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Cosmeri Rizzato,
Daniele Campa,
Raffaele Pezzilli,
Pavel Soucek,
William Greenhalf,
Gabriele Capurso,
Renata Talar-Wojnarowska,
Anette Heller,
Krzysztof Jamroziak,
Kay-Tee Khaw, [......],
Ludmila Vodickova,
Markus W Büchler,
Peter Bugert,
Pavel Vodicka,
John P Neoptolemos,
Jens Werner,
Jörg D Hoheisel,
Andrea S Bauer, Nathalia Giese,
Federico Canzian
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ABSTRACT: There is strong epidemiologic evidence indicating that common genetic variability could be implicated in pancreatic cancer risk and, to date, various loci have been proposed. In particular, there is increasing evidence of the involvement of ABO gene variability and pancreatic cancer risk. In a large multicentric study of 1,028 pancreatic ductal adenocarcinoma cases and 2,257 controls in the context of the PANcreatic Disease ReseArch (PANDoRA) consortium, we investigated the suggested association with increased risk for carriers of single nucleotide polymorphisms (SNPs) determining the A or B allele in comparison with the O allele, which encodes for a non-functional enzyme. Since glycosyltransferase activity, encoded by ABO, is higher for the A1 variant compared with the A2 variant, we investigated the hypothesis that A1 carriers were at an increased risk of pancreatic cancer. In our analysis, carriers of the A1 were indeed at greater risk of developing the disease. In addition, we investigated the possible influence that genetic variability at the ABO locus may have in pancreatic cancer survival, but we observed no effect in our population.
Oncology Reports 04/2013; 29(4):1637-44. · 1.84 Impact Factor
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ABSTRACT: Pancreatic adenocarcinoma (PaCa) being the deadliest cancer is partly due to early metastatic spread. Thus, we searched for PaCa initiating cell (PaCIC) markers with emphasis on markers contributing to metastatic progression. PaCIC were enriched from long-term and freshly-established lines by repeated selection for spheroid or holoclone growth in advance of evaluating PaCIC markers. Sphere and holoclone formation steeply increased by re-cloning and remained stable thereafter. Cells not forming spheres or holoclones died upon re-cloning. PaCIC enrichment in spheres and holoclones was accompanied by increased motility, anchorage-independence and up-regulated CXCR4 expression. After subcutaneous injection in NOD/SCID mice tumorigenicity and, impressively, recovery of metastasizing tumor cells in peripheral blood, spleen, bone marrow, lung and pancreas was strongly increased in spheres and holoclones. PaCIC-enrichment in spheres and holoclones was accompanied, besides CXCR4, by up-regulated CD44v6, alpha6beta4, weakly CD133 and tetraspanin Tspan8 expression. Notably, CD44v6, alpha6beta4, CXCR4 and Tspan8 expressing PaCa cells had a growth advantage in vivo and became dominating in migrating and in distant organs settled tumor cells. This is the first report showing that CD44v6, alpha6beta4, Tspan8 and CXCR4 are biomarkers in PaCIC allowing for long-term survival, expansion and migration in immunocompromised mice. The stability of the percentage of PaCIC in long-term and freshly-established lines after a roughly 8-fold enrichment by cloning indicates PaCIC, though required for long-term survival, concomitantly depending on support by non-CIC. © 2013 Wiley Periodicals, Inc.
International Journal of Cancer 01/2013; · 5.44 Impact Factor
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P Sivaramakrishna Rachakonda,
Andrea S Bauer,
Huaping Xie,
Daniele Campa,
Cosmeri Rizzato,
Federico Canzian,
Stefania Beghelli,
William Greenhalf,
Eithne Costello,
Michaela Schanne,
Anette Heller,
Aldo Scarpa,
John P Neoptolemos,
Jens Werner,
Markus Büchler,
Jörg D Hoheisel,
Kari Hemminki, Nathalia Giese,
Rajiv Kumar
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ABSTRACT: KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09-4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14-2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33-7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.
