMPAC cell line was characterized by its morphology, growth rate, tumorigenicity, and chromosomal analysis. Three known tumor suppressor genes, p16/CDKN2, BRCA2, and p53, all of which are important in the development of pancreatic carcinoma and frequently are involved in a variety of cancer syndromes, were analyzed. RESULTS: FAMPAC cells grew as an adhering monolayer in culture medium supplemented with 10% fetal bovine serum and formed tumors rapidly in nude mice. The doubling time ranged from 24 to 48 hours. Karyotype analysis demonstrated the complexity of chromosomal deletions and rearrangements. The cells were negative for ductal differentiation markers such as cytokeratin 7 and MUC1, indicating poor differentiation. Analysis of FAMPAC cells revealed overexpression of the mutated p53 gene (exon 5, codon 175: CGC --> CAC), the presence of a homozygous deletion in the p16 gene, and the presence of wild-type BRCA2 in the tested hot spots. CONCLUSIONS: To the authors' knowledge, FAMPAC is the first established human pancreatic carcinoma cell line associated with a familial background. FAMPAC is a tumorigenic cell line with a complex molecular pattern of mutations. These findings may be useful in understanding the mechanisms responsible for the development of sporadic or hereditary forms of pancreatic carcinoma.

Prevention of chemotherapy-related toxic side effects by infection with adeno-associated virus type 2.

Drug resistance and toxic side effects are major limiting factors in the clinical use of antineoplastic chemotherapy. Patients with pancreatic cancer generally do not benefit from chemotherapy. The nonpathogenic adeno-associated virus type 2 (AAV-2) has been shown to sensitize human tumor cells to gamma irradiation and chemotherapeutic drugs. In the present study, we characterized the therapeutic role of AAV-2 infection in combination with 5-fluorouracil (5-FU)-based chemotherapy on pancreatic cancer cells in an animal model. In Lewis rats bearing s.c. implants of syngeneic DSL6A pancreatic cancer cells, intratumoral infection with AAV-2 (MOI 10E8 i.u.) in combination with 5-FU (5 or 50 mg/kg body weight) resulted in significantly reduced tumor growth and prolonged survival time compared with 5-FU single therapy. Most surprisingly, AAV-2-infected rats remained in a much better physical condition compared to their noninfected counterparts. While rats treated with 5-FU single therapy lost weight, were sluggish and died within 4 months after tumor implantation, animals infected with AAV showed much better vigilance, with body weight, leukocyte number and hemoglobin levels similar to healthy rats. In particular, 5-FU-related side effects like thrombocytopenia and leukopenia were significantly reduced in animals treated with the combination regimen. By in vitro analysis, human (Capan-1 and DANG) pancreatic cancer cell lines were shown to be sensitized to 5-FU chemotherapy to an extent similar to DSL6A cells. AAV-2 infection enhanced 5-FU-induced apoptosis by a factor of 8 to 14 in both human and rat pancreatic cancer cell lines. The data suggest that infection with the nonpathogenic AAV-2 significantly improves both chemotherapy efficacy and physical appearance and offers a novel strategy in cancer treatment.

Prevalence of familial pancreatic cancer in Germany.

Based on several case-control studies, it has been estimated that familial aggregation and genetic susceptibility play a role in up to 10% of patients with pancreatic cancer, although conclusive epidemiologic data are still lacking. Therefore, we evaluated the prevalence of familial pancreatic cancer and differences to its sporadic form in a prospective multicenter trial. A total of 479 consecutive patients with newly diagnosed, histologically confirmed adenocarcinoma of the pancreas were prospectively evaluated regarding medical and family history, treatment and pathology of the tumour. A family history for pancreatic cancer was confirmed whenever possible by reviewing the tumour specimens and medical reports. Statistical analysis was performed by calculating odds ratios, regression analysis with a logit-model and the Kaplan-Meier method. Twenty-three of 479 (prevalence 4.8%, 95% CI 3.1-7.1) patients reported at least 1 first-degree relative with pancreatic cancer. The familial aggregation could be confirmed by histology in 5 of 23 patients (1.1%, 95% CI 0.3-2.4), by medical records in 9 of 23 patients (1.9%, 95% CI 0.9-3.5) and by standardized interviews of first-degree relatives in 17 of 23 patients (3.5%, 95% CI 2.1-5.6), respectively. There were no statistical significant differences between familial and sporadic pancreatic cancer cases regarding sex ratio, age of onset, presence of diabetes mellitus and pancreatitis, tumour histology and stage, prognosis after palliative or curative treatment as well as associated tumours in index patients and families, respectively. The prevalence of familial pancreatic cancer in Germany is at most 3.5% (range 1.1-3.5%) depending on the mode of confirmation of the pancreatic carcinoma in relatives. This prevalence is lower than so far postulated in the literature. There were no significant clinical differences between the familial and sporadic form of pancreatic cancer.