Lung Damage Induced By Albumin Administration in Acute Pancreatitis is Reversed by Nitric Oxide Synthase Inhibition
E. Abdo, A. Coelho, S. Sampietre, N. Molan, J. E. Cunha, L. D'Albuquerque, M. Machado. University of S. Paulo, Brazil.
Background/Aim: Previous study demonstrated that inhibition of nitric oxide synthase (iNOS) reverses the effect of albumin on lung damage in burn. The aim of this study was to evaluate whether inhibition of iNOS reverses the effect of albumin on lung damage in AP.
Methods: AP was induced in male Wistar rats by intraductal 2.5% taurocholate injection. To evaluate the effect of albumin on lung damage in AP, animals received IV saline (Group I) or human albumin (Group II) immediately after AP. To evaluate the effect of iNOS inhibition on lung damage in AP, iNOS specific inhibitor S-methylisothiourea (SMT)) was given to animals immediately after AP. The animals were divided into groups: Group III: saline was given after AP and SMT, and Group IV: albumin was given after AP and SMT. After 12 hours serum amylase levels, lung myeloperoxidase (MPO) activity, and pulmonary vascular permeability were determined.
Results: Serum amylase levels, lung MPO activities and vascular permeability of lungs were significantly increased after AP. Albumin administrated after AP increased lung permeability (70.92 ± 7.0 mg EB/g tissue) compared to saline administration (47.41 ± 4.62 mg EB/g tissue) (p<0.05). However, albumin administration with SMT reduced lung permeability (42.77 ± 9.49 mg EB/g tissue) compared to albumin administration without SMT (70.92 ± 7.0 mg EB/g tissue) (p<0.05). There were no significant differences in serum amylase and lung MPO activities among groups.
Conclusion: Restoration of extracellular fluid in AP with albumin increased the lung permeability. Inhibition of iNOS before albumin administration reduced this albumin induced damaging effect in AP.
Early Surgery for Necrotizing Pancreatitis: Primary Necrosectomy is not Necessary
Y. P. Abdullaev,1 S. I. Galeev,1 M. A. Rubtsov.21Department of Surgery, Saint-Petersburg State Medical Academy n.a. Mechnicov, Russia, 2Department of Surgery, Clinical Hospital of St. Luke, Saint-Petersburg, Russia.
Introduction: It's generally accepted: necrosectomy in early phase of severe pancreatitis increases mortality. At the same time, most of irreversible organ failures and deaths associated with infection resistant to conservative measures.
Objectives: To perform comparative analysis of treatment results in two series of patients: managed conservatively (group 1), underwent early open drainage of extrapancreatic necrosis with delayed necrosectomy (group 2).
Material and Methods: A retrospective study of 71 patient with severe pancreatitis (abscesses and cysts were excluded) in 9 year period (2000-2009) was done. 17 patients were treated without operation, other (54) underwent early (mean time from the disease onset - 6,1±7,1 days) drainage with further necrosectomy (mean time from primary operation - 12,5±6,0 days).
Results: Above-mentioned groups were comparable in regard to local complications severity (Balthazar index turned out to be equal, p=0,49; extended pancreatic necrosis (>50%) was diagnosed in 4 patients of group 1 (26,7%) and 8 of group 2 (19,5%), p=0,83). Mean admission APACHE II score between two groups didn't differ (8,9±5,4 vs. 14,5±7,8; p=0,24). We haven't observed early drainage negative effect on a course of the disease (mortality rates in groups were practically similar: conservative therapy - 4 patients died (23,5%); surgical treatment - 19 patients died (35,2%), p=0,37). Misfortune of surgery in most part of cases might be explained by late admission of patients with advanced septic complications.
Conclusion: Drainage and necrosectomy temporal separation seems to be beneficial sepsis-preventive tactic, equalizing results of conservative and surgical treatment.
New-Onset Diabetes in Pancreatic Cancer: A Study in the Primary Care Setting
G. Aggarwal,1 G. M. Petersen,2 S. T. Chari.11Division of Gastroenterology, 2Department of Epidemiology, Mayo Clinic, Rochester, MN.
Background: In epidemiologic studies, new-onset Diabetes Mellitus (DM) has been shown to precede the diagnosis of pancreatic cancer (PaC) by up to 36 months. In clinical practice, new-onset DM is diagnosed by primary care physicians.
Aim: To determine, in the primary care setting, the proportion of PaC patients that have new-onset DM, especially prior to onset of cancer symptoms.
Methods: We retrospectively reviewed the medical records of 100 consecutive patients (51 male, mean age 71 ± 14 years) followed in Mayo Clinic's primary care clinics between 1995-2007, who were eventually diagnosed with PaC. Onset of DM was defined by the first date fasting blood glucose was ≥126 mg/dl. The date the physician diagnosed DM was also noted.
Results: 40% of PaC diagnosed in the primary care setting had DM. Onset of DM was <36 months before the diagnosis of PaC in 26 (65%). Median duration of cancer specific symptoms (abdominal pain, back pain, jaundice, anorexia, weight loss) in patients with DM was 1 month (range, asymptomatic-6). In 22/26 (85%) patients with new-onset DM there were no cancer specific symptoms at the onset of DM; the median duration of DM prior to PaC diagnosis in this sub-group was 8.5 months (range, 0.5-35). However, in only 4/26 patients did the physician make a diagnosis of DM at its onset and in 12/26 patients, the diagnosis of DM was never made. The average delay between the onset of DM and physician diagnosis of DM in the remaining 10 patients was 3.4 months (range, 0.25-14).
Conclusions: Screening patients with new onset DM for PaC could potentially identify 22 % PaC prior to onset of symptoms. However, physicians frequently do not diagnose DM at its onset.
Effect of Pancreatic Cancer Resection on Glucose Homeostasis
G. Aggarwal,1 S. T. Chari,1 R. Pannala,2 R. Basu,3 A. Basu.31Division of Gastroenterology and 3Endocrinology, Mayo Clinic, Rochester, MN, 2Division of Gastroenterology, Virginia Mason Medical Center, Seattle, WA.
Background: Up to 85% of pancreatic cancer (PaC) patients have either impaired fasting glucose or diabetes mellitus (DM) which is frequently new-onset (<36 months) and resolves after tumor resection. Our goal was to delineate abnormalities of glucose metabolism in PaC before and after cancer resection.
Methods: We measured insulin and glucose levels at 22 time points after intravenous glucose infusion (Frequently Sampled Intravenous Glucose Tolerance Test) in 9 PaC patients (mean age 65.6 yrs, 6 males), before and after cancer resection. Using the MINMOD Millenium® model we determined the parameters of insulin secretion and action. Statistical comparisons of pre- and post-operative parameters were made using the Wilcoxon Sign-Rank test.
Results: PaC patients lost weight before resection [median, 11.4 (0-22.7) kg] and lost further weight [median, 3.8 (1.3-14) kg] after resection. PaC resection was associated with a reduction in fasting blood glucose [median, 118 (87-137) vs 102 (69-113) mg/dL; p=0.039], a rise in acute insulin response to glucose [median, 51.6 (-26.8-175.3) vs 75.4 (32.2-302.1) mU L-1 min; p= 0.05] and a decrease in insulin resistance [median, 2.2 (0.6-4.6) vs 0.78 (0.5-2.3) mM mU L-2; p= 0.008].
Conclusions: Pre-operatively, PaC patients have insulin resistance and an impaired acute insulin response to glucose despite weight loss, likely due to tumor secreted diabetogenic substances. Following PaC resection, glucose homeostasis improves with a rise in acute insulin response to glucose, a marked decrease in insulin resistance and post-operative weight loss.
Mist1-/- Mice Have Increased Sensitivity to an Alcohol Laced Diet
S. Alahari,1 C. Pin.1,21Department of Physiology and Pharmacology and 2Pediatrics, University of Western Ontario, Children's Health Research Institute, London, Canada.
Background: In humans, excessive alcohol consumption is a leading cause of pancreatitis. While alcohol has been shown to sensitize the pancreas to subsequent insult, the ethanol diet on its own does not cause pancreatitis. This suggests that additional factors play a role in dictating pancreatic injury in response to alcohol. Mice lacking the transcription factor MIST1 (Mist1-/-) show acinar cell disorganization, increased sensitivity to cerulein-induced pancreatitis and possibly increased susceptibility to alcohol induced pancreatitis. Here, we examined the cellular and molecular response of Mist1-/- mice to chronic feeding of an ethanol laced diet.
Methods: Wild type or Mist1-/- mice were fed either the Lieber-DeCarli liquid ethanol or control diet for 6 weeks. Body weight, caloric intake, serum amylase and pancreatic edema were assessed. Pancreatic morphology was evaluated by H&E staining while RT-PCR and Western Blotting assayed for gene and protein expression.
Results: No significant differences in caloric intake, serum amylase and tissue edema were found. Ethanol fed Mist1-/- mice gained significantly less weight than their control counterparts. Notably, they showed peri-ductal infiltration of inflammatory cells and adipocytes, in addition to the characteristic acinar disorganization evident in Mist1-/- mice. These accumulations are comparable to those observed in aged (12 and 18-month old) Mist1-/- mice.
Conclusion: These studies show that Mist1-/- mice are more sensitive to alcohol induced pancreatic damage compared to wild types. Whether this is due to an inability to activate protective mechanisms is unclear. However, these findings suggest that altered MIST1 function may be an underlying cause to pancreatitis susceptibility.
An Active AKT is Required for KRAS Modulation of GLI1 Activity in Pancreatic Cancer Cells
L. L. Almada, S. F. Elsawa, G. L. Lund, M. E. Fernandez-Zapico. Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN, USA.
Ectopic activation of the GLI transcription factors has been implicated in the growth and maintenance of a number of malignancies including pancreatic cancer. Several studies suggest that GLI proteins act as effectors of different oncogenic pathways. Here, we demonstrate the ability of AKT1 to activate GLI1-mediated transcription in pancreatic cancer cells. Luciferase reporter and expression assays show that AKT1 is able to promote GLI1 transcriptional activity in these cancer cells. Knockdown of AKT1 using RNA interference or dominant negative molecules inhibits GLI1-mediated regulation of its target genes as well as impairs KRAS activation of GLI1 in pancreatic cancer cells. Conversely, constitutively active mutant AKT synergizes with GLI1 in the activation of its promoters in pancreatic cancer cells. Further analysis of this molecular interaction shows that GLI1 protein sequence contains candidate AKT phosphorylation sites within domains that are essential for its transcriptional activity such as the coactivator binding domain, thus, suggesting potential molecular mechanisms underlying the modulation of GLI1 function by this kinase. Indeed, AKT1 phosphorylates GLI1 in vitro and mutations of these candidate phosphorylation sites diminish GLI1 transcriptional activity. Taken together, these results provide a novel insight into the mechanisms underlying the ongogenic networks of events that control pancreatic carcinogenesis. Moreover, the knowledge derived from this study presents new avenues for the development of therapeutic regimens for this dismal disease.
M.E.F.-Z. is supported by the Division of Oncology Research, CA136126, Mayo Clinic Pancreatic SPORE CA102701 and Mayo Clinic Center for Cell Signaling in Gastroenterology DK084567.
Continuous Subcutaneous Insulin Infusion (CSII) Therapy for Management of Apancreatic Diabetes Mellitus
K. Altaf,1 J. Adu,1 G. Morrison,2 Q. M. Nunes,1 C. Halloran,1 J. P. Neoptolemos,1 C. P. Weston,2 R. Sutton.11Liverpool NIHR Pancreas Biomedical Research Unit, Division of Surgery and Oncology, 2Department of Endocrinology, Royal Liverpool University Hospital, Liverpool, UK.
Background: Insulin-dependent diabetes mellitus caused by islet tissue loss secondary to pancreatitis or surgery is subject to significant swings in plasma glucose and carries a high risk of hypoglycaemia. We report on the previously undocumented use of CSII therapy in the management of apancreatic patients with poorly controlled ('brittle') diabetes mellitus.
Methods: Patients on multiple daily injections (MDI) of insulin with poorly controlled apancreatic diabetes mellitus subsequently commenced on CSII pump therapy were included in this study, and their diabetic management compared before and after CSII.
Results: Seven patients (5 males, 2 females, median age 47 y) developed diabetes mellitus after acute necrotising pancreatitis (n = 3) or pancreatic surgery (n=4), initially managed by MDI and subsequently by CSII. Median duration of MDI was 27 months (range 16-156), HBA1c 8.8% (7.0-10.4), daily insulin requirement 64U (range 22-104) and hypoglycaemic episodes 3/week (0-7). Upon conversion to CSII, pumps used were Accu-Chek Spirit (n=5), Paradigm real-time MMT-522 (n=1) and Animas 2020 (n=1). After 12 mo CSII, median HBA1c was 7.5% (6-8.4%; p<0.05), daily insulin requirement 31U (16.1-51.8) and hypoglycaemic episodes 1(0-4/wk). All patients improved on every parameter and no episodes of diabetic ketoacidosis were reported.
Conclusion: Use of CSII for 'brittle' apancreatic diabetes mellitus in selected patients resulted in a decrease in hypoglycaemic episodes and sustained improvement in glycaemic control, providing proof of principle for CSII in these patients.
Late Onset Pancreatic Necrosis in Acute Pancreatitis
K. Altaf,1,2 T. Odetoyinbo,3 N. Kibriya,3 R. Thomson,2 L. Moulton,2 C. Garvey,3 J. Evans,3 J. P. Neoptolemos,1,2 R. Sutton.1,21Liverpool NIHR Pancreas Biomedical Research Uni, 2Department of Surgery and Oncology, 3Department of Radiology, Royal Liverpool University Hospital, Liverpool, UK.
Background: The full extent of necrosis in acute pancreatitis usually occurs within 96 hours of symptom onset, with guidelines recommending contrast enhanced CT (CE-CT) two to 10 days after admission to assess extent. We sought to determine whether late onset necrosis is a significant clinical problem potentially requiring guideline modification.
Methods: Patients who had minimal or no pancreatic necrosis on an initial CE-CT seven or more days after symptom onset (6 days after admission) were identified from a prospectively maintained database of patients who had pancreatic necrosectomy between 2005 and 2010. All their serial CE-CTs were assessed for necrosis blindly by two independent investigators by a novel morphometric method.
Results: Eight patients (6 males, 2 females, median 72 y) were included. Aetiology was gallstones (5; one underwent sphincterotomy and stone extraction), alcohol (1) and idiopathic (2). New or progressive pancreatic necrosis (median 62%, range 36-80%; median 4.3-fold increase; P=0.01) was found at a median of 15 (6-36) days after admission. There were significant increases of necrosis in all parts of the pancreas (head P=0.01, body P=0.01, tail P=0.02). Head or body necrosis significantly increased total necrosis (head P<0.001, body P=0.003). Six patients underwent minimal access necrosectomy (median 3 procedures, range 1-7), two open. Median ITU/HDU stay was 7.2 (0-28) and hospital stay 93 (77-171) days, with no mortality.
Conclusion: Clinically significant late onset necrosis in acute pancreatitis requires particular consideration that may mandate additional CE-CT later than previously recommended.
Transformation of Pancreatic Epithelial Cells by BrafV600E
V. A. Appleman,1 D. S. Klimstra,2 B. C. Lewis.11Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, 2Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY.
The majority of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC), and activating mutations in the KRAS oncogene are present in over 90% of these tumors. Interestingly, one third of all KRAS wild-type PDAC harbor activating mutations in the gene encoding the downstream Kras effector Braf. We therefore sought to determine the consequences of expressing an activated Braf molecule in primary pancreatic duct epithelial cells (PDECs). Expression of BrafV600E results in the activation of the Mek-Erk and PI3K-Akt signaling cascades. Measurement of cell proliferation demonstrated that BrafV600E stimulates the proliferation of PDECs, albeit to a lesser extent than activated KrasG12D. BrafV600E also promotes the survival of PDECs after exposure to apoptotic stimuli. This survival is dependent on both the Mek-Erk and PI3K-Akt signaling cascades, as inhibition of these pathways abrogated Braf-induced survival. Interestingly, this survival is also dependent on IGF1R-stimulated signaling. Consistent with its ability to stimulate the proliferation and survival of PDECs, orthotopic transplantation of PDECs expressing BrafV600E, and deleted at the Ink4a/Arf and Trp53 tumor suppressor loci, resulted in tumor formation. Interestingly, cell lines derived from orthotopic tumors required the activation of the PI3K-Akt signaling axis, but not the Mek-Erk signaling pathway, for survival after exposure to apoptotic stimuli, suggesting differential roles for these pathways during pancreatic tumor initiation and progression. Thus, our studies provide evidence that activating Braf mutations lead to the transformation of pancreatic epithelial cells, and additionally suggest that downstream Ras effectors may play different roles during pancreatic tumor initiation and progression.
A Novel Cancer-Targeting Adenovirus Expressing Interferon Alpha for Pancreatic Cancer Therapy
L. Armstrong, J. Davydova, E. Brown, J. Han, M. Yamamoto, S. M. Vickers. Department of Surgery, University of Minnesota, Minneapolis, MN.
More effective systemic therapy is acutely needed in pancreatic adenocarcinoma due to its dismal prognosis. Interferon alpha (IFNα) is promising in multimodality therapy, but has a short half-life and systemic side effects. We have made an IFNα-expressing oncolytic adenovirus to locally express IFNα at high concentration with our most advanced oncolytic adenovirus backbone. The COX-2 promoter drives viral replication due to its known overexpression in pancreatic cancer, while avoiding replication in normal organs including liver. The Ad5/Ad3 chimeric fiber is used for optimal cancer cell infectivity. We incorporate the adenoviral death protein for enhanced viral spread, and placement of IFNα under control of adenovirus major late promoter enables localized expression of IFNα selectively in COX-2 positive cancer cells. In aggregate, this virus will allow for focused tumor treatment with limited systemic toxicity. In vitro testing shows a robust effect on highly aggressive metastatic pancreatic cancer cell lines, S2VP10 and S2O13. Both cell lines showed COX-2 expression in RT-PCR analysis, and analysis of COX-2 promoter strength using an E1 deleted luciferase vector showed the 1.5kb COX-2 promoter was active in both. Next, we analyzed cytolytic ability. When compared to 5/3 Ad wt (nonselective positive control virus), our virus has a similarly potent effect on S2VP10 cells, and is more powerful than 5/3 Ad wt on S2O13. The virus shows no toxicity to COX2-negative BT474 cells. Quantification of IFNα production revealed that this vector expresses IFNα in a viral replication dependent manner. These data indicate that we have made a very potent cancer selective IFNα expressing oncolytic adenovirus with great utility in pancreatic cancer.
Gemcitabine Induction of P21Cip/Waf Promotes Resistance to Apoptosis in Pancreatic Cancer
T. Arumugam,1 V. Ramachandran,1 T. Fujii,1 R. F. Hwang,2 W. Choi,3 D. J. McConkey,3 C. D. Logsdon.1Dept. of 1Cancer Biology, 2Surgical Oncology, 3Urology, UT MD Anderson Cancer Center, Houston, TX.
Background: The clinical standard of care for pancreatic cancer (PC) is treatment with gemcitabine (Gem). However, resistance to Gem is common. In order to identify resistance mechanisms, we investigated the effects of Gem on PC cell gene expression. We observed a rapid increase in p21Cip/Waf expression after Gem treatment. Therefore, we investigated the influence of this molecule on PC cell responses to Gem.
Methods: Six pancreatic cancer cell lines (MPanc96, MiaPaCa-2, Panc1, BxPc3, SU8686 and L3.6pl) were treated with Gem in vitro and then analyzed for multiple parameters, including mRNA expression using Illumina microarrays, senescence by □-gal expression, effects on apoptosis and cell cycle by FACS and, for expression of p21Cip/Waf by WB and ELISA. To analyze the effects of p21Cip/Waf on Gem responses, SiRNA mediated silencing was utilized.
Results: Gem treatment caused all PC cells to accumulate in S-phase and to express senescence □-gal. Sensitive cells (BxPc3, SU8686 and L3.6pl) also underwent apoptosis. In contrast, Gem resistant cells (MPanc96, MiaPaCa-2, and Panc1) became cytostatic but did not die. Gem treatment altered gene expression in all cells. One of the most highly induced genes was p21Cip/Waf. WB and ELISA confirmed Gem induction of p21Cip/Waf expression both in vitro and in vivo in mouse xenograft models. Furthermore, microarray analysis of resected tumor samples from patients that received either no pretreatment or pretreated with radiation and Gem indicated that p21Cip/Waf levels were elevated in the treated samples. p53 phosphorylation at s15, which monitors activity in the DNA damage-ATM-chk1/2 pathway, was also elevated by Gem treatment and may partially explain the increase in p21Cip/Waf expression. SiRNA against p21Cip/Waf sensitized all PC cells to Gem, including those that have high endogenous resistance, both in vitro and in vivo.
Conclusion: Gem treatment induces p21Cip/Waf expression causing cell cycle arrest and resistance to apoptosis. Strategies aimed at inhibiting the induction or actions of p21Cip/Waf may be useful for overcoming Gem resistance.
INGAP Induces In Vitro Endocrine Differentiation of Human Pancreatic Duct Epithelial Cells Grown in 3D-Cultures
B. Assouline-Thomas, D. Ellis, J. Makhlin, M. Petropavlovskaia, L. Rosenberg. Department of Surgery, McGill University, Montréal, QC, Canada.
Islet Neogenesis Associated Protein (INGAP) is a Reg3 protein discovered in the partially duct-obstructed hamster pancreas as a factor inducing islet neogenesis. A 15 amino acids bioactive fragment, INGAP peptide (INGAP-P), reverses diabetes in rodents and improves glucose homeostasis in patients with diabetes in clinical trials. In vitro, INGAP-P induces the differentiation of human islet-derived duct-like-structures (DLS) into fully-functional islets. We hypothesized that adult human pancreatic ductal cells retain a morphogenetic plasticity and can be induced by INGAP-P to undergo endocrine differentiation.
Monolayers of immortalized, nontumorigenic human pancreatic ductal epithelial (HPDE) cells were treated with 167nM INGAP-P (5 min to 24hr). RT-PCR data show the rapid activation of a cascade of the transcription factors Pdx-1, NeuroD, IA-1 and MafA reminiscent of that occurring during fetal ontogeny of the endocrine pancreas, followed by an increase in insulin expression. Immunofluorescence showed a decreased signal for the ductal marker CK19 and the translocation of Pdx signal from the cytoplasm to the nucleus as well as the appearance of a weak signal for C-peptide, suggesting the onset of differentiation towards a beta cell phenotype.
HPDE cells cultured in a 3D system form clusters. When treated for 1 week with 167nM INGAP-P, clusters showed a decreased CK19 signal and the translocation of Pdx-1 protein to the nucleus accompanied by a strong expression of the mature beta cell markers C-peptide and Glut2.
These results show that INGAP induces endocrine differentiation of human adult ductal cells and reveal the critical contribution of the 3D organization of the cells in the endocrine differentiation process.
Does the 3-Aminobenzamide Effect on Bacterial Translocation Affect Experimental Acute Necrotizing Pancreatitis?
S. Aydin,1 A. T. Isik,2 E. Cinar,3 O. Demir,2 C. Akay,4 M. Ozyurt,5 S. Deveci,6 M. R. Mas.21Department of Surgery, Guven Hospital, A. Ayranci, Ankara, Turkey, 2Department of Internal Medicine, Gulhane School of Medicine, Ankara, Turkey, 3Department of Infection Disease, Gulhane School of Medicine, Ankara, Turkey, 4Department of Pharmacology, Gulhane School of Medicine, Istanbul, Turkey, 5Department of Microbiology, Gulhane School of Medicine, Istanbul, Turkey, 6Department of Pathology, Gulhane School of Medicine, Ankara, Turkey.
Background: One of the most important complications of acute pancreatitis is the secondary bacterial infections of the pancreas and gut. Translocation of bacteria from the gut is accepted as being responsible for the development of septic complications in acute pancreatitis. In this study, our aim was to investigate the effect of PARP inhibition via 3-aminobenzamide on the bacterial translocation in acute pancreatitis.
Methods: Forty-five male Sprague-Dawley rats were randomly allocated into three groups. Group I (Sham+saline) received normal saline infusion into the common biliopancreatic duct. Acute pancreatitis was induced in Group II (acute pancreatitis+saline) and Group III (acute pancreatitis+ 3-aminobenzamide) by the retrograde injection of taurocholate into the common biliopancreatic duct. Six hours after induction of pancreatitis, the rats in Group I and II were treated with saline (1 ml, every 12 hours), while the rats in Group III were treated with 3-aminobenzamide (10 mg/kg/day every 12 hours), intraperitoneally. In the 54th hour of the study, blood and tissue samples were taken for biochemical, microbiological and histopathological analysis.
Results: Acute pancreatitis developed in Groups II and III. Pathologic score [median (25-75% percentiles)] of the pancreatitis in Group III [8 (7-9)] was significantly lower than in Group II [19 (18-21)] (p<0.001). Bacterial translocation to mesenteric lymph node (53.3%), peritoneum (60%) and pancreas (46.7%) in Group III was significantly lower than in Group II (100% for all) (p<0.02, p<0.03, p<0.005, respectively). Pancreatic tissue glutathione peroxidase, superoxide dismutase, and malondialdehyde levels were better in Group III compared to Group II (p<0.001 for all). Comparison of Groups II and III demonstrated reduced severity of inflammation of the gut in Group III (p>0.05). Improvement in bacterial translocation was correlated with reducing oxidative stress.
Conclusions: We demonstrated that 3-aminobenzamide therapy improved histopathologic score and oxidative stress in experimental pancreatitis. In addition, it was demonstrated microbiologically and histopathologically that 3-aminobenzamide therapy improves bacterial translocation. Further survival studies demonstrating the efficacy of 3-aminobenzamide therapy and explaining the potential mechanisms of bacterial translocation prevention in acute necrotizing pancreatitis will be beneficial.
Bacterial Translocation in Experimental Acute Necrotizing Pancreatitis: Effects of Infliximab
S. Aydin,1 A. T. Isik,2 B. Unal,3 B. Comert,2 M. Ozyurt,4 S. Deveci,5 G. Erdem,2 M. T. Unal,2 G. Ozgur,2 O. Cengiz,6 I. Tasci,2 M. R. Mas.21Department of Surgery, Guven Hospital, A. Ayranci, Ankara, Turkey, 2Department of Internal Medicine, Gulhane School of Medicine, Ankara, Turkey, 3Department of Surgery, University of Inonu, Malatya, Turkey, 4Department of Microbiology, Gulhane School of Medicine, Istanbul, Turkey, 5Department of Pathology, Gulhane School of Medicine, Ankara, Turkey, 6Department of Surgery, Numune Training Hospital, Sihhiye, Ankara.
Background: Translocation of bacteria from the gut is one of the most important factors in the development of septic complications and mortality in acute pancreatitis. The present study was designed to search the effects of infliximab treatment on BT in animal necrotizing pancreatitis.
Methods: Forty-five Sprague-Dawley rats were allocated into three groups. Acute pancreatitis was induced in group II (positive controls; n=15) and group III (Infliximab; n=15) by retrograde injection of taurocholate into the common biliopancreatic duct. Group I (Sham; n=15) received normal saline infusion into the common biliopancreatic duct as placebo. Group I and II were treated by normal saline and group III were treated with infliximab intraperitoneally on 6th, 30th and 54th hours after induction of pancreatitis. All surviving animals were killed 54 hours after the induction of pancreatitis, and specimens were collected for amylase measurement as well as histopathologic and microbiologic examinations.
Results: Edema, acinar cell necrosis, inflammatory infiltration, hemorrhage, fat necrosis and perivascular inflammation in group III were decreased with infliximab treatment when compared with group II (p<0.001). Bacterial translocation to mesentery lymph node in group I, II and III were 20%, 100% and 46%, respectively. Bacterial translocation to peritoneum and pancreas in group III were lower than group II (p<0.02).
Conclusions: Infliximab administration resulted in beneficial effects on bacterial translocation and histopathologic changes in the experimental necrotizing pancreatitis. Whether anti-TNF therapy has a role in prevention of complications of ANP needs to be established.
Systems Biology: Implications is accessing Drug Target Gene Signatures in Pancreatic Cancer
A. S. Azmi,1 R. M. Mohammad,2 Z. Wang,1 F. H. Sarkar.11Department of Pathology, 2Department of Internal Medicine, Wayne state University, Detroit MI, USA.
The major reason for low response to target therapies in pancreatic cancer (PC) is the incomplete understanding and validation of specific molecular targets at the gene level along with complexities of cross talk between crucial pathways in this disease. To overcome this barrier, one must fully understand the molecular interactions among key signaling pathways in PC. Recently we have found that specific, orally active murine double minute 2 (MDM2) inhibitor MI-219 can synergize with chemotherapeutic drug oxaliplatin that results enhanced anti-tumor effects in PC. Here we have used integrated genomic expression microarray profiling coupled with pathway network modeling to obtain crucial information in the synergistic efficacy of MI-219-oxaliplatin. Global gene analysis of capan-2 (wt-p53) cell shows that MI-219 alone induces alterations in 48 genes. On the other hand, oxaliplatin treatment results in alterations of 761 genes. However, their combination resulted in alteration of 767 genes with emergence of 286 synergy unique genes. Principal component analysis revealed that each drug treatment and time point had individual global gene signatures. Further analysis of the synergy unique genes revealed activation of several local gene networks of MDM2-p53 pathway including CREBBP, NF-κB, EGR1 and E-cadherin tumor suppressor module (verified at protein level), all positively affecting p53 re-activation that drives cells toward apoptosis. We believe that success in personalized medicine will require advances in our understanding of the true potential and concepts of this technology toward elucidating the role of most influential driver genes in drug-drug interaction that would positively impact treatment outcome.
Predicting Post-ERCP Pancreatitis (PEP) Before and After ERCP Using Accepted and Novel Risk Factors
D. D. Ballard, S. D. Saini, J. Spaete, E. J. Wamsteker, M. J. DiMagno. Dept of Medicine, GI Div, Univ. of Michigan, Ann Arbor.
We aimed to develop predictive risk models for PEP pre- and post-ERCP using risk factors we recently reported plus accepted and investigational risk factors. We identified 5254 patients who had 8264 ERCPs at the University between 6/30/1997-5/30/2009. PEP occurred in 228 patients. We randomly selected 348 controls for a 1.5:1 proportion of controls to cases. In a masked fashion we collected data for 6 known risk factors (age, female gender, suspected sphincter of Oddi dysfunction [SOD], ≥2 pancreatic injections, pancreatic sphincterotomy, moderate/difficult cannulation), pancreatic duct stenting and 8 investigational variables (alcohol use and consumption, cigarette smoking and pack years, Charlson comorbidity score, cardiovascular disease, aspirin).
Results: In a univariate model PEP frequency increased significantly with lower mean age, women, suspected SOD, pancreatic sphincterotomy, moderate/difficult cannulation, pancreatic stent placement, lower Charlson score and decreased with diabetes and current smoking. In the multivariate model, 5 variables independently predicted PEP with C-statistic=0.70. Three existed pre-ERCP (women [OR=1.6], non-current smoking [OR=1.8], suspected SOD [OR=1.9]) and 2 resulted from ERCP (pancreatic sphincterotomy [OR=3.1], moderate/difficult cannulation [OR=3.6]). Assuming a baseline 5% PEP probability, the PEP risk in the pre-ERCP model ranged from 5-24%. The two ERCP factors in the post ERCP model independently increased the PEP risk 5-31.2% but up to 48.6% when added to factors in the pre-ERCP model.
Conclusion: The pre-ERCP model identifies patients at risk for PEP, but sphincterotomy and/or difficult cannulation at ERCP impart(s) high independent PEP risk(s) and increase(s) the risk for patients identified in the pre-ERCP model.
Evidence for Anti-Tumor Activity and Improved Bioavailability of a Novel Curcumin Analog- CDF against Pancreatic Tumors in Vivo
S. Banerjee, S. Ali, Z. Wang, A. Azmi, A. Ahmad, S. Padhye, F. H. Sarkar. Department of Pathology, Karmanos Cancer Institute, Detroit, MI.
Purpose: Pancreatic cancer (PC) patients respond very poorly to widely acclaimed chemo preventive agent curcumin because of poor systemic bioavailability and rapid excretion. The current study was aimed to assess anti-tumor as well as, the pharmacokinetics and tissue distribution of a novel curcumin analog- curcumin-difluorinated (CDF) in mice compared to curcumin. In addition, its effect on COX-2 protein, NF-κB and PGE2 were determined.
Methods: Molecular docking was assessed by standard computer modeling. PGE2 in conditioned media was done utilizing immunoassay kit, while NF-κB was done by EMSA. Serum pharmacokinetics and tissue distribution were carried out using validated HPLC with tandem mass spectrometry (LC-MS/MS) methods. For anti-tumor studies, ICR-SCID mice were orthotopically implanted with PC tumor cells and randomized into treatment groups comprising: (a) untreated control; (b) Curcumin (250 mg/kg b.wt/day orally; and (c) CDF only (250 mg/kg body weight/day orally) for 21 days.
Results: Molecular docking showed that fluorocurcumin analogs- CDF do not introduce any major steric changes which were consistent with down-regulation of NF-κB and reduced PGE2. Pharmacokinetic parameters revealed better retention and bioavailability of CDF in the pancreas tissue being 10-fold higher compared to curcumin. Average pancreatic tumor weight revealed significant reduction in tumor weight in CDF group relative to untreated control and curcumin (p<0.05).
Conclusion: Our observations clearly suggest that the bioavailability of CDF is much superior compared to curcumin, suggesting that CDF would be clinically useful in the future, and thus further development is warranted.
Omega-3 Fatty Acid Decreases Proliferation of Pancreatic Precancerous Lesions By Attenuating AKT Phosphorylation
M. Barron, Y. Ding, B. Mullapudi, K. Adrian, M. Tsao, P. Grippo. Department of Surgery, Northwestern University, Chicago, IL.
The fatty acids (FA) ω-3 and ω-6 are known to have differential roles in cancer progression. We have previously shown that diets high in ω-3 FA mitigate, while diets high in ω-6 FA promote mutant Kras-induced pancreatic precancerous lesions; however the underlying mechanisms for the differential effect are poorly understood. Thus, we examined the effect of ω-3 and ω-6 on AKT signaling because of its role in tumorigenesis as a pro-survival factor, using in vitro and in vivo modeling systems. EL-Kras mice were fed one of three isocaloric diets with varying ω-3:ω-6 FA ratios including 2.5:1 (ω-3), 1:15 (control), and 1:118 (ω-6). Pancreata of EL-Kras mice fed the high ω-3 FA diet showed a decrease in pAKT, while those fed the high ω-6 FA diet showed no change in pAKT, relative to total AKT. Human pancreatic ductal epithelial cells overexpressing ras (HPDE-ras) were treated with docohexaenoic acid (DHA), an ω-3 FA, and linoleic acid (LA), an ω-6 FA. Similar to our previously published in vivo data, DHA treatment of HPDE-ras cells resulted in increased levels of apoptosis and decreased levels of proliferation, whereas LA had little effect on either. HPDE-ras cells treated with DHA showed a decrease in pAKT, relative to total AKT, while cells treated with LA showed little change. Addition of a PI3K inhibitor exhibited no change in the relative level of pAkt in DHA-treated cells. High levels of ω-3 FA inhibit the phosphorylation of AKT, inhibit proliferation, and increase apoptosis relative to the ω-6 FA treatments in vivo and in vitro. Overall, our findings suggest that ω-3 FA inhibits the growth of pancreatic precancerous lesions and the differential effect between ω-3 and ω-6 FA may be mediated by AKT signaling.
The Association of Blood Urea Nitrogen, Creatinine and Hematocrit with In-Patient Mortality in Acute Pancreatitis
M. J. Bartel, J. T. Kurtzman, M. G. Warndorf, M. Cox, S. Robinson, P. R. Burchard, S. R. Gordon, T. B. Gardner. Section of Gastroenterology and Hepatology, Dartmouth-Hitchcock Medical Center, Lebanon, NH.
Background/Aim: Acute pancreatitis (AP) causes death in 5-10% of all patients. Blood urea nitrogen (BUN), hematocrit (Hct) and creatinine (Cr) are markers of hemoconcentration shown to predict necrosis and organ failure in AP. We aimed to compare these markers in regards to their association with mortality in AP.
Methods: We performed a retrospective chart evaluation of all patients admitted to our medical center from 1985-2009 with AP. No transfer patients were included. Cut off values for Hct (>44, >45, >47%), Cr (>1.8, >2.0, >3.0 mg/dL) and BUN (>22, >25, >33 mg/dL) at admission as well as at 24, 48, and 72 hours post-admission were evaluated for their association with mortality, organ failure and pancreatic necrosis.
Results: 701 total charts were abstracted; 488 patients presented directly to our center and were included in the analysis. 15 patients died and 42, 33, 29 and 23 respectively had organ failure at admission and 24, 48, and 72 hours post-admission. Abnormalities in BUN at all time points had the highest OR for mortality compared to Cr (Range: OR 7.1-18.1 for BUN at >33 mg/dL vs. OR 1.7-4.1 for Cr >3mg/dL) Elevations in BUN and Cr were equal at predicting the development of necrosis (Range: OR 0.6-5.1 for BUN and 1.6-6.4 for Cr). Hct was not associated with mortality, organ failure or necrosis.
Conclusion: In patients admitted for AP, elevated BUN is associated with the highest risk for mortality. Highest odds are achieved within the first 48h post admission and with a BUN >25mg/dL. Hemoconcentration in AP is best assessed with BUN rather than creatinine or hematocrit.
Inhibition of Hedgehog Signaling in a PDAC Xenograft Model Inhibits the Tumor and Its Microenvironment
D. Bausch,1 S. Fritz,2 C. Fernández-del-Castillo,1 A. L. Warshaw,1 S. P. Thayer.11Department of Surgery, MGH & HMS, Boston, MA, 2Chirurgische Universitätsklinik, Heidelberg, Germany.
Introduction: A recent shift in the understanding of the role of the hedgehog (Hh) signaling pathway in PDAC focuses on the tumor microenvironment, where its inhibition affects the stroma and increases tumor vascularity. Previous reports have also suggested that Hh signaling is proangiogenic in non-tumor models. The aim of this study was to determine the effects of Hh pathway inhibition on the tumor cells and their stroma, as well as on tumor angiogenesis in a human PDAC xenograft model.
Methods: Mice bearing subcutaneously xenografted human PDAC (n=5/group) were treated with the Hh pathway inhibitor 5E1, an antibody inhibiting SHH. After treatment for 7 days, tumor growth, viability, Hh pathway activity, mean vascular area and mesenchymal content were evaluated.
Results: Hh pathway inhibition reduced tumor growth by 74 to 118%. This effect was observed in both stromal and adenomatous components. Viable gland density, a measure of viable tumor cells, was reduced by 45-51%. The stroma, which normally comprises 43% of the tumor, was reduced by 40-65.5%. There was also an 18.2 to 36.4% reduction in mean vascular area. Targeted inhibition of the Hh pathway was verified by reduction of GL1 expression in the stroma.
Conclusion: Inhibition of Hh signaling in PDAC affects both the tumor and its stroma. In contrast to previous reports, Hh pathway inhibition reduced tumor vascularity, suggesting Hh plays a role in the maintenance or formation of tumor vasculature. Whether the reduction in the tumor growth and viability seen in the epithelium is a direct consequence of Hh pathway inhibition, or indirectly caused by its effect on the mesenchyme and vasculature, remains to be evaluated.
Co-Secretion of Acid With Zymogens in the Exocrine Pancreas: Consequences for Physiology and Pathology
N. Behrendorff, P. Thorn. School of Biomedical Sciences, University of Queensland, St. Lucia, Australia.
Release of zymogens (digestive enzyme precursors) from the exocrine pancreas occurs via exocytosis. Although it is known that secretory vesicles are acidic in other organs, little is known about the pH of the zymogen granules of the pancreas. Our current work indicates that the secretory granules are acidic and that this acidity is released upon exocytosis (Behrendorff, et al. 2010). Using the pH-sensitive extracellular fluorophore HPTS, we measured extracellular changes in pH following exocytosis. In physiological stimulation with 20 pM CCK, drops down to pH 6.5 can be observed (Behrendorff, et. al, 2009).
What happens to these protons after exocytosis? Although the downstream ductal system of the pancreas is highly buffered by the bicarbonate secretion of the duct cells, little is known about the acinar luminal pH micro-environment. Previous work had suggested that an extracellular acid environment could influence endocytosis (Freedman, Kern, Scheele, 2001) however, our work using physiological pH changes found no influence on fusion pore closure.
This release of protons appears to influence intracellular calcium signalling and negatively regulates exocytosis. When the cells are pathologically stimulated with caerulein, a large amount of protons are released which do not diffuse immediately. This extended acidification appears to damage tight junctions and therefore may compromise paracellular permeability, leading to pancreatitis.
Quality of Life After Total Pancreatectomy and Islet Autotransplant for Chronic Pancreatitis in Children
M. D. Bellin,1 M. L. Freeman,2 S. J. Schwarzenberg,1 D. M. Radosevich,2 T. B. Dunn,2 G. J. Beilman,2 D. E. R. Sutherland.2University of Minnesota, Departments of 1Pediatrics and 2Surgery.
Background: Children with chronic pancreatitis (CP) that is refractory to medical and endoscopic therapies are candidates for total pancreatectomy and islet autotransplant (TP/IAT). Initial studies suggest that most experience significant pain relief, and half are insulin independent (II) at 1 year. Objective measures of quality of life are lacking.
Aim: The primary aim was to determine if quality of life is improved in pediatric patients undergoing TP/IAT.
Methods: Nineteen consecutive children (age 5-18 years) undergoing TP/IAT from 12/06 to 12/09 at the University of Minnesota were enrolled. Health questionnaires including the Medical Outcomes Study 36-item short form (SF-36) were administered at baseline and 3 months, 6 months, and annually after surgery. HbA1c, glucose, and C-peptide levels were measured. Insulin use was recorded.
Results: Prior to surgery, mean Physical Component Score (PCS) was 30.2 and a mean Mental Component Score (MCS) was 34.1 (standardized normal =50, standard deviation =10). Both PCS and MCS significantly improved significantly after surgery. By 1 year, mean PCS was 50 and mean MCS was 45.7. Mean scores on all 8 component subscales improved. Average islet yield was 3,513 ± 2,480 islet equivalents per kilogram body weight (IE/kg) but was substantially lower in the 6/19 patients with a prior history of Puestow procedure with or without distal pancreatectomy (1,218 ± 1,189 IE/kg vs. 4,457 ± 2,145 IE/kg, p=0.01). Seven patients maintained II and another 4 required minimal insulin (≤1 injection/day, <0.25 u/kg/day), all with HbA1c levels ≤6.5%. A prior Puestow was associated with a higher likelihood of insulin dependence (p=0.04).
Conclusions: This study provides the first objective evidence that quality of life is improved after TP/IAT in selected pediatric patients with severe chronic pancreatitis. Both physical and emotional summary component scores on the SF-36, which were nearly 2 standard deviations below the population normal score before surgery, normalized after TP/IAT in these patients.