PLoS ONE 01/2013; 8(4):e60870. · 4.09 Impact Factor
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Daniele Campa,
Cosmeri Rizzato,
Gabriele Capurso, Nathalia Giese,
Niccola Funel,
William Greenhalf,
Pavel Soucek,
Maria Gazouli,
Raffaele Pezzilli,
Claudio Pasquali, [......],
Daniela Basso,
Mario Plebani,
Paola Fogar,
Markus W Büchler,
Peter Bugert,
Pavel Vodicka,
Ugo Boggi,
John P Neoptolemos,
Jens Werner,
Federico Canzian
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ABSTRACT: Pancreatic cancer is the fourth leading cause of cancer deaths in the European Union and in the USA, but little is known about its genetic susceptibility. The PANcreatic Disease ReseArch (PANDoRA) consortium was established to unite the efforts of different research groups; its aim is to create a large bio-database to uncover new genetic factors for pancreatic cancer risk, response to treatment, and patient survival. So far 2220 cases of pancreatic adenocarcinoma, a smaller number of cases of endocrine pancreatic tumours (n=86), chronic pancreatitis (n=272) and 3847 healthy controls have been collected. As a collective effort of the consortium, SNPs associated with pancreatic adenocarcinoma risk from a genome-wide association study performed in Caucasians were replicated. The possibility that the same genetic polymorphisms may influence patient survival as well was also addressed. This collective effort is particularly important for pancreatic cancer because it is a relatively rare disease for which little is known about aetiopathogenesis and risk factors. The recruitment of additional collaborators and partner institutions is continuously on-going.
Digestive and Liver Disease 11/2012; · 3.05 Impact Factor
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ABSTRACT: Treatment options for pancreatic ductal adenocarcinoma (PDAC) are limited. Histone deacetylase inhibitors are a new and promising drug family with strong anticancer activity. The aim of this study was to examine the efficacy of in vitro and in vivo treatment with the novel pan-HDAC inhibitor belinostat on the growth of human PDAC cells.
The proliferation of tumour cell lines (T3M4, AsPC-1 and Panc-1) was determined using an MTT assay. Apoptosis was analysed using flow cytometry. Furthermore, p21Cip1/Waf1 and acetylated histone H4 (acH4) expression were confirmed by immunoblot analysis. The in vivo effect of belinostat was studied in a chimeric mouse model. Antitumoural activity was assessed by immunohistochemistry for Ki-67.
Treatment with belinostat resulted in significant in vitro and in vivo growth inhibition of PDAC cells. This was associated with a dose-dependent induction of tumour cell apoptosis. The apoptotic effect of gemcitabine was further enhanced by belinostat. Moreover, treatment with belinostat increased expression of the cell cycle regulator p21Cip1/Waf1 in Panc-1, and of acH4 in all cell lines tested. The reductions in xenograft tumour volumes were associated with inhibition of cell proliferation.
Experimental treatment of human PDAC cells with belinostat is effective in vitro and in vivo and may enhance the efficacy of gemcitabine. A consecutive study of belinostat in pancreatic cancer patients alone, and in combination with gemcitabine, could further clarify these effects in the clinical setting.
BMC Cancer 06/2012; 12:226. · 3.01 Impact Factor
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Ariane Groth,
Alexei V Salnikov,
Sabine Ottinger,
Jury Gladkich,
Li Liu,
Georgios Kallifatidis,
Olga Salnikova,
Eduard Ryschich, Nathalia Giese,
Thomas Giese,
Frank Momburg,
Markus W Büchler,
Gerhard Moldenhauer,
Ingrid Herr
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ABSTRACT: To enhance T-cell responsiveness toward cancer cells, we overexpressed TRAIL in lymphocytes, as this death ligand induces tumor-specific apoptosis. To increase contact time of lymphocytes with tumor cells and thereby of TRAIL with its death receptors, lymphocytes were linked to the CD3 arm of bispecific antibody EpCAMxCD3, to guide the lymphocytes to tumor cells positive for the cancer stem cell marker EpCAM/ESA.