Asparagine-Linked Glycosylation of Human Chymotrypsinogen C (CTRC)
M. Bence, M. Sahin-Tóth. Department of Molecular and Cell Biology, Boston University, Boston, MA, USA.
Background and Aims: Mutations in the digestive proenzyme chymotrypsinogen C (CTRC) increase the risk for chronic pancreatitis. The mutations cause misfolding and diminished secretion, thereby decreasing the protective trypsin-degrading activity of CTRC in the pancreas. In addition, misfolding of CTRC mutants can cause endoplasmic reticulum (ER) stress, which may contribute to acinar cell damage through induction of apoptosis. The aim of the present study was to determine whether or not human CTRC undergoes Asn-linked glycosylation and to examine the role of this modification in CTRC folding and function.
Methods: Potential sites of Asn-linked glycosylation (Asn-Xaa-Ser/Thr) in human CTRC were eliminated by mutating the Asn residues to Ser individually or in combination. CTRC mutants were expressed in HEK 293T and AR42J cells. PNGase F and endoglycosidase H were utilized to determine the glycosylation state of CTRC mutants.
Results: Human CTRC contains a single Asn-linked glycan on Asn52. Mutation of Asn52 (N52S) has no effect on CTRC activity but reduces CTRC secretion about 10-fold. Decreased secretion is probably due to misfolding and degradation of unglycosylated CTRC in the ER, as overexpression of the N52S CTRC mutant elicits ER stress in AR42J acinar cells. Despite its critical role, Asn52 is not conserved in rat CTRC, which is glycosylated on Asn90. Introduction of the Asn90 glycosylation site into the N52S human CTRC mutant restored full glycosylation of CTRC but increased secretion only by 4-fold.
Conclusion: Asn-linked glycosylation of human CTRC is required for efficient folding and secretion, however, the Asn-linked glycan is unimportant for activity or inhibitor binding. Comparative studies with rat CTRC revealed that the position of the Asn-linked glycan is critical for optimal folding, and it may vary among the otherwise highly homologous mammalian CTRC sequences.
Upregulated Micro-RNA 21 Targets PDCD4 and Inhibits Proliferation in Pancreatic Cancer
I. Bhatti, V. Lee, A. Lee, J. N. Lund, R. I. Hall, C. Tufarelli, D. Lobo, M. Larvin. University of Nottingham Medical School, Royal Derby Hospital, Derby, UK.
Pancreatic cancer (PaCa) is an aggressive, late presenting malignancy, and improved survival requires earlier diagnosis, staging and new therapies. Micro RNAs (miRNA) are short non-coding RNA sequences that regulate gene expression. miRNAs are dysregulated in many cancers, including PaCa. miRNA over-expression may inhibit tumour suppressor genes, promoting carcinogenesis. The aim was to investigate expression of candidate miRNAs in cell lines and tumours, followed by functional studies.
Samples were yielded from tumour and adjacent tissue in 24 PaCa patients. Expression levels of candidate miRNAs (miR-21, -148a, -375, -181b & -151) were measured in MIA-Pa-Ca-2, HUP-T3 & PSN-1 pancreatic cancer cell lines, in matched normal and tumour samples, and control pancreatic RNA. Relative expression was quantified using real-time quantitative PCR (RT-QPCR) following reverse transcription of target and control miRNAs.
miR-21 expression was significantly upregulated (p<0.0001), but miR-148a (p<0.0001) and -375 (p<0.0001) down regulated in cell lines and tumours. miR-21 levels significantly correlated with stage/resectability (p<0.001). The tumour suppressor gene PDCD4 is thought to form a direct miR-21 target, so downregulation was effected by transfecting MIA-Pa-Ca-2 with miR-21 hairpin oligonucleotide inhibitor. PDCD4 RNA message/protein levels were measured after 48h & 72h, by RT-QPCR and Western blotting. There was significant PDCD4 message and protein upregulation (p<0.001 & p<0.02) after 48h inhibition, with reduced rates of proliferation and cell death.
This study strengthens the view that miRNAs may form useful new diagnostic and prognostic biomarkers for pancreatic cancer, and potential therapeutic targets.
LEDGF, a Novel Transcriptional Partner of the Oncogene GLI1 in Pancreatic Cancer
K. L. Bledsoe, M. E. Fernandez-Zapico. Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN.
The GLI family proteins (GLI1, GLI2, and GLI3) are vertebrate zinc finger transcription factors, which can be activated by multiple oncogenic pathways. Alterations in GLI function can lead to tumor development in different tissues (e.g. pancreas). Knowledge of the mechanisms underlying GLI1 mediated transcription could help to understand the contribution of this transcription factor to the initiation and progression of pancreatic carcinogenesis. Our preliminary data shows the functional and physical interaction with lens epithelium derived growth factor (LEDGF) is critical for the transcriptional activity of GLI1. LEDGF is a chromatin tethering factor that could functions as a transcriptional co-activator for different target genes via its N-terminal PWWP domain and a C-terminal integrase binding domain that binds to HIV1 integrase, promoting retroviral integration. Here we demonstrate that GLI1 co-immunoprecipitates with LEDGF in pancreatic cancer cells and stromal cells. LEDGF promotes GLI-mediated transcriptional activity in these cells. We also found that LEDGF synergizes with GLI1 to activate transcription of two essential modulator of tumor microenvironment, TGFβ1 and IL-6. Future experiments are aimed at further defining GLI1-LEDGF interaction and establishing the biological significance of this molecular event. In summary, these data suggest that LEDGF functions as a GLI1 transcriptional co-activator, and expand the repertoire of molecules modulating the oncogenic functions of GLI1 in pancreatic cancer.
This study was supported by the Division of Oncology Research, CA136126, Mayo Clinic Pancreatic SPORE CA102701, and Mayo Clinic Center for Cell Signaling in Gastroenterology DK084567.
Role of Non-Oxidative Ethanol Metabolism in Pancreatic Acinar Mitochondrial Dysfunction and Cell Death
D. M. Booth,1 W. Huang,1,2 A. V. Tepikin,1 B. S. Kaphalia,3 R. Sutton,2 D. N. Criddle.11Physiological Laboratory, University of Liverpool, Liverpool, UK and 2Liverpool NIHR Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK, 3Department of Pathology, The University of Texas medical Branch, Galveston, TX, USA.
Background: Although fatty acid ethyl esters (FAEEs, non-oxidative metabolites of ethanol) have been strongly implicated in pancreatitis, the relative importance of oxidative and non-oxidative ethanol metabolism in pancreatic acinar cell injury is unclear.
Methods: Isolated murine pancreatic acinar cells were assessed by confocal microscopy for changes in [Ca2+]C (Fluo4), mitochondrial function (NAD(P)H/TMRM) and cell death induced by ethanol, fat and/or ethanol metabolites.
Results: Ethanol (10 mM) with palmitoleic acid (POA; 20 μM) produced oscillatory [Ca2+]C rises (61.5%) or no effect (15.4%). When oxidative metabolism was inhibited with 4-methylpyrazole (4-MP; 100 μM), sustained [Ca2+]C elevations prevailed, associated with NAD(P)H decreases (65.5%) and mitochondrial depolarization, abolished by the FAEE synthase inhibitor 3-Benzyl-6-chloro-2-pyrone (3-BCP; 10 μM). The ethanol/POA combination increased necrosis or apoptosis significantly when 4-MP was present (4.3 and 3.7-fold, respectively), but not when MP was absent. Inhibition of FAEE synthase or hydrolase activity with 3-BCP or bis-(4-nitrophenyl) phosphate (BNPP; 200 μM), respectively, reduced ethanol/POA/4-MP-induced necrosis.
Conclusions: Low concentrations of ethanol and fatty acid induce pancreatic acinar cell necrosis when oxidative metabolism is blocked, but not if FAEE generation is blocked. These results suggest that non-oxidative rather than oxidative ethanol metabolism induces pancreatitis.
Polymorphisms in Metabolic Genes are Associated With Early Onset Pancreatic Cancer
R. Brand,1 H. Brand,2 A. Lozano,1 A. Moore,1 B. Diergaarde,2 D. Whitcomb.11Departments of Medicine and 2Human Genetics, University of Pittsburgh, Pittsburgh, PA.
The identification of risk factors for pancreatic adenocarcinoma (PC) development and understanding their impact on age of disease onset is essential for the successful implementation of early detection and prevention strategies. Early age onset pancreatic cancer (EAPC) is responsible for about 5% of deaths worldwide and accounts for up to 30% of total number of years-of-life-lost by PC. Since smoking and alcohol are well established environmental risk factors for the development of pancreatic diseases, we hypothesized that variants of metabolic genes would be more common among EAPC (≤55 years).
Methods: Nineteen (putative) functional single-nucleotide polymorphisms (SNPs) from 14 different metabolic genes were genotyped in 259 PC cases (46 EAPC) and 259 frequency-matched healthy controls. We used logistic regression adjusting for gender to estimate associations between genotypes and EAPC risk.
Results: The variants of two SNPs, rs1695 in GSTP1 (I105V; P=0.01) and rs1799930 in NAT2 (R197Q; P=0.047) were statistically significantly more common among those with EAPC than among those who were diagnosed with pancreatic cancer after age 55. Case-control comparisons showed that among participants ≤55 years the minor allele of SNP rs1695 was associated with an increased risk for EAPC [per allele odds ratios and corresponding 95% confidence interval: 2.1 (1.1-4.3)].
Conclusions: Our results suggest that polymorphism in GSTP1 and NAT2 are related with risk of EAPC. Further investigations are warranted to determine if these SNPs should be incorporated into PC risk models.
Structure-Activity Relationships of Xanthines in Ca2+ Signalling, Phosphodiesterase Inhibition and Acute Cerulein-Induced Pancreatitis
M. Cane,1 W. Huang,1 R. Mukherjee,1 A. V. Tepikin,1 R. Sutton,2 D. N. Criddle.11Department of Physiology, University of Liverpool, UK, 2NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK.
Introduction: We have shown that caffeine ameliorates experimental acute pancreatitis, potentially due to inositol trisphosphate receptor (IP3R) inhibition. A range of mono-, di- and tri-methylated xanthines has now been examined for relevant signalling effects in vitro and in vivo.
Methods: Isolated murine pancreatic acinar cells were studied by confocal microscopy to measure cytosolic Ca2+ (Fluo-4). Phosphodiesterase (PDE) inhibition was determined using a PDE assay kit (Enzo Life Sciences, UK). Acute pancreatitis was induced by seven hourly injections of cerulein (ip). Xanthines were administered hourly for seven hours from the third cerulein injection and severity was assessed by standard assays at set time points.
Results: ACh-induced Ca2+ oscillations were inhibited 41±7.5% and 86±5% by 500 μM and 2 mM caffeine respectively. Paraxanthine showed the most potent inhibition of oscillations (87±2.6% at 500 μM), whereas mono-methyl xanthines showed the least. PDE inhibition was most marked with theophylline, similar to IBMX as a positive control. Caffeine significantly ameliorated pancreatitis in a dose-dependent manner; serum amylase was reduced from 46.3±4.7 U/L to 30.9±4.4 and 26.2±3.1 U/L at 10 and 25 mg kg-1 respectively. Conversely, neither paraxanthine nor theophylline significantly ameliorated pancreatitis.
Conclusions: Paraxanthine was the most potent inhibitor of IP3-mediated Ca2+ signals, whilst theophylline was the most potent PDE inhibitor. However, neither compound ameliorated pancreatitis in vivo. The specific protective role of caffeine in ameliorating acute pancreatitis remains elusive.
BMP2 Stimulates Inflammatory Responses in Pancreatic Acinar Cells
Y. Cao,1 M. Tyler,1 W. Yang,1 C. Chao,2 M. R. Hellmich,2 T. C. Ko.1,21Department of Surgery, UTHSC-Houston, 2UTMB, Galveston, TX.
Aim: To determine whether Bone Morphogenetic Proteins (BMP) 2 has proinflammatory activity in pancreatic acinar cells.
Background: Acute pancreatitis (AP) is a necrotizing inflammatory disorder. Previously, we have shown that the BMP signaling pathway is activated in CR-induced AP and pretreatment with a BMP antagonist attenuates CR-induced AP (Yang W et al. APA Annual meeting 2009). These suggest that BMP2 may promote CR-induced inflammation.
Methods: Primary pancreatic acinar cells were isolated from Swiss Webster mice and cultured overnight in DMEM with 10% FBS. The cells were then serum-starved for 4 hrs, followed by 6 groups of treatment for 18 hrs: (1) control, (2) BMP2 (250 ng/ml), (3) TGF-β1 (1 ng/ml), (4) POAEE (palmitoleic acid ethyl ester, an alcohol metabolite, 50 μM), (5) a combination of TGF-β1 (1 ng/ml) and POAEE (50 μM), and (6) a combination of BMP2 (250 ng/ml), TGF-β1 (1 ng/ml) and POAEE (50 μM). Whole cell lysates were prepared and subjected to immunoblotting using specific antibodies against the phospho-p65 of NF-κB and Cox2, two key inflammatory mediators in AP.
Results: Compared to vehicle control, treatment with BMP2, TGF-β1 and POAEE alone increased phospho-p65 level (by 1.30, 1.72 and 1.63 fold respectively), and Cox2 expression (by 6.02, 4.36 and 1.84 fold respectively). An additive effect on Cox2 expression was observed with the combined treatment of BMP2, TGF-β1 and POAEE (by 9.41 fold increase).
Conclusion and Discussion: BMP2, TGF-β1, and POAEE cooperatively stimulate inflammatory responses in pancreatic acinar cells. These data, together with our previous in vivo findings, suggest that activated BMP signaling in AP enhances inflammation, and alcohol, the most frequent risk factor in pancreatitis, exacerbates the proinflammatory activity of BMP2.
Evaluation of Sparse Stool Collections Compared With 72-Hour Collections as an Efficacy Measure of Pancreatic Enzyme Replacement Therapy in Patients With Exocrine Pancreatic Insufficiency
S. Caras,1 D. Boyd,1 L. Zipfel,1 S. Sander-Struckmeier.21Abbott, Marietta, GA, USA; 2Abbott, Hannover, Germany.
Background: The key efficacy measure of pancreatic enzyme replacement therapy (PERT) in the treatment of exocrine pancreatic insufficiency (EPI) is the coefficient of fat absorption (CFA). Although rarely performed in clinical practice, CFA measurement involves a 72-hour stool collection, which presents a logistical challenge due to hospitalization to ensure collection requirements in clinical studies.
Aim: To demonstrate suitability of sparse stool fat evaluation as an alternative to complete 72-hour collection and measurement of CFA in subjects with EPI due to cystic fibrosis (CF).
Methods: This was a data analysis of a double-blind, randomized, placebo-controlled, two-period cross-over trial in subjects aged 7-11 years with EPI due to CF. Percent fat (PF) data were compared with CFA as a dichotomous variable, and the analysis included evaluation of sensitivity, specificity, and positive predictive value (PPV). Area under the curve (AUC) values (1 = 100% accuracy) were obtained from receiver operating characteristic plots of sensitivity vs 1-specificity. Correlation analyses were performed to evaluate the association between PF and CFA as a continuous variable and verified with scatter plots.
Results: Twelve subjects provided samples (10 in both treatment periods and two in only one period). PF values below a 30% threshold were highly predictive for CFA values ≥80%, as shown by PPV, sensitivity, and specificity values ≥0.89, with high accuracy (AUCs >0.92). Correlations between PF and CFA as a continuous variable were high.
Conclusion: Sparse stool sampling to measure PF is a possible method to determine adequate response to PERT.
Supported by Abbott, Marietta, GA
The Addition of Erlotinib to Gemcitabine and Cisplatin Does Not Appear to Improve Median Survival In Metastatic Pancreatic Cancer
P. Carlson,1 D. Fogelman,2 B. Kelley,1 W. Qiao,2 M. Javle,2 M. Overman,2 G. Varadhachary,2 R. Wolff,2 J. Abbruzzese.21Baylor College of Medicine, Houston, TX; 2Department of Gastrointestinal Medical Oncology, M.D. Anderson Cancer Center, Houston, TX.
Introduction: Metastatic pancreatic cancer carries a poor prognosis, with median survival on the order of several months. The combinations of gemcitabine and erlotinib and gemcitabine with cisplatin may be superior to single agent gemcitabine in patients with good performance (PS 0-1). We retrospectively compared outcomes of patients treated with gemcitabine, cisplatin, and erlotinib (GCE) with that of patients treated with gemcitabine and cisplatin in order to assess the potential benefit of erlotinib.
Methods: We analyzed 112 patients who presented between 2006 and 2009 with previously untreated metastatic pancreatic cancer initially treated at the M.D. Anderson cancer center with either GC or GCE. Kaplan-Meier curves were used to estimate OS. Log rank tests were used to compare OS between groups. The Cox proportional hazards regression model was used to evaluate the ability of patient prognostic variables or treatment group to predict OS.
Results: 45 patients were treated with GCE, while 67 were treated with GC. Cox analyses found no difference in overall survival (median 6.9 vs. 5.6 months, respectively, p=.29). The presence of liver metastases (p=.05), poor PS (p=.001), and elevated baseline CA 19-9 (p=.02) correlated with worse survival.
Discussion: The addition of erlotinib to gemcitabine and cisplatin does not appear to improve median or six-month survival in patients with metastatic pancreatic cancer.
Acute Pancreatitis Accelerates the Initiation and Progression to Pancreatic Ductal Adenocarcinoma in Mice Expressing Oncogenic Kras in the Nestin Cell Lineage
C. Carrière,1 A. L. Young,1 D. S. Longnecker,2 M. Korc.11Departments of Medicine, and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH and the Norris Cotton Comprehensive Cancer Center at Dartmouth Hitchcock Medical Center, Lebanon, NH; 2Department of Pathology, Dartmouth Medical School, Hanover, NH.
Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing adult progenitors is sufficient for pancreatic intraepithelial neoplasia (PanIN) formation, but does not give rise to carcinomas possibly due to the limited life-span of the mice. We have shown previously that acute pancreatitis can accelerate PanIN initiation and progression to pancreatic ductal adenocarcinoma (PDAC) in a mouse model where oncogenic Kras is targeted to all pancreatic cell types. We now report that induction of two episodes of caerulein-induced acute pancreatitis in adult mice carrying a mutated Kras allele in the Nestin cell lineage also leads to increased PanIN formation, accelerated progression from low to high grade and with low frequency, PDAC. These data indicate that 1) the Nestin cell lineage is exquisitely sensitive to oncogene activation and can give rise to PDAC; 2) acute pancreatitis can act, at least in a mouse model, as a powerful promoter of neoplastic progression to cancer.
Preoperative Determinants of the Length of Hospital Stay in Patients Undergoing Surgery for Pancreatic Cancer
V. V. Chandrabalan, D. C. McMillan, R. Carter, J. Kinsella, T. Quasim, C. Pow, J. Edwards, J. Al-Refaai, A. McPhee, N. B. Jamieson, C. J. McKay, C. R. Carter, E. J. Dickson. West of Scotland Pancreatic Unit and University of Glasgow, Royal Infirmary, Glasgow, UK.
Background: Surgery for pancreatic cancer is associated with considerable morbidity. Surgeon and patient-related factors are important in determining outcome. As a consequence, most pancreatic surgery is now carried out in specialist centres. However, patient optimisation is complex since the factors that determine postoperative morbidity are poorly understood. This study compared preoperative measures of patient comorbidity and length of hospital stay in patients undergoing pancreatic surgery.
Methods: 123 patients who underwent surgery for pancreatic cancer (pancreaticoduodenectomy = 104, trial dissection with bypass = 19) were included in the study. POSSUM Physiology Score (PPS), systemic inflammation (Glasgow Prognostic Score, mGPS) and routine blood analysis results were collected. 63 patients underwent cardiopulmonary exercise testing (CPET) and their anaerobic thresholds (AT) were recorded.
Results: The median length of hospital stay (LOS) was 17 days and 46 patients stayed ≤ 14 days. LOS was not associated with body habitus, mGPS or blood analysis. In patients who had CPET, low AT (<11 mls/kg/min) was associated with LOS greater than 14 days (p=0.007). Prolonged LOS was associated with pancreaticoduodenectomy (p=0.027) and anastomotic failure (p<0.001).
Conclusions: CPET was the only pre-operative objective measure of patient physiology that correlated significantly with prolonged LOS. LOS is a useful surrogate marker of post-operative adverse events. Although CPET did not predict specific complications, it may determine the host response to major surgery and its immediate sequelae.
The Relationship Between the Systemic Inflammatory Response, Patient Physiology and Anaerobic Threshold in Patients Undergoing Surgery for Pancreatic Cancer
V. V. Chandrabalan, D. C. McMillan, R. Carter, J. Kinsella, T. Quasim, C. Pow, J. Edwards, J. Al-Refaai, N. B. Jamieson, C. J. MacKay, R. C. Carter, E. J. Dickson. West of Scotland Pancreatic Unit and University of Glasgow, Royal Infirmary, Glasgow, UK.
Background: The presence of an ongoing systemic inflammatory response is associated with poor long term outcome in patients undergoing surgery for pancreatic cancer. However, the basis of this systemic inflammatory response is not clear and may reflect comorbid disease. The aim of the present study was to examine the relationship between the systemic inflammatory response, preoperative physiology and anaerobic threshold (AT) in patients undergoing surgery for pancreatic cancer.
Methods: 63 patients who underwent surgery for pancreatic cancer and had preoperative evaluation of their systemic inflammatory response (Glasgow Prognostic Score, mGPS), comorbidity (physiological POSSUM, PPS) and cardiopulmonary function (cardiopulmonary exercise test, CPET) were studied.
Results: The mGPS was inversely associated with a low AT (<11mls/kg/min, p=0.017) and directly associated with bilirubin (>22mg/dl, p=0.002) but not associated with age (p=0.744), sex (p=0.194), BMI (p=0.347), smoking (p=0.316) or PPS (p=0.435). PPS was directly related to smoking (p=0.010) but not to other factors. Lower AT was associated with females (p=0.010), BMI (>25, p=0.004), elevated bilirubin (p<0.001) and low haemoglobin (<12 gm/dl, p=0.001) but not with the other factors.
Conclusions: The results of the present study would suggest that, compared with the PPS, the systemic inflammatory response is more closely associated with a poor anaerobic threshold. This may suggest an underlying hypoxic basis for the presence of a systemic inflammatory response in patients prior to surgery for pancreatic cancer.
Histology and Molecular Pathology Define Clinically Relevant Phenotypes in Carcinoma of the Ampulla of Vater
D. Chang, A. Johns, C. Scarlett, M. Pajic, A. Chou, M. Jones, J. Samra, S. Henshall, E. Musgrove, R. Sutherland, A. Gill, A. Biankin. Cancer Research Program, Garvan Institute of Medical Research, Sydney, Australia.
Purpose: Individuals with adenocarcinoma of the ampulla of Vater (AAV) demonstrate a broad range of outcomes, which is not surprising since carcinomas may arise from any 1 of 3 epithelia that converge at that location. Clinical decisions concerning the aggressiveness of therapy, including the appropriateness and type of adjuvant therapy are hampered as a result.
Methods: We assessed the relationship between histological and molecular subtypes based on the expression of CDX2 and MUC1, and outcome in 68 patients who underwent operative resection for AAV.
Results: Patients with a histopathological and molecular pancreatobiliary (PB) phenotype (CDX2 negative, MUC1 positive) carcinoma segregated into a group which had an independently poor prognosis (HR=4.26, 95% CI:2.06-8.77, P<0.0001). The only other independent poor prognostic factor was the presence of LN metastases. Stratification using LN status defined 3 clinically relevant phenotypes: 1. a non-PB (intestinal) phenotype without LN metastases had an excellent outcome (5-yr survival 92%, median survival 173 months); 2. a PB phenotype and LN metastases had a poor outcome (13%, 5.7 mo) and 3. the remainder (intestinal/LN pos or PB/LN neg) had intermediate outcome (46%, 45 mo).
Conclusion: These data identify 3 distinct clinically and biologically relevant phenotypes of AAV based on a combination of histopathological and molecular criteria. This distinction may be used to refine current therapeutic strategies, interpret past clinical trials, and be used for stratification in future clinical trials to better define the need, and type of adjuvant therapy based on the biology of an individuals carcinoma.
A Novel Role for EP4 Receptor in Regulating Pancreatic Stellate Cells Function
C. Charo,1 V. Ramachandran,1 V. Holla,1 P. Yang,1 R. N. Dubois,1 R. F. Hwang,2 C. D. Logsdon.1Depts. of 1Cancer Biology, and 2Surgical Oncology, UT MD Anderson Cancer Center, Houston, TX.
Background: An important feature that PDAC and chronic pancreatitis (CP) patients share is prominent fibrosis. Pancreatic stellate cells (PSC) are the predominant source of this fibrosis. COX-2 activity is high in both CP and PDAC. This study examines the role of EP4 receptor in PGE2 stimulation of PSC collagen Ia gene expression.
Methods: COX-2 expression was measured by RT-PCR. Prostaglandins were measured by LC MS MS mass spectrometry method. PSC were treated with PGE2 in the presence and absence of the EP4 receptor antagonist ONO AE 203 in vitro followed by measurements of proliferation (MTS), migration (Boyden chamber) and invasion (invasion chamber). Expression of stroma related genes (collagen IA, fibronectin and vimentin) and EP receptors 1-4 were measured by RT-PCR. Effects on COL1A1 gene transcription were analyzed using a promoter-luciferase reporter construct.
Results: PSC expressed COX2 and secreted high levels of PGE2. PGE2 dose-dependently stimulated PSC proliferation (18±0.44% vs. control, p<0.05), migration (93±0.53%, p<0.05) and invasion (92±0.62%, p<0.05) with maximal, effects observed with 100 nM PGE2. PGE2 also stimulated PSC expression of stromal genes with COL1A1 showing the greatest increase. All four EP receptors were present in PSC. Inhibition of EP4 using ONO AE 203 resulted in a decrease in migration (82±0.75% vs. control, p<0.05) and invasion (79±1.1%, p<0.05) and almost abolished COL1A1 expression. PGE2 stimulated activity of the COL1A1 gene promoter.
Conclusion: PGE2 acts through the EP4 receptor to stimulate PSC activities including regulation of collagen gene transcription. This provides a link between COX-2 activity and pancreatic fibrosis. Therefore, targeting EP4 receptor might be of therapeutic value in pancreatic diseases including CP and PDAC.
Integrin α6β4 Contributes to HGF-Stimulated Migration and Invasion by Promoting Autocrine EGFR Signaling
M. Chen,1 Z. Cruz-Monserrate,2 T. Knifley,1 K. L. O'Connor.11Markey Cancer Center, University of Kentucky, Lexington; 2Department of Cancer Biology, University of Texas, M.D. Anderson Cancer Center, Houston.
The lethality of pancreatic cancer is due to elevated incidences of invasion and metastasis. Previous studies from our lab and others show that pro-invasive integrin α6β4 is upregulated at an early stage in pancreatic tumors and is maintained during tumor progression and, in turn, integrin α6β4 promotes chemotactic migration and invasion toward HGF. To gain insight into mechanisms governing how integrin α6β4 promotes these processes, we performed Affymetrix Gene Chip analysis on Panc-1 clones with differing integrin α6β4 expression. After statistical processing, we found 582 genes altered by integrin α6β4 signaling. From these results, we found that high integrin α6β4-expressing Panc1 clones substantially enhanced expression of genes affecting epidermal growth factor receptor (EGFR) signaling. The genes upregulated by integrin α6β4 included EGFR, EGF-like ligands amphiregulin and epiregulin, ectodomain cleavage proteases ADAMTS1 and MMP1 and hyaluronan synthase 3. The upregulation of these genes was confirmed by real-time PCR, immunoblot analysis and/or ELISA. We further tested the role of autocrine EGFR signaling by pretreating cells with EGFR blocking antibodies (LA1) or EGFR-specific protein tyrosine kinase inhibitor (PD153035) and tested HGF-stimulated chemotaxis, chemo-invasion and invasive growth in three-dimensional culture. Interestingly, these inhibitors blocked integrin α6β4-dependent HGF-stimulated chemotaxis, invasion and invasive growth. In summary, our data suggest integrin α6β4 confers a motile and invasive phenotype to pancreatic carcinoma cells, in part, by coordinately regulating expression of key elements of autocrine EGFR signaling.
Eicosapentaenoic Acid Induces Apoptosis in Pancreatic Cancer Cells
M. C. Chen, H. Takahashi, J. L. Park, H. Pham, H. A. Reber, O. J. Hines, V. L. W. Go, G. Eibl. Department of Surgery, UCLA Center of Excellence in Pancreatic Diseases, David Geffen School of Medicine at UCLA, Los Angeles, California.
Introduction: We previously reported that the n-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) exhibited antitumor activities in pancreatic cancer. However, the mechanisms of the antiproliferative effect of EPA on pancreatic cancer (PaCa) cells remain elusive.
Aim: Our aim was to demonstrate whether the antiproliferative effect of EPA on PaCa cells involves induction of apoptosis.
Methods and Results: We first confirmed that EPA (0-100 μM) dose-dependently inhibited cell proliferation in three PaCa cell lines (BxPC-3, MIA PaCa-2, and Panc-1) at 48 hrs measured by MTT and BrdU assays, with an IC50 ranging from 50 μM to 75 μM. EPA dose- and time-dependently induced apoptosis determined by Cell Death Detection ELISA with a maximal 4-5 fold induction at 16 to 24 hrs (100 μM). In both BxPC-3 and MIA PaCa-2 cells, EPA (0-100 μM) dose-dependently increased the expression of the pro-apoptotic proteins (cleavage of PARP, Caspase-3/7) by Western blot analysis. Furthermore, pre-treatment with 100 μM of the pan-caspase inhibitor zVAD-fmk reversed EPA-induced apoptosis completely in BxPC-3 cells, indicating a caspase-dependent apoptosis by EPA. Pre-incubation with EPA (25 to 100 μM) attenuated exogenous n-6 PUFA arachidonic acid (1 μM) stimulated proliferation in BxPC-3 cells, corroborating our previous in vivo finding of the antitumor efficacy of a n-3 PUFAs enriched diet.
Conclusion: Our data suggest that the growth inhibitory effects of EPA are mediated by the induction of caspase-dependent apoptosis. Dietary supplementation of n-3 PUFAs either alone or with other chemotherapeutic drugs may prove a beneficial therapeutic agent in pancreatic cancer patients.
Minnelide: Single Chemotherapeutic Agent Against Pancreatic Cancer
R. Chugh, V. Sangwan, D. Borja-Cacho, V. Dudeja, S. M. Vickers, A. K. Saluja. Department of Surgery, University of Minnesota, Minneapolis, MN.
Background: Pancreatic cancer carries one of the most dismal prognosis, with a median survival of less than six months. Previous data from our group has shown that triptolide, a diterpenoid isolated from Tripterygium wilfordii, inhibits HSP70 expression leading to cell death in pancreatic cancer cells, as well as growth and loco-regional spread of pancreatic tumors in vivo. However, limitations arising from the insolubility of triptolide in water have delayed its progression into the clinic. We therefore developed a water-soluble prodrug of triptolide named Minnelide which converts to the parent compound both in vitro and in vivo.
Aim: To evaluate the efficacy of Minnelide as a potential therapeutic agent for pancreatic cancer.
Methods: Pancreatic cancer cells of varying metastatic potential (MIA PaCa-2/S2-VP10/S2-013) were incubated with different concentrations of Minnelide or vehicle (control). Cell viability was measured using an MTT-based assay, and apoptosis assessed using annexin V staining, caspase 3/9 activation and TUNEL staining in tumor samples. HSP70 Expression was measured in lysates from Minnelide treated cells as well as orthotopic tumors from Minnelide -treated mice by western blot and immunohistochemistry in tumor samples. In vivo efficacy of the drug was measured in an orthotopic model in nude mice, which were treated with either vehicle or Minnelide at different stages of tumor formation.
Results: Minnelide decreases cell viability of three different human pancreatic cancer cell lines tested in a dose- and time-dependent manner (viability values expressed as % of control (48h post-treatment: MIA PaCa-2 22.73 ± 1.55; S2-VP10 57.3 ± 8.8; S2-013 46.6 ± 3.8). Furthermore, cell death was associated with Annexin V positivity and activation of caspase 3/9, as well as positive TUNEL staining in Minnelide-treated tumors. Minnelide repressed HSP70 protein expression at 100nM, supporting our previous results which show that HSP70 plays an important role in tumor progression. In the in vivo orthotopic mouse model, Minnelide dramatically decreased tumor size and loco-regional tumor spread, and extended survival, both in the early (7 d post-inoculation; >83% decrease in tumor volume vs. control) and late (28 d post-inoculation; >70% decrease in tumor volume vs. control) stages of pancreatic cancer.
Conclusion: Minnelide holds great promise as a novel single-agent therapeutic strategy against pancreatic cancer. It is a potent, non-toxic anti-cancer agent, causing cell death via apoptosis and tumor regression in vivo. We are currently in process of initiating Phase I clinical trials for this drug.
The Vacuolar-ATPase (v-ATPase) Confers Invasive Properties to Human Pancreatic Cancer by Modulating Mmps
C. Chung, C. Mader, P. Protiva, J. Schmitz, A. Koleske, S. Crawford, F. Gorelick. Section of Digestive Diseases, Departments of Medicine and Surgery, VA CT Healthcare System, Yale University School of Medicine; Department of Pathology, Northshore Research Institute, University of Chicago Medical School.
Background & Aims: The vacuolar ATPase (v-ATPase) is a proton transporter found on many intra-cellular organelles and the plasma membrane (PM). The v-ATPase on PMs of cultured cancer cells may contribute to their invasive properties in vitro. The role of the v-ATPase in human cancer tissue remains unclear. The aim of this study was to determine whether the expression and cellular localization of v-ATPase corresponded to the stage of human pancreatic neoplasia. Since matrix metalloproteinases (MMPs) affect pancreatic cancer invasion and metastasis, and some require an acidic environment for activation, we investigated whether v-ATPase activation affected MMP activities in vitro.
Methods: Immunohistochemistry was performed on human pancreatic cancer specimens ranging from pancreatic intraepithelial neoplasia (PanIN) lesions to pancreatic ductal adenocarcinoma (PDAC). Labeling was graded based on intensity and cellular distribution. Components of the v-ATPase and proteins involved in cellular invasion such as cortactin were evaluated by immunofluorescence microscopy in human pancreatic cancer cells. Zymography and chemotactic assays assessed MMP-2 and -9 activities and invasion capacities using chemical inhibitors and RNA interference.
Results: V-ATPase intensity increased across the range of pancreatic histology from normal ducts to PDAC, p<0.001. Low-grade PanIN lesions displayed polarized v-ATPase labeling while high-grade PanIN lesions and PDAC demonstrated more intense and diffuse v-ATPase localization. In human pancreatic cancer cells, PM v-ATPase was prominent in Panc-1 cells but not in BXPC3 cells. In Panc-1 cells, the V1E subunit localized to PMs and co-localized with cortactin, a component of the cellular invasion apparatus. Treatment with concanamycin or shRNA targeting the V1E subunit significantly reduced MMP-9 activities in Panc-1 cells. In contrast, v-ATPase inhibition in cells with detectable MMP-2 activities (Panc-1) markedly increased protease activity, p<0.001. Cellular invasion and wound migration was significantly inhibited with v-ATPase blockade only in cells with predominantly MMP-9 activity, p<0.05.
Conclusions: Human PDAC demonstrate a loss of v-ATPase polarity and increased expression that correlates with increasing invasive potential. The v-ATPase selectively modulates MMP activities that may be linked to a more invasive cancer phenotype.
Central Role of Dendritic Cells in Pancreatitis
N. E. Cieza-Rubio, A. Bedrosian, A. Nguyen, J. Ibrahim, A. Malhotra, J. Henning, M. Connolly, R.M. Barilla, P. Tan, S. Cohen, G. Miller. Department of Surgery, New York University, New York, NY.
Introduction: Chronic pancreatitis is characterized by repeated episodes of intense inflammation ultimately resulting in organ fibrosis. Dendritic cells (DC) have a central role in initiating innate and adaptive immune responses and have been recently linked to the pathogenesis of organ fibrosis. We postulated that DC have a central role in the development of chronic fibro-inflammatory disease of the pancreas.
Methods: Chronic pancreatitis was induced in C57BL/6 mice using thrice weekly injections of cerulein (q1h × 7) for 3 weeks. To expand DC in pancreatitis we used daily injections of Flt3 ligand (10ug) or adoptive transfer of bone marrow derived DC. Bone marrow derived DC were generated by culturing fresh bone marrow aspirates in GM-CSF (20 ng/ml) for 8 days. Cellular analysis was determined using flow cytometry and IHC.
Results: DC expand from <1% to 10-15% of pancreatic leukocytes in chronic pancreatitis. Overall, the total number of pancreatic DC increased nearly 100-fold in chronic pancreatitis. Pancreatic DC also undergo a relative expansion of their myeloid compartment and an increase in phenotypic maturation in pancreatitis. To determine the effects of DC in pancreatitis, DC were further recruited to the pancreas using either Flt3L or DC adoptive transfer. DC expansion resulted in a 50-fold increase in recruitment of CD45+ leukocytes to the pancreas, increased intra- and inter-lobular pancreatic fibrosis, increased acinar and islet cell destruction, and overt diabetes. DC expansion resulted in an increase in pancreatic antigen specific CD4+ T cells with strong Th2 polarization. Depletion of Th2 cells abrogated the inflammatory and fibrotic effects of DC. Effects of DC, including expansion of Th2 compartment, were further potentiated in mice deficient in MyD88 signaling.
Conclusions: DC expand in chronic pancreatitis and exacerbate inflammation and fibrosis by activating pancreas-restricted Th2 cells. MyD88 blockade potentiates DC effects.
EUS-Guided Fine Needle Injection of a Chemotherapeutic Agent Into the Pancreas
S. Cizginer,1 A. R. Kambadakone,2 M. Mino-Kenudson,3 C. E. Macfarlane,4 W. R. Brugge.11GI Unit, 2Department of Radiology, 3Department of Pathology, MGH, Boston, MA USA; 4Biocompatibles UK Ltd, Farnham, Surrey, UK.
Background: LC Bead microspheres (Biocompatibles, UK Limited) are designed for the delivery of the chemotherapeutic agent, Irinotecan, into the liver for local treatment of hypervascularised tumors.
Aim: The aim of this study was to determine the feasibility of LC Bead microspheres (with/without Irinotecan) injection under Endoscopic Ultrasound (EUS)-guidance in the pancreas of pigs.
Methods: 12 pigs were injected under EUS-guidance with a 19-gauge needle into the tail of the pancreas. The pancreas was examined histologically at day 7. Pancreatitis was graded as localized (grade 1, 2) or diffuse (grade 3). The end points of this study were feasibility, clinical tolerance, radiologic and histologic determination of pancreatitis.
Results: 12 male Yorkshire pigs underwent injection of microspheres and Irinotecan (0, 50, 100, 200, 300mg). In 10 out of 12 animals, an intra-pancreatic, hyperechoic focus with an average diameter of 2.2 cm was visible by EUS and a hypodense area in the tail of the pancreas by contrast-CT. In 2 animals no beads were seen at necropsy. The animals were monitored for 7 days (N=12) after the injection. No fever, abdominal pain, vomiting or anorexia was observed during this period. At sacrifice, histological studies demonstrated a microspheres depot with Grade 1 or 2 (localized pancreatitis) tissue reactions. The dose of the administered drug did not correlate with the grade of the reaction.
Conclusions: The EUS-guided injection of LC Bead microspheres into the swine pancreas was technically successful. Low grade localized pancreatitis was observed near the injection site.
EUS-Guided Injection of Irinotecan Into the Pancreas
S. Cizginer,1 C. Karaca,1 Y. Konuk,2 A.R. Kambadakone,3 M. Mino-Kenudson,4 C. E. Macfarlane,5 W R Brugge.1Departments of 1Gastroenterology, 2Surgery, 3Radiology, 4Pathology, Massachusetts General Hospital, Boston, MA, USA, 5Biocompatibles UK Ltd, Farnham, Surrey, United Kingdom.
Background: LC Bead microspheres (Biocompatibles, UK Limited) are designed for the delivery of the chemotherapeutic agent, Irinotecan, into hypervascularised liver tumors.
Aim: Was to determine the feasibility of LC Bead microspheres (with/without Irinotecan) injection under Endoscopic Ultrasound (EUS)-guidance in the pancreas of pigs.
Methods: Twelve pigs were injected with LC Bead microspheres under EUS-guidance with a 19-gauge needle into the tail of the pancreas. The pancreas was examined histologically for the degree (grade 1-3) and distribution of pancreatitis (after 7 days).
Results: In ten out of twelve animals, an intra-pancreatic, hyperechoic focus with an average diameter of 2.2 cm was visible by EUS and a hypodense area in the tail of the pancreas by contrast-CT. At sacrifice, in 10 out of 12 animals, a depot of beads could be located in the tail of the pancreas grossly and radiologically. In these animals, cross sectional histological studies demonstrated a microspheres depot with Grade 1 or 2 (localized pancreatitis) tissue reactions. The dose of the administered drug did not correlate with the grade of the reaction. Clinically, the animals appeared to tolerate the procedure without sequelae.
Conclusions: The EUS-guided injection of LC Bead microspheres into the swine pancreas was technically successful. Low grade localized pancreatitis was observed near the injection site.
Hedgehog Responding Cells in the Pancreatic Cancer Microenvironment
M. A. Collins, B. Skendrovich, S. Rakshit, M. Pasca di Magliano. Departments of Surgery and Cell and Developmental Biology University of Michigan, Ann Arbor, MI.
Hedgehog signaling is one of the core pathways associated with the development of pancreatic adenocarcinoma, one of the most deadly human malignancies. Sonic Hedgehog (Shh), one of the pathway ligands, is secreted by the tumor cells and acts in a paracrine manner to activate the Hedgehog (Hh) pathway in the surrounding stroma. Binding of Shh to its transmembrane receptor Patched initiates a cascade of events that culminates in the activation of Gli transcriptions factors, and expression of downstream target genes. Gli1 is one of the target genes, and its expression can be taken as a readout of pathway activation. The nature of the Hh responding cells within the pancreatic cancer microenvironment is currently poorly understood, in part because most studies have relied on transplantation models that do not have an intact tumor microenvironment. We have set out to characterize Hh responding cells in a genetically engineered mouse model of pancreatic adenocarcinoma that closely resembles the human disease. The pancreatic cancer mouse model, p48-Cre;LSL-KrasG12D, or KC, was crossed into a Hedgehog reporter mouse strain that express the bacterial gene beta-galactosidase (LacZ) under the control of the Gli1 promoter. Therefore, in the compound transgenic mice, Hh responding cells express LacZ. In KC-Gli1LacZ/+, Lac-Z positive cells accumulate during PanIN formation. Interestingly, though, only a small subset of the components of the stroma are Lac-Z positive. Further characterization has shown that immune cells, vascular components and a subset of the stromal fibroblasts are among the Gli1-expressing cells. We are currently investigating the nature of Gli1 positive and Gli1 negative cells in the stroma, and determining their relative contribution to pancreatic cancer progression. Our work provides the first characterization of Hh responding cells in the intact pancreatic cancer microenvironment; therefore, it will provide us with clues to understand how Hedgehog inhibition might affect pancreatic cancer treatment in humans.
Splenic Artery Infiltration in Pancreatic Adenocarcinoma of the Body and Tail: Is Surgical Resection Worthwhile?
S. Crippa, S. Partelli, G. Barugola, D. Tamburrino, P. Capelli, C. Bassi, P. Pederzoli, M. Falconi. Department of Surgery, University of Verona, Italy.
Background: Tumor-involvement of celiac axis and superior mesenteric artery represent criteria of unresectability (T4) in pancreatic ductal adenocarcinoma (PDA) of the head. Few studies focused on the value of splenic artery (SA) invasion (which is classified as T3 tumor) in PDA prognosis.
Aim: To evaluate prognostic factors in PDA of the boby/tail, emphasizing the role of SA infiltration.
Methods: Between 1990 and 2008, 91 patients who underwent distal pancreatectomy with standard lymphadenectomy for histologically-proven PDA of the body/tail were analyzed. Clinico-pathological prognostic factors were evaluated.
Results: Postoperative morbidity was 33% with no mortality. Overall 5-year survival rate was 27% with a median survival of 33 months. Invasion of SA was observed in 18 patients (20%). Patients with SA invasion had a significantly worse prognosis compared with those without invasion (median survival: 16 vs 37 months, p=0.037). On multivariable analysis, poorly differentiation (G3/G4), R2 resection, lymph node metastasis, adjuvant therapy, and SA invasion were independent predictors of survival.
Conclusion: Along with other well-known prognostic factors, invasion of SA is an independent predictor of poor survival in PDA of the body/tail. In the presence of SA infiltration at preoperative imaging, neoadjuvant treatment instead of upfront surgery should be considered. SA infiltration might be reclassified from T3 to T4 tumor.
The Characteristics of Organ Injury in the Early Stage of Severe Acute Pancreatitis
N. Cui, E. Zhao, D. Zhang, S. Zhang, N. Xu. Tianjin Nankai Hospital, Tianjin China.
Objective: To investigate the incidence and characteristics of organ injury in the early stage of severe acute pancreatitis (SAP).
Methods: The data of 79 cases of SAP were analyzed retrospectively in Tianjin Nankai hospital from July 2007 to December 2009. All patients were hospitalized within 24 hours of onset and the clinical information was completely consistent with diagnosis and grading standards of SAP. The acute physiology and chronic health evaluation (APACHE)-II score ≥8, and patients with pre-existing coagulopathy and/or the original organ dysfunction were excluded. Lung, kidney, heart and liver injuries, gastrointestinal dysfunction and pancreatic encephalopathy were explored respectively, as well as the relationship between organ injury and age, etiology, APACHE-II score, CT score, onset of infection, hospital stay, and mortality.
Results: Organ injury occurred in 49 cases of 79 patients with SAP (62.02%). The average age of organ injury group and non-organ injury group were (45.0±16.3)y, and (39.9±14.2)y (p<0.05). APACHE-II scores of organ injury group and non-organ injury group were 10.2±4.8 and 8.0±3.4 (p<0.05), and there were 26 and 8 cases of APACHE-II score ≥10 in two groups (p<0.05). In organ injury group, the cases of CT score ≥ 6 and < 6 points were 39 and 19, respectively (p<0.05). Single organ injury was found in 34 patients (43.03%; 34/79), double organ injury in 8 patients (10.12%; 8/79), and multiple organ injury in 7 patients (8.86%; 7/79).The incidences of organ injury were lung 36.71% (29/79), liver 21.51% (17/79), heart 12.65% (10/79), kidney 11.39% (9/79), gastrointestinal tract 7.59% (6/79) and brain 3.79% (3/79). Organ injury in the early stage of SAP mostly occurred within 72h of the onset (80%; 39/49), especially in the first 24h (25.31%; 20/79). But most pancreatic encephalopathy occurred after 72h (2.53%; 2/79). The incidences of lung injury and heart injury were 68% (17/25) and 32% (8/25) in elderly group (25 cases, age≥60y), which were much more significant increase than 22.22% (12/54) and 3.7% (2/54) over younger group (54 cases, age<60y), p<0.01. The mortalities of organ injury group and non-organ injury group were 14.29%(7/49) and 0%(0/30), p<0.001. And the hospital stay of group with organ injury was (25.7±8.3)d, longer than (12.1±4.7)d of non-organ injury, p<0.05.
Conclusions: Age, high APACHE-II score (≥10) and CT score (≥ 6) were risk factors in complicated organ injury with SAP. In the early stage of SAP, it often accompanied by lung and liver injury within 72h. However, the elderly patients were more vulnerable to predispose to lung and heart injury. Our results demonstrate that organ injury was related to the SAP, and the increase in the number of organ dysfunction results in the higher mortality.
Three-Dimensional Collagen I Promotes Gemcitabine Resistance in Pancreatic Cancer Through Increased Membrane Type 1-Matrix Metalloproteinase-Mediated HMGA2 ESSxpression
S. Dangi-Garimella,1 S. B. Krantz,2 M. R. Barron,2 M. A. Shields,1 P. J. Grippo,2 D. J. Bentrem,2,3 H. G. Munshi.1,31Division of Hematology/Oncology, Department of Medicine; 2Division of Surgical Oncology, Department of Surgery, Feinberg School of Medicin; 3The Jesse Brown V.A. Medical Center; 3The Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago.
One of the hallmarks of human pancreatic ductal adenocarcinoma (PDAC) is its pronounced type I collagen-rich fibrotic reaction. Although recent reports have shown that the fibrotic reaction can limit the efficacy of gemcitabine chemotherapy, the underlying mechanisms remain poorly understood. In this report we show that the type I collagen allows PDAC cells to override checkpoint arrest induced by gemcitabine. Cells grown in 3D collagen microenvironment have minimal Chk1 phosphorylation and continue to proliferate in the presence of gemcitabine. Collagen increases membrane type 1-matrix metalloproteinase (MT1-MMP)-dependent ERK1/2 phosphorylation, resulting in an increased HMGA2 expression, to attenuate the effect of gemcitabine. Over-expression of MT1-MMP in the collagen microenvironment increases ERK1/2 phosphorylation and HMGA2 expression, and thereby further attenuates gemcitabine-induced checkpoint arrest. MT1-MMP also lets PDAC cells to continue to proliferate in the presence of gemcitabine in a xenograft mouse model. Clinically, human tumors with increased MT1-MMP demonstrate increased HMGA2 expression. Overall, our data demonstrate that collagen upregulation of MT1-MMP contributes to gemcitabine resistance in vitro and in a xenograft mouse model, and suggest that targeting MT1-MMP could be a novel approach to sensitize pancreatic tumors to gemcitabine.
Integrated Ras Activity Promotes the Development of Chronic Pancreatitis in Mice
J. Daniluk, Y. Liu, B. Ji, C. D. Logsdon. Department of Cancer Biology, UT M.D. Anderson Cancer Center, Houston, TX.
Background and Aims: K-Ras mutations are present in the pancreas of many normal adults and at least ∼30-40% of those with chronic pancreatitis (CP). The influence of mutant K-Ras on the cell signaling pathways activated by various stimuli are unknown. We hypothesized that extrinsic stimuli would greatly increase Ras activity and lead to the development CP in animals with mutant K-Ras.
Methods: Control (WT) or LSL-KRasG12D/Ela-CreER (LSL/Cre) mice, which express endogenous levels of mutant K-Ras in pancreatic acinar cells, were treated with cerulein (50μg/kg/injection, 5 injections on day 1 and one injection/day on day 2-5 and the injections were repeated 2 weeks later), lipopolysaccharide (LPS, 10mg/kg/injection, once a week for 4 weeks) or camostat (0.1% of solid food mass). The mice were sacrificed 4 weeks after beginning of the treatment. Histology, Ras activity and pancreatitis parameters were evaluated.
Results: Despite a lack of obvious phenotype, there was higher basal Ras activity in LSL/Cre mice compared with WT mice. Treatments with cerulein, camostat or LPS induced sustained elevations of Ras activity in LSL/Cre mice but not WT mice. Furthermore, repeated treatments with cerulein or LPS induced the development of fibrosis, inflammatory cell infiltration, and loss of acinar cells mimicking CP in LSL/Cre but not WT mice.
Conclusions: In concert with our previous findings, Ras activity levels determine the development of pancreatic diseases. Various extrinsic stimuli integrated with endogenous K-Ras mutation to increase Ras activity beyond a threshold leading to chronic inflammation which initiates a feed-forward loop of pathogenic Ras activity. Understanding the mechanisms of this feed forward loop may have preventive and therapeutic values for both CP and PDAC.
NIS-CRAD as a Novel Theranostic for Pancreatic Cancer
J. Davydova,1 M. Trujillo,2 S. McDonough,2 M. Oneal,2 E. Brown,1 J. Han,1 L. Armstrong,1 S. Vickers,1 J. Morris,2 M. Yamamoto.11Department of Surgery, University of Minnesota, Minneapolis, MN; 2Division of Endocrinology, Mayo Clinic, Rochester, MN.
More than 90% of pancreatic cancer patients develop metastases which are often missed by existing imaging modalities. As adjuvant chemotherapy and radiotherapy remain ineffective for metastatic disease, it remains clear that new interventions for pancreatic cancer are required.
Oncolytic adenovirus (Ad) therapy represents a novel cancer treatment platform. In this research, we develop a new generation infectivity-enhanced Conditionally Replicative Adenovirus (CRAd) expressing the sodium-iodide symporter (NIS). NIS induces uptake of radioactive iodine by tumor cells and can be directly imaged using noninvasive radioiodine scanning. NIS-based imaging will allow noninvasive staging of the disease, but more importantly, will allow therapy with 131I, enhancing the effect of oncolytic Ads.
In our in vitro studies, NIS-expressing Ads with 5/3 capsid and adenovirus death protein (ADP) modifications significantly outperformed the wild type Ad in killing S2VP10, S2013, AsPC1, and MiaPaca2 pancreatic cancer cells. When radioiodine uptake was assessed, this was shown to be time- and dose-dependent. To study in vivo NIS uptake and the effect of combination therapy with 131I, we established A549 subcutaneous tumors in nude mice. Oncolytic Ad-treated mice showed significantly improved radiotracer uptake than mice injected with a replication-defective NIS-Ad. Survival rate was improved when it was combined with radioiodine. We are currently assessing in vivo combined therapeutic effect and imaging capability of NIS-CRAds in a pancreatic cancer model.
A multimodal therapy with high clinical potential that combines virotherapy, radiation therapy, and imaging techniques can be developed based upon this novel approach.
Analysis of Risk Factors and Management of Biliary Strictures Following Pancreaticoduodenectomy
M. Dal Molin,1 G. Butturini,1 S. Partelli,1 D. Daskalaki,1 G. C. Mansueto,2 P. Pederzoli,1 C. Bassi.11Surgical and Anaesthesiological Department, Policlinico G.B. Rossi, University of Verona; 2Department of Radiology, Policlinico G.B. Rossi, University of Verona.
Background: The incidence and course of biliary strictures after Pancreaticoduodenectomy (PD) have been poorly documented, due to few long-survivors and to a small number of studies considering complications after hospital discharge.
Methods: From 1996 to 2006, 755 consecutive patients underwent PD in our institution for periampullary diseases. We performed a retrospective analysis of a prospective database and we considered a follow-up period of 3 years, to determine the incidence and outcome of biliary strictures following PD.
Results: The incidence of postoperative biliary stenosis after PD was 2.1%. The median time to stricture was 16.5 months. No differences were observed in the incidence of biliary stenosis after surgery for benign or malignant disease, nor in the median time to stricture formation. By univariate and multivariate analyses biliary strictures were associated with a history of cholecistectomy (p=0.01). Postoperative chemoradiotherapy in patients with malignant diseases was not associated with stricture formation. Local recurrent disease was found in 4 patients with malignancies; a single patient had a biliary stenosis due to recurrency in the perihilar nodes. All the strictures were managed conservatively with percutaneous biliary balloon dilatation and stenting. Surgical reintervention or redo biliary-enteric anastomosis were not required.
Conclusion: Postoperative biliary stricture after PD is an infrequent and usually benign complication. The only significant preoperative risk factor was cholecistectomy. Strictures can be managed with conservative techniques in most patients.
Deletion of Cationic Trypsinogen or Cathepsin B Gene in Mice Doesn't Influence Caerulein Induced Activation of Nfkb in Pancreas
R. Dawra, L. Rishi Delori, A. Bekolay, R. P. Sah, A. K. Saluja. Department of Surgery, University of Minnesota, Minneapolis MN.
Supramaximal stimulation with caerulein results in pancreatic acinar cells injury and development of pancreatitis. During the initial stages, both activation of trypsinogen to trypsin and nuclear factor kappa B (NFκB) activation is observed. While intra-acinar trypsin activation is considered important for acinar cell injury, NFκB translocation to the nucleus is known to play a role in initiating an inflammatory response. Protease inhibitors like AEBSF, TLCK and E64d have been shown to inhibit both intracellular trypsin activity and NF-κB activation indicating that trypsin activation might be involved in NFκB activation. However, nonspecific effects of these inhibitors can not be totally excluded.
Aim: Of the present study was to understand the relationship between trypsin activation and NFkB activation in pancreatic acinar cells and between acinar cell injury and inflammatory response during pancreatitis.
Methods: Trypsin activation and NFκB activation in pancreas after single i.p. injection of caerulein (50μg/kg) were evaluated. Pancreatic acinar cells prepared from cationic trypsinogen or cathepsin B deleted and wild type mice were stimulated using supra-maximal concentrations (100nM) of caerulein for 30 and 60 min. Trypsin activity was measured in the cell homogenates and NFκB activation was measured in nuclear extracts.
Results: There was a significant increase in trypsin activity in pancreatic homogenates prepared from wild type mice in response to supramaximal caerulein stimulation as compared to unstimulated controls. No significant trypsin activity was observed in homogenates prepared from cationic trypsinogen or cathepsin B gene deleted mice. Furthermore, there was a significant decrease in acinar cells necrosis in cationic trypsinogen or cathepsin B deleted mice as compared to wild type mice after caerulein induced pancreatitis. However, no significant difference was observed in either IKB degradation or NFκB activation in response to supramaximal stimulation of acinar cells from trypsinogen or cathepsin B deleted mice as compared to cells from wild type mice.
Conclusion: These findings indicate trypsin activation and NFκB activation observed after supramaximal caerulein stimulation of pancreas are independent events. Activation of trypsin is only involved in acinar cells injury and the initial inflammatory response evolves in absence of active trypsin.
Cigarette Smoke Extract (CSE) But Not Nicotine Enhances Secretagogue Evoked Pancreatic Acinar Necrosis
M. J. DiMagno, A. Shah, T. Tesmer, C. C. Chen, Y. Osawa. Depts. of Med & Pharmacology, Univ. of Michigan, Ann Arbor.
Cigarette smoking is an independent risk factor for acute pancreatitis (AP). In contrast, we recently observed that current smokers have a lower risk of post-ERCP AP and others reported that the nicotinic anti-inflammatory pathway ameliorates experimental AP. We investigated whether CSE directly increases acinar injury and oxidative stress and whether nicotine in doses approximating mean and peak plasma concentrations of smokers has similar effects.
Methods: We measured cerulein (100nM) evoked cell necrosis by LDH release and oxidative stress by glutathione release in dispersed pancreatic acini of mice preincubated 30 min with CSE (0 and 1- 3%) or nicotine (0 and 100nM-1mM).
Results: In a dose response study, increasing CSE concentrations (0, 1, 2 & 3%) augmented mean cerulein evoked LDH release (%) above basal at 3h (8.8+/-0.9 vs 8.4+/-1.1 vs 12.9+/-0.8 vs 16.0+/-2.0; ANOVA=0.01). In a 3h time-course, CSE 3% vs 0% increased cerulein evoked LDH release (%) above basal at 3h (16.0+/-2.0 vs 8.8+/-0.9; P<0.005) but not 1h (3.3+/-1.1 vs 1.3+/-0.9) or 2h (9.0+/-1.1 vs 3.2+/-1.1). All nicotine concentrations except 1mM had no significant effect on cerulein evoked LDH release at 3h. High 1mM vs no nicotine, however, reduced cerulein-evoked LDH release (%) above basal at 3h (5.2+/-0.9 vs 16.6+/-1.1; P<0.0005). As a marker of oxidative stress, % glutathione release above basal was greater after 3h in the cerulein treated 3% vs 0% CSE groups (60.6+/-7.7 vs 19.1+/-6.1; P<0.005).
Conclusion: CSE but not nicotine augments secretagogue evoked acinar necrosis, possibly by increasing oxidative stress. High dose nicotine, however, reduces acinar necrosis and illustrates how components of cigarette smoke may exert opposite effects on pancreatitis by direct and possibly vascular or neural influences.
Establishing the Final Stages of Acinar Cell Differentiation Utilizing Inducible Mist1 Expression
D. DiRenzo, S. F. Konieczny. Department of Biological Sciences and the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN.
Our studies focus on Mist1, a basic helix-loop-helix transcription factor that is important for efficient acinar cell secretion, cell-cell communication and apical-basal polarity. Defective cell polarity and communication are important events in disease progression and Mist1KO mice develop precancerous PanIN lesions at an increased rate and more severe pancreatitis than wildtype mice. These results suggest that Mist1 has a key role in maintaining the final stages of acinar cell differentiation and prevention of pancreatic disease. To determine if the Mist1KO phenotype is reversible and to identify genes affected by Mist1 expression, we generated an inducible mouse line (LSL-Mist1myc) in which Mist1 expression is activated through Cre-mediated recombination. When LSL-Mist1myc mice are crossed to Mist1Cre-ER mice, Mist1myc expression is induced in acinar cells upon Tamoxifen (TM) addition. Analysis of Mist1Cre-ER/Cre-ER; LSL-Mist1myc mice confirmed that Mist1myc expression is detected as early as 6 hr post-TM with zymogen granule organization and gap-junction protein expression being restored within 72 hr. Expression array analyses showed that roughly 1,000 genes were altered in Mist1KO pancreata compared to wildtype. Furthermore, induced Mist1 expression altered the transcript levels of 650 genes within 36 hr post-TM. We conclude that Mist1 controls the end stages of acinar cell differentiation and that induction of Mist1 in adult Mist1KO mice leads to the re-organization of pancreatic tissues. Currently, we are focused on identifying Mist1 regulated genes and establishing how these genes contribute to acinar cell differentiation, function, and disease suppression.
Inhibition of SIRT1 Expression: A Novel Therapeutic Strategy for Pancreatic Cancer
V. Dudeja, R. Chugh, V. Sangwan, N. Mujumdar, D. Borja-Cacho, R. Dawra, S. Vickers, A. Saluja. Department of Surgery, University of Minnesota, Minneapolis, MN, USA.
SIRT1 is a NAD-dependent protein deacetylase that regulates life span. Due to its pro-survival function, SIRT1 may play a role in pathogenesis of cancer. The aim of this study was to evaluate the role of SIRT1 in the pathogenesis of pancreatic cancer.
Methods: SIRT1 was downregulated in pancreatic cancer cell lines (MiaPaCa-2 & S2VP10) and cholangiocarcinoma cell line KMBC by SIRT1 siRNA. Cell viability was measured by MTT assay. The effect of SIRT1 downregulation on cell cycle and annexin V staining (marker for apoptosis) was evaluated by flowcytometry. Autophagy was measured by immunoblotting for LC3 protein expression, a marker for autophagy, and by immunofluorescence by evaluating the pattern of distribution of LC3II.
Results: SIRT1 downregulation by siRNA markedly reduced the viability of both pancreatic cancer and cholangiocarcinoma cells in a time dependent fashion. Viability at 96h (% of control) expressed as mean±SEM: MiaPaCa-2: 22.6±3.2, S2VP10 42.3±7.61. SIRT1 downregulation is not associated with annexin positivity i.e. apoptosis activation or cell cycle arrest. Down-regulation of SIRT1 is associated with increase in LC3 II from as measured by western blotting. Further down-regulation of SIRT1 leads to punctuate distribution of LC3 as compared to diffuse pattern in control cells, suggesting localization of LC3 to autophagosomes and activation of autophagy. Blocking autophagy by downregulating genes essential for autophagy viz. Beclin and ATG5 provide protection against SIRT1 downregulation induced cell death.
Conclusion: Silencing of SIRT1 expression activates cell death by autophagy in pancreatobiliary cancer cells. SIRT1 holds a great promise as a potential candidate for the drug development.
Adjuvant Radiotherapy After Pancreatic Cancer Resection in the Elderly: Is it Time to Reconsider Our Treatment Strategies?
V. Dudeja,1 E. B. Habermann,1 A. Abraham,1 W. Zhong,1 S. M. Vickers,1 M. C. Posner,2 P. W. T. Pisters,3 W. B. Al-Refaie.11Univ. of Minnesota and Minneapolis VAMC, 2Univ. of Chicago, Chicago, IL, 3Univ. of Texas, MD Anderson Cancer Center.
Background: Despite evidence supporting multimodality therapy after pancreas cancer surgery, we hypothesized that older age strongly influences receipt of adjuvant radiotherapy (RT) after pancreatectomy for adenocarcinoma in the US.
Methods: Using the 1988-2006 SEER database we identified 3,880 persons, age ≥ 65 years, who underwent pancreatectomy for non-metastatic pancreatic adenocarcinoma and survived three months or more. Multivariate analyses were used to examine the impact of older age on adjuvant RT, controlling for covariates.
Results: While over 56% of pancreas cancer surgeries were performed in those ≥ 65 years, only 34% of the patients in this group (≥ 65 years) received adjuvant RT. Multivariate analyses showed that age is the strongest predictor of delivery of adjuvant RT, even stronger than tumor size, lymph node status and race. Patients aged 65 to 69 yrs were four times more likely to receive adjuvant RT than those older than 80 years (OR= 4.21, 95% CI 3.24-5.46, p<0.0001). Further stratified analysis by extent of pancreatectomy continued to show that older age predicted receiving no adjuvant RT after pancreaticoduodenectomy.
Conclusions: This large population-based appraisal showed that older age is the strongest predictor of receiving no adjuvant RT after pancreatectomy for adenocarcinoma over two decades. While comorbidities and delayed recovery after pancreatectomy remain common barriers to timely completion of adjuvant therapies, these results lend support to trials of adjuvant and neoadjuvant systemic therapy for resectable pancreatic adenocarcinoma.
AGR2 Regulates the Invasion of Pancreatic Cancer Cells Through Cathepsins B and D
L. Dumartin,1 H. J. Whiteman,1 M. E. Weeks,2 J. F. Timms,1 T. A. Brentnall,3 M. P. Bronner,4 C. Brennan,5 N. R. Lemoine,1 T. Crnogorac-Jurcevic.11Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Centre for Molecular Oncology, Institute of Cancer, London, UK; 2Cancer Proteomics Laboratory, EGA Institute for Women's Health, University College London, Gower Street, London, UK; 3Division of Gastroenterology, Department of Medicine, University of Washington, Seattle, USA; 4Department of Anatomic Pathology, Cleveland Clinic, Cleveland, USA; 5Queen Mary University of London, School of Biological and Chemical Sciences, London, UK.
Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is highly lethal due to disseminated disease at the time of presentation. Here we studied the mechanisms by which anterior gradient 2 protein (AGR2) confers metastatic capability to PDAC cells.
Methods: AGR2 expression in human pancreatic cells and tissues was analysed by immunochemistry. 2D-DIGE was performed to identify proteins regulated following AGR2 expression. Both in vitro and in vivo assays (zebrafish embryos) were used to test changes in invasion and metastases in tumour cells with modified AGR2 expression.
Results: AGR2 protein was absent in normal pancreas but was invariably induced in pancreas precursor lesions, PDACs and their metastases. AGR2 was localised in the endoplasmic reticulum, lysosomes and at the surface of cancer cells; the invasiveness of which and in vivo metastatic ability were directly correlated to levels of AGR2 expression. Moreover, higher levels of cathepsins B and D were observed following AGR2 over-expression, and both proteases were critical for the AGR2-induced increase in invasion and metastases.
Conclusion: AGR2 is induced early in pancreatic cancer development and confers its pro-metastatic activity via cathepsins B and D.
Prognostic Molecular Imaging in Pancreatic Cancer Through Analysis of the Stromal Activity
M. Erkan,1 A. A. Fingerle,2 C. Reiser-Erkan,1 Jochen Gaa,2 Helmut Friess,1 Jörg Kleeff.1Departments of 1General Surgery and 2Radiology, Technische Universität München, Munich, Germany.
Aims: To identify tumor specific stromal changes in pancreatic cancer using MRI-imaging coupled with quantitative image analysis and immunohistochemistry.
Methods: Postoperative high resolution MRI imaging of the resected specimens using standard and diffusion-weighted sequences was carried out in 6 patients with chronic pancreatitis and 12 patients with pancreatic ductal adenocarcinoma. Stromal activity was assessed by IHC and quantitative image analysis in terms of α-smooth muscle-actin, CD31, collagen and periostin expression of the stromal tissue. Prognostic value of the identified stromal factors was tested separately on a cohort of 113 pancreatic cancer patients. Survival analysis was performed using the Kaplan-Meier and Log-Rank analyses. Prognostic factors were determined in a multivariable analysis using a Cox proportional hazards model.
Results: Postoperative high resolution T2-scanning correlated accurately with the histomorphological data. Among analyzed factors only periostin expression was significantly different in pancreatic cancer tissues compared to chronic pancreatitis tissues (lower in cancer, p=0.01). Collagen expression correlated negatively (p=0.03) whereas periostin expression correlated positively (p=0.002) with the microvessel density of the tumors. Pancreatic cancer patients with higher periostin expression survived significantly longer than the ones with low expression (29.7 months vs. 15.8 months, p=0.01). High periostin expression was an independent prognostic marker among pancreatic cancer patients (p=0.017, 95% CI: 1.1159 - 4.563).
Conclusions: MRI imaging is a promising method to assess the stromal activity in pancreatic cancer, thereby providing an important tool in the differential diagnosis and in the estimation of the prognosis of pancreatic cancer patients.
Pigment Epithelium-Derived Factor Increases Neuropathy and Fibrosis in Pancreatic Cancer
M. Erkan, T. Samkharadze, C. Reiser-Erkan, E. Demir, C. Michalski, H. Friess, J. Kleeff. Dept. of Surgery, Technische Universität München, Munich, Germany.
Background: Pigment Epithelium-Derived Factor (PEDF) is a noninhibitory member of the serine protease inhibitor gene family with neuroprotective, neuroproliferative, and anti-angiogenic functions. Its role in pancreatic fibrosis and neuropathy is unknown.
Materials and Methods: The expression and localization of PEDF were assessed by quantitative RT-PCR, IHC, and quantitative image analysis and correlated with neural and microvessel density in the normal pancreas (n=20) and pancreatic cancer (n=55). Primary human pancreatic stellate cells (PSC), primary human Schwann cells, primary human endothelial cells and mouse neuroblastoma cells were used for functional experiments. The effect of hypoxia on PEDF production in cancer cell lines and immortalized pancreatic ductal epithelial cells was assessed by quantitative RT-PCR and ELISA. The effect of recombinant PEDF on PSC was assessed by immunoblot analysis.
Results: PEDF expression was homogenous in the normal pancreas. Higher expression was found in the activated stroma of pancreatic cancer. Recombinant PEDF-protein increased PSC activity by 275%, periostin and fibronection secretions by 11-fold (p<0.0001), and 5.6-fold (p<0.0001), respectively. In pancreatic cancer, compared to the normal pancreas, there was a significant increase in neuropathy (p=0.0251) and periacinar fibrosis with a concomitant loss of the fine periacinar innervation. In cancer cell lines and in human immortalized pancreatic ductal epithelial cells, hypoxia increased PEDF mRNA up to 132-fold without a surge in its protein expression. Higher expression of PEDF in cancer cells was significantly correlated with better patient survival (median survival 22 mo vs. 17 mo, p=0.043).
Conclusion: PEDF increases PSC activity, thereby contributing to the desmoplasia of pancreatic cancer. PSC over-activation likely leads to periacinar fibrosis and degeneration of the fine acinar innervation. Increased focal PEDF expression in cancer cells correlates with neuropathic changes and better patient survival.
Salmonella Typhi (ST) Infects Pancreatic Ductal Cells in vitro
B. Farfán,1 M. L. Ramírez-Aguilar,2 M. Pelaez-Luna,1 G. Gutiérrez,1 C. Alpuche-Aranda,2 G. Robles-Díaz G.11Experimental Medicine Unit-School of Medicine, UNAM; 2Microbiology and Immunology Department, School of Medicine, UNAM. México City, Mexico.
Background: ST infects the gastrointestinal tract. Some patients become asymptomatic ST carriers. Reports indicate that ST can cause pancreatitis after gaining access to the pancreas by refluxing from the biliary tract into the main pancreatic duct, in ST carriers. ST pathogenic and infection mechanisms on the pancreas are unknown.
Aim: To study ST's infection mechanisms and intracellular survival in vitro.
Methods: CAPAN2 cells were incubated with ST for 1h (MOI 1:100) or LPS (10-1000nm) for 24 hours. ST's cellular internalization was verified using phase contrast microscopy and epifluorescence. Intracellular ST survival was assessed by registering intracellular colony forming units (CFU/ml) at 3, 6 and 24 hours post-infection. ST's cell cytotoxicity was assessed by measuring DHL in culture supernatants. Cellular viability of infected cells and of those exposed to LPS was assessed with flow citometry, using anexin V and 7AAD markers.
Results:ST cellular internalization: Microscopy and epifluorescence showed ST but no inert particles inside CAPAN2 cells. Intracellular ST survival: 3.6±1×107, 2.6±0.6×107 and 3.6±0.7×107 CFU/ml from infected cells were counted at 3, 6 and 24 hours respectively. ST citoxicity: DHL activity percentage in infected and LPS exposed cell cultures was comparable at 3, 6 and 24 hours. Cellular viability:89±4.2 cells (from both, ST infected and LPS exposed cells) survived at least 24 hours.
Conclusions: ST is able to infect pancreatic ductal cells in vitro. ST and infected pancreatic cells survived 24 hours after infection. LPS did not induced apoptosis or cellular necrosis. ST's pathogenic mechanisms on the pancreas are yet to be clarified.
Biomarkers for the Diagnosis of Autoimmune Pancreatitis and in Distinguishing it From Pancreatic Cancer
K. Felix,1 O. Hauck,1 F. Fakelman,1 M. Schnölzer,2 T. Kempf,2 M. W. Büchler,1 J. Werner.11Department of General Surgery, University of Heidelberg, Heidelberg Germany; 2Protein Analysis Facility, German Cancer Research Center, Heidelberg, Germany.
Autoimmune pancreatitis (AIP) is defined by a characteristic lymphoplasmacytic infiltrate and pancreatic enlargement or mass that can mimic pancreatic cancer (PaCa) and the distinction between AIP and PaCa can be challenging. It is extremely important to diagnose AIP to choose the appropriate treatment and avoid unnecessary surgery. Mass spectrometry (MS), and 2D-DIGE has been applied to analyze serum protein alterations associated with AIP and PaCa, and to identify biomarkers indicative for the diseases. Patients sera were either equalized using peptide library beads and the elution fractions were analyzed onto CM-10 arrays or alternatively immunodepleted from the 20 most prominent serum proteins prior further 2D-DIGE analysis. The identity of the biomarkers detected was determined by a combination of protein fractionation techniques, chromatographic purification steps, PAGE, and MS. Data analysis revealed 28 proteins able to discriminate between the groups. 7 proteins were purified and characterized by peptide mapping and PSD-MALDI-TOF-MS. Apo A-II, apo-A-I, TTh were identified and found as 2-3-fold decreased in PaCa sera. Identification of proteins that emerged from our 2D-PAGE/WB and MS-based analysis revealed procarboxypeptidaseA2, α-enolase, succinyl-CoA-synthetase β-subunit, transferrin-precursor, leukocyte elastase inhibitor and GRP 78 as AIP-autoantigens. Further identification of proteins that emerged as prominent markers in AIP/PaCa is in progress. We have identified 15 serum proteins as AIP markers which can easily be assessed by simple tests of patient sera and can be used to ensure that PaCa is not misdiagnosed.
Role of Pancreatic Stellate Cells in Pancreatic Cancer: The Urokinase Plasminogen Activator System
E. Fiala-Beer, Z. Xu, P. Phillips, L. Yang, D. Goldstein, R. Pirola, J. Wilson, M. Apte. Pancreatic Research Group, University of New South Wales, Sydney, Australia.
The Urokinase Plasminogen Activator (uPA) system [comprising urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), the uPA receptor (uPAR), and the plasminogen activator inhibitors (PAI-1 and PAI-2)] has been implicated in cancer progression. However, it is not fully characterised in pancreatic cancer (PC), particularly in the stromal reaction of PC. We have shown that pancreatic stellate cells (PSCs) produce the stromal reaction of PC and facilitate PC progression by inducing tumour growth and metastasis.
Aims: To i) identify uPA system components in human PSCs (hPSCs) and in MiaPaCa2 (human PC cells); ii) assess the effect of hPSCs on uPAR expression in MiaPaCa2; and iii) assess whether hPSC-induced MiaPaCa2 proliferation is modulated by the uPA system.
Methods: i) hPSCs isolated from resected PC tissue (cancer associated hPSCs, CAhPSCs). mRNA and protein for uPA system assessed by qRT-PCR and immunoblotting respectively. ii) uPA system mRNA levels assessed in MiaPaCa2 cells after co-culture with CAhPSC. iii) MiaPaCa2 proliferation assessed after incubation with CAhPSC secretions ± amiloride (uPA inhibitor).
Results: i) CAhPSCs express mRNA and protein for uPA, tPA, uPAR, PAI-1 and PAI-2. MiaPaCa2 exhibit mRNA for all uPA components. ii) CAhPSCs induce uPAR mRNA in MiaPaCa2 (1.87 fold, p<0.05, n=4 separate CAhPSC preparations). iii) Amiloride significantly inhibits CAhPSC-induced MiaPaCa2 proliferation by 23±4.4% (p<0.05, n=3 separate CAhPSC secretions).
Conclusions: This is the first demonstration of uPA system expression in hPSCs. Notably, hPSCs induce uPAR expression in PC cells and hPSC-stimulated PC cell proliferation is mediated in part via uPA.
Implication: Modulation of the uPA system may help to disrupt stromal-tumour interactions and inhibit PC progression.
Clinical and Morphological Aspects of Autoimmune Pancreatitis Associated With Ulcerative Colitis
L. Frulloni, A. Amodio, A. Katsotourchi, T. Viaro, R. Niensted, T. Tumelero, L. Benini, M. Pellicciari, I. Vantini. Department of Medicine, University of Verona, Italy.
Background: Ulcerative colitis (UC) is associated with type II AIP and low relapse rate has been described.
Aim: To evaluate aspects of patients suffering from AIP with (AIP-UC+) or without UC (AIP-UC-).
Patients and Methods: From our prospective AIP database, we selected 64 pts (44 M, 20 F, mean age 47±17 yrs) with a definitive diagnosis of AIP in the period 2003-2010, all in follow-up. Clinical findings at onset, clinical outcome, imaging, need for surgery, serum IgG4 levels were evaluated.
Results: 12 out of 64 AIP patients (19%) had a histological diagnosis of UC (5 M and 7 F, mean age 30±12 yrs), 4 with focal and 8 with diffuse AIP. In the main features of AIP-UC- Vs AIP-UC+ the age at onset, sex, jaundice, biliary involvement, serum IgG4 higher than 135 mg/dl were significantly different between the 2 groups at clinical onset. Surgery was performed only in AIP-UC- group, but the difference was not statistically significant. On the contrary, pancreatic relapses documented at instrumental investigation (MR or CT) were similar in both groups. However, immune-suppressant drugs were used only in AIP-UC- group.
Conclusions: AIP associated with UC presents peculiar epidemiological and morphological aspects and seems to be a less aggressive form of AIP.
Evaluation of M-ANNHEIM Classification in an Italian Series of Patients Suffering From Chronic Pancreatitis
L. Frulloni, A. Amodio, A. Katsotourchi, T. Viaro, R. Niensted, T. Tumelero, M. Pellicciari, L. Benini, I. Vantini. Department of Medicine, University of Verona, Italy.
Background: M-ANNHEIM classification is the first attempt to correlate etiological, clinical, functional and radiological findings in CP.
Aim: To evaluate M-ANNHEIM classification in a consecutive series of patients suffering from CP in Italy.
Patients and Methods: Patients with a definitive, probable or borderline diagnosis of CP in the period 2007-2010 were enrolled. Data on etiological, clinical, functional and radiological aspects were achieved, following criteria previously published.
Results: 302 pts (194 M, 108 F, mean age 48.3±15.4 yrs) were enrolled. In 123 pts (41%) we found a single etiologic factor, 158 (52%) more than one and in 21 (7%) none. CP was definitive in 225 pts (75%), probable in 37 (12%) and borderline in 40 (13%). Frequency of male sex was higher in pts with definitive diagnosis of CP (70%), compared with those with probable (54%) or borderline (42%) diagnosis (p=0.002). Age at onset was different in pts with definitive (43±16 yrs), probable (39±16 yrs) or borderline (36±15 yrs) diagnosis of CP (p=0.006). Clinically, 51 pts (17%) were in stage 0, 131 (43%) in I, 75 (25%) in II, 39 (13%) in III and 6 (2%) in IV. Disease duration was different in the 5 groups (p<0.0001). 125 pts (41%) were in disease severity stage A, 97 (32%) in B, 55 (18%) in C, 22 (7%) in D and 3 (1%) in E. Disease duration was different in the 5 groups (p<0.0001). Clinical and severity stages did not differ in absence or presence (single and multiple) of the etiologic factors, except for autoimmune pancreatitis.
Conclusions: M-ANNHEIM classification is able to evaluate the clinical and severity stages of CP. The number of clinical and severity stages may be reduced for a better assessment of the disease.
A Metastatic Model of Pancreatic Cancer Using NOD/Shi-scid, IL-2Rγnull(NOG) Mice
T. Fujii,1 Y. Matsuda,1 K. Yamahatsu,1,2 H. Akiyama,1 K. Teduka,1 T. Yamamoto,1 T. Ishiwata,1 E. Uchida,2 Z. Naito.11Department of Pathology, Integrative Oncological Pathology, Nippon Medical School, Tokyo, Japan; 2Surgery for Organ and Biological Regulation, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of liver metastasis. Examining the mechanisms of liver metastasis of pancreatic cancer cells is important for understanding remote metastasis. Injection of human pancreatic cancer cells in the spleens of nude mice is one of the major liver metastasis models. However, it forms few metastatic nodules in the livers. NOG mice have severe immunodeficiency, including defects of T, B, NK and dendritic cell functions. Human colorectal and pancreatic cancer cells are reported to easily proliferate and metastasize to other organs in NOG mice. We examined whether PDAC cells metastasize to other organs in NOG mice.
Methods: Experimental liver metastases were generated in 6-week-old male NOG mice (N=3) through intrasplenic injection of 1×105 of PANC-1 cells, human PDAC cell line. Eight weeks later, the mice were sacrificed and major organs were excised, and processed for H&E staining and immunostaining for human E-cadherin.
Results: All mice exhibited multiple metastatic pancreatic cancer nodules in their livers and lungs, and the cancer cells formed large tumors in their spleens. One animal showed metastatic nodules in the kidney. Microscopically, many vascular invasions of pancreatic cancer were observed in livers, lungs and kidneys. Expression levels of E-cadherin in pancreatic cancer cells were reduced in the following order: spleens>livers>lungs.
Conclusion: Pancreatic cancer metastasis models using NOG mice showed many remote metastases in various organs. This new model may contribute to resolving the mechanisms of remote metastasis of pancreatic cancer. Reduced expression levels of E-cadherin in metastatic cancer cells may indicate that E-cadherin levels closely correlate to the remote metastasis of pancreatic cancer.
Clinical Implication of CXCL1 mRNA Expression Levels in Pancreatic Ductal Adenocarcinoma
H. Fujita,1 K. Ohuchida,1 K. Mizumoto,1,2 K. Nakata,1,3 J. Yu,1 T. Kayashima,1 H. Sakai,1 M. Onimaru,1 T. Manabe,1 T. Ohtsuka,1 M. Tanaka.11Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Kyushu University Hospital Cancer Center, Fukuoka, Japan; 3Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background and Aim: A member of CXC chemokine family, CXCL1, was reported to be secreted by several kinds of tumor cells. The aim of this study is to elucidate clinical implication of CXCL1 mRNA expression levels in resected pancreatic ductal adenocarcinoma (PDAC) tissues.
Methods: We obtained formalin-fixed paraffin embedded (FFPE) tissue samples from 94 patients with PDAC who underwent pancreatectomy at our institution from 1992 to 2007. Thirty-nine of 94 patients received gemcitabine-based adjuvant chemotherapy (AC). We measured CXCL1 mRNA expression levels by quantitative real-time RT-PCR (qRT-PCR), and investigated the association of their expressions with clinicopathological parameters and survival time using univariate and multivariate analyses. Additionally, we performed immunohistochemical analysis of a total of 40 sections from 94 PDAC patients to detect the localization of CXCL1.
Results: CXCL1 expression was detected exclusively in the PDAC cells and correlated well with the CXCL1 mRNA levels [Spearman's correlation coefficient (ρ) = 0.790; P < 0.0001]. Although there was no statistically significant correlation between CXCL1 mRNA levels and prognosis of the patients, the high CXCL1 group had a significantly longer survival in gemcitabine treated patients (P = 0.032).
Conclusions: Quantitative analyses of CXCL1 mRNA expressions using FFPE tissue samples were useful to predict the gemcitabine-sensitivity of patients with PDAC.
Novel Model of Acute Pancreatitis Severity and Outcome Prediction
S. I. Galeev,1 Y. P. Abdullaev,1 M. A. Rubtsov.11Department of Surgery, Saint-Petersburg State Medical Academy n.a. Mechnicov, Russi; 2Department of Surgery, Clinical Hospital of St. Luke, Saint-Petersburg, Russia.
Introduction: There are few relevant methods for predicting of fatal outcome and prolonged ICU stay in acute pancreatitis. According to literature data, most scoring systems (Ranson, APACHE etc.) serve as predictors of severity and outcome simultaneously.
Objectives: To detect and compare precursors associated with severe pancreatitis and unfavorable disease outcome.
Material and Methods: A retrospective study of 89 patients with acute pancreatitis in 9 year period (2000-2009) was done. 71 patients suffered from severe disease, 17 had mild pancreatitis confirmed by CT with contrast-enhancement. There were different clinical, laboratory and instrumental data analyzed for detection severity and outcome markers.
Results: Univariate analysis showed that 5 signs are useful for severe pancreatitis prediction: APACHE ≥ 8, CRP ≥ 120 mg/l, serum amylase < 250 U/l, procalcitonin ≥ 0,5 ng/ml, pleural effusion (on chest X-ray). Outcome prediction model also included 5 variables, conjoined into 3 groups: 1. early pancreatitis-specific organ failure (PO2/FiO2 < 250, creatinine > 150 mcmol/l, pressure-adjusted heart rate ≥ 10); 2. CT data (extended pancreatic necrosis); 3. infected necrosis. Outcome prediction accuracy of this model amounted to 88,3% (sensitivity-93,3%; specificity-84,2%).
Conclusion: Our data suggest: severity predictors and precursors of outcome in acute pancreatitis are different. Early outcome prediction seems to be more important clinical problem than severity prognosis. So, rational prognostic model in acute pancreatitis aimed to solve two separate problems: prediction of severity and mortality.