Lymphocytes were transduced with TRAIL lentivirus and the antitumor effect in presence and absence of EpCAMxCD3 was evaluated in vitro and in xenograft studies using epithelial cell adhesion molecule (EpCAM)-positive pancreatic and prostate cancer cells.
Compared with control lymphocytes, TRAIL-lymphocytes increased cytotoxicity and further induced expression of several apoptosis-related molecules. Cotransplantation of TRAIL-lymphocytes and tumor cells in mice or peritumoral injection of TRAIL-lymphocytes in larger xenografts retarded growth and induced apoptosis. Combination of TRAIL-lymphocytes with EpCAMxCD3 potentiated tumor eradication by enhancing antiapoptotic and antiproliferative signaling and by decreasing tumor vasculature. Intratumoral cyst formation was involved and associated with enhanced chemokine secretion and infiltration of mouse macrophages, suggesting contribution of an inflammatory host response. Most importantly, tumorigenicity of pancreatic cancer cells with cancer stem cell features resistant to conventional chemotherapy was strongly reduced.
This gene-immunotherapeutic approach may be a new tool to support endogenous immune responses toward cancer even in its advanced stages.
Clinical Cancer Research 02/2012; 18(4):1028-38. · 7.74 Impact Factor
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Andrea S Bauer,
Andreas Keller,
Eithne Costello,
William Greenhalf,
Melanie Bier,
Anne Borries,
Markus Beier,
John Neoptolemos,
Markus Büchler,
Jens Werner, Nathalia Giese,
Jörg D Hoheisel
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ABSTRACT: A solid process for diagnosis could have a substantial impact on the successful treatment of pancreatic cancer, for which currently mortality is nearly identical to incidence. Variations in the abundance of all microRNA molecules from peripheral blood cells and pancreas tissues were analyzed on microarrays and in part validated by real-time PCR assays. In total, 245 samples from two clinical centers were studied that were obtained from patients with pancreatic ductal adenocarcinoma or chronic pancreatitis and from healthy donors. Utilizing the minimally invasive blood test, receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) analysis demonstrated very high sensitivity and specificity of a distinction between healthy people and patients with either cancer or chronic pancreatitis; respective AUC values of 0.973 and 0.950 were obtained. Confirmative and partly even more discriminative diagnosis could be performed on tissue samples with AUC values of 1.0 and 0.937, respectively. In addition, discrimination between cancer and chronic pancreatitis was achieved (AUC = 0.875). Also, several miRNAs were identified that exhibited abundance variations in both tissue and blood samples. The results could have an immediate diagnostic value for the evaluation of tumor reoccurrence in patients, who have undergone curative surgical resection, and for people with a familial risk of pancreatic cancer.
PLoS ONE 01/2012; 7(4):e34151. · 4.09 Impact Factor
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ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is frequently associated with fibrosis and a prominent inflammatory infiltrate in the desmoplastic stroma. Moreover, in PDAC, an epithelial-to-mesenchymal transition (EMT) is observed. To explore a possible connection between the infiltrating cells, particularly the polymorphonuclear neutrophils (PMN) and the tumor cell transition, biopsies of patients with PDAC (n = 115) were analysed with regard to PMN infiltration and nuclear expression of β-catenin and of ZEB1, well-established indicators of EMT. In biopsies with a dense PMN infiltrate, a nuclear accumulation of β-catenin and of ZEB1 was observed. To address the question whether PMN could induce EMT, they were isolated from healthy donors and were cocultivated with pancreatic tumor cells grown as monolayers. Rapid dyshesion of the tumor cells was seen, most likely due to an elastase-mediated degradation of E-cadherin. In parallel, the transcription factor TWIST was upregulated, β-catenin translocated into the nucleus, ZEB1 appeared in the nucleus, and keratins were downregulated. EMT was also induced when the tumor cells were grown under conditions preventing attachment to the culture plates. Here, also in the absence of elastase, E-cadherin was downmodulated. PMN as well as prevention of adhesion induced EMT also in liver cancer cell line. In conclusion, PMN via elastase induce EMT in vitro, most likely due to the loss of cell-to-cell contact. Because in pancreatic cancers the transition to a mesenchymal phenotype coincides with the PMN infiltrate, a contribution of the inflammatory response to the induction of EMT and-by implication-to tumor progression is possible.