Targeting Pancreatic Stellate Cells (PSC) to Treat Pancreatic Cancer
V. Gonzalez-Villasana,1,5 G. N. Armaiz-Pena,1 R. Hwang,5 C. Rodriguez-Aguayo,1 A. K. Sood,2,3,4 C. Logsdon,2 G. Lopez-Berestein.1,2,41Departments of Experimental Therapeutic; 2Cancer Biolog; 3Gynecologic Oncology and the 4Center for RNAi and Non-coding RNA, and 5Surgery University of Texas, M.D. Anderson Cancer Center, Houston, TX; 5Universidad Autonoma de Nuevo Leon, San Nicolas de los Garza, Nuevo Leon, Mexico.
Background: PSCs are the major source of extracellular matrix and cytokine production in chronic pancreatitis and pancreatic cancer. The fibrosis produced by PSCs may serve as a protective barrier preventing drugs from accessing cancer cells as well as perpetuating the inflammatory response. Moreover, PSCs create an environment that stimulates cancer cell growth and invasion. We observed that PScs share many characteristics with the monocyte/macrophage lineage.
Aim: We tested whether pamidronate, a macrophage differentiation inhibitor, would inhibit PSC viability and activity.
Methods: Western blots were carried out for alpha smooth muscle actin (a marker of PSC activation), unprenylated Rap1a (a marker for pamidronate effectiveness), and TG2 (a marker for macrophage differentiation). Autophagy was determined by acridine orange staining and apoptosis by annexinV staining.
Results: Pamidronate (0.1-100 mM) inhibited PSCs alpha smooth muscle actin expression, and led to cell cycle arrest in G1, Pamidronate also induced PSCsto undergo autophagy and apoptosis. This latter effect was most likely related to the inhibition of prenylation among membrane associated proteins. Maximal effects were observed at 72 h after PSC exposure to 100μM pamidronate. These doses are comparable to those previously reported for pamidronate effects on osteoclasts and macrophages.
Conclusion: Pamidronate inhibited proliferation and inactivated PSCs in vitro through inhibition of protein prenylation, and increased cell apoptosis and cell cycle arrest in G1. These data indicate that macrophage modulators influence PSC. Pamidronate may be useful for inhibition of tumor growth and progression in pancreatic cancer.
Characterization of Unique Polycomb Repressive Complexes in Pancreatic Cancers
A. Grzenda, D. Moise, R. Urrutia. Laboratory of Chromatin Dynamics and Epigenetics, Mayo Clinic, Rochester, MN USA.
The exact mechanism of Polycomb Repressive Complexes (PRCs) in long-term gene silencing remains poorly understood. The Polycomb Repressive Complex 2 (PRC2) is comprised of four core components: EZH2, EED, SUZ12, and RBBP4/7, which is responsible for tri-methylating lysine-27 of histone 3 (H3-K27). EZH2 confers the methyltransferase ability responsible for this mark, however it lacks catalytic activity in isolation, requiring EED and SUZ12. The resultant H3-K27 mark functions to recruit Polycomb Repressive Complex 1 (PRC1), a maintenance complex that solidifies the repressed state of target genes via an equally poorly defined mechanism. As many Polycomb proteins exist in multiple isoforms, a variety of alternative PRC2 complexes have been described with unique functions in differentiation and development. Recently, PRC2 proteins have been studied for their potential oncogenic role in pancreatic cancer. EZH2 overexpression has been well characterized in other cancers and is associated with increased proliferation and malignant transformation via silencing of critical tumor suppressors. PRC2 overexpression has been implicated in silencing of p16, the most frequently lost tumor suppressor in pancreatic cancer. Here we report that: 1) Two unique PRC2 complexes are present in the pancreas. PRC2a is the canonical PRC2, whereas the newly identified PRC2b includes EZH2, EED, and RBBP4/7 but excludes Suz12; 2) Both PRC2a and PRC2b are catalytically active and are localized exclusively to nuclei; 3) Both PRC2a and PRC2b are capable of oncogenic transformation, and 4) Both PRC2a and PRC2b are overexpressed in pancreas cancer cell lines. Together, these results enhance our understanding of chromatin dynamics in pancreatic cancer and afford exciting new targets for therapeutic intervention.
Evaluating Absence of Rcan1, a Feedback Inhibitor of CN-NFAT, in Hormone-Driven, Adaptive Pancreatic Growth
G. T. Gurda, S. J. Crozier, J. A. Williams. University of Michigan School of Medicine, Ann Arbor, MI.
Introduction: Acinar proliferation is regulated by GI hormones, notably CCK. Our work showed CCK activates NFATs (Nuclear Factor of Activated T-cells) via calcineurin (CN) and alters expression of many genes, notably ∼100× increase in Regulator of Calcineurin 1 (Rcan1), a feedback inhibitor of CN-NFAT. Moreover, pharmacological inhibition of CN by FK506/CsA and acinar-specific overexpression of Rcan1, are sufficient to block pancreatic growth. Interestingly, low level of Rcan1 may be needed for cellular targeting or activity of CN. Given the dual role of Rcan1, we characterized the pancreatic phenotype and evaluated CCK-driven pancreatic growth in Rcan1 KO mice.
Methods: Pancreatic growth was induced by chow containing protease inhibitor (PI), which increases secretion of CCK. Rcan1 KO mice were on mixed C57bl/6 background (Vega et al, PNAS 2003), showed neurological deficits, but the GI-tract was not examined.
Results: The exocrine pancreas of Rcan1 KO mice appeared grossly and histologically normal. Likewise, no aberrations were noted with short-term (2hr) or long-term (2-7d) increase in CCK induced by PI. Short term PI-feeding in Rcan1 KO mice had no effect on Rcan1 mRNA by qPCR and protein by westerns vs fasting, but shows robust, >10× increase in wt littermates, comparable to outbred ICR mice. Rcan1 deficiency does not affect gene expression in early pancreatic growth, as fold change in PI-fed vs fasted mice for Lif, Nfil3, FGF21, Nedd9, cFos and Socs3 mRNA by qPCR was no different in Rcan1 KO vs wild type. Most notably, relative increase in pancreatic size, protein and DNA content, all more than doubling by day 7 of PI-feeding, appeared comparable in Rcan1 KO vs wild type. We conclude Rcan1 is not necessary for CCK-mediated pancreatic growth.
Extrapancreatic Malignancies in Pancreatic Cancer Patients - Epidemiological Observations and Possible Clinical Consequences
T. Hackert, C. Tjaden, U. Hinz, W. Hartwig, O. Strobel, MD, M. W. Büchler, J. Werner. Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg, Germany.
Introduction: Tumor syndroms are known in hereditary colorectal cancer and multiple endocrine neoplasia. Exocrine pancreatic cancer (PDAC) has not been associated with other malignancies. Aim of the study was the characterization of additional extrapancreatic tumors in PDAC patients and long-term outcome.
Methods: 1316 patients who underwent surgery for PDAC in our institution were analyzed regarding former tumor manifestations with type of tumors, epidemiological data, risk factors, PDAC tumor stage. and long-term survival.
Results: 175 PDAC patients (13.3%) had a history of 204 malignant tumors, 23 patients had 2 preceding malignancies and 3 patients showed a history of 3 cancers. Leading tumors were breast cancer (45 patients) and prostate cancer (34 patients), followed by colorectal, urothelial and gynecologic tumors (27, 26 and18 patients, respectively). No significant difference in overall survival was found between PDAC patients with and without preceding malignancies.
Conclusions: PDAC seems to be related with other tumors in a large number of patients. Especially glandular organs show a disposition for extrapancreatic tumor manifestations implying predisposing genetic disorders. Preceding malignancies did not affect the overall survival in PDAC patients. Further epidemiological and genetic investigations are necessary to clarify possible tumor syndromes and identify risk populations for the development of PDAC.
Anti-Inflammatory Activity of Apelin in Acute Pancreatitis
S. Han,1 E. W. Englander,1 C. Rastellini,1 T. C. Ko,3 M. R. Hellmich,1 C. Chao,1 J. F. Aronson,2 T. Quertermous,4 R. Kundu,4 G. H. Greeley Jr.1Dept. of 1Surgery, 2Pathology, Univ. of Texas Medical Branch, Galveston, TX; 3Dept. of Surgery, The Univ. of Texas Medical School at Houston, Houston, TX; 4Dept. of Medicine-Cardiovascular Medicine, Stanford Univ. School of Medicine, Stanford, CA.
Introduction: In rodent models of acute pancreatitis (AP) NF-κB plays a key role in activation of inflammation. Apelin is the endogenous ligand for the APJ receptor; both are expressed in the gut and pancreas. Apelin exerts an anti-inflammatory activity in the gut since nuclear accumulation of NF-κB is enhanced in apelin gene knock out (APKO) mice. The aims of this study were to define pancreatic apelin expression during cerulein-induced AP and the extent to which pancreatitis severity is enhanced in APKO mice.
Methods: Experiment 1 (Exp1): pancreatic apelin expression was measured after induction of AP in mice. Exp2: Pancreatitis severity (edema, pancreas weight (panc wt), protein nitration) and NF-κB activation were monitored in wild type (WT) and APKO mice after 4 or 7 cerulein injections. NF-κB activation was measured by EMSA.
Results: Exp1: Pancreatic apelin expression levels increased dramatically (∼4-fold) after induction of AP. Exp2: In APKO mice, gross edema, panc wt and protein nitration were accentuated (edema: WT: 2/11 vs APKO: 7/9; panc wt: WT: 261±20 vs APKO: 360±20mg, P<0.05). Nuclear NF-κB accumulation is higher (∼0.5-1.0-fold, P<0.05) in APKO mice.
Conclusions and Discussion: Pancreatic apelin expression is activated by AP. Increased apelin expression may function to exert an anti-inflammatory action since nuclear accumulation of NF-κB is enhanced in the pancreas of APKO mice. Our results imply that apelin treatment is a novel way to reduce inflammation in patients with AP.
Excellent Survival of Low-Risk Patients With Resected Pancreatic Adenocarcinoma
W. Hartwig,1 T. Hackert,1 A. Gluth,1 U. Hinz,1 F. Bergmann,2 M. W. Büchler,1 J. Werner.1Departments of 1General Surgery and 2Pathology, University of Heidelberg, Germany.
Background: Surgery is the only therapy with curative intention in potentially resectable pancreatic cancer. The present analysis aims to determine prognostic parameters in a large single-center patient cohort with resected pancreatic adenocarcinoma.
Methods: Between 10/01 and 08/09, 1070 consecutively resected patients with pancreatic adenocarcinoma were prospectively collected in an electronic database. All patients were classified according to the UICC 2010 cancer system. Parameters tested for survival prediction in univariate analysis included patient, tumor, resection, and co-morbidity characteristics. The parameters with significant results were used for multivariate survival analysis (Cox model). Identified parameters with positive or negative prognostic effect were used to define risk groups and to assess the effects on patient survival.
Results: Age, ASA-Score, CEA and CA19-9 levels, preoperative insulin-dependent diabetes, T-, N-, M-, R-, G- tumor classification, and lymph-node ratio were all significant in univariate analysis, whereas gender, BMI, insurance status, and postoperative pancreatic fistula were not. In multivariate analysis among patients with complete data, an eventually palliative resection (defined as T4, M1, or R2), Age ≥70 years, G3/4, lymph node ratio >0.2, preoperative diabetes, and CA19-9 levels >400U/ml were significantly associated with bad prognosis, whereas G1, T1/2, and R0 (revised definition) where significantly associated with a good prognosis. Using the number of these risk factors, patients were stratified into 4 risk-groups with significantly (P<0.0001) different prognosis: median and 5-year survival of 54.6 months and 43.7% in group 1 (n=82), 28.5 months and 17.0% in group 2 (n=172), 19.5 months and 6.6% in group 3 (n=433), and 10.4 months and 0% in group 4 (n=137), respectively.
Conclusions: A newly-defined risk profiling allows the classification of patients with resected pancreatic adenocarcinoma and good survival prediction. A group of patients with a median survival of >54 months and a 5-year survival of >43% has been identified.
Superiority of a Revised R-Definition to Predict Long-Term Survival in Patients With Resected Pancreatic Adenocarcinoma
W. Hartwig,1 T. Hackert,1 A. Gluth,1 U. Hinz,1 F. Bergmann,2 M. W. Büchler,1 J. Werner.1Departments of 1General Surgery and 2Pathology, University of Heidelberg, Germany.
Background: Curative resection has been demonstrated to be one of the most important factors influencing survival of patients with pancreatic carcinoma. The aim of the present study was to assess the validity of a revised R-definition first described by Esposito et al. (Ann Surg Oncol 2008).
Methods: 1070 consecutive patients undergoing resection for pancreatic adenocarcinoma at our institution between 10/2001 and 08/2009 were prospectively collected in an electronic database and were analyzed. Unlike before 07/2005 (n=363), pathologic reporting in the second period (n=707) included a standardized examination of resection specimens with inking of the resection margins, and R1 defined as a distance of the tumor from the resection margin of ≤1mm. In both periods, survival of R0, R1, and R2 resection groups were compared.
Results: Patient cohorts of both periods were comparable regarding age, gender, and tumor stage. Solely the type of resection differed, with pancreatic head resection, distal pancreatectomy, and total pancreatectomy in 77%, 15% and 8% of patients, respectively, in the first period, and 61%, 20%, and 19% in the second period (p<0.0001). With a median follow-up of 17 months, overall survival was comparable in the two periods. In the first period, survival of R2-resected patients (n=35) was significantly worse than that of R0 (n=258) and R1 resections (n=50) (p=0.0014), with no significant differences between R0 and R1 (p=0.70). Using the revised R1-definition, median and 3-year survival of the R0 resection group (n=232) were significantly better (p<0.001) compared to the R1 resection group (n=371) (31 months and 43% vs. 19.5 months and 17%, respectively). Furthermore, median survival of R2 resections (n=14) was worse than that of R1 resections (10.2 vs. 19.5 months, respectively).
Conclusions: The revised R1-definition shows excellent survival discrimination in patients with resected pancreatic adenocarcinomas and is superior to the conventional classification.
The Effects of Ethanol, Bile Acids and TNF-α on Expression of Aquaporins in Human Pancreatic Duct Cells
P. Hegyi,1 Z. Rakonczay Jr.,1 L. Kemény Jr.,1 Á. Zvara,2 L. Puskás,2 V. Venglovecz.31First Department of Medicine, University of Szeged; 2Laboratory of Functional Genomics, Biological Research Cente, 3Department of Pharmacology and Pharmacotherapy, University of Szeged, Szeged, Hungary.
Background: Bile acids, ethanol and TNF-α play an important role in the pathogenesis of acute pancreatitis which are known to affect the functions of both pancreatic acinar and ductal cells. The before mentioned toxic agents in high concentrations inhibit pancreatic ductal bicarbonate and fluid secretion. However, no information is available concerning the effects of the above mentioned factors on the regulation of Aquaporins (AQPs) expression.
Methods: Human pancreatic duct cells (CAPAN-1) were treated with ethanol (EtOH; 1, 10, 100 mM), chenodeoxycholate (CDC; 0.1, 0.3, 0.5 mM), glycochenodeoxycholate (GCDC; 0.1, 0.3, 0.5 mM) or TNF-α (0.2, 20 ng/ml) for 6, 12, 24 and 48 h and the mRNA expression of AQP isoforms (AQP1-12) was analyzed by real-time RT-PCR.
Results: All 12 AQPs were expressed in the CAPAN-1 cell line to a certain degree. AQP1, 3, 5, 6 and 11 were expressed at the highest levels while AQP2 and 4 were hardly detectable. In the CDC-treated group, the expression of AQP1, 3, 5, 6 and 11 decreased dose- and time-dependently. 24h treatment with 0.3 or 0.5 mM CDC highly downregulated the expression of AQPs (the expression rate was less than 10% vs. the controls). Administration of 0.5 mM GCDC or high concentration of EtOH (100 mM) for 24h reduced the expression of AQPs (to 20-30 and 30-40 % vs. the controls, respectively). Notably, there was a substantial recovery of the expression of AQPs at 48h in the GCDC and EtOH treated groups but not in the CDC treated group. 24h treatment with 20 ng/ml TNF-α downregulated the expression of AQP1, 5 and 6 (to 20-40% vs. the controls).
Conclusion: AQP1, 3, 5, 6 and 11 are strongly expressed in CAPAN-1 cells. Toxic agents downregulate the expression of AQPs. The role of AQPs in the pathogenesis of acute pancreatitis needs further investigation. Supported by OTKA, NKTH-TAMOP and MTA.
Cancer/Testis Antigens and the Activation of the Gametogenic Expression Program in Pancreatic Cancer
A. Heller,1 T. Giese,2 S. Gnjatic,3 K. Giorgadze,1 A. Berger,4 I. Zörnig,4 D. Jäger,4 F. Bergmann,5 M. W. Büchler,1 J. Schmidt,1 J. Werner,1 N. A. Giese.11Department of Surgery, 2Institute of Immunology; 3Ludwig Institute for Cancer Research Ltd, Memorial-Sloan Kettering Cancer Center, New York, NY, 4Department of Medical Oncology/NCT and 5Institute of Pathology, University Hospital Heidelberg, Germany.
Aim: Cells which enter tumorigenesis or gametogenesis share common features such as immortalization, migration, invasion, and immune evasion. The discovery of Cancer/Testis Antigens (CTA) - proteins which are preferentially expressed in germ cells, trophoblasts and tumors, let us to hypothesize that CTA expression in pancreatic cancer may reflect reactivation of the silenced gametogenic program, with specific CTA bearing pathogenic significance and serving as novel targets for immune therapy.
Methods: Expression of 42 CTA in different cancerous pancreatic specimens and cell lines was compared to that in gametogenic and somatic tissues. Analysis of mRNA expression (QRT-PCR) has been supplemented by the studies of protein distribution in tissues (immunohistochemistry) and by profiling of corresponding autoantibodies in patients' sera (ELISA).
Results and Conclusion: The germ-line gene expression was neither restricted to gametogenic organs nor generally silenced in somatic tissues. In pancreatic cancer, the reactivation of the gametogenic program did not appear to proceed in a linear fashion. Rather, the CTA could be grouped as: i) ubiquitous (3 genes), ii) differential (15 genes expressed in a subset of cell lines and tissues) and iii) absent. The manner of relation of the expression data to clinico-pathological parameters and to emergence of autoantibodies indicated that CTA may represent novel tools serving diagnostic and therapeutic purposes in pancreatic cancer.
Diabetes is Associated to Malignant Pancreatic Tumors and Serves as Prognostic Factor for Ductal Adenocarcinoma
R. Hennig,1 A. Kis,1 U. Hinz,1 J. Kleeff,2 J. Schmidt,1 H. Friess,2 M. W. Büchler.11Department of Surgery, University of Heidelberg, Germany; 2Department of Surgery, Klinikum rechts der Isar, TUM, Munich, Germany.
Introduction: Studies have demonstrated connections between pancreatic cancer and diabetes suggesting that the diabetes is caused by the tumor, while others have identified diabetes as a risk factor.
Material and Methods: This is a retrospective study including 489 patients undergoing surgery because of a tumor in the pancreas between 10/2001 and 9/2004.
Results: The majority of patients (n=386) was diagnosed with ductal adenocarcinoma. In 122 of these patients (31,8%) type II and in 2 patients (0,5%) type I diabetes was evident before surgery. In 68% of the patients the diabetes was diagnosed within 5 years before tumor diagnosis. An insulin dependent diabetes type II (IDDM) significantly worsened the prognosis for R0 resected patients. In the group of 39 patients with IPMN 7 patients had diabetes, none with IPMN adenoma (0/8), only 1 with borderline IPMN (1/9) but 6 with an IPMN invasive carcinoma (6/22). In addition, 35 patients had neuroendocrine tumors and 7/25 with a malignant tumor had diabetes.
Conclusion: Patients undergoing surgery because of a pancreatic tumor, about 30% of patients with malignant disease are diabetic before surgery. Patients with benign IPMNs and neuroendocrine tumors were non-diabetic and therefore, could benefit from preoperative assessment of diabetes regarding diagnosis and operative strategy. Compared to normal population, diabetes occurs more frequently in pancreatic cancer patients. In two third of patients diabetes type II is diagnosed within 5 years, implicating that this type of diabetes might be caused by the tumor and a possible early symptom. In addition, IDDM serves as an independent prognostic factor.
Xbp1 Ablation in the Adult Murine Pancreas has Long-Term Effects on Pancreatic Morphology
D. A. Hess,1 S. Humphrey,1 A-H. Lee,2 L. Glimcher,2 S. F. Konieczny.11Department of Biological Sciences and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN; 2Department of Immunology and Infectious Disease, Harvard University, Boston, MA.
The exocrine pancreas consists primarily of acinar cells that produce large volumes of zymogen granules used in digestion. This places extreme demands on the protein synthetic machinery of the cells, often necessitating the activation of stress pathways that can regulate aberrant protein folding, collectively termed the unfolded protein response (UPR). One component of the UPR, the IRE1/Xbp1 pathway, plays an important role in regulating protein folding during times of endoplasmic reticulum stress. Here we present preliminary investigations of the role of the IRE1/Xbp1 component of the UPR in maintenance of pancreatic homeostasis. We have generated a mouse line in which the Xbp1 is ablated in adult pancreatic acinar cells. Time course studies reveal an initial drop in Xbp1 target gene expression accompanied by increased expression of ER stress indicators including BiP and CHOP. Pancreata subsequently show a substantial drop in acinar hallmark gene expression including amylase and the transcription factor Mist1. Apoptotic onset was observed in large portions of the pancreas, followed by an apparent recovery in which non-apoptotic acinar cells reestablish themselves. These reestablished acinar cells show an uncharacteristic expansion of the zymogen compartment and enlargement of both nuclear and cell size that persists up to eight weeks after onset of apoptosis. These data suggest that ablation of Xbp1 results in long-term alterations in pancreatic morphology. Future studies will elucidate the mechanism of the observed acinar cell morphological changes and long-term effects of Xbp1 ablation on pancreatic disease onset.
Laparoscopic vs. Open Distal Pancreatectomy: A Case Control Comparison
M. Howard, T. Bradish, J. O'Connell, R. M. Charnley, S. A. White, J. J. French. Department of HPB Surgery, Freeman Hospital, Newcastle, UK.
Introduction: Distal pancreatectomy has traditionally been performed as an open procedure. In an attempt to improve patient outcome such procedures have recently been performed utilising a laparoscopic technique. The aim of this study was to compare patients undergoing distal pancreatectomy via either the open or laparoscopic technique.
Methods: 15 consecutive patients undergoing laparoscopic distal pancreatectomy at the Freeman Hospital HPB unit were identified for study from a prospectively managed database (group 1). These patients were matched with 15 historical patients who underwent open procedures, but would have been suitable for laparoscopic surgery (group 2). Data was collected from patients' medical records and trust databases. Parameters studied included indication, size of lesion, resection margin, mortality, morbidity, operative time, blood loss, post-operative analgesia and time to mobilisation, hospital stay, and total cost.
Results: There was no statistical difference in indication, size of lesion, margin, or splenectomy rate between each group. There was no mortality in either group. Lower morbidity was seen in the group 1 (p=0.03). Post operative analgesia and total hospital stay were both significantly less in group 1 (p=0.02). Median intra-operative blood loss was lower in group 1 (200 vs 1600mls, p=0.0047), and median post-operative mobilisation was faster both for sitting in a chair (p=0.0005) and aided walking (0.0096). Group 1 had a longer median operative time (315 vs 250 mins, p=0.0288). Total hospital stay was shorter (p=0.004) and total cost less (p=0.001) in group 1.
Conclusions: In anatomically favourable patients, and in centres supporting advanced laparoscopic techniques, laparoscopic distal pancreatectomy should be offered as the method of resection.
Experimental Acute Pancreatitis in Reg3α (PAP2) Knockout Mice and Reg1 Knockout Mice
C. Huan,1 M. Mueller,1 A. Stanek, C. M. DeMarinis,1 H. Okamoto,2 A. Sugawara,2 M. E. Zenilman.11Department of Surgery, State University of New York, Downstate Medical Center, Brooklyn, NY; 2Department of Advanced Biological Science for Regeneration, Tohoku University Graduate School of Medicine, Sendai, Japan.
Aim: To examine and compare the roles of Reg3α (PAP2) and Reg1 in acute pancreatitis.
Background: Acute pancreatitis (AP) can cause calamitous systemic inflammation as a result of incompletely understood mechanisms. A family of pancreatitis associated proteins (PAPs) derived from pancreas has been proven to be protective during the disease via inhibition of inflammation. In rodents, the PAP family consists of PAP1, PAP2 and PAP3. Regenerating islet-derived 1(Reg1) produced by acinar cells shares similar sequences and structures with PAP, but has an unclear role in AP. We have reported dissimilar induced expression profiles of individual PAPs and Reg1 in the early stage of AP, implying that they may have non-overlapping anti-inflammatory roles. To delineate the functions of Reg3α and Reg1 in AP, we studied cerulein induced AP in Reg3α knockout mice and Reg1 knockout mice.
Methods: AP is induced in mice with 7 ip injections of cerulein (50ug/kg) 1 hour apart. 16hr after the last injection, serum was collected for lipase and amylase assays, and pancreata were harvested for histologic and immunohistochemical studies.
Results: Compared to WT, edema and inflammatory infiltrates of AP in Reg3α KO mice were more severe, and serum amylase and lipase levels were significantly higher. In contrast, Reg1 KO mice showed similar inflammatory changes as well as serum amylase and lipase levels as their WT littermates in the early stage of AP.
Conclusions: Our results indicate that Reg3α is a critical protector in AP via inhibition of inflammation. This is the first discovery of a physiological role of Reg3α using Reg3α KO mice. In contrast to Reg3α, Reg1 is not a direct anti-inflammatory protein, suggesting that it has other functions in AP.
A Novel Model of Acute Alcoholic Pancreatitis by Concomitant Administration of Ethanol and Fatty Acid
W. Huang,1,3 R. Mukherjee,1,3 V. Elliott,2 D. M. Booth,3 A. Tepikin,3 D. N. Criddle,3 R. Sutton.1,21Division of Surgery and Oncology and 2NIHR Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK; 3Physiological Laboratory, University of Liverpool, Liverpool, UK.
Introduction: At present no simple, reliable model of murine alcoholic pancreatitis exists. Non-oxidative metabolites of alcohol, fatty acid ethyl esters, are synthesized in the pancreas by enzymes which conjugate ethanol (EtOH) to fatty acids and have been implicated as mediators of acute pancreatitis. We have characterised a novel acute pancreatitis model induced by concomitant administration of EtOH and fatty acid (palmitoleic acid: POA) in mice.
Methods: CD1 mice (30-35 g) were injected intraperitoneally twice one h apart, either with normal saline (50 μl), or 1.32 g/kg pure EtOH, or 1.32 g/kg EtOH + 0.25 mg/kg POA, or 1.32 g/kg EtOH + 2 mg/kg POA. The severity of acute pancreatitis was assessed at 2, 6, 12, 24 and 48 h after the first injection. The peak serum EtOH concentration was quantified by enzymatic assay.
Results: The peak serum EtOH concentration achieved was 35.6 ± 4.9 mM in the EtOH alone group. Saline, EtOH alone, or EtOH with the lower concentration of POA caused no discernible pancreatic injury. In contrast, EtOH with the higher concentration of POA induced significant increases of serum amylase (peak at 12 h), pancreatic trypsin and myeloperoxidase activity, accompanied by marked histopathological changes that peaked at 24 h. All parameters decreased at 48 h.
Conclusion: Our results indicate that concomitant administration of EtOH and POA causes acute pancreatitis. Since this novel model may parallel clinical acute alcoholic pancreatitis, it could be a useful tool to study alcohol-induced acute pancreatitis.
Stellate Cell-Derived Dickkopf-3 (DKK3) Induces PDAC Tumor Progression and Chemoresistance
H. Husted,1 T. Moore,1 D. Deng,2 V. Ramachandran,2 T. Arumugam,2 C. D. Logsdon,2 R. F. Hwang.1Depts. of 1Surgical Oncology and 2Cancer Biology, The University of Texas-M.D. Anderson Cancer Center, Houston TX.
Introduction: Dickkopf-3 (DKK3) is a Wnt inhibitor reported to be a tumor suppressor in several cancers. We previously found that DKK3 is expressed by human pancreatic stellate cells (HPSCs). In this study, we investigated the role of DKK3 in PDAC tumor progression and chemoresistance and determined the signaling mechanisms involved.
Methods: The effects of DKK3 on HPSC and PDAC proliferation, migration and invasion and on PDAC response to gemcitabine (Gem) were investigated by treatment with recombinant DKK3 or by silencing DKK3 in HPSCs or PAC using shRNA. Activation of the Wnt, MAPK, Akt, JNK and NFkB pathways by DKK3 was determined using WB and luciferase reporter assays. The in vivo role of HPSC-derived DKK3 in tumor growth and metastasis was examined in an orthotopic PAC model.
Results: DKK3 significantly increased proliferation, migration and invasion of both PDAC cells and HPSCs. Silencing of DKK3 in chemoresistant PDAC (Panc1 and HS776T) rendered cells significantly more sensitive to Gem. Likewise, overexpression of DKK3 in chemosensitive cells (L3.6pl and BxPC3) increased Gem resistance. DKK3 activated the Wnt and MAPK pathways in HPSCs but not in PDAC cells. In contrast, the NFkB pathway was strongly activated in both cell types. Tumors in mice injected with Bxpc3+HPSC-shDKK3 were significantly smaller than those in mice with Bxpc3+HPSC-shControl.
Conclusion: In contrast to its reported tumor suppressive role of DKK3 in other cancers, our data suggest that DKK3 promotes tumor progression and chemoresistance in PDAC. The signaling mechanisms activated by DKK3 are cell type-dependent. Targeting DKK3 may be an effective therapeutic approach influencing both pancreatic cancer and its microenvironment.
Characterization of CD24 Expression in Intraductal Papillary Mucinous Neoplasms and Ductal Carcinoma of the Pancreas
N. Ikenaga, K. Ohuchida, H. Fujita, K. Mizumoto, M. Tanaka. Departments of Surgery and Oncology, Kyushu University, Fukuoka, Japan.
Backgrounds & Aims: CD24 is a molecule involved in cell adhesion and tumor metastasis. The aims of this study were: (1) to evaluate the association between CD24 expression and the progression of intraductal papillary mucinous neoplasms of the pancreas (IPMNs); and (2) to investigate the association between CD24 expression in pancreatic cancer and the prognosis of patients who underwent curative pancreatectomy.
Methods: Immunohistochemical analysis of CD24 was performed for 95 IPMNs and 83 pancreatic cancers. We investigated the association between CD24 expression and the histological grade of IPMNs, clinicopathological parameters of pancreatic cancers and the survival time of pancreatic cancer patients who underwent pancreatectomy.
Results: The positive rates of CD24 expression in intraductal papillary mucinous adenoma (IPMA), borderline IPMN (IPMB), non-invasive intraductal papillary mucinous carcinoma (IPMC) and invasive IPMC were 5/24 (20%), 12/25 (48%), 10/23 (43%) and 15/23 (65%), respectively. The CD24 positive rates were significantly higher in IPMB and IPMC compared with that in IPMA (P = 0.046 and P = 0.007, respectively). The staining scores, which were determined by the percentage of stained cells and the staining intensity, were significantly higher in invasive IPMC than in non-invasive IPMC (P = 0.043). In pancreatic cancers, the higher tumor stage (P = 0.007), greater number of nodal metastasis (P = 0.021) and higher-grade of the tumors (P < 0.001) were more frequent in the CD24-positive group compared with the CD24-negative group. CD24 expression was associated with shorter survival by univariate analysis (P = 0.028) However, based on multivariate analysis, the CD24 expression was not associated with survival.
Conclusions: CD24 is involved in the progression of IPMNs and in the malignant behavior of pancreatic cancers.
Impact of S-1 for Patients With Pancreatic Cancer Relapse
K. Ishido, Y. Toyoki, D. Kudo, A. Kimura, T. Miura, S. Tsutsumi, H. Ogasawara, S. Narumi, K. Hakamada. Department of Gastrointestinal Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
Background: Since the CONKO-001 report, adjuvant chemotherapy with gemcitabine has been the standard treatment for patients who have undergone resection of pancreatic cancer in Japan. However, few reports have described the recommended regimens for patients who show disease relapse after adjuvant chemotherapy. We evaluated retrospectively the efficacy and safety of S-1, an oral fluoropyrimidine derivative, as a second-line chemotherapy for patients with disease relapse after adjuvant chemotherapy with gemcitabine.
Patients and methods: Between 1991 and 2007, 41 patients with pancreatic cancer relapsed after curative resection, and were given adjuvant chemotherapy with gemcitabine. For 20 patients (S-1 group), S-1 was administered orally twice daily after meals at a dose of 100 mg/m2/day for 14 consecutive days, followed by a 7-day rest. The other 21 patients (Control group) did not receive S-1, and continued with gemcitabine as long as possible.
Results: During a median follow-up time of 35 months, there was a significant difference in overall survival between the S-1 group and the Control group (median OS, 20.9 vs 13.7 months; p=0.031, log-rank test). Furthermore, there was also a significant inter-group difference in survival after relapse (SAR) (median SAR, 11.7 vs 6.02 months; p=0.0026, log-rank test). No increase in grade 3/4 hematologic and non-hematologic toxicity was seen in the S-1 group.
Conclusion: Second-line chemotherapy with S-1 and adjuvant chemotherapy with gemcitabine could be an efficient strategy, providing increased benefit for patients with pancreatic cancer.
CanSto Pancreas: An Information Management System for Clinico-Pathological and Biospecimen Data
A. Johns,1 G. Hammond,2 M. Thomas,1 A. Biankin,1 J. McBride.21Cancer Research Program and 2Peter Wills Bioinformatic Centre, Garvan Institute of Medical Research, Sydney Australia.
Increasingly, information management systems designed to efficiently archive and manage biospecimen and associated patient data are being utilized for translational research studies. We set out to design an application that would facilitate access and management of multiple sources of information. These included information relating to patient outcome, histopathology, biospecimen processing and management and molecular pathology.
Using our research information architecture, we have designed and delivered a comprehensive information system that manages all of the above information sources. The information system is called CanSto Pancreas and is in use across the four current sites of the Australian Pancreatic Cancer Genome Initiative (APGI).
Researchers are able to manage these information sources through a single, secure interface optimised for data entry. A powerful research query tool and a rich research programming environment are available to extract data, including patient and biospecimen accrual and survival reports.
Using CanSto Pancreas, collaborators can efficiently manage their clinical data collections and linked biospecimen, accumulation, allowing molecular pathology data to be easily correlated with clinical endpoints. The use of a common information model facilitates data sharing and management of large-scale translational research studies across different geographical sites.
Simultaneous Isolation of DNA, RNA and Proteins for Genetic, Epigenetic and Transcriptomic Analysis of Pancreatic Cancer
A. Johns,1 C. Toon,1,2 M. Jones,1 A. Mawson,1 M. Brancato,1 A. Gill,3 S. Grimmond,4 A. Biankin.1,51Cancer Research Program, Garvan Institute of Medical Research, Sydney Australia; 2Department Anatomical Pathology, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney Australia; 3Department of Anatomical Pathology, Royal North Shore Hospital, Sydney Australia; 4Queensland Centre for Medical Genomics, University of QLD, Brisbane Australia; 5Department of Upper GI Surgery, Bankstown Hospital, Sydney Australia. For the Australian Pancreatic Cancer Genome Initiative.
The availability of high-quality biospecimens has been recognised as a rate-limiting factor for translational research projects in the genomic era. High quality DNA and RNA analytes are required for projects such as the International Cancer Genome Consortium (www.icgc.org) where whole genome sequencing will be performed on 400 pancreatic cancers who have undergone surgical resection. It is crucial that these biospecimens are collected and processed under stringent conditions and accurately represent the spectrum of pancreatic cancer. For this reason there is an increasing requirement for simultaneous isolation of DNA, RNA and protein from appropriately qualified tumour tissues. We present a methodological approach using the AllPrep Extraction method (Qiagen, Hilden Germany) which allows for efficient isolation of RNA, DNA and protein from a single tissue sample, embedded in OCT Compound with pathological review. The quantification and qualification of the isolated analytes were assessed by the NanoDrop method for measuring concentration and purity, and subsequently related back to clinico-pathological parameters such as percentage neoplastic cellularity of parent tissue specimen, histological grade, age and sex.
This method ensures maximal recovery of each analyte from precious biospecimens and overcomes limitations in traditional extraction methods by eliminating the variation inherent in prepping these analytes from different samples. A methodological approach streamlines workflow, ensures comparable data and increases tumour bank sustainability.
CPET (Cardiopulmonary Exercise Testing) for Assessment of Risk Prior to Pancreaticoduodenectomy
M. A. Junejo,1 A. J. Sheen,1 D. Atkinson,2 J. Moore,2 P. Foster,2 M. J. Parker,2 A. K. Siriwardena.11Regional Hepatobiliary Unit; 2Departments of Anaesthesia and Critical Care, Manchester Royal Infirmary, Manchester UK.
Introduction: Pancreaticoduodenectomy (PD) is the standard of care for non-metastatic tumours confined to the pancreatic head and can be undertaken with low in-hospital mortality. As pancreatic cancer is predominantly a disease of later life, many with the disease have considerable cardiovascular co-morbidity and this is likely an increasing trend. CPET offers a dynamic, combined, functional test of cardiopulmonary reserve. This study evaluates CPET for cardiopulmonary risk assessment test prior to PD.
Methods: In the 28 months from 1st September 2007 to 31st December 2009, 83 patients underwent pancreaticoduodenectomy. Data were collected prospectively and the study was undertaken with institutional board approval. Patients with known co-morbidity and those aged over 65 were evaluated preoperatively with CPET according to a pre-defined protocol. Younger patients and those with minimal co-morbidity proceeded directly to surgery after staging. 44 (53%) underwent CPET prior to surgery and 41 of these underwent PD (3 patients had advanced disease at operation). Median age in CPET patients was 66 (45-78) and in the non-CPET 42 patients median age was 50 (37-74) [P<0.0001 Mann-Whitney U-test]. There was 1 death in the CPET group and 2 in the non-CPET group (overall mortality 3.6%).
Results: Anaerobic threshold (AT) did not predict cardiovascular complications, post-operative morbidity or outcome (for comparison, neither did revised cardiac risk index). However a pre-operative VE/VCO2 > 34 was strongly predictive of post-operative death with a receiver operator curve area under curve of 0.974. VO2/HR was predictive of High Dependency Unit stay (p 0.039).
Conclusion: Contemporary perioperative assessment must offer accurate risk assessment to address the high morbidity in a low mortality operation. These preliminary data suggest that CPET may offer an objective test of peri-operative risk for pancreaticoduodenectomy.
Identification of Post-Translational Modified (PTMs) Proteins in Secretin Stimulated Endoscopic Pancreas Function Test (ePFT) Collected Pancreas Fluid
V. Kadiyala,1 J. Paulo,1,2 P. A. Banks,1 H. Steen,2 D. L. Conwell.11Center for Pancreatic Disease, Division of Gastroenterology, Endoscopy and Hepatology, Brigham and Women's Hospital, Boston MA; 2Proteomics Core Lab, Children's Hospital Boston, Boston, MA; Harvard Medical School, Boston, MA.
Background: PTMs (phosphorylation, glycosylation) are crucial to protein function and as such may prove to be useful biomarkers for understanding mechanisms of disease (ie., CA 19-9). Pancreas fluid (PF) is a rich source of secreted proteins that may be biomarkers of disease mechanisms and pathophysiology. To date, PTMs of PF remain largely unexplored in patients with chronic pancreatitis (CP). The ePFT safely collects large quantities of PF that can be processed for proteomic analysis.
Aim: Characterize PTM proteins in ePFT collected PF of controls and CP.
Method: We analyzed 11 samples of secretin stimulated ePFT collected pancreas fluid: 6 CP and 5 control samples.
Sample processing and analysis for PTM:
- 1) Determine protein concentration using Pierce BCA (bicinchoninic acid) Protein Assay.
- 2) Protein extraction by trichloroacetic acid precipitation.
- 3) Protein separation by SDS-PAGE and stain protein with Coomassie dye.
- 4) Identify glycosylated proteins using Pierce Glycoprotein Stain.
- 5) Identify phosphorylated proteins through phosphoprotein enrichment and Pro-Q Diamond Phosphoprotein Gel Stain.
Results: We found no significant phosphoprotein staining in both the CP and control PF samples. We found significant, and similar, glycoprotein staining among samples in both CP and control PF samples.
- Endoscopically collected pancreas fluid can be processed for PTM analysis.
- Pancreas fluid was rich in post-translationally modified glycosylated proteins.
- Further investigation of PF for PTMs is warranted.
Metabolic Bone Disease in Chronic Pancreatitis: A Systematic Review
V. Kadiyala,1 S. Yu,1 M. Muir,2 P. A. Banks,1 D. L. Conwell.11Center for Pancreatic Disease, Division of Gastroenterology, Endoscopy and Hepatology; 2Brigham and Women's Hospital Medical Library, Brigham and Women's Hospital; Harvard Medical School, Boston MA.
Background: A number of GI diseases have been shown to be associated with low bone mineral density and metabolic bone disease (MBD). Several small reports describe a potential relationship between chronic pancreatitis (CP) and low bone density. The true association of MBD and CP has not been well explored.
Aim: Use a meta-analysis to quantitatively estimate the association between MBD and CP.
Methods: Literature searches in Embase and PubMed in collaboration with a medical librarian yielded twenty one articles. Three studies were amenable to data extraction and chosen for attempted meta-analysis; these were from Argentina (1997), Denmark (2000) and the Czech Republic (2008). A data collection form was developed and patient counts extracted. The proportion of CP patients with MBD was calculated for each study.
Results: A total of 72/145 (49.7%) CP patients in the three studies had low bone density. CP was defined by endoscopic and/or abdominal imaging criteria. Bone mineral density was measured by DEXA (Z and T score) at various skeletal sites. The lab, demographic, risk factor and comorbidity data was inconsistently reported, and not combinable for meta-analysis. A systematic review is reported: prevalence of MBD in each study was Czech Republic 39.7% [29, 51.3]; Denmark 62.1% [49.1, 73.8] and Argentina 92.9% [69.5, 99.6].
Conclusion: The heterogeneity of the studies and lack of controls prevented meta-analysis. A systematic review will be reported. A prospective observational study of CP with controls, accounting for risk factors (age, gender, smoking, exocrine insufficiency) for MBD, is needed to determine the true relationship between CP and MBD.
Expression Patterns of Cytoskeletal Proteins in Human Pancreatic Cancer Cells in a Novel Three-Dimensional Spheroidal Culture
Y. Kawamoto, Y. Matsuda, T. Ishiwata, T. Yamamoto, T. Suzuki, K. Kawahara, Z. Naito. Department of Pathology, Integrative Oncological Pathology, Nippon Medical School, Tokyo, Japan.