Clinical and Developmental Immunology 01/2012; 2012:720768. · 1.84 Impact Factor
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ABSTRACT: Timely and accurate diagnosis of pancreatic ductal adenocarcinoma (PDAC) is critical in order to provide adequate treatment to patients. However, the clinical signs and symptoms of PDAC are shared by several types of malignant or benign tumors which may be difficult to differentiate from PDAC with conventional diagnostic procedures. Among others, these include ampullary cancers, solid pseudopapillary tumors, and adenocarcinomas of the distant bile duct, as well as inflammatory masses developing in chronic pancreatitis. Here, we report an approach to accurately differentiate between these different types of pancreatic masses based on molecular analysis of biopsy material. A total of 156 bulk tissue and fine needle aspiration biopsy samples were analyzed using a dedicated diagnostic cDNA array and a composite classification algorithm developed based on linear support vector machines. All five histological subtypes of pancreatic masses were clearly separable with 100% accuracy when using all 156 individual samples for classification. Generalized performance of the classification system was tested by 10 × 10-fold cross validation (100 test runs). Correct classification into the five diagnostic groups was demonstrated for 81.5% of 1,560 test set predictions. Performance increased to 85.3% accuracy when PDAC and distant bile duct carcinomas were combined in a single diagnostic class. Importantly, overall sensitivity of detection of malignant disease was 92.2%. The molecular diagnostic approach presented here is suitable to significantly aid in the differential diagnosis of undetermined pancreatic masses. To our knowledge, this is the first study reporting accurate differentiation between several types of pancreatico-biliary tumors in a single molecular analytical procedure.
Journal of Molecular Medicine 11/2011; 90(4):457-64. · 4.67 Impact Factor
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Jan Sperveslage,
Sunna Frank,
Carola Heneweer,
Jan Egberts,
Bodo Schniewind,
Malte Buchholz,
Frank Bergmann, Nathalia Giese,
Johanna Munding,
Stephan A Hahn,
Holger Kalthoff,
Günter Klöppel,
Bence Sipos
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ABSTRACT: CCR7 expression on tumor cells promotes lymphatic spread in several malignant tumors. However, a comprehensive characterization of the CCL19/CCL21-CCR7 axis in pancreatic ductal adenocarcinoma (PDAC), which is known for its high rates of lymph-node metastases, is still lacking. CCR7 mRNA and CCR7 protein were found to be expressed in spheroid cultures of all six examined PDAC cell lines. In migration assays, CCR7 expressing PDAC cells showed enhanced migration toward CCL19 and CCL21, the two ligands of CCR7. In an orthotopic nude mouse model, CCR7-transfected PT45P1 cells gave rise to significantly larger tumors and showed a higher frequency of lymph vessel invasion and lymph-node metastases than mock-transfected cells. In an analysis using quantitative real-time PCR, CCR7 showed fourfold overexpression in microdissected PDAC cells compared to normal duct cells. Moderate-to-strong immunohistochemical CCR7 expression, found in 58 of 121 well-characterized human PDACs, correlated with high rates of lymph vessel invasion. Conversely, PDACs completely lacking CCR7 expression showed only low rates of lymph vessel invasion and lymph-node metastases. The evaluation of CCL21 expression by immunofluorescence staining revealed a significant upregulation of CCL21 in peritumoral and intratumoral lymph vessels compared to lymph vessels in disease-free pancreata. In conclusion, our study revealed strong evidence that lack of CCR7 impairs the metastatic potential of PDAC. Lymph vessel invasion by CCR7 expressing PDAC cells may be additionally enhanced by upregulation of CCL21 in tumor-associated lymph vessels, representing a previously unknown factor of lymphatic spread.