Introduction: Pancreatic cancer cells grown on conventional two-dimensional (2D) culture systems differ in their morphology, cell-to-cell and cell-to-matrix adhesions, and cellular differentiation from those growing in vivo. However, the cancer cells cultured in three-dimensional (3D) cell culture systems are expected to show similar patterns of cell morphology and signaling pathways to the cells in vivo. In the present study, we used the recently developed NanoCulture plate, which has a specific microsquare pattern on the bottom of the plate, allowing the formation of 3D spheroids without the need for any gel, matrix, or scaffold. Morphological changes and different expression patterns of cytoskeletal proteins of pancreatic ductal adenocarcinoma (PDAC) cells in 2D and 3D cell cultures were analyzed.
Methods: We determined the spheroid-forming ability of four PDAC cell lines (PANC-1, KLM-1, MIAPaCa-2 and PK-45H) in a NanoCulture plate. Alterations in morphology and alignment of actin filaments and α-tubulin in 2D and 3D cultures were examined using phase-contrast microscopy and immunocytochemistry.
Results: All the examined PDAC cells have grown as monolayers in 2D culture. PANC-1 and KLM-1 formed spheroids in 3D culture, while PK-45H and MIAPaCa-2 did not. Strong expression of F-actin was observed at the attached sites on the culture plate. F-actin was detected on the grids of the NanoCulture plate in PANC-1 cells, but not in PK-45H. The levels of α-tubulin expression in the PDAC cells were higher in 3D culture than in 2D culture.
Conclusion: PDAC cells showed morphological changes, spheroid formation, and alterations of expression patterns of cytoskeletal proteins in 3D culture. The 3D spheroidal culture system is a useful method for the examination of intracellular proteins with contrast phase microscopy and confocal microscopy.
A Modified 13C-Mixed Triglyceride Breath Test Detects Moderate Pancreatic Exocrine Insufficiency
J. Keller, P. Layer. Dept. Internal Medicine, Israelitic Hospital, Hamburg, Germany.
Background: The noninvasive 13C-mixed triglyceride breath test (13C-MTG-T) diagnoses severe but not moderate pancreatic exocrine insufficiency reliably.
Hypothesis: Sensitivity of the 13C-MTG-T can be increased by 1. strict limitation of physical activity to reduce variability of endogenous CO2 production; 2. correction for gastric emptying velocity; and/or 3. increase in the lipid dose, thereby exceeding lipolytic capacity and postponing 13C-MTG digestion and absorption in individuals with lower enzyme secretion.
Methods: In 10 healthy volunteers (HV) and 9 patients with suspected pancreatic disease a secretin test (S-T) was performed as reference method for evaluation of pancreatic exocrine function (collection of duodenal aspirates during 30 min basal period and 60 min stimulation with 1 U/kg*h secretin iv). Bicarbonate concentrations and activities of lipase, amylase, trypsin and chymotrypsin were measured in duodenal juice. In addition, a modified 13C-MTG-T (250 mg 13C-MTG, 26 g fat, breath samples over 8 hours) and a 13C-octanoic acid gastric emptying test were performed in all participants. Subjects remained strictly seated during breath testing.
Results: (HV vs. patients, mean±SD): S-T: Basal lipase outputs (459±650 vs. 204±314 U/min, p=0.300) and secretin-stimulated lipase outputs (850±403 vs. 617±493 U/min, p=0.274) were similar in HV and patients. In both groups, lipase outputs increased significantly in response to secretin (p≤0.016). 13C-MTG-T: 2, 4, 6 and 8 hours postprandially, cumulative 13C-exhalation was always significantly higher in HV than patients (8h-cumulative 13C-excretion: 51.7±15.4 vs. 31.3±15.3 % of dose, p=0.012). Compared with normal values, 1 HV and 5 patients had moderately decreased lipase outputs. Sensitivity of the 13C-MTG-T (parameter: 6h-cumulative 13C-exhalation <27% of dose) for detection of decreased lipase output was 100%, specificity was 92%. Gastric emptying parameters were similar in patients and controls and correction for these did not improve accuracy of the 13C-MTG-T.
Conclusions: The modified 13C-MTG-T detects moderate pancreatic exocrine insufficiency.
Glia Maturation Factor is Expressed in the Mouse Pancreas and Lung
D. Kempuraj, Z. Yuan, I. Samuel. Surgery, VAMC and the University of Iowa Carver College of Medicine, Iowa City, IA.
Glia Maturation Factor (GMF) is a novel intracellular signaling molecule first identified in neurons where it was shown to regulate stress kinase activation and cytokine production. Investigators have also shown GMF expression in renal tissues. Our laboratory was the first to demonstrate GMF expression in the rat pancreas and its induction in ligation-induced acute pancreatitis. In view of potential studies in the mouse using transgenic variants, it was important to determine GMF expression in the mouse pancreas. Here we investigated the expression of GMF in the mouse using immunoblotting and a sensitive immunohistochemistry technique: the high sensitivity ImmPRESS polymerized reporter enzyme staining system. Pancreata of mice killed 5 or 48 hr after distal pancreatic duct ligation showed progressive expression of GMF in the exocrine pancreas compared to sham-operated controls, while negative controls showed no staining. Expression of GMF in the mouse pancreas was confirmed with immunoblotting. Interestingly, GMF was also identified in the mouse lung with immunoblotting. Immunohistochemistry of mouse pancreas using specific antibody to p38 MAPK showed increased expression of p38 MAPK with 48 hr of duct ligation compared to sham, while negative controls showed no staining. We conclude that GMF is expressed in the mouse pancreas and that GMF is induced in pancreata of mice with duct ligation-induced acute pancreatitis. The parallel induction of GMF and p38 MAPK in the pancreas of pancreatic mice may indicate a signaling relationship with GMF regulating p38 MAPK induction as seen in the neurological system. The presence of GMF in pulmonary tissues may have relevance in the pathogenesis of acute lung injury that we have observed in previous studies in this experimental model.
Immunochemotherapy with 5-Fluorouracil and Interferon-α Enhances Innate and Adoptive Immune Responses Against Pancreatic Cancer
H. Khallouf,1,2 S. Serba,1 A. Märten,1,2 D. Jäger,2 J. Schmidt.11 University Hospital Heidelberg, 2National Center for Tumor Diseases, Heidelberg, Germany.
Pancreatic cancer has the poorest prognosis of all cancers urging the necessity of new therapeutic approaches. Clinical data show encouraging results for combining Interferon-α (IFN-α) immunotherapy with 5-Fluorouacil (5-FU) chemotherapy. However, the role of IFN-α in this Immunochemotherapy scheme is still elusive. Here we investigate in vivo the relevance of different immune subsets in the anti-cancer immune response mediated by IFN-α + 5-FU.
Luciferase-transfected Panc02 cells were implanted in the pancreas of C57BL/6 mice. Five days later, mice were treated with 5-FU alone, 5-FU + IFN-α or with a vehicle control. In parallel, CD4+ T cells, CD8+ T cells and NK cells were depleted by neutralizing antibodies. Depletion of CD11c+ dendritic cells (DCs) was performed using transgenic CD11c.DTR mice. On day 21, tumor volume was measured and splenic NK cells were isolated then used as effectors against Panc02 cells in a standard chromium release assay. Tumors were frozen for further immunohistochemistry analysis to evaluate the immune cell infiltration.
Our data show that treatment with IFN-α + 5-FU has significantly decreased tumor volume in comparison with control or 5-FU treatments. This decrease in tumor volume was remarkably abolished by depleting CD8+ T cells or NK cells. Furthermore, NK cells isolated from IFN-α + 5-FU treated mice showed enhanced cytotoxicity against Panc02 cells. Studying the role of CD4+ T cells and DCs besides immunohistochemistry analysis is still ongoing. To conclude, this study gives more insight about the mechanism of action of INF-α in combination with 5-FU and introduces CD8+ T cells and NK cells as main players in the observed anti-cancer immune response.
Evaluation of Cerulein (CR)-Induced Acute Pancreatitis (AP) in Mice With Micro-Computed Tomography (MCT) Imaging
S. O. Kim,1,3 C. Chao,1,3 C. Rastellini,1,3 J. Wei,2 I. Patrikeev,2 M. Motamedi,2 J. Aronson,4 G Greeley,1 T. C. Ko,6 Y. Cao,6 M. Falzon,5 B. M. Evers,7 H. Saito,7 M. R. Hellmich.1,31Department of Surgery; 2Center for Biomedical Engineering, 3The Animal Models of Pancreatic Disease Core; 4Department of Pathology; 5Department of Pharmacology and Toxicology, University of Texas, Galveston, TX; 6University of Texas Health Science Center at Houston, Houston, TX; 7Department of Surgery, University of Kentucky, Lexington, KY.
Introduction: Understanding the induction and resolution of pancreatitis in rodent models have advanced our knowledge of the pathobiology of pancreatitis in humans. Extracellular signal regulated kinase (ERK), the prototypic member of the family of mitogen-activated protein (MAP) kinases, is activated in AP. The Aims of this study were 2-fold: 1) to assess if inhibition of ERK activation would attenuate the severity of CR-induced AP, and 2) to determine if MCT could be used as a non-invasive method to evaluate AP severity in mice since radiographic assessment of AP in mice have not been described.
Methods: AP was induced in Swiss Webster mice by injecting CR (50 μg/kg) IP Q1h for 9h (n =2 mice/group). ERK was inhibited in vivo by injecting U0126 (U) (3mg/kg) IP. Fenestra LC contrast was injected by tail-vein. Mice were imaged using Inveon micro-CT scanner (Siemens) under anesthesia (1.5% isoflurane). Images were collected at 107 mm/pixel isotropic resolution and 3D reconstruction of the entire pancreas was performed. Pancreatic tissue was harvested after imaging for histology. T-tests were used to compare experimental results. Data are expressed as mean±SEM.
Results: CR increased pancreatic pERK levels (ERK activation) by immunohistochemistry, verifying AP induction. Inhibition of ERK signaling with U blocked pancreatic pERK. CR+DMSO mice (74±0.9mm3) was compared to U- treated control mice (58±11), p=NS due to small sample size; CR+DMSO pancreas measured larger compared to the pancreas of CR+U treated mice (65.3±1.7), p=0.04. Histologic findings correlated with changes in pancreatic volume measurements.
Conclusion: Inhibition of the MEK/ERK signaling axis attenuates the severity of CR-induced AP. MCT can be used as a non-invasive method for monitoring of the induction and attenuation of AP in small animals. Use of MCT should allow for each mouse to be its own control thus reducing experimental variation while at the same time reducing the number of mice/experiment.
Apigenin Induces Pancreatic Cancer Apoptosis Via p53 and PUMA
J. C. King, Q.-Y. Lu, L. Zhang, G. Li, A. Moro, H. A. Reber, V. L. W. Go, G. Eibl, O. J. Hines. UCLA Center for Excellence in Pancreatic Diseases, Los Angeles, CA.
Background: Pancreatic cancer (PC) is among the deadliest malignancies despite advances in detection, resection, and adjuvant therapy. Flavonoids such as apigenin are attractive therapeutics due to their low toxicity and varied actions. Based on previous work we hypothesized p53 may be a mediator of apigenin anticancer effects.
Methods and Results: PC cell lines (MiaPaCa-2, BxPC-3) were treated with apigenin±pifithrin (PFT; p53 DNA binding inhib) or vehicle. Apigenin induced p21 and PUMA expression by western blot that was reversible with PFT (-31.6±7.5% and -29.5±1.7%, P<0.05) indicating reversal of transcription dependent p53 function. Apigenin induced apoptosis in PC cells detected by ELISA that was not reversible with PFT (4.7±0.1 vs 4.7±0.3-fold vs DMSO, P=0.94) suggesting a transcription independent mechanism. Mitochondrial extracts showed mitochondrial translocation of p53 (+31.0% vs DMSO) and cytochrome c release as seen by immunofluorescence. Immunoprecipitation showed apigenin-induced PUMA/BclXL (-44.1%) and p53/BclXL dissociation (-71.7±6.8%, P<0.05) and p53/PUMA/Bak binding (+84.3%), supporting a p53 transcription independent model of cell death. Nude mice received orthotopic injection of PC cells and were fed control or 0.2% apigenin diet for 6 weeks. Apigenin-fed mice had smaller tumors (0.7±0.1 vs 0.9±0.1 g, P=0.058) and this correlated with tumor apigenin level (HPLC; Pearson correlation -0.65, P<0.05). TUNEL stain of tumors demonstrated increased apoptotic cells in treated mice (56.3±6.2 vs 33.2±8.3 cell/HPF; P<0.05).
Discussion: Apigenin abrogates PC growth by a transcription independent pathway of p53-mediated cell death in vitro and causes increased apoptosis yielding smaller tumors in vivo. Apigenin may be useful as an adjunct for this deadly disease.
Surgical Impact of Remnant Pancreatic Volume and Body Composition on Pancreatic Anastomotic Failure After Pancreatoduodenectomy
Y. Kirihara,1 N. Takahashi,2 Y. Hashimoto,1 G. Sclabas,1 S. Khan,1 J. Sakagami,3 M. G. Sarr,1 M. B. Farnell.11Division of Gastroenterologic and General Surgery; 2Department of Radiology; 3Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
Aim: To determine if pancreatic remnant volume, subcutaneous/visceral fat area (SVF) ratio and skeletal muscle index (SMI) calculated from pre-operative abdominal computed tomography (CT) can predict pancreatic anastomotic failure (PAF) after pancreatoduodenectomy (PD).
Background: High body mass index (BMI) is a known predictor of PAF after PD. Effect of pancreatic volume and body composition on PAF has not been studied.
Methods: 173 consecutive patients who underwent pre-operative CT and PD at Mayo Clinic between 2004 and 2009 were included. Skeletal muscle area, subcutaneous and visceral fat cross-sectional area at L3 were calculated using pre-operative CT. Muscle and fat were identified semi-automatically using the CT Hounsfield threshold method. Remnant pancreatic volume was calculated from CT as a sum of pancreatic tissue area to the left of presumed surgical cut margin over multiple slices. SMI was calculated as skeletal muscle area/height2. Pancreatic duct size and pancreatic stiffness were recorded at surgery.
Results: PAF occurred in 14%. Using univariate analysis, remnant pancreatic volume ≥34 cm3, SMI ≤38.5 cm2/m2 for women and ≤52.4 cm2/m2 for men, BMI ≥30 kg/m2, SVF ratio ≤0.8 pancreatic duct size <3 mm, and soft pancreatic texture were all predictors of PAF. Multivariate analysis identified four predictors of PAF: pancreatic volume (odds ratio (OR): 28.4, P<0.001), SMI (OR 14.0, P<0.001), BMI (OR 7.5, P<0.05) and SVF ratio (OR 6.3, P<0.001).
Conclusion: Larger remnant pancreatic volume, lower SMI, and lower SVF ratio measured on preoperative CT were significant predictors of PAF after PD.
Targeting the Synergistic Crosstalk Between Insulin and G Protein-Coupled Receptor Signaling by Metformin Potently Inhibits Pancreatic Cancer Cell Proliferation in vitro and in vivo
K. Kisfalvi,1 J. Sinnett-Smith,1 G. Eibl,2 E. Rozengurt.11Department of Medicine, 2Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles.
Current therapies offer poor survival rates in pancreatic cancer patients. More effective therapies will likely arise from targeting synergistic crosstalks in the cancer cells that promote their unrestricted proliferation.
Results: We identified a crosstalk between insulin/IGF-1 and G protein-coupled receptor (GPCR) agonist signaling pathways that synergize to strikingly enhance cell proliferation and DNA synthesis in human pancreatic cancer cells. This synergistic interaction depended on the function of mammalian target of rapamycin (mTOR) Metformin, the most widely used drug for type-2 diabetes, is thought to activate AMP kinase (AMPK), which negatively regulates mTOR. We found that metformin abrogated the enhanced DNA synthesis, anchorage-dependent and independent growth induced by insulin and GPCR agonists in pancreatic cancer cells, including PANC-1 and MIAPaCa-2. Metformin induced sustained AMPK activation and AMPK inhibitor reversed the effects of metformin. Next we examined how metformin affects pancreatic cancer growth in vivo using PANC-1 and MIAPaCa-2 tumor xenografts in nude mice. Metformin, given daily intraperitoneally (250 mg/kg) markedly decreased the rate of growth of both PANC-1 and MIAPaCa-2 xenografts (p=0.007 in PANC-1, p=0.005 in MIAPaCa-2). We then administered metformin orally, as currently available for human patients, in drinking water (2.5mg/ml). Metformin again strongly inhibited the growth of the tumor xenografts (p=0.014 in PANC-1, p=0.009 in MIAPaCa-2).
Conclusion: These results raise the possibility that metformin could be a candidate in novel treatment strategies for human pancreatic cancer.
Haplotypes in Melanoma Inhibitory Activity 2 Correlate With Survival and Chemoresistance in Pancreatic Cancer
B. Kong, C. W. Michalski, N. Valkovska, S. Rieder, X. Hong, S. Streit, H. Friess, J. Kleeff. Department of Surgery, Technische Universität München, Munich, Germany.
Background: Response rates of pancreatic ductal adenocarcinomas (PDAC) to chemotherapy are low; identification of a subgroup of patients who are more likely to benefit from chemotherapy might constitute a rational approach to improve prognosis.
Methods: Expression of melanoma inhibitory activity 2 (MIA2) was examined in pancreatic tissues and pancreatic cancer cell lines. MIA2 polymorphisms were defined by conventional PCR sequencing and high-resolution melting curve analysis. To assess the functional relevance of MIA2, loss-of-function and gain-of-function studies were performed in pancreatic cancer cell lines.
Results: MIA2 staining in cancer cells was found in 71% (43 out of 61) of the PDAC tissues. Though MIA2 expression was found in ASPC-1, Capan-1 and Colo-357, MIA2 was secreted only by ASPC-1 and Capan-1. Sequencing results revealed a homozygous Met141/His547 allele in Colo-357 but a homozygous Ile141/Asp547 allele in ASPC-1 and Capan-1 cells. PDAC patients heterozygous or homozygous for Met141/His547 (n=75/223 patients) survived significantly shorter than patients homozygous for Ile141/Asp547 (15 vs. 21 months). Functionally, silencing of MIA2 Ile141/Asp547 in Capan-1 and ASPC-1 conferred resistance to gemcitabine treatment, while silencing of MIA2 Met141/His547 in Colo-357 had no such effect. Correspondingly, overexpression of MIA2 Ile141/Asp547 in Su86.86 cells increased sensitivity to gemcitabine which was specifically rescued by MIA2 RNAi. However, overexpression of the MIA2 Met141/Asp547, Ile141/His547 or Met141/His547 variants had no such effect. Furthermore, the different MIA2 polymorphisms were associated with specific activation patterns of AKT and ERK signalling pathways following gemcitabine treatment.
Conclusion: Haplotypes in MIA2 are associated with survival and chemoresistance in pancreatic cancer.
Endoscopic Ultrasound During Acute Pancreatitis
V. Kotwal, R. Talukdar, M. J. Levy, S. S. Vege. Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic College of Medicine, Rochester, MN, USA.
Background and Aims: There are limited data on the role of endoscopic ultrasound (EUS) during the episode of acute pancreatitis (AP). Our aims were to study indications for EUS during AP, findings on EUS, correlation with outcomes and impact on management.
Methods: Patients admitted to Mayo clinic hospitals for AP between July 2004 to August 2009, and had EUS done during stay were identified, charts reviewed and following information extracted: Indications for EUS, findings on EUS (visualization of pancreas, hypo- and hyperechogenicity, peripancreatic fluid), outcomes (hospital stay, interventions and ICU transfer).
Results: We studied 22 patients (M:F=8:14). Indications for EUS were to confirm bile duct stones (n=6), detect tumor (n=5), idiopathic pancreatitis (n=4), and in patients with contraindication to CT scan or doubtful diagnosis on CT (n=3). In 2 patients EUS was done for peripancreatic collection drainage, in one each to guide ERCP to drain PD and as liver enzymes increased post ERCP. Findings on EUS: peripancreatic fluid in 10 (45%), hypoechogenicity in 8 (36%), non-visualization in 5 (22%), hyperechogenicity in 2 (9%) patients. No EUS finding correlated with patient outcomes. When compared to CT, EUS could not differentiate between interstitial and necrotizing pancreatitis but correlated well with CT in patients with peripancreatic inflammation/fluid. EUS prevented diagnostic ERCP in 5/6 patients with suspected biliary pancreatitis. EUS detected main duct IPMN in 1/5 patients with suspected tumor.
Conclusion: The most common indications for EUS in AP were suspected biliary pancreatitis. EUS had a role in preventing diagnostic ERCP. EUS could not differentiate between interstitial and necrotizing pancreatitis and the findings did not have any prognostic significance.
Epithelial to Mesenchymal Transition is Involved in the Radioresistance of Pancreatic Cancer Cells
S. Kozono, K. Ohuchida, T. Ohtsuka, K. Mizumoto, M. Tanaka. Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Objectives: Pancreatic cancer is a devastating disease that is characterized by a marked resistance to radiotherapy. In this study, we investigated the involvement of epithelial to mesenchymal transition (EMT) in resistance to radiation therapy in pancreatic cancer cells.
Methods: To investigate the involvement of EMT in radioresistance of pancreatic cancer cells, we generated radioresistant pancreatic cancer cell lines (CFPAC-1) by repeated exposures to radiation. Then, we performed colony formation assay to assess the 50% survival radiation dose in radioresistant CFPAC-1 and 8 pancreatic cancer cell lines and also compared the expression levels of EMT-related makers in these cell lines using real-time RT-PCR and immunohistochemistry.
Results: Radioresistant CFPAC-1 underwent morphologic change from epithelial cobblestone phenotype to elongated fibroblastic phenotype. Radioresistant CFPAC-1, KP-2, Suit-2, Panc-1 and Mia Paca-2 showed the significantly higher 50% survival radiation dose than Nor-P1, Capan-1, CFPAC-1 and Capan-2. Radioresistant pancreatic cell line group showed low expression levels of E-cadherin mRNA and protein and high expression levels of vimentin, Zeb-1 mRNA and protein compared to radiosensitive ones. We observed a significant correlation (R2=0.618; p=0.0069) between the 50% survival radiation dose and Zeb-1 mRNA expression.
Conclusions: The pancreatic cancer cell lines showing EMT-like phenotype were resistant to radiationtherapy. EMT may maintain radioresistance in human pancreatic cells.
Overexpression of the Collagenase MT1-MMP in KrasG12D Mice Leads to Pancreatic Tumors With Pronounced Fibrosis
S. B. Krantz,1 S. Dangi-Garimella,2 M. A. Shields,2 E. C. Cheon,1 M. R. Barron,1 P. J. Grippo,1,3 D. J. Bentrem,1,3,4 H. G. Munshi.2,3,4Departments of 1Surgery and 2Medicinel; 3Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL; 4Jesse Brown VA Medical Center, Chicago, IL.
Pancreatic cancer is associated with a pronounced fibrotic reaction that was recently shown to limit the delivery of chemotherapy. To identify potential therapeutic targets to overcome this fibrosis, we examined the interplay between fibrosis and a key proteinase required for growth and invasion in the collagen-rich microenvironment, namely membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14). Compared to control KrasG12D mice that express an activating mutation necessary for pancreatic cancer development, mice that express MT1-MMP in addition to KrasG12D developed a greater number of large lesions that were similar to human intraductal papillary mucinous neoplasms (IPMNs). These lesions were associated with a significant amount of surrounding fibrosis, increased α-smooth muscle actin positive cells in the stroma, indicative of activated myofibroblasts, and a greater amount of Smad2 phosphorylation, demonstrating increased TGF-β signaling. In vitro, MT1-MMP overexpression in pancreatic cancer cells increased collagen production by pancreatic stellate cells in a TGF-β dependent manner. We therefore demonstrate that MT1-MMP can promote fibrosis in pancreatic neoplasias in vivo, and in vitro, MT1-MMP can activate pancreatic stellate cells through increased TGF-β signaling. Targeting MT1-MMP may thus provide a new treatment strategy to address the significant challenges of delivering therapeutic agents through the fibrotic microenvironment in pancreatic cancer.
Resection of Non-Functional Neuroendocrine Carcinoma of the Pancreas With and Without Synchronous Liver Metastasis Provides Excellent Survival
B. Kulemann, F. Makowiec, O. Sick, G. Seifert, U. T. Hopt, T. Keck. Department of Surgery, University of Freiburg, Germany.
Non-functional pancreatic neuroendocrine carcinoma are rare, usually indolent malignant tumors, for which there is no effective, systemic therapy to date. To identify potential prognostic factors, we evaluated the outcome after resection in patients with primary malignant pancreatic neuroendocrine tumors (NET) performed in a single institution.
Methods: Since 1996, 37 / 1086 pancreatic resection at our institution were performed for non-functional NETs of the pancreas. The mean follow-up period was 5 years. 21 patients received distal, and 13 pancreatic head resections. In three patients, a tumor enucleation was performed. The median tumor diameter was 30 mm (range 2-83 mm). Six patients had liver metastases at the time of surgery, four of which were resected simultaneously. Seven patients (19%) showed a margin-positive R1 resection. The median postoperative follow-up was 3.5 years. A survival analysis was performed using Kaplan-Meier estimation with log-rank-statistics. All but two patients had well differentiated tumors.
Results: Mortality was 3%, complications occurred in 47%. The cumulative 5-year-survival-rate was 71%. Survival in patients with tumors larger than 30 mm was clearly significantly reduced (37% vs 86% (<30 mm) after 5 years). Analysis of survival showed a trend to worse prognosis in the small group with liver metastases with a 5-year-survival-rate of only 50% compared to 76% in the group without metastases. The resection margin, body mass index (BMI) and nodal status did not influence survival in our study group.
Conclusion: Survival in our series was comparable with large national series (SEER). In our patients, the prognosis was mainly influenced by tumor size and liver metastases. For patients with large pancreatic NETs and/or liver metastases a multimodal therapy ought to be considered to increase long-term survival after resection.
High Number of Acinar Cells in the Cut Edge of Pancreas Increases the Risk for Immediate Postoperative Complications After Pancreaticoduodenectomy
M. Laaninen,1 M. Bläuer,1 K. Vasama,2 S. Räty,1 J. Sand,1 I. Nordback,1 J. S Laukkarinen.11Dept. of Gastroenterology and Alimentary Tract Surgery, and Tampere Pancreas Laboratory, 2Dept. of Pathology, Tampere University Hospital, Tampere, Finland.
Introduction: Soft pancreas may be one risk factor for postoperative pancreatitis after pancreaticoduodenectomy. Pancreatitis, in turn, may increase the risk for anastomotic leakages and other complications. Earlier it has been suggested that increased amount of intrapancreatic fat is critical. Our aim was to analyse the amount of different cell types of cut edge of pancreas (CEP), and to study the possible correlation to early postoperative complications (also other than fistulas) after pancreaticoduodenectomy.
Materials and methods: In 40 Whipple-operated patients, HE stained section of CEP was electronically photographed and areas of different cell types were calculated. Positive urine trypsinogen (early detector of pancreas irritation / inflammation), was measured on 1.-6. p.o. days and drain amylase on 3. p.o. day. All complications were prospectively recorded. Anastomotic leakages were graded according to ISGPF classification and delayed gastric emptying (DGE) according to ISGPS classification.
Results: High number of acinar cells in CEP was a risk factor for positive u-trypsinogen (p<0.05), high drain amylase (p<0.05), DGE (p<0.05) and leakages (NS; total 5%). High amount of fibrosis, in turn, was protective for positive u-trypsinogen, drain amylase release and wound infections. Intra- and extrapancreatic fat played no role.
Conclusions: Not intrapancreatic fat, but high amount of acinar cells of CEP seems to be critical to increase the risk for postoperative complications after pancreaticoduodenectomy. High amount of fibrosis of CEP, in turn, seems to be protective for immediate postoperative complications.
Generation of Polyclonal Human IgG Directed Against Pancreatic Ductal Adenocarcinoma Cell Lines From Peripheral Mononuclear Cells of Healthy Donors
E. Langenmair, G. Wolff-Vorbeck, U. T. Hopt, U. A. Wittel. Department of General- and Visceral Surgery, Universitätsklinik Freiburg, Freiburg, Germany.
Introduction: While in many solid tumors antibody based chemotherapy regimens have found entry into the clinical routine, in pancreatic adenocarcinoma (PDAC) antibodies show little effects. Since cells of PDAC might present different antigens we examined the possibility to develop antibodies directed against PDAC cell lines without bias toward the immunogenic antigen.
Materials and Methods: Plasma-membrane fractions from the Panc-1 cell line and peripheral blood mononuclear cells (PBMC) from healthy donors were prepared by density centrifugation. PBMCs were co-incubated with and without antigen in the presence of IL-4, IL-21, and monoclonal mouse anti-CD40 receptor antibody. After 7 days antigen was re-administered and after 14 days the cell culture supernatants were collected for further examination. ELISAs were performed with plasma-membrane preparations and methanol fixed Panc-1 and MiaPaCa-2 cells as solid phase. Immunofluorescence was performed with methanol fixed Panc-1 cells.
Results: In all experiments high levels of IgG directed against the stimulation antigen were detected (no antigen vs. antigen 0.14±0.09 vs. 3.87±0.32; p<0.02). In 50% of the experiments binding of the IgGs to methanol fixed Panc1 cell-surfaces and in one experiment binding was directed toward Panc1 as well as the surface of MiaPaCa-2 cells. Immunofluorescence with cell culture supernatants confirmed cell surface binding of the human IgG molecules.
Conclusion: Human IgG directed against antigens displayed on the tumor cell surfaces of PDAC cell lines can be generated in vitro from healthy donors by co-incubation of mononuclear cells with antigen derived from the pancreatic tumor celline Panc-1.
Development and Validation of a miRNA-Based Laboratory Developed Test for Pancreatic Cancer
L. S. Lee,1 A. M. Bellizzi,1 D. L. Conwell,1 M. Sanders,2 D. C. Whitcomb,2 G. Rateb,3 C. Menard,3, J. A. Morisset,3 V. Shivakumar,4 S. Hahn,5 G. J. Tsongalis,6 M. Lloyd,7 B. Andruss,7 A. Adai,7 A. E. (Szafranska) Schwarzbach.71Division of Gastroenterology, Brigham & Women's Hospital, Boston, MA; 2Division of Gastroenterology, University of Pittsburgh Medical Center, Pittsburgh, PA; 3Department of Gastroenterology, University of Sherbrooke, Fleurimont, Canada; 4H Lee Moffitt Cancer Center & Research Institute, Tampa, FL; 5Department of Pathology & Internal Medicine, Rühr University, Bochum, Germany; 6Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, NH; 7Asuragen, Austin, TX.
Introduction: Differential diagnosis between chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC) can be challenging due to similar clinical presentation and imaging features. Previously we reported a laboratory developed test based on a difference in raw expression between miR-196a and miR-217. This test allows distinguishing PDAC from CP with sensitivity (Se) and specificity (Sp) of ∼95% and was validated in formalin fixed paraffin embedded (FFPE) specimens with masses occupying ≥60% area or those that could be enriched to this content. There is additional clinical interest in identifying small pancreatic malignancies in a background of pancreatitis.
Materials and Methods: Expression levels of 11 miRNAs were interrogated in 216 frozen, FFPE and fine needle aspirate (FNA) specimens using TaqMan® RT-qPCR. A new multi-miRNA signature was developed to estimate the probability of PDAC detection in specimens with low tumor content.
Results: The multi-miRNA signature improved Se of PDAC detection by 2-3 fold in a set of FFPE PDAC specimens with 10% - 88% tumor area, as compared to the miR-196a/miR-217 assay. Performance of this signature on FNA specimens will be discussed.
Novel Function of FUT-175 as Apoptosis Inducer
J. Ling,1 Z. Li,1 T. Uwagawa,1 P. J. Chiao.1,2,31Department of Surgical Oncology, 2 Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas, USA, and 3Graduate Program in Cancer Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, 6655 Travis, Houston, Texas, USA.
Pancreatic ductal adenocarcinoma (PDAC) is the 4th most common cause of adult cancer death, and the 5-year survival rate has remained 1%-3% as measured 25 years ago. To increase the survival rate of PDAC patients, novel therapeutic agents are desperately needed. Constitutive activation of nuclear factor-κB (NF-κB) is a frequent molecular alteration in PPDAC and a number of studies suggest that constitutive NF-κB activity plays a key role in chemoresistance of this disease. To identify an effective therapeutic agent for targeting NF-κB signaling in PDAC, we studied the effect of FUT-175, a synthetic serine protease inhibitor, in induction of apoptotic responses.
In a time- and dose-dependent manner, FUT-175 inhibited NF-κB activation and concurrently upregulated the expression of tumor necrosis factor receptor-1 (TNFR1), which in turn activated the proapoptotic caspase-8 and Bid pathways and induced apoptosis in PDAC cells. To further study the action of FUT-175 as a potential chemotherapeutic agent for PDAC, we compared the anti-cancer activities of FUT-175 with Smac mimetics in 12 PDAC cell lines. FUT-175 is a much more potent apoptosis inducer than Smac mimetics and all the PDAC cells are sensitive to the treatment of FUT-175 alone.
These results suggested a possible mechanism by which FUT-175 may disrupt inter-connected signaling pathways by both suppressing the NF-κB antiapoptotic activity and inducing TNFR-mediated apoptosis, and this synthetic serine protease inhibitor is a potential novel therapeutic agent for PADC.
Beta III- and Beta IVB-Tubulin Mediate Sensitivity to Chemotherapeutic Drugs in Pancreatic Cancer Cells
J. Liu,#1 J. McCarroll,#2 M. V. Apte,1 J. Youkhana,1 M. Kavallaris,2 P. A. Phillips.1#These authors contributed equally to the study. 1Pancreatic Research Group, University of New South Wales, Sydney, Australia; 2Children's Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, Sydney, Australia.
Pancreatic cancer (PC) has a poor prognosis, most commonly due to resistance to chemotherapeutics. β-tubulin isoforms (βII, βIII and βIVb) have been implicated in chemoresistance in other cancers. We demonstrated that silencing βIII-tubulin sensitizes lung cancer cells (in vitro and in vivo) to tubulin binding agents (TBAs) and DNA damaging agents (Cancer Res, 2007, 2010). βIII-tubulin is highly expressed in pancreatic ductal adenocarcinoma samples and in PC cell lines. However, the role of β-tubulins in the resistance of PC cells to chemotherapy drugs is unknown.
Aim: To determine the effect of siRNA silencing of βII, βIII or βIVb-tubulin in PC cells on drug sensitivity.
Methods: MiaPaCa-2 cells ± β-tubulin specific siRNA were treated with gemcitabine, taxol or vincristine (n=4-5). Seven days later colonies from clonogenic assays were counted (surviving fraction).
Results: Silencing βIII-tubulin significantly sensitized MiaPaCa-2 cells to the anti-metabolite gemcitabine (at doses of 1.25-7nM; p<0.05) and to the TBAs, taxol (1.25-2.5nM; p<0.05) and vincristine (0.25-1nM; p<0.05). Silencing βIVb-tubulin significantly sensitized the cells to vincristine (0.25-1nM; p<0.05) and gemcitabine (0.625-2.5nM; p<0.05). Silencing βII-tubulin had no effect on drug sensitivity of MiaPaCa-2.
Conclusion: This is the first study to show that suppression of βIII and βIVb-tubulin increases drug sensitivity in PC cells. These novel data support the concept that specific tubulin isotypes have a unique function in regulating drug sensitivity in PC.
Implication: Targeting βIII or βIVb tubulin may improve chemotherapy response in PC.
A Single Nucleotide Polymorphism in Tumor Suppressor Gene SEL1L as Predictive and Prognostic Marker for Pancreatic Ductal Adenocarcinoma
Q. Liu,1* J. Chen,1* B. Mai,2 C. Amos,1 A. M. Killary,3 S. Sen,4 M. L. Frazier.11Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, Texas; 2Program in Molecular Genetic Technology, The University of Texas MD Anderson Cancer Center, School of Health Professions, Houston, Texas; 3Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas; 4Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas; *These authors contributed equally to this work.
SEL1L is a putative tumor suppressor gene that is frequently downregulated in pancreatic ductal adenocarcinomas (PDAs). We hypothesized that the single nucleotide polymorphism (SNP) rs12435998 in SEL1L may influence the clinical outcomes of PDA patients. To test this, we retrospectively analyzed DNA samples from 575 patients with pathologically confirmed primary PDA. Of them, 105 had been enrolled in a clinical trial of neoadjuvant chemoradiotherapy and 85 had subsequently undergone pancreaticoduodenectomy (PD). The target region was amplified by polymerase chain reaction (PCR), and then the genotypes were analyzed by pyrosequencing. We performed Kaplan-Meier analysis to evaluate the correlation between different SNP genotypes and age at diagnosis, survival time after PD, and time to tumor progression after PD. In nonsmokers, we found a significant difference in median age at diagnosis between patients with variant genotypes (AG/GG) and those with wild-type genotype (AA) (59 and 62 years, respectively; log-rank test, P = 0.020); patients with variant genotypes also showed an increased hazard ratio (HR) of 1.39 (95% confidence interval [CI], 1.05-1.84). Among the clinical trial patients, those with variant genotypes had a median post-PD survival time that was 34.7 months shorter (log-rank test, P = 0.011; HR = 1.96; 95% CI, 1.10-3.32), and a median post-PD time to tumor progression that was 5.2 months shorter (log-rank test, P = 0.040; HR = 1.60; 95% CI, 1.02-2.50) Our results suggest that SEL1L has a role in modifying age at diagnosis of PDA in nonsmokers. We also report for the first time that SNP rs12435998 may serve as a prognostic marker in PDA patients who undergo the same treatments as the clinical trial.
A Novel PI3K-AKT-GLI3 Axis Activates the Machinery Controlling Autophagy in Pancreatic Cancer Cells
A. E. Lo Ré,1,2 L. L. Almada,2 R. Pardo,1 M. I. Molejón,1 S. F. Elsawa,2 M. I. Vaccaro,1 M. E. Fernandez-Zapico.21Department of Biological Sciences, School of Biochemistry, University of Buenos Aires, Buenos Aires, Argentina; 2Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, MN.
Autophagy is an evolutionarily conserved degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells. It has been suggested that autophagy can acts in tumor promotion and progression, however, the molecular mechanisms underlying this phenomenon have not been elucidated. Here, we have found that the transmembrane protein VMP1, a key mediator of autophagy, is upregulated in pancreatic cancer cells undergoing starvation. Analysis of the mechanism revealed that GLI3, an effector of the Hedgehog pathway regulates the expression and promoter activity of VMP1. Chromatin immunoprecipitation assays demonstrated that GLI3 binds to the VMP1 promoter. We have found that the histone acetyltransferase p300 complex with GLI3 and cooperates with this transcription factor to activate VMP1 expression. RNAi knockdown of p300 impairs starvation as well as GLI3-induced activation of this promoter. Finally, we identified the PI3K-AKT axis as the signaling mediator modulating the activity of this novel GLI3-p300 transcriptional complex. This pathway is activated in pancreatic cancer cells undergoing starvation and promotes GLI3-mediated VMP1 transcriptional activity. Thus, together these data provide evidence of a new regulatory mechanism modulating autophagy and integrates this cellular process into the network of events promoting pancreatic carcinogenesis.
This study was supported by the Division of Oncology Research, CA136126, Mayo Clinic Pancreatic SPORE CA102701, Mayo Clinic Center for Cell Signaling in Gastroenterology DK084567 and CONICET.
HP1 Binds to KLF11 to Regulate CXCR4, a Key Mediator of Metastasis and Angiogenesis in Pancreatic Cancer
G. Lomberk, A. Mathison, M. Fernandez-Zapico, C. Demars, N. Buttar, R. Urrutia. Division of Gastroenterology, Mayo Clinic, Rochester, MN 55905, USA.
Previous studies have characterized the binding of HP1 to known tumor suppressor proteins. However, direct evidence supporting the participation of HP1 in the tumor suppressive activity of these proteins is lacking. Here, we address this gap in existing knowledge by characterizing the physical and functional interaction between HP1α and the tumor suppressor protein, KLF11. An HP1-interacting domain in the C-terminus of KLF11 was confirmed by immunoprecipitation and biomolecular fluorescence complementation. Notably, disruption of HP1 interaction abolishes key tumor suppressive properties of KLF11, including its ability to suppress transformation and senescence as well as to increase apoptosis. While KLF11 suppresses pancreatic tumorigenesis in vivo, mutation of its HP1 binding motif completely abolishes this activity. Using microarray and RT-PCR validation in pancreatic cancer cells, we find that loss of HP1 binding to KLF11 significantly upregulates expression of CXCR4, an important mediator of metastasis and angiogenesis in pancreatic cancer. Moreover, KLF11 occupies the CXCR4 promoter in vivo with HP1α and its histone methyltransferase, SUV39H1. However, the KLF11 HP1-binding mutant fails to recruit HP1α and SUV39H1 to the CXCR4 promoter and significantly upregulates its promoter activity via luciferase assay. Finally, treatment of mice harboring xenografts from the HP1-binding KLF11 mutant with AMD3100, a CXCR4 inhibitor, inhibits tumor growth. Thus, this study reports a novel KLF11/HP1/histone methylase complex that regulates a key gene, CXCR4, involved in pancreatic cancer progression, further supporting the role of KLF11 as a tumor suppressor gene working via chromatin remodeling systems.
Triptolide Induces Expression of miR-129* and miR-142-3p in Pancreatic Cancer Cells
T. N. MacKenzie,1,2 N. Mujumdar,1 V. Thayanithy,1 A. Sarver,1 R. Chugh,1 S. Subramanian,1,3 V. Sangwan,1 S. Vickers,1 A. Saluja.1,2,31Department of Surgery; 2Department of Pharmacology; 3Masonic Cancer Center, University of Minnesota, Minneapolis, MN.
Triptolide inhibits cancer cell growth in vitro and blocks growth and metastatic spread in vivo. Our lab has shown that triptolide triggers apoptosis via inhibition of HSP70 expression. As small non-coding RNAs that negatively regulate gene expression, miRNAs are now recognized as powerful gatekeepers of apoptosis. Understanding the effect of a drug on miRNA expression has lead to the identification of miRNAs that mediate apoptosis and chemosensitivity. We hypothesize that triptolide inhibits HSP70 expression via a novel mechanism of inducing the expression of miRNAs that target HSP70.
Methods: We performed miRNA and mRNA microarray analysis of human pancreatic cancer cells (MIA PaCa-2 and S2-013) prior to and following different time points of triptolide treatment and concurrently monitored cell viability. We profiled tumors of these cells when grown in mice to address whether triptolide modulation of miRNAs and mRNAs in vitro may correlate in vivo.
Results: Both miR-142-3p and miR-129* potentially target HSP70 and expression of these miRs increases immediately after treatment, before loss of cell viability in our miRNA microarray analysis. These data suggest that increased expression of miR-142-3p and miR-129* is a direct result of triptolide treatment. We have validated these results via qRT-PCR.
Conclusions: Altering miR-142-3p and miR-129* expression may enhance chemosensitivity or impart chemoprotection from triptolide. Understanding how triptolide affects microRNA and mRNA expression allows us to elucidate the molecular mechanism of action of triptolide so that we may maximize triptolide's effectiveness.
Video Submission: Laparoscopic Frey Procedure
M. A. Makary, Tejwant Datta, V. Singh, P. Okolo, A. Kalloo, D. K. Andersen. Johns Hopkins Multi-disciplinary Pancreatitis Clinic, Baltimore, MD.