International Journal of Cancer 10/2011; 131(4):E371-81. · 5.44 Impact Factor
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Andreas Keller,
Petra Leidinger,
Andrea Bauer,
Abdou Elsharawy,
Jan Haas,
Christina Backes,
Anke Wendschlag, Nathalia Giese,
Christine Tjaden,
Katja Ott, [......],
Johannes Dietl,
Sylvia Hofmann,
Hans-Peter Lenhof,
Stefan Schreiber,
Hugo A Katus,
Wolfgang Rottbauer,
Benjamin Meder,
Joerg D Hoheisel,
Andre Franke,
Eckart Meese
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ABSTRACT: In a multicenter study, we determined the expression profiles of 863 microRNAs by array analysis of 454 blood samples from human individuals with different cancers or noncancer diseases, and validated this 'miRNome' by quantitative real-time PCR. We detected consistently deregulated profiles for all tested diseases; pathway analysis confirmed disease association of the respective microRNAs. We observed significant correlations (P = 0.004) between the genomic location of disease-associated genetic variants and deregulated microRNAs.
Nature Methods 09/2011; 8(10):841-3. · 19.28 Impact Factor
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Cosmeri Rizzato,
Daniele Campa, Nathalia Giese,
Jens Werner,
P Sivaramakrishna Rachakonda,
Rajiv Kumar,
Michaela Schanné,
William Greenhalf,
Eithne Costello,
Kay-Tee Khaw,
Tim J Key,
Afshan Siddiq,
Justo Lorenzo-Bermejo,
Barbara Burwinkel,
John P Neoptolemos,
Markus W Büchler,
Jörg D Hoheisel,
Andrea Bauer,
Federico Canzian
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ABSTRACT: Pancreatic cancer has one of the worst mortality rates of all cancers. Little is known about its etiology, particularly regarding inherited risk. The PanScan project, a genome-wide association study, identified several common polymorphisms affecting pancreatic cancer susceptibility. Single nucleotide polymorphisms (SNPs) in ABO, sonic hedgehog (SHH), telomerase reverse transcriptase (TERT), nuclear receptor subfamily 5, group A, member 2 (NR5A2) were found to be associated with pancreatic cancer risk. Moreover the scan identified loci on chromosomes 13q22.1 and 15q14, to which no known genes or other functional elements are mapped. We sought to replicate these observations in two additional, independent populations (from Germany and the UK), and also evaluate the possible impact of these SNPs on patient survival. We genotyped 15 SNPs in 690 cases of pancreatic ductal adenocarcinoma (PDAC) and in 1277 healthy controls. We replicated several associations between SNPs and PDAC risk. Furthermore we found that SNP rs8028529 was weakly associated with a better overall survival (OS) in both populations. We have also found that NR5A2 rs12029406_T allele was associated with a shorter survival in the German population. In conclusion, we found that rs8028529 could be, if these results are replicated, a promising marker for both risk and prognosis for this lethal disease.
PLoS ONE 01/2011; 6(11):e27921. · 4.09 Impact Factor
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Wei Zhou,
Georgios Kallifatidis,
Bernd Baumann,
Vanessa Rausch,
Jürgen Mattern,
Jury Gladkich, Nathalia Giese,
Gerhard Moldenhauer,
Thomas Wirth,
Markus W Büchler,
Alexei V Salnikov,
Ingrid Herr
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ABSTRACT: According to the cancer stem cell hypothesis the aggressive growth and early metastasis of pancreatic cancer may arise through dysregulation of self-renewal of stem cells in the tissue. Since recent data suggest targeting of cancer stem cells by some dietary agents we studied the effect of quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Using in vitro and in vivo models of pancreatic cancer stem cells we found quercetin-mediated reduction of self-renewal as measured by spheroid and colony formation. Quercetin diminished ALDH1 activity and reverted apoptosis resistance as detected by substrate assays, FACS and Western blot analysis. Importantly, combination of quercetin with sulforaphane, an isothiocyanate enriched in broccoli, had synergistic effects. Although quercetin led to enhanced binding of the survival factor NF-kappaB, co-incubation with sulforaphane completely eliminated this pro-proliferative feature. Moreover, quercetin prevented expression of proteins involved in the epithelial-mesenchymal transition, which was even stronger in presence of sulforaphane, suggesting the blockade of signaling involved in early metastasis. In vivo, quercetin inhibited growth of cancer stem cell-enriched xenografts associated with reduced proliferation, angiogenesis, cancer stem cell-marker expression and induction of apoptosis. Co-incubation with sulforaphane increased these effects and no pronounced toxicity on normal cells or mice was observed. Our data suggest that food ingredients complement each other in the elimination of cancer stem cell-characteristics. Since carcinogenesis is a complex process, combination of bioactive dietary agents with complementary activities may be most effective.
International Journal of Oncology 09/2010; 37(3):551-61. · 2.40 Impact Factor
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ABSTRACT: To study secretion patterns of pro- and anti-inflammatory cytokines, and activation of various cellular subsets of leukocytes in peripheral blood.
We have conducted a prospective observational study. One hundred and eight patients with a diagnosis of acute pancreatitis and onset of the disease within last 72 h were included in this study. The mRNA expression of 25 different types of cytokines in white blood cells was determined by quantitative real time polymerase chain reaction. Levels of 8 different cytokines in blood serum were measured by enzyme linked immunosorbent assay. Clinical data and cytokine expression results were subjected to statistical analysis.
Severe and necrotizing acute pancreatitis (AP) is characterized by the significant depletion of circulating lymphocytes. Severe acute pancreatitis is associated with a typical systemic inflammatory response syndrome and over-expression of pro-inflammatory cytokines [interleukin (IL)-6, IL-8, macrophage migration inhibitory factor (MIF)]. Serum IL-6 and MIF concentrations are the best discriminators of severe and necrotizing AP as well as possible fatal outcome during the early course of the disease.
Deregulation of cellular immune system is a key event leading to severe and necrotizing AP. IL-6 and MIF could be used as early predictors of complications.
World Journal of Gastroenterology 04/2010; 16(15):1845-53. · 2.47 Impact Factor
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ABSTRACT: The process of extracting comprehensive proteome representations is a crucial step for many proteomic studies. While antibody microarrays are an evolving and promising methodology in proteomics, the issue of protein extraction from tissues for this kind of analysis has never been addressed. Here, we describe a single-step extraction buffer for the isolation of proteins from mammalian tissues under native conditions in an effective and reproducible manner. Protein was extracted from cell lines BxPC-3 and SU.86.86, rat organs (pancreas, liver, heart and lung) and human pancreatic cancer tissues using several buffer systems that contained individual nonionic or zwitterionic detergents in comparison to commercial extraction buffers. Also, detergent combinations were used that included at least one polymeric phenylethylene glycol, a long-chain amidosulfobetaine, cholate and a zwitterionic detergent. Extracts were analyzed for protein quantity and quality. The detergent cocktails exhibited superior extraction capacity. Additionally, they demonstrated a substantially higher recovery of membrane and compartmental proteins as well as much better preservation of protein functionality. Also, they did not interfere with subsequent analysis steps such as labeling. In Western blot and antibody microarray assays, they outperformed the other buffer systems, indicating that they should also be useful for other types of proteomic studies.