Introduction: Local resection of the pancreatic head with longitudinal pancreatico-jejunostomy (LR-LPJ), or Frey procedure, is increasingly being offered to patients with chronic pancreatits.
Methods: We describe the Frey procedure performed using a totally laparoscopic technique. We assessed the technical feasibility, operative time and clinical result of this procedure in a 42 year-old female with chronic pancreatitis and symptomatic pancreatic ductal stones.
Results: The laparoscopic Frey procedure was successfully performed using sharp dissection to open the pancreatic duct and resect the pancreatic head. Multiple stones in the pancreatic head and tail were manually extracted. A stapled technique for the jejunojenostomy and a freehand, hand-sewn technique for the pancreaticojejunostomy was used. The operative time was 225 minutes. There were no operative or postoperative complications. The patient was discharged on postoperative day four. Follow-up was complete at three months at which point the patient was noted to have continued resolution of preoperative symptoms.
Conclusion: The laparoscopic Frey procedure is a feasible surgical option and can be performed with excellent results. Further research is needed to evaluate this procedure in comparison to its open equivalent operation.
Clinical Outcome After the Frey Procedure: The Paradox of Symptom Resolution and the Severity of Chronic Pancreatitis
M. A. Makary, V. Singh, Y. Cui, P. Okolo, A. Kalloo, F. Eckhauser, D. K. Andersen. Johns Hopkins Multi-disciplinary Pancreatitis Clinic, Baltimore, MD.
Introduction: The local resection of the pancreatic head with longitudinal pancreatico-jejunostomy (LR-LPJ), or Frey procedure, has been shown to be as effective as pancreatico-duodenectomy (Whipple) or duodenum-preserving pancreatic head resection (Beger procedure) for the relief of pain due to chronic pancreatitis (CP). Controversy persists over the selection of patients for these surgical options, and the impact of the extent of underlying fibrosis on the postoperative outcome is poorly understood.
Methods: We analyzed 31 patients who underwent the Frey procedure for documented CP accompanied by persistent pain from 2006-2010 (mean follow-up=22 months). All had duct dilatation due to one or more strictures, pseudocysts, or intraductal stones, and all had documented chronic inflammation on gross or microscopic analysis of the excavated tissue removed from the pancreatic head. We evaluated symptomatic outcomes in patients with severe or extensive fibrosis (SEF) and those with minimal or mild fibrosis (MIF).
Results: 30 of 31 patients survived the postoperative period and were followed to assess symptom resolution or persistence, medication need, and disability. Of the 20 SEF patients, 18 (90%) had complete or near-complete resolution of symptoms and disability. Of the 10 MIF patients, 6 (60%) had complete or near-complete resolution of symptoms and disability. Continued opioid use was common (14/20 and 8/10) in both groups.
Conclusion: Complete or near-complete symptom resolution after the Frey procedure is more likely in the setting of severe or extensive fibrosis due to CP. Despite ductal dilatation, the presence of mild or minimal fibrosis may decrease the likelihood of symptom resolution after the LR-LPJ procedure.
The Chemical Chaperon 4- Phenylbutyric Acid Increases Amylase Secretion, Reduces Trypsin Activation and Endoplasmic Reticulum Stress in Pancreatic Acini
A. Malo,1 C. Schafer,2 B. Goke,1 C. H. Kubisch, MD.11Department of Medicine II, University of Munich, Germany; 2Department of Gastroenterology, Clinic Neumarkt, Germany.
Background: Endoplasmic reticulum (ER) stress leads to accumulation of un- or misfolded proteins inside the ER and initiates the unfolded protein response (UPR). Several UPR components are physiologically involved in pancreatic development and are activated during acute pancreatitis. However, the exact role of ER stress in exocrine pancreatic acini is mainly unclear. The present study examined the effects of 4-phenylbutyric acid (4-PBA), a known ER-chaperone, on acinar function and UPR components.
Materials & Methods: Isolated rat pancreatic acini were stimulated with cholecystokinin (CCK) (10 pM-10 nM). Half of the cells were preincubated with 3,75mM 4-PBA. Components of the UPR, including BiP expression, PERK phosphorylation, XBP1 splicing, CHOP expression and JNK phosphorylation were measured, as were effects on amylase secretion and trypsin activation.
Results: CCK generated a biphasic secretion dose-response curve. Preincubation with 4-PBA was followed by an increase of about 68 % of amylase secretion at 300pM CCK stimulation, whereas trypsin activation with supraphysiological CCK doses war significantly reduced (40% at 10 nM CCK). CCK in supraphysiologic doses increased BiP levels, PERK phosphorylation, XBP1 splicing, CHOP expression and JNK phosphorylation. All of this, except XBP-1 splicing, was significantly decreased after preincubation of the cells with 4-PBA.
Conclusions: The ER function and stress response appears to be involved in pancreatic physiology and pathophysiology. By preincubation with an ER-chaperone, that might be beneficial in diabetes mellitus and cystic fibrosis, it was possible to increase acinar cell secretion and suppress trypsin activation within acinar cells. Further it was possible to reduce elements of the UPR and several proapoptotic pathways emitting from the ER. Future efforts should be directed at understanding the roles of these mechanisms in the pancreas.
Insulin Protects Against Oxidant-Induced Impairment of Ca2+ Signalling and Plasma Membrane Ca2+-ATPase (PMCA) Inhibition in Pancreatic Acinar Cells
P. Mankad, T. Leggett, A. James, J. Bruce. Faculty of Life Sciences, University of Manchester, Manchester, UK.
Introduction: Oxidant - induced impairment of cytosolic calcium ([Ca2+]i) signalling and an irreversible increase in [Ca2+]i (Ca2+ overload) has been implicated in acute pancreatitis (AP). Our studies have shown that this Ca2+ overload response is due in part to inhibition of the PMCA, and coincided with mitochondrial depolarisation. In experimental models of AP, insulin was found to be protective. Therefore the aim of the current study was to test the effects of insulin on oxidant-mediated impairment of [Ca2+]i signalling and inhibition of the PMCA in pancreatic acinar cells (PAC).
Methods: PACs were freshly isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. In addition, an in situ [Ca2+]i clearance assay was used in which the PMCA was pharmacologically isolated.
Results: Oxidative stress induced by hydrogen peroxide (H2O2) caused a concentration dependent Ca2+ overload response and inhibition of the PMCA. Pre-treatment with insulin (100nM for 30 minutes) prevented the H2O2 - induced Ca2+ overload and attenuated H2O2 - induced inhibition of the PMCA. However, insulin had no effect on H2O2 - mediated mitochondrial depolarisation (TMRM imaging). Furthermore, insulin (100nM) activated Akt signalling, confirmed by western blotting with pAkt S473 antibody.
Conclusion: In summary, these data suggest that insulin may protect PAC against oxidant-induced Ca2+ overload and PMCA inhibition independent of mitochondrial depolarisation; possibly via Akt mediated cell survival pathways.
Disordering of Lysosomal and Autophagic Pathways in Nonalcoholic and Alcoholic Experimental Pancreatitis
O. A. Mareninova,1 I. Yakubov,1 S. J. Pandol,1 F. S. Gorelick,2 A. S. Gukovskaya,1 I. Gukovsky.11VAGLAHS, University of California Los Angeles & Southern California Research Center for ALPD and Cirrhosis; 2VAMC & Yale University, West Haven, CT.
Introduction: We recently showed that nonalcoholic pancreatitis causes impaired autophagy, mediating key pathologic acinar cell responses. Here we investigate lysosomal defects underlying the impaired autophagy. Further, we test the hypothesis that both induction of autophagy and its impairment (due to lysosomal dysfunction) are critical for alcoholic pancreatitis.
Methods: We used rodent models of pancreatitis induced by cerulein (CR), L-arginine, and in vitro CCK-8 hyperstimulation of acinar cells. For alcoholic pancreatitis, rats were fed ethanol diet followed by a low-dose CR (0.5 μg/kg).
Results: Both nonalcoholic and alcoholic pancreatitis caused defective maturation of cathepsins B and L (shift toward partially processed forms) and their decreased activity in lysosome-enriched subcellular fractions. Pancreatic levels of key lysosomal membrane proteins, i.e. LAMP-2, decreased. LAMP decrease mediates not only impaired cathepsin processing and autophagy but also dramatic redistribution of free cholesterol we observed with filipin staining - from normally plasma membrane to vesicular (endolysosomal) localization. Ethanol feeding alone, which does not cause pancreatitis, induced pathologic alterations in lysosomes similar to pancreatitis; however, it did not stimulate autophagy. In contrast, autophagy was both induced and impaired with ethanol plus low-dose CR, resulting in acinar cell vacuolation and trypsinogen activation.
Conclusion: We propose that a combination of 2 "hits": 1) ethanol-induced lysosomal dysfunction and 2) autophagy induction by another factor - leads to defective autophagy and thus alcoholic pancreatitis.
The Transcription Factor PAX6 Activates Expression of the Chemokine Receptor CXCR4 in Pancreatic Cancer
J. B. Mascarenhas, J. D. Kubic, A. E. Ludvik, D. Lang. Department of Medicine, University of Chicago, Chicago, IL.
Pancreatic ductal adenocarcinoma (PDAC) tumors are a major cause of cancer death and have among the poorest prognosis of any malignancy. The carcinogenic mechanisms that lead to PDAC, as well as the molecular pathways involved in invasion and metastasis, remains poorly understood. We find the transcription factor PAX6 to be expressed in primary PDAC tissues (29/43, 67.4%). PAX6 is expressed in the pancreatic bud during embryogenesis but expression becomes restricted to the endocrine cells and a minority of ductal cells in the adult pancreas. CXCR4 is a cytokine receptor that is expressed in PDAC as well as in an overlapping expression pattern with PAX6 during development. We find CXCR4 expression in 83.7% (36/43) of PDAC tumors, and the majority of the PAX6 positive samples are also CXCR4 positive (27/29, 93%). Due to the overlap of expression during development and in the tumor samples, and that PAX6 works as a transcription factor, we tested whether PAX6 directly activates CXCR4. Indeed, this hypothesis is supported with the finding of a putative intronic enhancer element in the CXCR4 locus containing several potential PAX binding sites. This enhancer was identified by phylogenetic footprinting through comparison of orthologous regulatory regions of the human and mouse genomes. PAX6 was able to drive expression from reporter constructs containing this enhancer region 2.8 fold +/- 0.7 fold over vector alone controls in luciferase assays. The ability of PAX6 to activate this reporter construct was abolished when the enhancer was deleted. Our findings support a model where PAX6 promotes the expression of down-stream effectors, such as CXCR4, thereby promoting the PDAC cancer phenotype of local invasion, metastasis, and tumor cell growth.
Molecular Mechanisms of L-Arginine Induced Experimental Acute Pancreatitis
O. Masood,1,2 A. K. Siriwardena,1 J. Bruce.21 Hepatobiliary Unit, Manchester Royal Infirmar; 2Faculty of Life Sciences, University of Manchester.
Impairment of cytosolic Ca2+ ([Ca2+]i) signaling and Ca2+ overload has emerged as a possible mechanism that precipitates acute pancreatitis (AP). In the L-arginine (Larg) induced experimental model of AP nitric oxide (NO) has been implicated however L-arg-induced AP is largely unaffected by nitric oxide synthase (NOS) inhibitors. In addition L-ornithine (Lorn), a NOS-independent metabolite of Larg, also induces pancreatitis. Both Larg and Lorn are known to activate calcium-sensing like receptors such as the GPRC6A which may be responsible for initiating the associated Ca2+ overload. Pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured using fura-2 imaging. Oxidative stress was measured using dichlorofluorescein (DCF) and cell death using trypan blue exclusion.
Larg and Lorn (100mM) induced spike-like, reversible increases in [Ca2+]i in 46% and 74% of cells and Ca2+ overload in 11% and 26% cells respectively. At 500 mM both induced Ca2+ overload in all cells however at this concentration this was also seen with the negative osmotic control, mannitol. Isosmotic Larg and Lorn (100mM) induced only reversible increases in [Ca2+]i. Neither Larg nor Lorn had significant effects on CCK-evoked [Ca2+]i oscillations. Both Larg and Lorn induced oxidative stress responses of 22% +/- 5 and 37% +/- 6 (of a maximum response seen with 3mM H202.) respectively. Both Larg and Lorn caused cell death in 76% +/- 4 and 89% +/- 7 at 3hours respectively, compared to 35% +/- 4 and 40% +/- 3 with controls (Hepes, Glycine).The data suggest that the Larg and Lorn causes a significant increase in oxidative stress and cell death, although the induced Ca2+ overload is most likely due to non-specific hyper-osmotic effects.
Novel Tumor Suppressor Pathway in Pancreatic Cancer Derived From Selective Remodeling of Desmoplasia
A. J. Mathison, A. Liebl, C. J. DeMars, U. Shergill, G. A. Lomberk, V. Shah, N. Buttar, R. A. Urrutia. Division of Gastroenterology, Mayo Clinic, Rochester, MN.
Stellate cells are emerging as key regulators of processes in several organs, including for the production and remodeling of pancreatic desmoplasia. Additionally, there is evidence that stellate cells regulate and control the growth, progression and metastasis of cancer. KLF11 is an induced, early response transcription factor to TGFβ signaling and an essential mediator in preventing epithelial cancer cell transformation. Here, we evaluated the relationship among stellate cells, KLF11, fibrosis and cancer. Transcript profiles of extracellular matrix and adhesion genes show that several collagen transcripts, collagens 1a1, 1a2, 3a1, 5a1, and 11a1, are downregulated in stellate cells overexpressing KLF11. Conversely, matrix metalloproteinases, MMP1, 3, 7, 8, 9, 10, 12, 13, are upregulated. Overall, the response indicates an anti-fibrogenic response. To test this, wild type and KLF11-/- mice were injected with fibrosis-inducing agents. Greater fibrotic deposition was observed in KLF11-/- mice, supporting KLF11 as an essential regulator of fibrotic deposition and degradation. Also, stellate cells expressing KLF11 inhibit pancreatic cancer growth in xenografts compared to control stellate cells which enhance tumor growth. Luciferase assays and ChIP indicate that KLF11 interacts directly with collagen promoters to repress their expression, while it also binds MMP promoters, leading to their activation. Thus, KLF11 has a vital role in stellate cells mounting an anti-fibrogenic response. Since KLF11 works with many chromatin proteins, this work suggests that inhibitors of histone acetylases, methylases, and deacetylases may be beneficial in treating pancreatic cancer by targeting remodeling of the tumor microenvironment.
Calcium Phosphate Nanoparticles for Selective Targeting of Human Pancreatic Cancers
G. Matters,1 C. McGovern,1 B. Barth,2 E. Altınoğlu,3 J. Adair,3 M. Kester,2 J. Smith.11Depts. of Medicine and 2Pharmacology, Pennsylvania State University, Hershey, PA; 3Dept. of Materials Science Engineering, Pennsylvania State University, University Park, PA.
Introduction: We have previously demonstrated that human pancreatic cancers over-express the CCK-2/ gastrin receptor. The aim of this study was to evaluate the ability of fluorescent target-specific nanoparticles to image pancreatic cancer in vivo. Calcium phosphate nanoparticles (CPNPs) are ∼20nm diameter non-toxic vehicles for the delivery of therapeutic or imaging agents in biological systems.
Methods: CPNPs were embedded with near the infra-red imaging agent indocyanine green (ICG) for imaging and conjugated via PEG-maleimide coupling with decagastrin (gastrin-10) for targeting the CCK receptors. Sensitivity and specificity of targeted versus non-targeted CPNPs were examined using human BxPC-3 pancreatic cancer cells in vitro with fluorescent microscopy and flow cytometry. In vivo imaging was conducted in nude mice bearing BxPC-3 orthotopic tumors after an injection of gastrin-labeled CPNPs compared to non-target specific uptake.
Results:In vitro, cancer cells exposed for 60 mins to gastrin-10-PEG-CPNP displayed fluorescent staining. Flow cytometry confirmed the increased binding to pancreatic cancer cells using target specific CPNP compared to unlabeled particles. In vivo, fluorescent imaging of the orthotopic tumor was readily visualized at 7 and 24 hrs post injection of fluorescent gastrin-10-PEG-CPNP but not with non-targeted CPNP.
Conclusion: Utilization of the CCK-2 receptors on human pancreatic cancer increases the target-specificity of nanoparticles. Biodegradable gastrin-targeted CPNP may be useful vehicles for early detection and /or treatment delivery vehicles for pancreatic cancer. NIH R01 CA117926
Multifocal IPMNs: Pathology and Field Defect
H. Matthaei,1 A. Norris-Kirby,1 K. Olino,1 C. L. Wolfgang,1 R. D. Schulick,1 J. L. Cameron,1 K. D. Jackson,1 M. dal Molin,1 P. Capelli,2 G. Zamboni,2,3 L. Bortesi,3 T. Furukawa,4 A. C. Tsiatis,1 S. M. Hong,1 M. Goggins,1 J. R. Eshleman,1 R. H. Hruban,1 A. Maitra.11The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins University, Baltimore, USA; 2Department of Pathology, University of Verona, Verona, Italy, 3Sacro Cuore Hospital, Negrar, Verona, Italy, 4International Research and Educational Institute for Integrated Medical Sciences, Tokyo Women's Medical University, Tokyo, Japan.
Background: IPMNs are increasingly diagnosed cystic precursors of pancreatic cancer and tumor multifocality is common. The underlying pathology and clonality are unclear. A field defect has been hypothesized.
Methods: We included 38 patients with multifocal IPMNs from 3 high volume hospitals for pathological re-review. A selection of 30 IPMNs from 13 patients was analyzed for clonality. DNA from tumor and matched normal cells was subjected to KRAS pyrosequencing and LOH analysis (Chr6q and 17p).
Results: Synchronous IPMNs occurred in 30 patients (82%) and 8 patients (18%) had metachronous IPMNs. All patients had at least 2 physically distinct IPMNs (range 2-6 lesions). The majority of multifocal IPMNs demonstrated low dysplastic branch duct neoplasms predominantly exhibiting a gastric-foveolar subtype. Clonality analysis showed clearly discordant LOH profiles in 2 of 10 patients (20%); 4 of 13 patients (31%) had distinct KRAS mutations within their multifocal IPMNs. In summary, these results demonstrate the presence of multiclonality in the pancreata of at least 5 of 13 patients (38%). The remaining cases showed overlapping mutations, but these were all common mutations.
Conclusion: Multifocal IPMNs are mostly represented by branch duct lesions of low dysplasia and gastric-foveolar subtype. Focusing on frequent molecular alterations we identified distinct patterns in IPMNs within 5 patients. This proves multiclonality and a field defect why follow-up for metachronous disease following resection of an IPMN is indicated.
Autophagy is Neither a Prerequisite for - nor a Consequence of - Early Trypsin Activation in Experimental Pancreatitis
J. Mayerle, S. Malla, B. Krüger, T. Wartmann, M. Sendler, F. U. Weiss, F. S. Gorelick, W. Halangk, M. M. Lerch. Department of Medicine A, University of Greifswald, Division of Medical Biology, University of Rostock, Division of Experimental Surgery, University of Magdeburg, Germany, and Yale University Medical School, New Haven, CT, USA.
Introduction: Intracellular protease activation, an initiating event for pancreatitis, was suggested to begin in autophagosomes. We tested whether intracellular trypsin activation depends on autophagy, or autophagy on trypsin activation, in the early phase of pancreatitis.
Methods: We used mice expressing the fluorescent autophagy marker LC-3-GFP to prepare acini and to induce pancreatitis (7 i.p. caerulein injections, 50μg/kg). Organelles were separated by percoll gradient centrifugation at 50.000g (densities 1.03-1.16 g/ml) and autophagy identified using specific antibodies against ATG16 or a shift to membrane bound LC3-II (fluorescent on live imaging of acini) indicating autophagosomes. Trypsin activation after 10nM CCK was imaged confocally (Ile-Pro-Arg-AMC) with or without gabexate-mesilate.
Results: Autophagosome formation during pancreatitis was confirmed by the appearance of fluorescent vesicles in the pancreas of LC3-GFP-mice and a shift of LC3 to its membrane-bound LC3-II form. Significant levels were reached at 4 h. Premature trypsin activation also developed during pancreatitis but as early as 1h. Simultaneous confocal imaging of autophagosome formation (LC3-fluorescence) and trypsin activation (Ile-Pro-Arg-AMC-fluorescence) in isolated acini indicated that the two events arise in different compartments. In subcellular fractions of 1h pancreatitis animals the trypsin activation-compartment contained cathepsin-B but did not correspond to autophagosomes. Trypsin inhibition with gabexate-mesilate did not prevent autophagosome formation in response to supramaximal stimulation.
Conclusions: While autophagy has been shown to be important in regulating the ultimate levels of intrapancreatic protease activity and pancreatitis severity, the initial activation of trypsin develops neither in autophagosomes nor does it depend on autophagy. Onset of autophagy in the pancreas, in turn, does not require trypsin activation.
The Role of EUS Guided Elastography to Diagnose Solid Pancreatic Mass Lesions
J. Mayerle,2 P. Simon,2 E. J. Dickson,1 M. M. Lerch,2 C. R. Carter,1 C. J. McKay.11Lister Departmen of Surgery, Glasgow Royal Infirmary, Glasgow, Scotland, UK; 2Department of Medicine A, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Introduction: EUS-guided realtime-Elastography examines tissue stiffness. Calculation of the reconstructed strain field (stiffness) can be assessed quantitatively and is expressed as strain ratio (SR). We evaluated whether EUS-guided transient elastography would increase the diagnostic accuracy compared to EUS-FNA for pancreatic mass lesions.
Methods: In a prospective cohort study (10/2008-10/2009) we recruited 89 consecutive patients with a solid pancreatic mass and performed EUS, EUS-guided elastography with a linear Pentax EUS-scope and the HITACHI-EUB-7500 as well as EUS-guided-FNA using a 22G Cook needle. Definite diagnosis by cytology or histology was regarded as the gold standard.
Results: 71 patients had malignant lesions and 18 patients presented with benign lesions. Median SR of benign lesions was 16 (±13.86 95%CI) compared to 44.4 (±8.8 95%CI) for malignant lesions (p<0.001). Optimal Cut-Off as of ROC analysis was 24.8. Elastography detected malignant lesions with a sensitivity of 96%, a specificity of 42%. Overall accuracy was 84% with an AUC of 0.76. EUS-FNA detected malignant lesions with a sensitivity of 84%, a specificity of 100%. Accuracy was 88%. B-Mode EUS in the hands of an experienced endosonographer achieved a sensitivity of 93%, a specificity of 68% and overall accuracy here was 88%.
Conclusion: At present, elastography of pancreatic mass lesions has a low specificity for discriminating benign from malignant pancreatic lesions and therefore cannot yet replace FNA. While elastography is a promising technique SR should not overrule the assessment by an experienced endosonographer. However, in less experienced hands SR might help in identifying malignant lesions.
Tobacco Carcinogen NNK (4-[Methylnitrosamino]-1-[3-Pyridyl]-1-Butanone) Induces Zymogen Activation In In-Vitro And In-Vivo Models of Acute Pancreatitis
S. Minervini, A. Raoof, C. Shugrue, F. Gorelick, T. Kolodecik, E. Thrower. Section of Digestive Diseases, VA Connecticut Healthcare, West Haven, & Yale University School of Medicine, New Haven, Connecticut.
A recent clinical study has demonstrated that cigarette smoking is a substantial risk factor for developing acute pancreatitis. The main carcinogen found in tobacco is NNK (4-[methylnitrosamino]-1-[3-pyridyl]-1-butanone). NNK has been shown to be a potent agonist of several signaling pathways. We hypothesized that NNK could directly mediate pancreatitis responses. The effects of NKK (0.1 nM-100 nM) were examined on isolated rat pancreatic acinar cells. We found that, at higher concentrations (10 and 100 nM) it caused a 3-fold and 5-fold activation of trypsinogen and chymotrypsinogen, respectively, over control. Furthermore, NNK pre-treatment sensitized acinar cells to cerulein (100 nM)-induced trypsinogen and chymotrypsinogen activation. NNK did not significantly affect amylase secretion when given alone or with cerulein. Similar effects of NNK on zymogen activation were seen in acinar cells treated with hyperstimulatory concentrations of the muscarinic agonist carbachol (1 mM). Potential targets of NNK include non-neuronal nicotinic acetylcholine receptors and β−adrenergic receptors; both types of receptors were detected in the acinar cell using PCR. Preliminary studies suggest that NKK may mediate acinar cell responses through a nicotinic and/or β−adrenergic receptor. Studies done in-vivo showed that NNK given to rats by IP injection (100mg/kg body weight) caused a 3-fold increase in both trypsinogen and chymotrypsinogen activation within 3 hours. These studies suggest that NKK might act directly on the pancreatic acinar cell to initiate pancreatitis responses.
The Proteome of Mesenteric Lymph in Health, Acute Pancreatitis and Shock: Implications for Clinical Trials
A. Mittal, M. Middleditch, K. Ruggiero, B. Loveday, J. A. Windsor, A. J. P. Phillips. Department of Surgery and School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Background: These studies are based on the concept that factors transported via the protein fraction of mesenteric lymph (ML) contribute to the development of organ dysfunction during critical illness. The aim was to define the ML proteome (MLP) in health (fed and fasted), acute pancreatitis (AP) and haemorrhagic shock (HS).
Methods: ML proteomic analysis was undertaken using iTRAQ™ and liquid chromatography -tandem mass spectrometry. The first experiment defined the MLP in health, the second in experimental AP and the third in HS.
Results: The first experiment identified 150 proteins including 26 hypothetical proteins. The most abundant protein classes identified in ML differed significantly from those reported for plasma and included protease inhibitors (16%) and proteins related to innate immunity (12%). In the second and thirds set of experiments 245 proteins were identified, including 35 hypothetical proteins. Eight proteins had a significant increase in their relative abundance in AP and 7 of these were pancreatic catabolic enzymes. Sixty proteins had a significant increase in their abundance in HS-ML. Potentially toxic pancreatic enzymes had increased abundance post-shock. A bioinformatics approach allowed for the identification of key pathways and a number of highly connected proteins.
Conclusions: This is the first detailed description of the proteome of normal and disease conditioned ML. This study provides the rationale for further research to investigate the efficacy of enteral protease inhibition in the treatment of AP. This study also identified key pathways and potentially important highly connected proteins in HS which will form the focus of future research.
Is the Prognosis Better After Resection for Invasive IPMN than for Pancreatic Cancer?
T. Moriya, L. W. Traverso. Department of General Surgery, Virginia Mason Medical Center, Seattle, WA.
Background: Some believe the prognosis after resection is better for invasive pancreatic intraductal papillary mucinous neoplasm (IPMN) than for pancreatic adenocarcinoma (Pan Ca).
Objective: To identify clinic-pathological features that might support a more favorable survival for invasive IPMN.
Methods: Between 1993 and 2009 we analyzed 210 patients who underwent pancreatic resection for IPMN of which 46 had invasive IPMN. We then compared their survival with 116 Pan Ca cases resected during the same time period using Log rank and Cox regression analysis. Finally a survival analysis was performed between cases with IPMN (n=40) versus Pan Ca (n=40) matched for tumor location and pTNM stage (AJCC pTNM 7th edition).
Results: For the matched analysis for location and TNM stage there was no significant difference in actuarial survival between the two groups (p=0.26) even though matched analysis showed more favorable histologic features for invasive IPMN: less poor tumor differentiation (18% for invasive IPMN vs. 38% for Pan Ca, p<0.05), lymphatic invasion (13% vs. 33%, p<0.05) and venous invasion (13% vs. 30%, p<0.05). After a median follow-up of 25 mo the 5 year actuarial survival was better for invasive IPMN (Invasive IPMN 57% vs. Pan Ca 30%, p=0.03). Invasive IPMN were resected in an earlier TNM stage (Stage IA, B; 26%, II A; 26%, II B, 46%) than Pan Ca (9%, 22%, 66%, p<0.01). After univariate analysis (Log rank) invasive IPMN had better survival than Pan Ca (p=0.03) which did not persist after multivariate analysis (Cox regression, p=0.076, 95% CI; 0.95-2.74).
- Invasive IPMN showed less aggressive histologically features than PancCa, even when matched by stage.
- Actuarial survival is better for invasive IPMN.
- Although Invasive IPMN was a significant prognostic variable for survival after univariate analysis, only a trend was observed after multivariate analysis.
Mucin Expression in Pancreatic Cancer and its Correlation With Aggressiveness and Cell Survival
N. Mujumdar, V. Sangwan, S. M. Vickers, A. K. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN.
Introduction: Pancreatic cancer, the fourth leading cause of cancer-related deaths in the United States, is one of the most lethal human malignancies. Hence, efforts to gain information on the pathobiology of pancreatic cancer and on molecular mechanisms related to invasion and metastasis are essential to generate effective treatments. Recent studies have shown that mucins that maintain the integrity and protect epithelial surfaces are overexpressed in carcinomas derived from epithelia including pancreatic cancer. The carboxy terminal of MUC1 (MUC1-C) inhibits apoptosis by translocating to the mitochondria and preventing the release of cytochrome c. However, the mechanism by which MUC1-C translocates to the mitochondria is unclear.
Aim: The aims of this study were to investigate: (i) whether the expression pattern of MUC1 correlates with the aggressiveness of pancreatic cancer, (ii) to examine the effect of downregulation of MUC1 on survival of pancreatic cancer cells, and (iii) to elucidate the mechanism by which MUC1 aids in survival of pancreatic cancer cells.
Methods: Expression of MUC1 was studied in different pancreatic cancer cell lines: MiaPaCa-2, Capan-1, BxPC-3, Hs766T, S2-013, S2-VP10 and Aspc-1 by q-PCR and by western blotting. MUC1 expression was down regulated using siRNA and assayed for cell viability (CCK8 Assay) and apoptosis (Caspase-3 activation).
Results: The present study shows that MUC1 is expressed in all the pancreatic cancer cell lines tested by both qPCR and western blotting. The levels of MUC1 are higher in the aggressive cell lines: S2-013, S2-VP10 and Aspc-1 than the others. A knock-down of MUC1 shows a decrease in cell viability after 48 h of transfection. The decrease in cell viability is associated with a slight increase in the parameter of apoptosis, caspase-3 activation.
Conclusions: Thus, our study shows that MUC1 is expressed in pancreatic cancer cell lines at both RNA and protein levels. The expression pattern correlates with the aggressiveness of the cell lines; more aggressive cell lines (S2-013, S2-VP10 and Aspc-1) show a higher expression of MUC1. Also, MUC1 expression is essential for the survival of pancreatic cancer cells. Future studies will be aimed at elucidating the mechanism by which MUC1 expression inhibits cell death in pancreatic cancer cells which will help to develop therapeutic strategies.
Protective Effects of Caffeine in an Experimental Biliary Pancreatitis Model
R. Mukherjee,1,3 W. Huang,1,3 M. Cane,3 V. Elliott,2 A. Tepikin,3 D. N. Criddle,3 R. Sutton.1,21Division of Surgery & Oncology; and 2NIHR Pancreatic Biomedical Research Unit, Royal Liverpool University Hospital, UK; 3Department of Physiology, University of Liverpool, UK.
Introduction: Our previous work showed that caffeine protects against cerulein-induced pancreatitis & also inhibited pathological cytosolic Ca2+ ([Ca2+]C) spikes in isolated acinar cells hyperstimulated by cholecystokinin. Our present study investigated the effects of caffeine on acinar cell [Ca2+]C responses to taurolithocholic acid 3-sulfate (TLC-S) & the severity of experimental acute biliary pancreatitis.
Methods: Confocal fluorescence microscopy was used to assess changes in [Ca2+]C, in isolated murine pancreatic acinar cells in response to 500μM TLC-S. Acute pancreatitis was induced in CD1 mice by retrograde pancreatic duct perfusion of 3mM TLC-S. Caffeine at a dose of 25 mg/kg/h×7 was given at 1 h after induction of pancreatitis & severity was assessed by standard biomarkers & histopathology. Serum caffeine levels were also measured at various time points using mass spectrometry.
Results:In vivo, caffeine significantly reduced the severity of pancreatitis (p<0.05) as evidenced by decreased serum amylase level, pancreatic trypsin activity, pancreatic myeloperoxidase activity & blinded histopathological scores. The peak serum caffeine concentration tested was ∼ 1mM. In vitro, caffeine at 10mM (a known concentration to inhibit inositol 1,4,5-trisphosphate receptor) greatly inhibited the [Ca2+]C plateau induced by bile salt hyperstimulation, whereas 1mM had no significant effect.
Conclusion: Like cerulein induced pancreatitis, caffeine is also protective in experimental biliary pancreatitis. This effect may not to be through the modulation of [Ca2+]C but rather caffeine's other inhibitory effects.
Alisporivir (Deb025) Prevents Necrosis in and Decreases the Severity of Experimental Pancreatitis
R. Mukherjee,1,2 W. Huang,1,2 A. V. Tepikin,1 A. Gukovskaya,3 D. N. Criddle,1 R. Sutton.21Physiological Laboratory, University of Liverpool, 2NIHR Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK; 3VA Greater Los Angeles Healthcare System and University of California Los Angeles, CA, USA.
Introduction: Cytosolic calcium overload induces the mitochondrial permeability transition pore (mPTP) and a decline of ATP production in acute pancreatitis. We have previously reported that mPTP inhibition by cyclophilin-D knockout prevents pancreatic acinar cell necrosis and ameliorates acute pancreatitis. Here we have tested the novel non-immunosupressive cyclophilin inhibitor Alisporivir (DEB025) in experimental murine acute pancreatitis.
Methods: Pancreatitis was induced by retrograde ductal bile acid infusion or by seven cerulein injections (7×50 μg/kg ip), either without or with Alisporivir (DEB025) (10 mg/kg or 100 mg/kg) one h after bile acid or on the third cerulein injection. A standard range of parameters was assessed at set time points thereafter (n≥5 all groups), including plasma amylase, IL6, pancreatic trypsin, myeloperoxidase and pancreatic histopathology.
Results: All parameters were significantly reduced by Alisporivir (DEB025) in both models (p<0.05), more effectively by the lower dose. Plasma amylase, pancreatic necrosis and overall histopathology scores were no or little different from normal values.
Conclusion: MPTP inhibition with Alisporivir (DEB025) prevents necrosis in and greatly reduces the severity of acute pancreatitis.
Combined Inhibition of Src and EGFR Abrogates Tumor-Stromal Interaction and Angiogenesis in Pancreatic Cancer In Vivo
N. S. Nagaraj, F. Revetta, M. K. Washington, N. B. Merchant. Departments of Surgery and Pathology, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN.
Background: Evidence exists for interactions between pancreatic cancer cells and stromal fibroblasts that affect angiogenesis and the invasive phenotype of pancreatic cancer. Oncogenic collaboration between Src and EGFR in cell transformation is well established. The purpose of this study was to determine whether Src and EGFR inhibition can regulate tumor-stromal interaction and angiogenesis in pancreatic cancer.
Methods:In vivo effects of the Src inhibitor dasatinib and EGFR inhibitor erlotinib treatment was determined on mouse xenografts of BxPC3 cells. Immunohistochemical staining for secreted protein acidic and rich in cysteine (SPARC)/Osteonectin, fibronection, VEGF, phospho-STAT3 and CD31 was performed on pancreatic cancer xenografts treated with Src and EGFR inhibitors in vivo.
Results: Combined inhibition of Src and EGFR with gemcitabine inhibits constitutively activated STAT3 signaling and decreases nuclear expression of phosphorylated STAT3 in vivo. Blocking STAT3 activation decreases expression of VEGF as well as fibronectin which promote angiogenesis. Furthermore, significant decreases in CD31 expression and microvascular density are also seen. SPARC, which strongly characterizes the tumor-associated stroma was also significantly decreased in treated xenografttissue compared with vehicle treated controls.
Conclusion: These results provide evidence that combined targeted biological therapy targeted against Src and EGFR in addition to cytotoxic chemotherapy can inhibit tumor-stromal interaction and angiogenesis in pancreatic cancer through a STAT3 mediated mechanism.
Serous Cystadenoma of the Pancreas Showing Enlargement on Imagins: Report of a Case
M. Nakamoto, N. Minagawa, A. Wada, A. Higure, K. Yamaguchi. Department of Surgery 1, School of Medicine, University of Occupational and Environmental Health, Japan.
We report a rare case of serous cystadenoma (SCA) of the pancreas that showed enlargement in size, from 24mm to 35mm, on imaging during 14- month follow-up.
In January of 2009, a 58-year old Japanese woman underwent total hysterectomy and bilateral oophorectomy for thecoma of the ovary. Preoperative abdominal computed tomography showed massive ascites (Meigs syndrome) and a multilocular cyst of the head of the pancreas, 24 mm in diameter, without mural nodules therein. In March of 2010, follow-up CT displayed an enlargement of the cyst of the pancreas, up to 35 mm. There was no ascites. Ultrasonography and magnetic resonance images showed a multilocular cyst in the head of the pancreas, 35 mm in the diameter. Endoscopic retrograde cholangiopancreatography (ERCP) showed a displacement of the main pancreatic duct by the cyst and no communication between the cyst and pancreatic ductal system. Endoscopic ultrasonography showed mural nodules within the cyst. Pylorus preserving pancreatoduodenectomy was performed under the tentative diagnosis of possible intraductal papillary mucinous neoplasm (IPMN) of branch duct showing enlargement in size and the appearance of mural nodule therein. Pathological examination showed multilucular cyst lined by a single layer of simple cuboidal epithelium having glycogen in the cytoplasm. Mitoses were rarely seen. The final pathological diagnosis was SCA of the pancreas.
In the present case, SCA of the pancreas showed the enlargement in size, from 24 mm to 35 mm, by the follow-up imaging during 14 months. The enlargement of the cyst on imaging may be related with the decompression of the abdomen by the disappearance of massive ascites (Meigs syndrome).
Outcome of Laparoscopic Spleen Preserving Distal Pancreatectomy With Special Reference to the Lateral Approach
M. Nakamura, T. Ohtsuka, S. Takahata, S. Shimizu, M. Tanaka. Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Aim: To evaluate the outcome of laparoscopic spleen-preserving distal pancreatectomy (Lap-SPDP) with special reference to the lateral approach.
Backgrounds: Lap-SPDP preserving the splenic artery and vein is complicated and challenging, because the splenic vein is often embedded in the sinus on the posterior side of the pancreatic body. Here we show the lateral approach of SPDP (LA-SPDP), and the outcome of Lap-SPDP.
LA-SPDP: Based on the anatomical feature that the pancreatic tail makes no sinus around the splenic vessels, the removal of the pancreas from the splenic artery and vein is started from the lateral end of the pancreas. The pancreatic artery is temporarily obliterated by a vascular clamp, and pancreatic parenchyma is cut by a linear stapler.
Patients: Eighteen patients underwent Lap-SPDP in Kyushu University. The splenic artery and vein were preserved in 11 patients (AV-pre) and excised in 7 patients (AV-ex; Warshaw's procedure).
Results: Eleven AV-pre patients did not show any dilated gastric mural veins (GMVs), while 5 out of 7 AV-ex patients showed engorgement of GMVs by CT. All the patients with engorged GMVs underwent endoscopic examination. Unexpectedly, only one patient showed mild gastric varices by endoscopy.
Discussion: SPDP preserving the splenic vessels is a physiological method, while SPDP excising the splenic vessels may cause GMV varices. However, CT signs of engorged gastric veins are not correlated with endoscopic findings. The clinical significance of GMV varices may be less than their appearance on CT.
Conclusion: Vessel-preserving SPDP is a realistic method, although it is not easy for laparoscopic surgery. Our LA-SPDP technique may contribute to make it easier for laparoscopic surgery.
The Role of Innate Immunity in the Pathogenesis of Experimental Autoimmune Pancreatitis in Mice
A. Nishio, M. Yamashina, S. Nakayama, K. Uchida, T. Fukui, K. Okazaki. Third Department of Internal Medicine, Kansai Medical University, Moriguchi, Japan.
Background & Aim: Autoimmune pancreatitis (AIP) is a discrete entity of pancreatitis that is characterized by a steroid-responsive, fibroinflammatory condition that often involves multiple organs. However, little is known about the precise pathogenesis of AIP. The aim of the study was to determine the role of innate immunity in the development of AIP using murine models in which pancreatitis was induced by toll-like receptor (TLR) stimulation.
Methods: Six-week-old female MRL/Mp mice were injected intraperitoneally with polyinosinic polycytidylic acid (poly I:C) or lipopolysaccharide (LPS) at doses of 5 mg/kg body weight twice weekly for 12 weeks. The mice were killed and the severity of pancreatitis was graded using a histological scoring system. Serum cytokine levels of mice with pancreatitis and mice that were given a single injection of TLR ligands were measured using enzyme-linked immunosorbent assays. The effect of TLR stimulation on the development of pancreatitis was also examined using C57BL/6 interleukin (IL)-10-deficient mice.
Results: Administration of poly I:C accelerated the development of pancreatitis in MRL/Mp mice, but LPS did not. Serum levels of IL-10 and IL-12 were significantly elevated in mice with AIP. A single injection of LPS markedly increased serum levels of IL-10, interferon-γ, tumor necrosis factor-α and IL-12 compared with those of poly I:C-treated mice. Treatment with poly I:C and LPS induced pancreatitis in IL-10-deficient mice, but not in wild-type mice.
Conclusions: Repeated stimulation of innate immunity induces autoimmunity in the pancreas of mice via an imbalance between proinflammatory and anti-inflammatory cytokines.
Clinical Significance of Serum Alkaline Phosphatase Level in Advanced Pancreatic Cancer
I. Ohno,1 S. Mitsunaga,1 K. Nakachi,1 S. Shimizu,1 H. Takahashi,1 H. Okuyama,1 Y. Kojima,2 A. Ochiai,1 H. Ueno,3 C. Morizane,3 S. Kondo,3 T. Okusaka,3 M. Ikeda.11Division of Hepatobiliary and Pancreatic Oncology, National Cancer Center Hospital East, Kashiwa, Japan; 2Department of Gastroenterology, National Center for Global Health and Medicine, Tokyo, Japan; 3Division of Hepatobiliary and Pancreatic Oncology, National Cancer Center Hospital, Tokyo, Japan.
Background: Alkaline phosphates (ALP) is a enzyme elevated by various hepatobiliary diseases. And it has been reported to be an important prognostic factor for several cancers and often thought to indicate bile stasis caused by liver metastasis. The aim of the study was to determine the significance of elevated serum ALP as a prognostic factor in patients with advanced pancreatic cancer (APC).
Methods: Serum ALP levels were measured in 393 patients with APC receiving gemcitabine monotherapy before treatment, and according to those levels, patients were subgrouped. Then we analyzed clinical data of each group to see characteristics of high ALP patients and examined the relationship between ALP level and survival, response to the treatment.
Results: High ALP group included worse performance status (PS>1) patients (41.3%, p=0.001), and associated with low serum albumin (3.31±0.38, p<0.01). High ALP group (Median Survival Time(MST) 112days) showed significantly worse prognosis compared to the normal ALP group (MST 217days)(HR=2.27, P<0.001). Multivariate analysis revealed elevated ALP (HR=2.08, P<0.001), CRP (HR=1.81, P<0.001), and ascites (HR=1.79, P<0.001) were independent prognostic factors. Similar results were seen in liver metastasis free patients without jaundice.