Journal of Proteome Research 02/2010; 9(2):963-71. · 5.11 Impact Factor
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Armin Kolb,
Simon Rieder,
Diana Born, Nathalia A Giese,
Thomas Giese,
Gottfried Rudofsky,
Jens Werner,
Markus W Büchler,
Helmut Friess,
Irene Esposito,
Jörg Kleeff
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ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with an overall 5-year survival rate of less than 5%. This dismal prognosis is largely due to the advanced stage of the disease at presentation, i.e., the late diagnosis. Therefore, early detection would have the potential to significantly improve the overall prognosis of PDAC patients. Diabetes mellitus (DM) has a high prevalence in PDAC patients and is frequently of new onset. The aim of this study was to analyze whether DM can be utilized as an early disease marker in PDAC. Quantitative RT-PCR analysis and immunohistochemistry for insulin and glucagon was performed in 22 PDAC and 16 normal pancreas tissues. Blood samples of 66 patients suffering from PDAC, 35 DM type 2 patients, and 29 healthy donors were analyzed for insulin, glucagon, C-peptide and glucose levels. Quantitative RT-PCR showed a two-fold increase of the glucagon/insulin ratio in pancreatic cancer tissues in comparison to the normal pancreas. By immunohistochemistry a shift in the expression pattern of glucagon and insulin, i.e., a higher glucagon/insulin ratio was found in PDAC associated islets compared to islets in the normal pancreas. Fasting insulin levels in PDAC patients were lower compared to DM patients. The calculated serum glucagon/insulin ratio was significantly different between PDAC and DM patients. At a cut-off of 7.4 ng/mU glucagon/insulin, pancreatic cancer induced new-onset DM could be discriminated from type 2 DM with 77% sensitivity and 69% specificity. In conclusion, the suggested serum glucagon/insulin ratio showed significant differences in patients with PDAC related DM and type 2 DM. Therefore, this analysis might help to identify PDAC in patients with new-onset DM in the age group at risk. Larger clinical trials have to confirm these findings.
Cancer biology & therapy 09/2009; 8(16):1527-33. · 2.64 Impact Factor
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ABSTRACT: Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect, but their complexity in structure and concentration, their close interaction, and their instability represent major challenges. For protein isolation in tissues, the breakdown of cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different processes for a compartmental protein preparation from pancreatic tissue, one of the most challenging tissues for protein isolation because of its high protease content. Success of the different procedures varied greatly. Based on a scheme of tissue-slicing and subsequent cell isolation, we established a reliable workflow for the fractional extraction of cytosolic proteins, membrane and organelle proteins, nuclear proteins, and cytoskeletal filaments. The tissue slices also allow for a representative confirmation of individual samples' cellular status by histochemical processes, and a proper separation or mixing of cellular material from across a tumor if required.
BioTechniques 05/2009; 46(4):297-304. · 2.67 Impact Factor
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ABSTRACT: Abstract
Background
Costimulatory signaling has been implicated as a potential regulator of antitumor immunity in various human cancers. In contrast to the negative prognostic value of aberrant B7-H1 expression by pancreatic cancer cells, the role of B7-H3 is still unknown. Therefore, we investigated the expression pattern and clinical significance of B7-H3 expression in human pancreatic cancer.
Methods
B7-H3 expression was evaluated by immunohistochemistry in 68 patients with pancreatic cancer who underwent surgical tumor resection. Expression data was correlated with clinicopathologic features and with the number of tumor-infiltrating T cells.
Results
B7-H3 expression was significantly upregulated in pancreatic cancer compared to normal pancreas (p < 0.05). In 60 of 68 examined tumors B7-H3 protein was detectable in pancreatic cancer cells. Patients with high tumor B7-H3 levels had a significantly better postoperative prognosis than patients with low tumor B7-H3 levels (p = 0.0067). Furthermore, tumor B7-H3 expression significantly correlated with the number of tumor-infiltrating CD8+ T cells (p = 0.018).
Conclusion
We demonstrate for the first time that B7-H3 is abundantly expressed in pancreatic cancer and that tumor-associated B7-H3 expression significantly correlates with prolonged postoperative survival. Our findings suggest that B7-H3 might play an important role as a potential stimulator of antitumor immune response in pancreatic cancer.
BMC Cancer. 01/2009;
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ABSTRACT: Pim-1 is a proto-oncogene involved in cell survival, differentiation and proliferation in several hematologic and epithelial malignancies. Clinically, absence of Pim-1 expression correlates with poor prognosis in prostate cancer. In the present study, the expression of Pim-1 is analyzed in pancreatic cancer and correlated to clinicopathological parameters.
Compared to benign, inflammatory and pre-malignant conditions (i.e., the normal pancreas, chronic pancreatitis and benign intraductal papillary mucinous neoplasm), expression of Pim-1 mRNA and protein increased significantly in pancreatic malignancies. Absence of Pim-1 immunopositivity in cancer cells strongly correlated with a poor prognosis (median survival 13.8 vs. 23.4 months, p = 0.0016). In vitro, rapidly dividing (high versus low serum concentrations) and hypoxic cells displayed higher Pim-1 mRNA and protein levels.
Pim-1 mRNA and protein was evaluated with quantitative real-time RT-PCR, immunofluorescence and immunocytochemistry analyses. Ex vivo expression analysis using semi-quantitative immunohistochemistry was performed using human pancreatic tissues of the normal pancreas (n = 10), chronic pancreatitis (n = 30), pancreatic ductal adenocarcinoma (n = 59) and other pancreatic tumors (n = 42). In consecutive sections HIF1-alpha was used as a marker of hypoxia. Survival of patients (n = 35) was compared using the Kaplan-Meier method and a log-rank test. In vitro analyses were performed using cultured pancreatic cancer cell lines (n = 8) and primary human pancreatic stellate cells.
Hypoxia is a novel inducer of Pim-1 expression. Compared to non-malignant tissues Pim-1 significantly increases in pancreatic cancer. However, the presence of Pim-1 in cancer cells has a positive prognostic impact.
Cancer biology & therapy 07/2008; 7(9):1352-9. · 2.64 Impact Factor
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ABSTRACT: The peptide hormones guanylin and uroguanylin and their receptor, guanylate cyclase C (GC-C), are expressed in pancreatic duct cells. In colon cancer, guanylin peptides are shown to exert strong anti-tumor activity through the GC-C pathway. The objective of this study was to analyze the role of guanylin and uroguanylin in human pancreatic cancer.
Quantitative real-time polymerase chain reaction (QRT-PCR) was used to show the expression of guanylin, uroguanylin and GC-C in specimens of human pancreatic cancer, chronic pancreatitis donor and in pancreatic tumor cell lines. The presence of guanylins and GC-C in tumor cell lines and in pancreatic cancer tissues was shown by immunofluorescence and immunohistochemistry. The effect of guanylin and uroguanylin on cell cycle and cell death of pancreatic cancer cells was investigated by fluorescence activated cell sorter (FACS) analysis using annexin and propidium iodide. In addition, the growth inhibitory effect of guanylins on pancreatic cancer cells was assessed using the MTT assay.
Guanylin, uroguanylin and GC-C were expressed at mRNA and protein levels in pancreatic cancer and cancer cell lines. As shown by QRT-PCR, GC-C expression was significantly up-regulated in pancreatic cancer compared with that in healthy pancreatic tissues (p<0.00001) and chronic pancreatitis (p<0.05). Guanylin and uroguanylin were not up-regulated in pancreatic cancer. The MTT assay revealed significant inhibition of pancreatic cancer cell proliferation by uroguanylin in a dose-dependent fashion, whereas Panc1 and Capan1 cell lines were significantly inhibited already at the lowest uroguanylin concentration (2 nM, p<0.05).
Our data suggest therapeutic properties of uroguanylin in pancreatic cancer via GC-C-dependent mechanisms. In addition, determination of GC-C expression might be a useful marker for differentiation between pancreatic cancer and chronic pancreatitis.
Scandinavian journal of gastroenterology 01/2008; 43(4):447-55. · 2.08 Impact Factor