Conclusion: High serum ALP level correlated with poor performance status and low serum albumin. ALP was also the independent prognostic factor in patients with APC.
Predictive Factors for Lymph Node Metastasis in Intraductal Papillary Mucinous Neoplasms of the Pancreas With Mural Nodules
T. Ohtsuka, Y. Sadakari, K. Kobayashi, S. Takahata, M. Nakamura, K. Mizumoto, M. Tanaka. Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: Little is known about the frequency of lymph node metastasis in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas, and we have not been able to determine how much lymph node dissection is necessary in individual cases. The aim of this study was to investigate the predictive factors for lymph node metastasis in IPMNs.
Methods: Medical records of 120 patients pathologically diagnosed as having IPMN were reviewed, and 16 possible predictive factors regarding lymph node metastasis were analyzed.
Results: Lymph node metastasis was observed in 7 patients (6%), all of whom were diagnosed as having mural nodules preoperatively. Sensitivity, specificity, and accuracy of preoperative imaging for detecting mural nodules of IPMNs in this study were, 84%, 97%, and 90%, respectively. Univariate analysis using 61 patients having mural nodules preoperatively revealed that the size of mural nodules >/=10mm and positive imaging findings for invasive tumor and possible lymph node metastasis were significant predictive factors for lymph node metastasis. Multivariate analysis demonstrated that only imaging finding for invasive tumor was an independent significant predictive factor. Positive and negative predictive values of the preoperative imaging finding for invasive IPMNs for lymph node metastasis were 50% and 98%, respectively.
Conclusions: Standard lymph node dissection would be recommended in IPMNs with mural nodules demonstrating preoperative imaging findings for invasive carcinomas.
Patients With Acute Pancreatitis (AP) Complicated by Organ Dysfunction Show Abnormal Lymphocyte Signaling
J. Oiva,1 H. Mustonen,1 M.-L. Kylänpää,1 L. Kyhälä,1 S. Siitonen,2 E. Kemppainen,1 H. Repo,2 P. Puolakkainen.11Department of Surgery, Helsinki University Central Hospital; 2Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland.
Aim: To outline aberrations in lymphocyte signaling profiles in AP patients with organ dysfunction.
Patients and methods: Study comprises 13 patients with AP admitted to intensive care unit at Helsinki University Central Hospital. All patients had organ dysfunction (acute respiratory distress syndrome in 12, renal dysfunction in eight). 13 healthy volunteers served as reference subjects. Levels of phosphorylated NF-kB (pNF-kB), p38 (pp38) and STAT3 (pSTAT3) were studied by whole blood flow cytometry.
Results: Patients, as compared to healthy subjects, showed lower levels of pNFκB induced by TNF in all lymphocytes (5.3±0.4 RFU vs 10.9±1.2 RFU, p<0.001), CD4+ lymphocytes (4.9±0.8 RFU vs 7.6±0.7 RFU, p<0.05) and CD8+ lymphocytes (6.2±1.0 RFU vs 10.0±0.8 RFU, p<0.05), while pp38 levels were higher in patients' lymphocytes (3.4±0.5 vs 2.5±0.1 RFU, p<0.05). In non-stimulated samples patients' lymphocytes had higher pSTAT3 levels than did control cells (5.8±1.0 RFU vs 2.4±0.1 RFU, p<0.001). The histograms were biphasic indicating presence of activated cells and subgroup analysis showed constitutive STAT3 activation in CD4+ and CD8+ but not in CD19+ lymphocytes.
Conclusion: Lymphocytes of AP patients with organ dysfunction show impaired NFκB activation, which may increase susceptibility to secondary infections, increased p38 activation capacity, which may sustain systemic inflammation and constitutive STAT3 activation, denoting on-going anti-inflammatory reaction. Monitoring of lymphocyte signaling may aid to find new therapeutic approaches and predictors of outcome of AP with organ dysfunction.
Pathologic Protease Activity is Reduced in Calcineurin-A Beta Knockout Mice
A. I. Orabi, M. U. Ahmad, A. U. Shah, Z. M. Mannan, S. Z. Husain. Department of Pediatrics, Yale University School of Medicine, New Haven, CT.
The premature activation of digestive proenzymes within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathologic rise in cytosolic Ca2+. Further, we have previously shown that a target of aberrant Ca2+ in acinar cells is the Ca2+/calmodulin-dependent phosphatase calcineurin (CN). CN inhibition reduced the severity of pancreatitis both in vitro and in vivo. In this current study, we hypothesized that CN within acinar cells is responsible for the initiation of the pancreatitis response. We obtained mice deficient in the pancreas-predominant isoform -A beta and examined whether acinar cells from these mice were protected from pancreatitis. Acinar cells were freshly isolated from calcineurin-A beta (CN-Aβ) deficient mice. PCR was performed on RNA extracts. Live cells were stimulated for 1 hr with high concentrations of the Ca2+-activating secretagogues caerulein (100 nM; CCK analog) and carbachol (1 mM; Ach analog). Acinar cells lacking CN-Aβ exhibited a 60% and 93% reduction in chymotrypsin activity when stimulated with caerulein and carbachol, respectively (n=3; P<0.05). No significant differences in amylase secretion were observed. Interestingly, total enzyme content per volume of cells was reduced as well as zymogen granule area in pancreatic tissue sections. Nevertheless, chymotrypsin activity relative to total chymotrypsin content was still significantly less in the CN-Aβ knockout mice compared with wildtype. These data suggest that the acinar cell predominant CN isoform, CN-Aβ, mediates pathologic protease activity during pancreatitis and may serve as an important target of the aberrant acinar cell Ca2+ rise associated with this disease.
Inositol 1,4,5-Trisphosphate Receptor Type 2 Deficiency Leads to Accumulation of Zymogen Granules in Pancreatic Acinar Cells
A. I. Orabi, Z. M. Mannan, A. U. Shah, S. Z. Husain. Department of Pediatrics, Yale University School of Medicine, New Haven, CT.
Calcium signals are critical to physiologic secretion of pancreatic enzymes from the pancreatic acinar cell. The primary calcium channel in the secretory, apical pole of the acinar cell is the intracellular ER-bound inositol 1,4,5-trisphosphate receptor, or IP3R. The type 2 isoform, or IP3R2, contributes about half of IP3R expression. In this study, we examined the role of IP3R2 in maintaining acinar cell secretion by using mice deficient in IP3R2. On hematoxylin and eosin staining as well as electron microscopy of pancreatic tissues, IP3R2-deficient acinar cells had a greater number of zymogen granules (ZGs). They also appeared to occupy a larger cross-section of the acinar cell. By immunoblot, amylase was increased by 2 fold, compared to wildtype (P<0.05), whereas insulin levels were unchanged. By activity measurement, amylase content was similarly increased in these cells. However, acinar cell amylase secretion after secretagogue stimulation (with either a CCK analog or an Ach analog) or at baseline within a one hour incubation period was unchanged between IP3R2-deficient and wildtype cells. Nevertheless, electron microscopy showed morphological evidence of basolateral exocytosis. Further, serum levels of amylase at baseline in the IP3R2 deficient mice were 40% higher than in wildtype mice (P<0.05). In summary, we demonstrate that IP3R2 deficiency causes an accumulation of zymogen granules. The effect is likely a result of reduced exocytosis over a long term period, but our short term secretion studies could not identify this defect. The defect results in pathologic basolateral exocytosis. In conclusion, these data implicate IP3R2 in calcium-dependent pancreatic enzyme secretion.
C-Src Resides on the Golgi in Acinar Cells and is Involved in Caerulein Mediated Trypsinogen Activation
L. S. Orlichenko,1 C. J. Baty,2 J. Karlsson,2 V. P. Singh.1Departments of 1Medicine; 2Cell Biology & Physiology, University of Pittsburgh, Pittsburgh, PA.
Introduction: Pancreatic acinar cells express several members of the Src non receptor tyrosine kinase family. Intra-acinar trypsinogen activation can damage these cells during pancreatitis. Our preliminary studies showed that Src inhibition reduces caerulein mediated trypsinogen activation. We therefore studied the mechanisms by which Src mediates this phenomenon and its role in acinar cell injury.
Methods: Mouse pancreatic acinar cells were treated with caerulein (CER) or 100□M pervanadate (PV) with and without dasatinib (10 □M), a specific Src inhibitor. Src activation was measured in immuno precipitates using an active Src antibody by western blotting (WB). Amylase release, trypsinogen activation (arbitrary units/□g DNA) and lactate dehydrogenase (LDH) leakage were studied. Intracellular calcium (Cai) levels were studied using Fura-2AM. Golgi morphology was studied by immunostaining (P115). Src family members were localized using immunostaining and WB on subcellular fractions.
Results: Supramaximal CER (100nM) and PV induced Src activation, Golgi expansion, trypsinogen activation (2.6, 3.7 fold basal respectively) and LDH leakage (2.8, 1.8 fold basal respectively). PV did not affect Cai. Dasatinib significantly reduced trypsinogen activation (75% and 80% respectively), LDH leakage (55% and 70% respectively) and minimized Golgi morphology changes without affecting caerulein induced amylase release. C-Src but not Yes localized to the Golgi by immunostaining and subcellular fractionation.
Conclusions: Src activation results in, and its inhibition prevents trypsinogen activation and acinar cell injury via altering the Golgi. C-Src localization to the Golgi and may support its role in these phenomena.
Brefeldin A Causes Retrograde Collapse of the Golgi, Prevents Co-Localization, Trypsinogen Activation, and Reduces Severity of Pancreatitis
L. S. Orlichenko, V. P. Singh. Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, PA.
Background: While the role of trypsin generation is well established in the pathogenesis of pancreatitis, the organelle/s contributing to this are unclear. Trypsin generation depends on cathepsin B and previous studies show that they co-localize in vacuoles adjacent to the trans-Golgi. We therefore used brefeldin A (BFA) to determine the role of the Golgi in trypsinogen activation.
Methods: Mice Pancreatic acini were pre-incubated with or without 50μM BFA, and then stimulated with caerulein (CER). Trypsin generation (arbitrary units/μg DNA), Golgi morphology (P115 immuno-staining), Cathepsin B localization (sub-cellular fractionation), F-actin localization (TRITC-phalloidin staining), amylase secretion were determined. BFA (25 mg/kg) or vehicle (0.1ml 50% DMSO) were given intraperitoneally (IP) and severity of CER (50μg/kg, hourly × 10, IP) induced pancreatitis was determined by serum amylase, pancreatic edema, histology.
Results: BFA significantly (p<0.05) reduced 100nM CER induced trypsin generation (2.27 vs.1.03 fold basal), reduced the zymogen to lysozomal ratio of cathepsin B (1.08 vs. 0.52), while causing retrograde collapse of the Golgi, without affecting CER induced basolateral F-actin reorganization or amylase dose response curve. In vivo, BFA dramatically reduced serum amylase, pancreatic edema and pancreatic necrosis (12.9 vs.5.2 fold basal, 83.8% vs. 76%, 15% vs. 1.4% with CER+ vehicle and CER+BFA respectively, p<0.01).
Conclusions: Antegrade trafficking from the Golgi contributes to intra-acinar trypsin generation. This suggests that post Golgi vesicular compartments contribute to formation of the "co-localized vacuole". Targeting these ameliorates pancreatitis.
Impact of EUS-FNA on Surgical Decision Making in Pancreatic Neuroendocrine Tumors (PNETs)
A. Panossian,1 K. Gill,1 H. Bertani,1 C. De Angelis,3 E. Dabizzi,1 J. Stauffer,2 M. Grewal,2 J. Nguyen,2 M. Wallace,1 T. Woodward,1 M. Raimondo.11Division of Gastroenterology & 2Division of Transplant Surgery, Mayo Clinic Florida, Jacksonville, FL; 3Division of Gastroenterology, Ospedale Le Molinette, Turin Italy.
Background & Aim: PNETs are rare and usually require surgical excision. Our aim was to evaluate the influence of EUS-FNA on the surgical decision-making in PNETs.
Methods: Databases at 2 Institutions were reviewed between 2000 and 2009 to identify all patients with PNETs. EUS findings were compared to CT, MRI, and nuclear scan findings.
Results: 68 patients (mean age 62 yrs, [28-85], 33 male) underwent EUS-FNA of pancreatic lesions. Mean tumor size on EUS was 21.6 mm (2.7-103 mm). Tumor location was: 9 uncinate process, 24 head, 26 body, 22 tail. 35 patients underwent surgery (12 whipples, 19 distals, 1 partial head resection, 3 enucleations); 33 had no surgery (16 metastatic disease, 2 observation, 1 died, 13 awaiting surgery). 18% of CT scans, 14% of MRI's, and 50% of nuclear scans failed to show any tumor. FNA was negative or inconclusive in 12 patients, 9 of whom had NET on surgery while 3 did not have surgery. In 8 patients (12%) who were shown to have a PNET by EUS, no other imaging technique showed a tumor. One patient underwent surgery after EUS excluded metastasis suspected on CT, and 3 patients did not have surgery after EUS showed vascular invasion or metastasis undetected by CT.
Conclusion: EUS-FNA impacted the surgical management of PNETs in 12/68 (18%) patients by detecting tumors undetected by other imaging modalities in 8 patients, excluding metastases in 1 patient, and detecting metastases in 3 patients. Our data suggests that despite improvements in radiological techniques, EUS significantly influences the clinical decision making of suspected PNETs.
Self-Expandable Metalstent for Walled-Off Necrosis
Á. Pap, M. Burai, T. Tarpay. Department of Gastroenterology, National Institute of Oncology, Budapest, Hungary.
There are 2 ways to treat pancreatic pseudocysts with endoscopy: transmural and transpapillary. In case of walled-off necrosis with debris or pus it is recommended to implant at least two 10F stents, and a nasocystic drain transmurally for continuous lavage. A common complication is the occlusion of the drains.
Case reports: A 35 years old male presented with acute pancreatitis. In some weeks a 10cm diameter fluid collection developed with presence of debris. EUS guided drainage was performed with balloon dilation and implantation of two 10F pigtail stents, a nasocystic drain and a jejunal feeding tube. No regression but high fever occurred soon thus another ERCP with further two 10F stents placement and sampling for bacterial culture were done. In spite of tailored antibacterial treatment the fever remained, so we decided to change the stents for a 2.2cm wide uncovered intestinal metal stent with nasocystic drain. After 8 weeks with continuous lavage the walled-off necrosis regressed and we removed the stent without complication.
A 75 years old female was admitted to the hospital for acute necrotising pancreatitis some weeks earlier. Walled-off necrosis developed at the tail of pancreas with sepsis, At EUS the irregular fluid collection was punctured at the fundus evacuating pure pus. Using a stiff guidwire we have dilated the opening with a 6-10F dilator and placed a 10mm diameter covered metal stent then a 5F nasocystic drain into the cyst for lavage. At the same time another guidewire was introduced into the jejunum through the working channel of the scope for jejunal feeding tube placement. The patient become feverless immediately and recovered gradually.
In case of walled-of necrosis with/without sepsis a large communication between the intestine and cyst should be sustained with selfexpandable metal stent. Supplementary nasocystic drain and jejunal feeding tube implantation with tailored, systemic antibiotic treatment should be considered, too.
S100A10 Increases Pancreatic Cancer Cell Resistance to Gemcitabine Treatment by the Inhibition of VMP1-Mediated Autophagy
R. Pardo, D. Grasso, M. I. Molejon, J. L. Iovanna, M. I. Vaccaro. Pathophysiology, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina.
Autophagy is a degradation process of cytoplasmic cellular constituents. The pancreatitis-induced vacuole-membrane-protein-1 (VMP1) triggers autophagy in mammalian cells. Recently we showed that Gemcitabine, the standard chemotherapy agent for pancreatic cancer, induces VMP1-mediated autophagy in human pancreatic cancer cells leading to apoptotic cell death. To study this VMP1 pathway we used the two-hybrid strategy. We found that VMP1 interacts with S100A10, a member of the S100 family proteins, which is overeexpressed in pancreatic cancer. VMP1-S100A10 interaction was confirmed using pulldown assay. The real time RT-PCR assay showed that S100A10 mRNA expression is higher in MIAPaCa-2 cells than in HeLa cells. S100A10 was induced under starvation and gemcitabine treatment in HeLa cells, but not in MIAPaCa-2 cells, suggesting that its expression is unregulated in pancreatic cancer cells. The effect of S100A10 overexpression on VMP1-induced autophagy in MIAPaCa-2 cells was evidenced by the recruitment of microtubule-associated protein-1 light-chain-3 (LC3) and we found that S100A10 overexpression reduces the formation of autophagosomes induced by gemcitabine treatment or by VMP1 overexpression. Overexpression of S100A10 modified VMP1 subcellular localization, changing the punctated pattern to a reticular distribution resembling endoplasmic reticulum structure. S100A10 knockdown using shRNA increased apoptosis in gemcitabine-treated cells evidenced by flow cytometry after annexin V/7AAD labeling and by caspase-3 activity assay. We conclude that the expression of S100A10 reduces VMP1-mediated autophagy and increases human pancreatic cancer cell resistance to gemcitabine treatment.
Differences in Migration Between PDAC Stem Cells and Non-Stem Cells
J. Park, D. Irimia, M. Toner, A. Liss, C. Fernández-del Castillo, A. L. Warshaw, S. P. Thayer. Dept. of Surgery, MGH & HMS, Boston, MA.
Background: Cancer stem cells are likely responsible not only for tumor initiation and growth, but possibly also for metastasis. Their ability to migrate may be a key mechanism promoting metastasis. Our aim was to determine whether PDAC stem cells are more capable of migration than non-stem cells and to determine the characteristics of that migration.
Methods: Using a microfluidic device, cell migratory characteristics were measured in non-stem cells vs. stem cells derived from PDAC cell lines. PDAC cells were sorted by FACS to identify two populations of putative stem cells, CD133+ESA+ or CD44+CD24+ESA+. Cell migration/mobility was captured by time-lapse photography every 20 min over 48 hrs.
Results: The percentages of cells migrating were significantly higher in PDAC non-stem cells compared to PDAC stem cells. Only 0.3% of CD133+ vs. 43% of CD133-cells were capable of migration. Similarly, 0.5% of CD24+CD44+ vs. 44% CD24-CD44-cells were capable of migration. Stem cells also had statistically significant lower velocities. Median velocities for stem cells were 2μm/hr vs. the non-stem cell compartments, where median velocities were 36μm/hr (p=0.02). Stem cells also had a significant time delay prior to migration. Non- stem cells had a median delay of 3hrs vs. 48hrs in the stem cell compartment (p=0.001).
Conclusion:Stem cells and non-stem cells retain the ability to migrate. PDAC non-stem cells had significantly higher numbers of cells capable of migration, faster velocities and shorter time delays to migration. This phenomenon implies that PDAC non-stem cells may play the major role in migration and metastasis of cancer cells. Further examination of the migratory characteristics of these populations may provide useful insights into the biology of cancer cell dissemination.
Comparative Proteomic Analysis (GeLC-MS/MS) Identifies Differentially-Expressed Proteins in Secretin Stimulated Endoscopic (ePFT) Collected Pancreatic Fluid
J. Paulo,1,2,3 P. A. Banks,1,3 D. L. Conwell,1,3 H. Steen.2,31Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital, Boston, MA; 2Proteomics Core Lab, Children's Hospital Boston, Boston, MA; Harvard Medical School, Boston, MA.
Background: The diagnosis of chronic pancreatitis (CP) is based currently on defined functional, morphological, and histological features. Proteomics may offer an alternate strategy to further affirm traditional CP diagnoses. Variations in the protein expression in pancreatic fluid may reflect the underlying mechanisms of disease that have yet to progress to fibrosis and/or pancreatic insufficiency, thus providing a means for the amelioration or retardation of the disease.
Aim: We aim to identify differentially-expressed proteins in secretin stimulated ePFT-collected pancreatic fluid from individuals with non-pancreatitis-related chronic abdominal pain (CAP) and those with CP.
Method: We performed a mass spectrometry (GeLC-MS/MS)-based differential proteomic analysis of pancreatic fluid from cohorts with CAP (n=9) and advanced CP (n=9).
- 1) We identified a total of 1389 proteins in 18 pancreatic fluid samples.
- 2) Spectral counting and statistical analysis thereof revealed 41 and 93 proteins as statistically up- and down regulated, respectively, in the pancreatic fluid of individuals with CP.
- 3) As expected, the largest percentage of differentially-regulated proteins are secreted / extracellular in origin.
- 4) Proteins down-regulated in our CP cohort were mainly classified as proteases, corresponding to the pancreatic insufficiency that is associated with chronic pancreatitis.
Conclusions: Our workflow resulted in the identification of potential biomarkers of chronic pancreatitis for use in further validation and provides a high-throughput approach for the further study of the pancreatic fluid proteome.
Mass Spectrometry-based Proteomic Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) Pancreatic Tissue
J. Paulo,1,2,3 P. A. Banks,1,3 H. Steen,2,3 D. L. Conwell.1,31Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; 2Proteomics Core Lab, Children's Hospital Boston, Boston, MA; Harvard Medical School, Boston, MA.
Background: Formalin-fixed and paraffin-embedded (FFPE) tissue is the standard method of sample preservation for hospital pathology departments. For retrospective biomarker investigation, FFPE tissue banks offer a vast resource of histologically-characterized specimens.
Aim: We aim to establish LC-MS/MS analysis of FFPE pancreatic tissue as a suitable option for the study of the pancreas proteome.
Method: We investigated the proteomic profile of FFPE pancreatic tissue specimens, using LC-MS/MS, from 9 archived samples that were histologically-classified as: no pancreatic disease (NP; n=3), chronic pancreatitis (CP; n=3) and pancreatic cancer (PC; n=3).
- We identified 525 non-redundant proteins from the 9 samples.
- Using our filtering criteria, 44, 13, and 18 unique proteins were identified in normal, chronic pancreatitis, and pancreatic cancer specimens, respectively.
- Unique NP proteins include: spink 1, retinol dehydrogenase, and pancreatic enzymes (elastase, colipase, carboxypeptidase A1, pancreatic triglycerol lipase).
- Unique CP proteins include: collagen α1 and α3, as well as immunoglobulin SNC73.
- Unique PC proteins include: annexin 4A, filamin A, and fibronectin.
Conclusions: We have shown that mass spectrometry-based FFPE tissue analysis is applicable to the study of pancreatic tissue. In addition, we have identified differentially-expressed proteins among FFPE tissues specimens originating from individuals with different pancreatic histologies.
Sample Preparation of Secretin Stimulated Endoscopic (ePFT) Collected Pancreatic Fluid for Proteomic Analysis
J. Paulo,1,2,3 Bechien Wu,1 P. A. Banks,1,3 D. L. Conwell,1,3 H. Steen.2,31Center for Pancreatic Disease, Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women's Hospital, Boston, MA; 2Proteomics Core Lab, Children's Hospital Boston, Boston, MA; Harvard Medical School, Boston, MA.
Background: The standardization of methods for human body fluid protein isolation is a critical initial step for proteomic analyses aimed to discover clinically-relevant biomarkers. Several caveats have hindered pancreatic fluid proteomics, including the heterogeneity of samples and protein degradation.
Aim: We aim to optimize sample handling of pancreatic fluid that has been collected using a safe and effective endoscopic collection method (ePFT).
Method: Using SDS-PAGE protein profiling, we investigate:
- 1) precipitation techniques to maximize protein extraction,
- 2) auto-digestion of pancreatic fluid following prolonged exposure to a range of temperatures,
- 3) effects of multiple freeze-thaw cycles on protein stability, and
- 4) the utility of protease inhibitors.
Results: Our experiments revealed that:
- 1) trichloroacetic acid (TCA) precipitation resulted in the most efficient extraction of protein from pancreatic fluid of the 8 methods we investigated,
- 2) although auto-digestion of proteins is prevalent at 23°C and 37°C, incubation on ice significantly slows such degradation,
- 3) when the sample is maintained on ice, proteolysis is minimal during multiple freeze-thaw cycles, and
- 4) the addition of protease inhibitors is assay-dependent.
Conclusions: Application of our optimized methodology to pancreatic fluid from various pancreatic disease states revealed robust and reproducible protein banding patterns via SDS-PAGE analysis. Our optimized sample preparation methods can be utilized in future studies further investigating the proteome of pancreatic fluids.
PRSS1, SPINK1 and CFTR Mutations in Families and Unrelated Subjects With Idiopathic Early Onset Chronic (CP) or Recurrent Acute Pancreatitis (RAP) in Mexico City
M. Pelaez-Luna,1 G. Robles-Diaz,1,2 S. Canizales,3 M. T. Tusie-Luna.31Experimental Medicine Unit, School of Medicine-Universidad Nacional Autonoma de Mexico (UNAM), Mexico City; 2Gastroenterology Department, INCMSZ, Mexico City; 3Biomedic Research Institute, UNAM, Mexico City, Mexico.
Background: Mutations in the PRSS1, SPINK1 and CFTR genes have been associated to CP and RAP. The frequency and nature of these mutations varies among populations.
Aim: To identify mutations in the PRSS1, SPINK1 and CFTR genes in Mexican families and unrelated subjects with early onset idiopathic CP or RAP.
Methods: PRSS1 (exons 2 and 3), SPINK1 (exon 3) and CFTR (16 mutations) genes were sequenced in members of 2 families with suspicion of hereditary pancreatitis and in 19 (14 males) unrelated patients with idiopathic early onset CP or RAP and in 50 healthy controls.
Results:Family 1: no PRSS1, SPINK1 of CFTR mutations were found. Family 2: Only the SPINK1 N34S mutation was found in 6 out of 7 members. Both parents and 1 sibling were healthy heterocygotes, 2 siblings (1 healthy and 1 with diabetes mellitus) and the index case were homocygotes. The remaining sibling was healthy, with normal genotype. Unrelated patients: A total of 5 (26%) cases presented an abnormal genotype. PRSS1: Two new mutations in exon 2 (V39A and N42S) were found in 2 cases. SPINK1: The N34S change was found in 2 cases and a new mutation (exon 3 V39A) was found in other subject. No CFTR mutations were identified. All controls had normal genotype.
Conclusions: SPINK1 N34S mutation does not segregate with the disease, supporting its disease modifier role. We found 2 new PRSS1 1 SPINK1 mutations; this suggests a great genetic and population heterogeneity of the disease. The new SPINK1 V39A mutation might be a risk factor for CP in our population.
Results of 546 Consecutive Pancreatic Operations at Helsinki University Hospital, Finland
H. Pelli,1 A. Juuti,1 H. Mustonen,1 S. Nordling,2 J. Sirén,1 C. Haglund,1 T. Kiviluoto.11Department of Surgery; 2Department of Pathology, Helsinki University Central Hospital, Finland.
Introduction: Pancreatic surgery has been associated with high postoperative mortality and morbidity. However, recent results in high-volume centres show low mortality rates. The aim of our study was to evaluate the results of pancreatic operations in our hospital at the era of centralisation.
Material and Methods: In this retrospective study we included all patients undergoing pancreatic operation in Helsinki University Hospital 2000-2009. Survival data of the patients were obtained from patient records and the Population Registry in Finland. The operations were performed by two operators. Diagnosis was obtained from pathologic records and pancreatic cancer diagnosis was re-evaluated from histological specimens by our pathologist. Life tables were calculated according to the Kaplan-Meier product limit method.
Results: There were 546 patients undergoing pancreatic operation between the 10 year time period. 393 patients underwent pancreaticoduodenectomy, 103 distal pancreatic resection, 19 total pancreatic resection and 31 other operation. Histological diagnosis was pancreatic ductal adenocarsinoma in 192, invasive IPMN in 23 and papillary cancer in 45 patients. Seven patients died postoperatively leading to 1.3 % hospital mortality. Disease specific 1-, 2- and 3 year survival for patients radically operated (R0) on pancreatic ductal adenocarsinoma (n=192) was 69%, 45 % and 28%. Patients who had ductal adenocarsinoma had significantly longer survival when the operation was R0 or there were no lymph node metastases.
Conclusion: Pancreatic surgery is safe operation in high volume centres. In our hospital the hospital mortality and long term survival are comparable to the others reported from centralised hospitals.
Frequency and Significance of Calcification in IPMN
R. Perez-Johnston,1 O. Narin,1 M. Mino-Kenudson,2 T. Ingkakul,3 A. L. Warshaw,3 C. Fernandez-del Castillo,3 V. D. Sahani.1Depts. of Radiology1, Pathology2 and Surgery3, Massachusetts General Hospital, Boston, MA.
The frequency and significance of calcifications in IPMN is unknown. We examined calcifications by CT in a large cohort of IPMNs and correlated them with clinicopathologic characteristics.
Methods: Preoperative imaging studies of 286 patients with surgically resected IPMN were retrospectively reviewed. Of these, 164 had contrast-enhanced CT. Morphologic characteristics of IPMN, presence and type of calcifications (punctate, coarse, eggshell), their location (wall, septae, ductal or solid component), the degree of dysplasia (adenoma, borderline, carcinoma in-situ, invasive carcinoma) and the epithelial subtype (gastric, intestinal, pancreato-biliary, oncocytic) were recorded. Symptoms at the time of diagnosis, history of smoking and alcohol consumption were obtained from medical records.
Results: Of the 164 IPMNs, 68 were branch-duct type (Br) and 96 main-duct or combined type (MD); 78 (48%) had a malignant component (CIS and Invasive). Calcifications were present in 33 cases (20%); of these, 16 were punctate, 11 coarse, and 9 eggshell. 15 were mural, 3 septal, 2 ductal, 1 in the solid component, and 13 in multiple locations. Calcifications were seen more frequently in MD type (66%, p=0.04), larger lesions (47mm vs 32 mm p= 0.007), and when there was MPD dilation (70% vs 45%, p=0.023). There was no association between presence of calcification and malignancy, epithelial subtype, age, gender, symptoms, smoking, or alcohol consumption. However, malignancy was present in 9/11 IPMN with coarse calcification (p=0.04), suggesting this may be a worrisome feature.
Conclusion: Calcification in a variety of forms is found in 20% of IPMNs, and is more common in larger lesions and in MD type. Although its overall presence has no correlation with malignancy or epithelial subtype, coarse calcification may be a radiologic sign for malignancy.
Ly-6Chi Monocytes Regulate Acute Pancreatitis Severity by Generating TNF-α
G. Perides,1 J. M. Laukkarinen,1 J. S. Duffield,2 M. L. Steer.11Dept. of Surgery, Tufts Medical Canter and Tufts University School of Medicine, Boston, MA; 2Dept. of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA.
Introduction: The morbidity and mortality of acute pancreatitis are directly related to its severity but events which regulate pancreatitis severity are poorly understood. We previously showed that the Ly-6Chi inflammatory monocyte sub-set plays a critical pro-injurious role in regulating pancreatitis severity and others have shown that TNF-α plays an important pro-injurious role in pancreatitis.
Aim: To determine if Ly-6Chi monocytes promote pancreatitis severity by generating TNF-α.
Methods: We selectively depleted Ly-6Chi monocytes by administering diphtheria toxin (DT) to transgenically modified mice that express the human DT receptor coupled to the CD11b promoter (CD11b-hDTR mice) and we repleted Ly-6Chi monocytes in DT-treated CD11b-hDTR mice by adoptive transfer of Ly-6Chi-Ly-6G- bone marrow cells harvested from TNF-α+/+ or TNF-α-/- mice. Pancreatitis was induced by supramaximal caerulein stimulation and severity determined by quantitating edema and cell injury/necrosis.
Results: Pancreatitis severity and TNF-α levels in mice depleted of Ly6C monocytes were reduced by administration of DT. Pancreatitis severity and TNF-α levels were restored by transfer of Ly-6Chi cells harvested from TNF-α+/+ but not TNF-α-/- donor mice. Pancreatitis severity was reduced in TNF-α-/- mice when compared to TNF-α+/+ controls but that severity was restored when Ly-6Chi cells from TNF-α+/+ donor mice were adoptively transferred into TNF-α-/- recipients.
Conclusion: Ly-6Chi inflammatory monocytes pro-injuriously regulate pancreatitis severity by generating TNF-α and interfering with this phenomenon may represent a suitable therapeutic target in acute pancreatitis.
Classification of the Severity of Acute Pancreatitis: An Argument for a 'Critical' Category
M. S. Petrov, S. Shanbhag, M. Chakraborty, A. R. Phillips, J. A. Windsor. Department of Surgery, The University of Auckland, Auckland, New Zealand.
Introduction: The Atlanta classification of acute pancreatitis (1992) defined 'mild' and 'severe' categories of acute pancreatitis. A new classification with four categories of severity (mild, moderate, severe, and critical) has been proposed.
Aim: To investigate whether the presence of both organ failure and infected pancreatic necrosis identifies a subgroup of patients with 'critical' course of acute pancreatitis.
Methods: A systematic review of three electronic databases (MEDLINE, EMBASE and Scopus) was done to select studies that included data on infection status of pancreatic necrosis and mortality in patients with acute pancreatitis and organ failure. The summary estimates of the effect associated with the infection status of pancreatic necrosis were calculated using the Mantel-Haenszel model and presented as risk ratio and 95% confidence interval.
Results: There were a total of 474 patients with organ failure from 14 studies, comprising 213 (45%) patients with infected pancreatic necrosis (of whom 92 (43%) died) and 261 (55%) patients with sterile pancreatic necrosis (of whom 57 (22%) died). The presence of infected pancreatic necrosis was associated with a significantly increased risk of mortality in patients with organ failure (risk ratio 1.84; 95% confidence interval 1.40 to 2.41; P < 0.0001). There was no significant heterogeneity between the studies (I2 = 0%; P = 0.50).
Conclusion: The presence of infected pancreatic necrosis is associated with more than an 80% increase in mortality risk in patients with acute pancreatitis and organ failure. This finding argues for a critical category of patients with acute pancreatitis, those with both organ failure and infected necrosis.
Patient Reported Outcomes After Pancreaticoduodenectomy for Different Diseases: A Follow-up Study
R. Pezzilli,1 M. Falconi,3 A. Zerbi,5 R. Casadei,2 L. Valli,3 R. Varale,4 G. Armatura,3 C. Felicani,1 C. Ricci,2 A. M. Morselli-Labate.1Departments of 1Digestive Diseases and Internal Medicine and 2Surgery, S. Orsola-Malpighi Hospital, Bologna; 3Department of Surgery, GB Rossi Hospital, Verona; 4Department of Surgery, S. Raffaele Hospital, Milan; 5Department of Surgery, Scientific Institute Humanitas, Rozzano-Milan. Italy.
Aim: To evaluate clinical features and quality of life (QoL) in a 2-year follow-up study in subjects who underwent pancreatic head resection (PHR).
Patients: 197 patients with benign and malignant diseases.
Investigations: A dedicated clinical form and the EORTC QLQ-C30 questionnaire were administered immediately before and 6, 12, 18 and 24 months after surgery.
Reference group: A sample of 197 sex- and age-matched norms.
Results: The final pathological diagnoses were: 145 pancreatic neoplasms (73.6%; mainly represented by 97 ductal carcinoma and 35 IPMN), 33 neoplasms of the papilla of Vater (16.8%), 8 chronic pancreatitis (4.1%), 11 other periampullary neoplasms (5.6%). Malignant diseases were present in 164 patients (83.2%) and benign in 33 (16.8%). Before surgery, global health was significantly lower (P=0.001) in the study population as compared to the norms. Sixty-two (31.5%) patients died during the follow-up (all of them with malignant disease) while thirty-three patients (16.8%) were lost. There was a significant progressive improvement of the global health during the follow-up. At the end of the study, the QoL was not significantly different from the norms, even if the 30 patients with benign disease had a significant better QoL than the 72 patients with malignant diseases.
Conclusions: The QoL before PHR was impaired as compared to the normative population, while it significantly improves in the 2 years following surgery.
Risk of Recurrence After Biliary Pancreatitis With and Without Preventive Measures
A. Popowicz, E. Wirén, G. Sandblom, Å. Andrén-Sandberg. Center of Gastroenterology, Karolinska University Hospital/Huddinge.
Background: According to guidelines at Karolinska University Hospital Huddinge patients suffering from biliary pancreatitis should be treated with ERCP with sphincterotomy or cholecystectomy immediately after the pancreatitis subsides to prevent recurrence. The aim of this study was to compare recurrence rates for patients treated according to the guidelines with those who were not.
Methods: A review of patients treated with diagnosis acute pancreatitis at Karolinska University Hospital in 2007 was performed. Patients with biliary pancreatitis were divided with regards to measures taken during hospital stay, i.e. ERCP with sphincterotomy or cholecystectomy, and measures taken after discharge with subdivision into measures scheduled before or after discharge.
Results: Altogether 52 patients were treated for biliary pancreatitis, 31 women and 21 men. No significant association between age, gender or etiology was found. Mean age was 54 years. Fifteen patients underwent cholecystectomy and 33 ERCP during hospital stay. Some patients received both treatments. Eleven patients underwent cholecystectomy and seven ERCP after discharge following decision before discharge. Ten patients underwent cholecystectomy and 40 ERCP after discharge following decision after discharge. Two cholecystectomies and two ERCP were planned but not performed. A total of 21 recurrences were noted, seven of which after ERCP or cholecystectomy was performed. In these seven relapses despite treatment according to guidelines etiology was biliary (N=4), alcohol (N=2) and other (N=1). Two relapses were seen after cholecystectomy and five after ERCP.
Conclusions: A standardised program for management of biliary pancreatitis with early intervention in terms of cholecystectomy/ERCP with sphincterotomy most likely reduces the recurrence risk.
Infected Necrosis Increases Severity of Experimental Necrotizing Pancreatitis in Mice
P. J. Poxleitner, S. C. Richter, U. T. Hopt, U. A. Wittel. Department of General- and Visceral Surgery, Universitätsklinik Freiburg, Freiburg, Germany.
Introduction: Infection of pancreatic necrosis in necrotizing pancreatitis increases the lethality of patients with acute pancreatitis. To examine the mechanisms underlying this observation, we developed and tested a model of primary infected necrosis in which primary infection of necrosis is achieved in taurocholate induced pancreatitis in mice.
Materials and Methods: Sterile necrosis (SN) of acute necrotizing pancreatitis was induced by retrograde injection of 4% taurocholate in the common bile duct of Balb/c mice. Primary infected pancreatic necrosis (IN) was induced by co-injecting 108 CFU E. coli. After 6, 12, 24, 48 and 120 hours animals were sacrificed and serum as well as organs related to pancreatitis associated SIRS were examined.
Results: Despite the presence of bacteria in the pancreas 24h after injection of 108 CFU E. coli (TN) (TN 1.8 106±6.8 105 vs. TB 4.9 107±6.4 106 CFU; p<0.001), acute pancreatitis was only induced when taurocholate was co-injected (TB) (histology score: TN 4.2±0.4 vs. TB 21.7±2.3; p<0.001). Infection of pancreatic necrosis increased the pancreatic damage with a marked increase in PMN leukocyte infiltrate (histology score 24h: SN 17.8±2.6 vs. IN 23.7±2.2; p<0.001). Systemically, infection of pancreatic necrosis increased the pulmonary vascular leak (albumin in bronchoalveolary lavage 6h: SN 151.0±57.7 μg/ml vs. IN 219.5±76.2 μg/ml; p<0.05). Serum glucose concentrations as parameter of the hepatic function were reduced in infected necrosis (24h: SN 167.0±35.6 mg/dl vs. IN 106.9±12.7 mg/dl; p<0.001).
Conclusion: Primary infection of pancreatic necrosis with E. coli induces increased pancreatic damage and worsens pulmonary and hepatic systemic complications in acute necrotizing pancreatitis in mice.
Pancreatic Cancer Cell Invasion is Increased by Hypoxia Induced MMP9 mRNA-Expression
P. Puolakkainen,1,3 H. Mustonen,1 S. Vainionpää,1 E. Kemppainen,1 H. Repo,2 H. Pelli.11Department of Surgery, Helsinki University Central Hospita; 2Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki; 3Department of Surgery, Turku University Central Hospital, Finland.
Background: Hypoxic environment in pancreatic cancer cell culture accelerates the cancer invasion. Matrix metalloproteinases (MMP) have the ability to degrade extra cellular matrix (ECM) in general. Further MMP9 mediates neo-angiogenesis in pancreatic cancer and in its metastasis.
Aim: Our aim was to analyze, how the MMP2 and MMP9 expression levels influence the cell interaction on invasion of cancer cells incubated in hypoxic as compared to normal conditions.
Material and Methods: We used Panc-1 and MiaPaCa-2 pancreatic ductal adenocarcinoma cell lines. Cells were incubated either in 5% CO2 in air at 37 °C (normal conditions) or in hypoxic environment (2% O2, 5% CO2, 93% N2, at 37 °C) for 24 h. MMP2 and MMP9 mRNA-expression levels were measured with RT-PCR.
Results: The invasion rate of Panc-1 and MiaPaCa-2 increased after the incubation in hypoxic conditions. Both cell lines expressed higher MMP9 mRNA-expression levels in hypoxic than in normal conditions. In Panc-1 cells MMP9 mRNA-expression levels were 2.32-3.76 times and in MiaPaCa-1 cells 3.78 times higher when incubated in hypoxic conditions than in the room air. MMP2 mRNA-expression levels in Panc-1 cell line were 0.45-0.54 times lower and MiaPaCa-1 expression levels the same in hypoxia than in the room air.
Conclusions: In in-vitro studies with two different pancreatic cancer cell lines, the increased invasion in hypoxic conditions is partially mediated by MMP9 but not by MMP2.
Mitochondial Injury Precedes NF-κB and Premature Trypsinogen Activation in L-lysine-induced Acute Pancreatitis in Rats
Z. Rakonczay Jr.,1 G. Biczó,1 S. Dósa,2 N. Shalbuyeva,3 Z. Hracskó,4 Z. Kukor,5 V. Venglovecz,6 I. S. Varga,4 B. Iványi,2 T. Wittmann,1 A. Gukovskaya,3 T. Takács,1 P. Hegyi.11First Dept. of Medicine, 2Dept. of Pathology, 4Dept. of Biochemistry and Molecular Biology, 6Dept. of Pharmacology and Pharmacotherapy, University of Szeged, Szeged, Hungary, 3Veterans Affairs Greater Los Angeles Healthcare System, University of California, Los Angeles, CA, USA; 5Dept. of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.
Introduction: Large intraperitoneal (i.p.) doses of basic amino acids, such as L-arginine, L-ornithine or L-lysine, have been shown to cause pancreatic acinar cell injury.
Aims: To determine the mechanisms by which L-lysine damages the pancreas.
Methods: Rats were injected i.p. with 2 g/kg L-lysine and sacrificed 0-168 h afterwards. Biochemical and histological parameters of pancreatitis were determined. In particular, we characterized the kinetics of L-lysine-induced pancreatic mitochondrial injury, trypsinogen and nuclear factor-κB (NF-κB) activation which are commonly thought to play a crucial role in the pathogenesis of acute pancreatitis.
Results: We showed that administration of L-lysine induced severe acute necrotizing pancreatitis. Early pancreatic mitochondrial damage (from 1 h) preceded the activation of trypsinogen (12-48 h) and NF-κB (24-168 h). Our data demonstrate that L-lysine impairs ATP synthase activity of isolated pancreatic, but not liver, mitochondria. Serum amylase and lipase activities were significantly increased, whereas pancreatic amylase activity was decreased. Administration of L-lysine also induced pancreatic oxidative stress and heat shock protein 72 expression. Elevated pancreatic myeloperoxidase activity was detected from 18 to 72 h.
Conclusions: Early pancreatic mitochondrial injury caused by large doses of L-lysine may lead to the development of acute pancreatitis independently of trypsinogen and NF-κB activation.
Supported by OTKA, MTA and NKTH.
C4.4A Mediates AGR2 Induced Aggressiveness of Pancreatic Cancer Cells
V. Ramachandran,1 T. Arumugam,1 C. D. Logsdon.1,2Depts. of 1Cancer Biology, 2Medical Oncology, UT MD Anderson Cancer Center, Houston, TX.
Background: We recently reported that anterior gradient 2 (AGR2) is an autocrine factor in PDAC that facilitates aggressive tumor growth, metastasis and resistance to therapies. But, the receptor responsible for mediating the effects of AGR2 has been unknown. In this study we have identified C4.4A, a GPI-linked receptor as the functional receptor of AGR2 and have characterized its signaling complex and mechanisms.
Methods: Immunoprecipitation was conducted using AGR2 antibody and western blotted for candidate receptors C4.4A, UPAR, CD59 and DAG-1. Gene silencing was conducted using siRNA. Recombinant AGR2 was used for analysis of proliferation (MTS assays), migration (Boyden's chamber assay) and invasion (Invasion chamber assay) of pancreatic cancer cell lines (MPanc96/BxPC-3/MiaPaCa-2). Apoptosis after siRNA silencing with and without gemcitabine (Gem) (10μmol/L) and AGR2 (10nM) was conducted by FACS analysis. ERK and AKT activation were analyzed using phospho-specific antibodies.
Results: AGR2 immunoprecipitated with LY-6 super family receptors - C4.4A, UPAR and CD59, but not with DAG-1. Silencing of C4.4A significantly reduced the endogenous (basal) proliferation (MPanc96-by 25% & MiaPaCa-2- by 30%) and completely blocked extracellular AGR2 mediated pancreatic cancer cell proliferation, migration and invasion (p<0.05). In contrast, silencing of UPAR or CD59 had no effect. Silencing of C4.4A itself induced significant apoptosis (2 fold) and also significantly increased Gem-mediated apoptosis in PDAC cells (p<0.05). Exogenous addition of AGR2 reduced the cell responses to Gem treatment and silencing C4.4A blocked this protective effect of AGR2. Silencing of laminins 1(1.5 fold) & 5 (2 fold) and integrins α3β1 (1.3 fold) increased the level of Gem-mediated apoptosis and blocked the ability of AGR2 to promote gem resistance (p<0.05). Exogenous AGR2 stimulated ERK and AKT phosphorylation within 5mins.
Conclusion: C4.4A is the first functional receptor identified for AGR2. Silencing C4.4A reduced the aggressiveness and improved the chemosensitivity of pancreatic cancer. C4.4A forms a signaling complex with laminins 1&5 and integrins α3β1. The AGR2/C4.4A autocrine loop may be a reasonable target for new therapeutics against PDAC.
Pancreatic Metaplasia Involves Changes in Both Cell Identity and Architecture
K. C. Ray,1 S. A. Blaine,1 M. Gannon,1,2,3 G. Gu,3 M. K. Washington,4 A. L. Means.1,31Depts. Of Surgery, 2Medicine, Cell and 3Developmental Biology, and 4Pathology, Vanderbilt University Medical Center, Nashville, TN.
In both pancreatitis and pancreatic cancer, the pancreas undergoes dramatic changes in cellular composition. In affected areas, most acinar cells are depleted while abnormal, often hyperplastic, ducts arise. Endocrine cells, normally present in the islets of Langerhans, are found within these hyperplastic ducts. We have used genetic lineage tracing in mouse models to determine the cell of origin for hyperplastic ducts, including PanIN-like lesions, and the endocrine cells found within these ducts. We used tissue-specific Cre transgenes to introduce genetic labels or to activate the KrasG12D oncogene and followed the fate of the cells in those tissues. We have found amazing plasticity within the exocrine pancreas, with both acinar cells and ductal cells giving rise to different types of hyperplastic ductal lesions, but the fate of the endocrine pancreas appears to be confined in the adult pancreas, able to change its architectural arrangement but not its cellular identity.
How Accurate is the Chronic Pancreatitis (CP) ICD-9-CM (577.1) Code?
N. Reddy, S. Nangia, M. J. DiMagno. Dept Int Med, GI Div, U of Michigan, Ann Arbor.
Valid clinical and epidemiologic studies require the accurate diagnosis of CP, often determined by the ICD-9-CM code (577.1) for CP, which is of uncertain reliability. We determined the accuracy of the ICD-9 code between 10/01/05-11/01/08 by comparing it to 3 diagnostic criteria: Ammann's criteria, the Mayo Score, and the Japanese Pancreas Society (JPS) guidelines.
Results: 1,345 patients had an ICD-9 code for CP. Of these 655 (48.7%) had CP, defined as fulfilling any of 3 sets of criteria. Frequencies of CP were similar among Mayo (48%), Ammann (42%) and JPS (42.8%) criteria. The diagnosis of CP was determined by 3 criteria in 82.9%, by 2 criteria in 7.5% and by 1 criterion in 9.6%. Etiologies of CP were 52.2% nonalcohol, 26.9% alcohol, 6.7% suspected alcohol, 5.5% suspected nonalcohol and 8.7% not reported. In the 690 without CP (non-CP), 93.8% had imaging to assess Cambridge and/or EUS score, 13.5% had 4 abnormal EUS features (possible CP) and 62.6% had only suggestive symptoms of CP by JPS guidelines. The mean ages of the CP and non-CP groups were similar (54.2+/-15.5 yrs vs 51.5+/-16.3 yrs) but CP had greater frequencies of women (56% vs 45.9%), chronic/recurrent abdominal pain (80.2% vs 59.3%), weight loss (31.4% vs 16.2%), steatorrhea (6.6% vs 0.7%), diabetes mellitus (35.0% vs 17.2%), pancreatic calcifications (42.9% vs <1%) ever smoking (57.1% vs 46.4%) and ever drinking alcohol (64% vs. 54.3%). Patients in the non-CP group had ≥1 diagnoses, including acute pancreatitis (47.0%), recurrent pancreatitis (17.2%), chronic abdominal pain (19.6%), pseudocyst (9.3%), IBS/functional dyspepsia (7.5%) and others.
Conclusion: The diagnosis of CP by ICD-9-CM code (577.1) is <50% accurate for definite CP and using it as the sole basis for diagnosis of CP will contribute to erroneous epidemiological data and conclusions.
Low pH Uncouples Gap Junctions in the Pancreatic Acinar Cell
A. Reed,1 S. Husain,2 M. Nathanson,1 F. Gorelick.11Department of Medicine, Yale University, New Haven, CT, 2Department of Pediatrics, Yale University, New Haven, CT.
Background: Pancreatic acinar cells are coupled through gap junctions, and perturbation of this coupling has been implicated in the pathogenesis of acute pancreatitis. Low extracellular pH (pHe) sensitizes the acinar cell to pancreatitis responses. However, the effect of low pHe on gap junctional coupling in the acinar cell is unknown.
Aim: To examine the effects of low pHe on gap junctional communication in the pancreatic acinar cell.
Methods: Isolated rat pancreatic acinar cells were treated with 10 pM cerulein in the presence of HEPES buffer at pH 7.4 and 7.0. To measure Ca2+ signal asynchrony, Ca2+ signals from each cell within an intact acinus were measured by confocal microscopy after loading with the Ca2+ dye, fluo-4 (6 μM). Next, intra-acinus variability of Ca2+ oscillation frequency was calculated. To measure fluorescence recovery after photobleaching (FRAP), cells were loaded with 5 μM 5(6)-CFDA, an individual cell was photobleached, and parameters of fluorescence recovery (rate constant and %recovery) were assayed.
Results: There was significantly more variability in the frequency of cerulein-induced Ca2+ oscillations in the cells treated at pH 7.0 (32%) compared to the cells treated at pH 7.4 (14%). In FRAP experiments, the percent recovery and rate constant of cells treated with buffer at pH 7.0 were significantly lower than cells treated with pH 7.4 buffer. The percent recovery and rate constant of cells treated with 10 pM cerulein at pH 7.0 was significantly lower than cells treated with buffer at 7.4, buffer at 7.0, and cerluein at 7.4.
Conclusion: Low pHe inhibits gap junctional intercellular communication in the pancreatic acinar cell.
Membranous Claudin-10 Expression is a Novel Positive Prognostic Factor in Pancreatic Cancer
C. Reiser-Erkan, M. Tardio, M. Erkan, W. Wang, T. Samkharadze, H. Friess, J. Kleeff. Chirurgische Klinik und Poliklinik Klinikum rechts der Isar Technische Universität München, München.
Background and Aims: Claudins are a family of proteins essential for the formation of tight junctions in epithelial and endothelial cells. The role of Claudin-10 in pancreatic cancer is evaluated.
Material and Methods: Claudin-10 mRNA and protein expression was evaluated by qRT-PCR and IHC analyses. Semi-quantitative immunohistochemistry was performed on tissues of the normal pancreas, chronic pancreatitis, PanIN tissue microarray (n=194) and pancreatic cancer. Survival of patients (n=47) was compared using the Kaplan-Meier method and log-rank analyses.
Results: Claudin-10-mRNA expression in pancreatic cancer decreased significantly (50%, p<0.0001) compared to the normal pancreas. Similarly, there was a significant reduction of Claudin-10 protein expression in PanIN lesions (PanIN 1=80%, PanIN 3= 40%), and in pancreatic cancer (9%). This reduction was also paralleled by a shift from membraneous to cytoplasmic expression. Two year mortality of pancreatic cancer patients with cytoplasmic Claudin-10 expression was 75% (median survival= 15.6 months), whereas 0% (median survival= 21.8 months, p=0.04) in patients with membraneous Claudin-10 expression.
Conclusion: Membraneous CLDN-10 expression is a novel prognostic marker in pancreatic cancer.
BHLHB2 Overexpression Is a Positive Prognostic Factor Indicating Gemcitabine Responsiveness in Pancreatic Cancer
C. Reiser-Erkan, W. Wang, M. Erkan, C. W. Michalski, Helmut Friess, J. Kleeff. Dept, of Surgery, Technische Universität München, Munich, Germany.
Aims: The cyclic adenosine monophosphate-inducible basic helix-loop-helix (bHLH) domain containing class-B2 transcriptional factor BHLHB2 is differentially expressed in a number of human malignancies. In the present study, the expression, regulation, and functions of BHLHB2 in pancreatic cancer were investigated.
Methods: Expression analyses were carried out in tissues of the normal pancreas (n=10) and pancreatic ductal adenocarcinoma (n=77) as well as in eight pancreatic cancer cell lines using quantitative RT-PCR, semiquantitative immunohistochemistry, and immunoblot analyses. In vitro functional experiments were conducted using siRNA transfection, hypoxia, serum starvation, apoptosis induction with gemcitabine and actinomycin-D, and invasion assays. Survival analysis was carried out using Kaplan-Meier and log-rank analyses.
Results: The weak/absent nuclear staining in normal pancreatic ducts and acinar cells was replaced by moderate to strong nuclear/cytoplasmic staining in PanIN lesions and pancreatic cancer cells. Patients with weak/absent nuclear BHLHB2 staining had significantly worse median survival compared to those with strong staining (13 months vs. 27 months, p=0.0025, HR= 2.275, 95% CI= 1.396 - 4.795). BHLHB2 mRNA and protein expressions were strongly induced by hypoxia and by serum starvation in pancreatic cancer cell lines. BHLHB2 silencing with RNAi had no significant effects on growth and invasion but increased apoptosis resistance against gemcitabine by reducing caspace-3 cleavage.
Conclusions: Hypoxia-inducible BHLHB2 expression correlates with better prognoses of pancreatic cancer patients and increased chemosensitivity towards gemcitabine.
Reverse Engineering of Gene Regulatory Networks Governing Cell-Cell Communication in the Pancreatic Cancer
Z. Rogon,1 A. Bauer,2 H. Busch,1,3 T. Giese,4 E. Soyka,5 A. Szabowski,1 J. Hoheisel,2 M. W. Büchler,5 J. Werner,5 R. Eils,1 N. A. Giese.51Theoretical Bioinformatics and 2Functional Genomics, German Cancer Research Center/DKFZ, Heidelberg, 3FRIAS, Albert-Ludwigs-University, Freiburg, 4Institute of Immunology and 5Department of Surgery, University Hospital Heidelberg, Germany.
Aim: Communication between tumor cells and surrounding stroma conveys aggressive behavior and resistance to chemo-, radio- and immunotherapy in the pancreatic cancer. Mathematical modeling of the experimental data and identification of patterns of interactions governing gene regulatory networks may provide a way of deciphering the double-paracrine information flow between pancreatic tumor and stellate cells.
Methods: The in vitro modeling of tumor-stellate cell interactions was performed by genome-wide RNA profiling of cells co-cultered under different conditions in a time-resolved manner. ODE-based high-through put complexity reduction CTRNN approach was applied to assess processing of information in each cell type and to identify specific clusters and patterns. Ranking of genes led to a selection of central-hub elements then used for TFBS analyses, reverse engineering and in silico simulations.
Results and Conclusion: Our global gene expression analysis, ranking and modeling allowed to i) capture network dynamics and major regulatory elements in tumor and stellate cells and ii) to create models simulating intercellular interactions. Identification of key players delivered a set of potential targets for experimental testing towards development of the novel therapeutics and personalized screening tools in pancreatic cancer.
Germline Mutations of Centrobin Gene Likely Predispose Patients to Pancreatic Cancer
W. S. Rubinstein,1 C. Zou,1 R. H. Koneru,3 K. Vogel,1 V. Wang,2 K. Kaul,2 R. E. Brand,4 Q. Gao.1Department of 1Medicine, 2Pathology, NorthShore University HealthSystem, 3MBP Program, Northwestern University, Evanston, IL; 4Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh Medical Center, Hillman Cancer Center, Pittsburgh, PA.
For the majority of pancreatic cancer-prone families, the responsible germline mutations have not been identified. In the subset of families that have an identified germline mutation, BRCA2 mutations account for the largest percentage. Components of the BRCA2 pathway are likely direct targets of tumorigenesis, and some may confer pancreatic cancer susceptibility through an inherited mutation. The aim of this study was to screen for germline mutations in centrobin, a gene encoding a BRCA2 binding proteins, in 276 participants in our Pancreatic Cancer Family Registry. We identified one potential mutation in a splicing donor site and three non-synonymous potential point mutations that are not present in 480 control subjects with no reported history of any cancer in themselves and their first-degree relatives (average age: 64.7 yrs). The mutation in the splicing donor site is predicted to disrupt the splicing donor site by NNsplice, which would lead to a deleterious mutation. The three non-synonymous potential point mutations are predicted to be potentially damaging by Polyphen, one of which was recurrent in four pancreatic cancer patients and one patient's family member. These findings suggest that these variants are potential disease-causing mutations. We are now in the process of determining whether loss of heterozygosity is present in the mutation carriers' pancreatic cancer samples. Family members of cases with putative disease-causing mutations will also be recruited for co-segregation analysis with pancreatic cancer.
Secretin Is Not Required For Pancreatic Growth Induced By Feeding Protease Inhibitor
M. E. Sabbatini,1 M. Dolors Sans,1 I. Nishijima,2 J. A. Williams.11Department of Molecular and Integrative Physiology, University of Michigan; 2Nationwide Children's Hospital, Columbus Ohio.
Introduction: Feeding trypsin inhibitors such as Camostat (FOY-305) is known to induce CCK release and stimulate pancreatic growth. However, Camostat has also been reported to stimulate secretin release and, because secretin often potentiates the action of CCK, it could participate in the growth response.
Aim: Our aim was to test the role of secretin in pancreatic adaptive growth through the use of C57BL/6 mice with genetic deletion of secretin or secretin receptor (Hum Mol Genetics 15:3241-3250, 2006).
Results: The lack of secretin or the secretin receptor in the whole pancreas, as well as in pancreatic acini was confirmed by RT-PCR. Other related components such as VPAC receptors (VPAC1 and VPAC2) and adenylate cyclase isoforms expression were not affected. Secretin increased cAMP levels in acini from wild-type (WT) mice but had no effect on acini from secretin receptor deleted mice while vasoactive intestinal polypeptide and forskolin, a direct activator of adenylate cyclase, still induced a normal response. The pancreas of mice without secretin was of normal size and histology indicating that secretin is not necessary in the mouse for normal pancreatic differentiation or maintenance. Secretin receptor deficient mice also had a normal sized pancreas. When WT mice were fed 0.1 % Camostat mixed in powdered chow the pancreas doubled in size in one week. This is accompanied by parallel increases in protein and DNA. When both littermate secretin and secretin receptor deleted mice were fed Camostat the increase in pancreatic mass was similar to WT mice.
Conclusion: These results indicate that secretin is not required for normal pancreatic maintenance or growth in response to feeding Camostat.
Expression of Multiple Adenylate Cyclase Isoforms in Mouse Pancreatic Acini, Islets and Ducts
M. E. Sabbatini, J. A. Williams. Department of Molecular and Integrative Physiology, University of Michigan.
Introduction: Secretin and VPAC receptors are responsible for the activation of cAMP pathway in pancreatic ducts and acini. However, nothing is known about the adenylate cyclase isoforms involved in this event. There are nine different adenylate cyclase isoforms activated by Gαs and each one has its own pattern of expression and regulation by calcium and other intracellular signals.
Aim: In this study we sought to establish which membrane-associated adenylate cyclase isoforms are expressed in the whole pancreas, as well as in purified pancreatic acini, islets of Langerhans and ducts.
Results and Conclusion: Using RT-PCR we found four different adenylate cyclase mRNAs, ADCY1, ADCY4, ADCY6 and ADCY9, expressed in the whole pancreas and in a standard isolated mouse pancreatic acinar preparation. Although ADCY2, ADCY3, ADCY5, ADCY7 and ADCY8 mRNAs were expressed in their respective positive control tissues, none of them were expressed in the pancreas. Since the preparation of isolated pancreatic acini has a low percentage of pancreatic ducts and islets of Langerhans, we further studied which isoforms are expressed in each isolated cell type. Using both laser capture microdissection and Accudenz® gradient separation followed by manual selection we found that multiple adenylate cyclase isoforms are expressed in each cell type: ADCY6 and ADCY9 mRNAs are expressed in pancreatic acini, islets and ducts whereas ADCY1 mRNA is only expressed in acini and ADCY4 mRNA is only expressed in pancreatic ducts. The different isoforms may allow the integration of multiple signals in addition to Gαs. For example, ADCY1, ADCY4 and ADCY6 are regulated by PKC while ADCY9 is known to be inhibited by calcium/calcineurin. The significance of the multiple isoforms in a single pancreatic cell type remains to be determined.
Role of Cathepsin B in the Pathogenesis of Chronic Pancreatitis
R. P. Sah, R. Dawra, A. Bekolay, A. Saluja. Division of Basic and Translational Research, Department of Surgery, University of Minnesota Medical School, Minneapolis, MN.
Background: Cathepsin B is known to be crucial in the early injury in acute pancreatitis (AP). It is generally believed that Cathepsin B is involved in intra-acinar cell activation of trypsinogen. Mutations related to pathologic trypsinogen activation have been linked with hereditary pancreatitis. However, the role of Cathepsin B in the pathogenesis of chronic pancreatitis (CP) is unclear.
Methods: We used a mouse model of CP based on repetitive episodes of AP (twice a week for 10 weeks) induced by caerulein (50μg/kg IP every hour X 6). We compared CP in wild type (WT) mice and Cathepsin B knockout (CB-/-) mice.
Results: After 10 weeks, whole pancreas weight (normalized to body weight) measured 49% and 60% of that of saline injected controls in WT and CB-/- respectively (p=0.03). On histology, comparable acinar atrophy, lymphocytic infiltration and duct dilatation was seen in both groups with about 500% increase in fibrosis measured by Sirius Red stained area. Tubular structures per 10x field (cytokeratin 19 immunostaining) increased from 3 in controls to 28±2 in WT and 31±2 in CB-/- groups indicating considerable ductular metaplasia. Significant but similar increase in stellate cell population (measured by desmin expression) and activation (measured by alpha smooth muscle actin expression) was seen in both groups. Immunostaining for Ki-67 antigen, a marker of proliferation revealed 108±5 positive cells per 10x field in WT vs 98±6 in CB-/- with only a few scattered positive cells in controls.
Conclusion: This is the first study to explore the role of Cathepsin B in CP. The results show that CP in CB-/- mice is comparable to WT mice implicating that unlike acute pancreatitis, Cathepsin B is not crucial in the pathogenesis of chronic pancreatitis. Furthermore, CB-/- mice lack significant trypsinogen activation which suggests that trypsinogen activation may not be necessary in the pathogenesis of chronic pancreatitis.
Nutritional Assessment of Autoimmune Pancreatitis as Studied By Multifrequency Bio-Electric Impedance Analysis
J. Sakagami, H. Yasuda, Y. Sogame, N. Suzuki, H. Hasegawa, K. Kataoka, T. Yoshikawa. Division of Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, JAPAN.
Background & Aim: Autoimmune pancreatitis (AIP) is a distinctive type of chronic pancreatitis (CP) probably linked to autoimmune mechanisms. Exocrine function impairment was reported to occur in about one half of the AIP patients. Recent reports also suggest AIP that is the pancreatic manifestation of a systemic disease called IgG4-associated systemic disease. However, little is known about systemic nutritional aspect of AIP. Our aim was to investigate nutritional difference between AIP and CP.
Methods: Ten patients with AIP were enrolled in this study (Age; 67.2+/-4.5, M:F; 9:1). Age and gender matched 20 patients with CP were also randomly selected. Intracellular water (ICW), extracellular water (ECW), skeletal muscle mass (SMM), body fat mass (BFM), and basal metabolic rate (BMR) were measured by multifrequency bio-electric impedance analysis. Body mass index (BMI) was also calculated.
Results: There were no significant differences between AIP and CP group with respect to BMI (22.2+/-0.8 vs. 21.8+/-0.6kg/m2; P=0.826), SMM (26.6+/-0.9 vs. 23.9+/-0.9kg; P=0.108), BFM (14.2+/-1.6 vs. 13.4+/-1.3kg; P=0.878), and ICW (21.9+/-0.7 vs. 19.9+/-0.7L; P=0.113). However, BMR (1422+/-31 vs. 1321+/-32kcal; P=0.043) and ECW (14.1+/-0.4 vs. 12.7+/-0.4L; P=0.025) of AIP group were significantly greater than those of CP group.
Conclusions: These findings indicate that degree of obesity and body fat/muscle contents are equivalent between AIP and CP groups, but basal metabolism is relatively maintained in AIP patients.
Murine Pancreatic Duct Ligation Causes Acute Lung Injury and 100% Mortality: A Novel Model for Investigating Acute Pancreatitis
I. Samuel, Z. Yuan, D. Meyerholz, E. Twait, D. Williard, D. Kempuraj. Surgery, VAMC/Univ of Iowa, Iowa City, IA.
Severe acute pancreatitis is a systemic disease with multiorgan dysfunction. Acute lung injury (ALI) substantially increases the risk of death from acute pancreatitis of any cause. However, suitable experimental models of gallstone pancreatitis with ALI are limited. Pancreatic duct ligation (PDL) in rats causes mild inflammation and no mortality. Surprisingly, PDL in mice has been completely overlooked. We developed a novel murine model of PDL-induced acute pancreatitis associated with ALI and 100% mortality. Laparotomy was done on C57/BL6 mice under isoflurane anesthesia (n=10/group). The diseased group had PDL close to the duodenum. Sham controls had the duct dissected but not ligated. Bile duct ligation (BDL) controls had common bile duct ligation without PDL. Only mice with PDL developed morphological evidence of acute pancreatitis and, moreover, had 100% mortality within 4 days. Mice with either PDL or BDL showed evidence of pulmonary neutrophil infiltration. As BDL controls had no mortality, we extended our studies to compare pulmonary characteristics. Pulmonary compliance was significantly reduced by 20% in the PDL group but not the BDL group. In addition, the bronchoalveolar lavage fluid neutrophil count and IL-1β concentration were significantly elevated in the PDL group but not the BDL group. Suggestive of systemic inflammation in the PDL group, stress kinases (p38, ERK, JNK) were activated in the pancreas and lung, and serum TNF-α and IL-1β were increased. The NF-κB pathway was activated in the pancreas within 1hr of PDL.
Conclusion: PDL-induced acute pancreatitis in mice is associated with acute lung injury, systemic inflammation, and death. The development of this novel model is an exciting and notable advance in the field.
Pancreatic Cancer Cells Respond to Triptolide via Early Activation of Cell Signaling Pathways
V. Sangwan,1 R. Chugh,1 N. Mujumdar,1 S. Banerjee,1 T. N. Mackenzie,1 V. Dudeja,1 S. Vickers,1,2 A. Saluja.1,21Department of Surgery; 2Masonic Cancer Center, University of Minnesota, Minneapolis, MN.
Background: Pancreatic cancer is the fourth-leading cause of cancer-related deaths in the United States, with a 5-year survival of <5%. We have previously shown that the diterpenoid, triptolide, causes cell death in pancreatic cancer cells and reduces the growth as well as loco-regional spread of pancreatic tumors in an orthotopic model. Although triptolide is an effective anti-cancer agent, very little is known about its mechanism of action. We therefore investigated the early response of pancreatic cancer cells to triptolide, as well as the events required for triptolide to function as a chemotherapeutic agent against pancreatic cancer.
Aim: To investigate the signaling events taking place in pancreatic cancer cells in response to triptolide.
Methods: Pancreatic cancer cells of varying metastatic potential (MIA PaCa-2/S2-VP10/Aspc-1) were incubated with 100nM triptolide or vehicle (control), for different time intervals (15'-24h). Cell viability was measured using an MTT-based assay, and apoptosis assessed using annexin V staining and caspase 3/9 activation. Activation of signaling pathways was assessed using phosphorylation status of pERK and pAkt using the Licor Odyssey Imaging System.
Results: Triptolide decreased the cell viability of MIA PaCa-2 and S2-VP10 24h post-treatment, but no effect of the compound was observed at earlier time points. However, triptolide activates the pERK signaling pathway 15' post-stimulation, and causes a decrease in pAkt levels. Intriguingly, triptolide-treated Aspc-1 cells do not exhibit decrease in cell viability under serum free conditions. Since these cells express high levels of the epidermal growth factor (EGF) and Hepatocyte growth factor (HGF) receptors, we studied the effect of their ligands, EGF and HGF on triptolide-induced cell death. Forty eight hours post-treatment with triptolide, cell viability decreased to 40% in the presence of EGF, and 60% in the presence of HGF in Aspc-1 cells.
Conclusion: Triptolide is a potent anti-cancer agent; treatment with triptolide alters pERK and pAkt levels in cells undergoing death. We show that triptolide action requires the presence of either EGF or HGF in some pancreatic cancer cell lines.
Selective Non-Resectional Management of Pancreatic Cystic Tumors: A Cohort Study Based On a Cystic Tumor Registry
S. Sanyal,1 A. J. Sheen,1 A. K. Siriwardena.1Regional Hepatobiliary Surgery Unit, Manchester Royal Infirmary, Manchester UK.
Introduction: Pancreatic cystic tumors carry a cancer risk that differs according to type with mucinous neoplasms being regarded as pre-malignant, serous cystic lesions as low risk and intraductal papillary lesions (IPMN) as being intermediate (high risk if main duct). This study reports the outcome of management in a dedicated regional registry with emphasis on selective resection.
Methods: 41 consecutive patients with non-inflammatory cystic tumors were recruited from clinic referrals (24 females; mean age at presentation 64 [39-89] years). 22 (53%) were incidentally discovered asymptomatic lesions. Commonest symptom was abdominal pain (n=15, 37%). Work-up included contrast-enhanced computed tomography in 100% and endoscopic ultrasound +/- fine needle aspiration in 27% (66%). Outcome after completion of staging is reported.
Results: 14 (34%) underwent resection with cancer being present on final histology in 5 (35%) (two were unresectable at surgery). 27 (66%) were managed without surgery. In 5, surgery was recommended but not undertaken because of metastases and/or co-morbidity. This left a final population of 22 patients with cystic tumors in whom a deliberate non-resectional policy was adopted. This group comprised 9 branch duct IPMN, 5 main duct IPMN, 3 mucinous cystic tumors, 3 serous cystic tumors and 2 incorrectly classified pseudocysts. During follow up (average 16.3 months [range 3-91 months]), interval restaging was undertaken in 18 and there have been no interval cancers.
Conclusion: Caution must be exercised in extrapolating from small volume series; however these data suggest that provided careful staging is undertaken, selected patients with pancreatic cystic tumors can be managed without resection.
A Selective COX-2 Inhibitor Inhibits CXC Chemokine Production in Human Pancreatic Cancer Cells
M. Satake,1,2 N. Kuroda,1 N. Uyama,1 T. Hirano,1 T. Okada,1 G. Eibl,2 V. L. Go,2 O. J. Hines,2 H. Reber,2 J. Fujimoto.11Departments of Surgery, Hyogo College of Medicine: Nishinomiya, Hyogo, Japan; 2David Geffen School of Medicine at U.C.L.A: Los Angeles, CA.
Background: Pancreatic cancer (PaCa) is critical. It is well demonstrated that COX-2 expression of PaCa cell lines are related with PGE2 production from arachidonic acid (AA). AA and PGE2 stimulate PaCa growth in COXs dependent manner, and this stimulation are suppressed by selective COX-2 inhibitor (Coxib). On the other hand, the current study defines a role of COX-2 in the over-production of ELR-positive CXC chemokines (CXCL8 and 5). COX-2 expression may relate with CXCLs production. ELR+CXCL show growth stimulation response via CXCR2. However, in PaCa, the relation of COX-2 dependent CXCL production has not been identifying. The aim of this study is to establish the profiles of COX-2 depending CXCLs production. Moreover, we use Coxib to detect their inhibitory effect in COX-2 dependent CXCL-production.
Methods: COX-2 positive cell line, BxPc-3(B), KMP-4 (K4) and COX-2 negative cell lines, Mia-PaCa-2 (MP) and KMP-3 (K3) were analyzed for CXCL8 and 5 by ELISA and Immunofluorescence studies (IMF). 24 hours after stimulated with AA, PGE2 and AA with Coxib, CXCLs were measured with ELISA.
Results: In COX-2 positive cells, CXCL5 and 8 are significantly higher when compared with COX-2 negative cells. AA and PGE2 are enhanced CXCLs production in COX-2 positive cells, and significantly suppressed by Coxib.
Conclusion: Our data demonstrate that the production of ELR+CXCL is correlated with COX-2 expression in PaCa. The production of ELR+CXCL is enhanced by AA and PGE2, and is suppressed by Coxibs. Since, CXCLs production may be regulated by COX-2 dependent manner; Coxibs may become new preventive approach for PaCa.
High S100A2 Calcium-Binding Protein Expression Defines a Metastatic Phenotype in Pancreatic Cancer
C. J. Scarlett, D. K. Chang, E. S. Stoddart, E. K. Colvin, A. Mawson, M. D. Jones, P. E. Tobelmann, M. Pajic, R. J. Daly, A. V. Biankin. Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW, Australia.
Background: High expression of S100A2 calcium binding protein (S100A2) is associated with a poor response to pancreatectomy for pancreatic cancer (PC), suggesting that S100A2 may play a key role in the development of a metastatic phenotype. This study aimed to define the functional role of S100A2 in PC metastases in vitro and in animal models of PC.
Methods: We examined altered S100A2 expression in vitro, and in vivo using xenograft models of pancreatic cancer, and assessed tumour growth and metastases using immunohistochemistry and phosphotyrosine-based proteomic profiling.
Results: PC xenografts in NOD/SCIDgcnull mice stably overexpressing S100A2 grew larger than empty vector controls and these mice developed extensive liver metastases that expressed high levels of S100A2. The predominant tyrosine kinases with increased activation in S100A2 xenografts were the Src family kinases (SFKs) YES/FYN/FGR, the SFK substrate Paxillin, and the proto-oncogenes ABL1 and UFO/AXL. Gain of S100A2 function in vitro revealed no difference in proliferation for S100A2 overexpressing PC cells compared to empty vector controls, however increased cell motility was observed for S100A2 overexpressing Panc 10.05 cells using wound healing assays.
Conclusion: These data suggest that high S100A2 expression defines a specific pancreatic cancer phenotype and plays a key role in metastasis. S100A2 is a potentially important molecular target for the development of novel therapeutic strategies for PC.
Marine Sponge Extract for Targeted Elimination of Pancreatic Cancer Stem-like Cells
S. Schlesinger,1 G. Kallifatidis,1,2 A. Klöppel,3 F. Brümmer,3 I. Herr.11Molecular OncoSurgery Group, Department of General Surgery, University of Heidelberg and German Cancer Research Center, Heidelberg, Germany, 2Translational Immunology Unit, German Cancer Research Center, Heidelberg; Germany; 3University of Stuttgart, Institute of Biology, Department of Zoology, Stuttgart, Germany.
Background: Despite intense efforts to develop treatments against pancreatic cancer, agents that cure this highly resistant and metastasizing disease are not available. Considerable attention has focused on marine organisms, which produce many undiscovered secondary metabolites with bioactivity against cancer cells. If these compounds are also effective against cancer stem cells is unknown.
Material and methods: The effects of methanolic extracts of ten different marine sponges and one freshwater sponge were screened. In vitro models of pancreatic tumor cells with stem-like phenotype were used to evaluate CSC-marker expression, ALDH1 activity, spheroid-forming and clonogenic capacity, migratory activity, apoptosis induction, multi-drug-resistance, viability and cell cycle arrest.
Results: While each sponge extract in 1:10 dilution diminished viability, the extract of one sponge was still effective in a dilution of 1:1000 against pancreatic cancer stem cells, while sparing normal cells like primary fibroblasts and endothelial cells. This special sponge extract efficiently and specifically inhibited cancer stem cell characteristics along with inducing apoptosis and preventing proliferation. This was due to inhibition of self-renewal activity and sensitization to apoptosis by inhibition of caspases, clonogenicity, spheroid-forming, migratory activity and downregulation of anti-apoptotic and EMT-related proteins.
Conclusions: Our data suggest that sponge extract may be suited for specific targeting of cancer stem cell characteristics. In our ongoing studies we aim to isolate the pure active substances for further evaluation alone or combined with standard chemotherapy.
Analysis of the Etiology in 458 Pancreatitis Patients According to the M-ANNHEIM Multiple Risk Factor Classification
A. Schneider,1 J. M. Löhr,2 M. V. Singer.11Dept. of Medicine II, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, German; 2Karolinska Institute, Stockholm, Sweden.
Background: Chronic pancreatitis (cP) results from the interaction of various risk factors. The M-ANNHEIM classification stratifies multiple (M) etiological risk factors such as Alcohol (A), Nicotine (N), Nutrition (N), Heredity (H) (including idiopathic disease), Efferent duct factors (E), Immunity (I), Miscellaneous factors (M), differentiates various disease stages and defines different degrees of disease severity.
Objectives: To identify the interaction and frequency of known etiological risk factors in pancreatitis patients.
Methods: Patients (n=458 with sufficient data, exclusion of biliary pancreatitis) from our clinic in Germany (1997-2008) were classified according to the M-ANNHEIM classification.
Results: In n=172/458 patients (38%, n=49 borderline cP, n=123 definite or probable cP), only up to one etiological risk factor was found. In n=286/458 patients (62%, n=62 borderline cP, n=224 definite or probable cP), two or more risk factors were identified. Alcohol was observed as a risk factor in n=266/465 (58%) patients. Nicotine was found in n=311/458 (68%) individuals. Alcohol was found in n=25/172 (15%) and Nicotine in n=44/172 (26%) patients with up to one risk factor. Alcohol was found in n=241/286 (84%) and Nicotine in n=267/286 (93%) patients with multiple risk factors. Interaction of Alcohol with Nicotine was found in n=236/286 (83%) patients with multiple risk factors. Idiopathic disease without any known etiological risk factor was identified in only n=56/458 (12%).
Conclusion: Nicotine is more frequently found as a risk factor of pancreatic inflammation than Alcohol. Idiopathic pancreatitis with absence of any known risk factor is rare.
Comparison of the Mannheim Diagnostic Criteria of Autoimmune Pancreatitis with other Diagnostic Criteria Systems
A. Schneider,1 J. M. Löhr,2 M. V. Singer.11Dept. of Medicine II, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, German; 2Karolinska Institute, Stockholm, Sweden.
Background: Different diagnostic criteria for autoimmune pancreatitis (AiP) have been developed in various centers from Europe, USA and Asia. We recently developed the Mannheim AiP Diagnostic Criteria. A consensus about the different diagnostic systems has not been reached.
Objectives: To compare the Mannheim AiP Diagnostic Criteria with other diagnostic systems.
Methods: Patients with non-alcoholic pancreatitis from our clinic (1997-2009) were studied. "Mannheim Definite AiP" is diagnosed in patients with negative pancreatic cancer work-up and fulfilling Mayo HISORt or Asian AiP Criteria; or simultaneously presenting with either typical imaging findings (CT or MRI), elevation of serum IgG4 or other autoantibodies, and disease response to steroids; or simultaneously presenting with idiopathic pancreatic disease, other autoimmune disease or features suggestive of AiP (e.g. Sjögren's syndrome), and disease response to steroids. In patients with "Mannheim Definite AiP", we compared the Mannheim AiP Diagnostic Criteria with Japan-, Korean-, Asian-, Mayo HISORt-, Revised Mayo HISORt- and Italian-Criteria.
Results: We detected "Mannheim Definite AiP" in n=21 patients. In n=5/21 patients, pancreatic histology was obtained by surgery. In only these patients, diagnosis of AiP could be established by any diagnostic system. In n=8/21 patients, the diagnosis of AiP was only achieved with the Mannheim AiP Diagnostic Criteria. In this cohort of patients, all individuals responded to steroid medication.
Conclusions: The Mannheim AiP Diagnostic Criteria allow the diagnosis of AIP in atypical forms of the disease.
R1 Rate and Lymph Node Status in Pancreatic Cancer is Determined by Intratumoral Factors
R. Segersvärd, A. Leifler, S. Ghazi, F. Jonsson, M. Nilsson, J. Permert. Dept. of Surgical Gastroenterology, CLINTEC Karolinska Institutet at Karolinska University Hospital Stockholm, Sweden.
Introduction: Microscopic residual disease (R1) and lymph node (LN) yield after pancreaticoduodenectomy (PD) for pancreatic cancer (PC) are commonly regarded as a function of surgical technique (i.e. sampling of the disease) and high quality pathology (i.e. analysis of the sample). The impact of intrinsic tumor factors on R1 rate and LN-status are less studied.
Aim: To evaluate the impact of intra-vascular (V), lymphatic (L) and perineural (PN) tumor growth on R1 rate and LN status.
Methods: 100 consecutive PD for PC from our prospective registry after implementation of standardized pathology. Venous resections (VR), R1 rate, tumor size (Tsize), differentiation (diff), and the presence (1) or absence (0) of V, L, PN growth, regional (N) and extra regional LN metastases (M1 LN) were analyzed. LN ratio (LN-met/LN) was calculated.
Results: Median Ts 30 mm, VR 31%, R1-rate 62 %. 54 % poor diff. V1 56%, L1 51%, PN1 87%. N1 82%, median LN-yield 21, LN ratio 0.2. M1LN 30%. In univariate analysis presence of V1 significantly increased R1, N1, LN ratio and M1LN. Presence of L1 did not affect R1 but increased N1, LN-ratio and M1LN. PN1 increased R1 and N1 but not M1LN. In multivariate analysis V1 was an independent risk factor for R1 (OR 7.2), N1 (OR 12.8) and M1LN (OR 10.2). PN1 was a contributing risk factor for N1 (OR 8.4). No impact of L1 was observed.
Summary: Intra-vascular growth (V1) is an independent risk factor for non-radical resection and presence of metastatic disease.
Conclusion: Intrinsic tumor factors determine local tumor clearance and nodal status. Pancreatic cancer should be considered primarily "biologically" non-resectable. Consequently, neoadjuvant strategies should be considered.
Clinical Implications of Multi-Color Co-Detection of microRNA and Protein Biomarkers in Pancreatic Cancer
L. F. Sempere,1 M. Preis,1 M. Korc.1,21Department of Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH; 2Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH.
Background: High-throughput profiling experiments have linked altered expression of microRNAs to different types of cancer, including pancreatic adenocarcinoma (PDAC). PDAC tissue specimens are a heterogeneous mixture of not only cancer cells, but also supportive and reactive tumor microenvironment elements. To clarify the clinical significance of altered microRNA expression in PDAC, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize microRNA accumulation within individual cells in formalin-fixed paraffin-embedded clinical specimens. This ISH method was implemented to be compatible with routine clinical immunohistochemical (IHC) assays to enable detection of microRNAs and protein markers.
Experimental design: We utilized this combined ISH/IHC assay to study a subset of PDAC-associated microRNAs, including microRNAs frequently detected at low (miR-126 and miR375) and high (miR-21 and miR-155) levels in a panel of matched normal pancreas and PDAC tissues.
Results: While altered expression of miR-21 was predominantly manifested within cancer cells, that of miR-126, miR-155 and miR-375 was confined to endothelial, immune and endocrine cells, respectively. These results suggest a heterogeneous participation of microRNAs in carcinogenesis by intrinsically affecting cancer cell biology or by modulating stromal, vascular and immune responses.
Conclusions: This rapid and sensitive multi-color ISH/IHC assay could be broadly applied as an investigational tool to better understand the etiological relevance of altered microRNA expression in PDAC, which has both diagnostic and therapeutic implications.
Nociceptor Cell Sprouting: Implication for Pancreatic Pain Perception
S. Seo,1,2 G. A. Lomberk,2 A. J. Mathison,2 N. Buttar,2 S. Brimijoin,3 A. Windebank,4 R. Urrutia.21Mayo Graduate School, 2Division of Gastroenterology, 3Department of Pharmacology and Experimental Therapeutics, 4Department of Neuroscience, Mayo Clinic, College of Medicine, Rochester, MN, USA.
Pancreatic cancer is a dismal disease with severe pain, which is processed through nociceptors, sensory neurons that respond to harmful stimuli. More than 80% of the neurons in dorsal root ganglion (DRG) are nociceptors. NGF released from the target tissue plays a critical role in survival and differentiation of these nociceptors. During chemotherapy with platinum-based compounds, there is a severe lesion of nociceptors with increase in pain. Thus, discovering the molecular mechanisms that lie downstream of NGF during nerve terminal formations by nociceptors may be helpful for designing new therapeutics to ameliorate this symptom. Here, we used DRG andPC12 cells as model systems. We have identified two transcription factors, KLF10 and KLF11, to be involved in the differentiation and maturation of nociceptors. KLF10 mRNA level increases upon NGF treatment, while that of KLF11 is decreased, suggesting that both may be involved in different subroutines of neuronal cell differentiation and nociceptor terminal sprouting. Measurement of neurite lengths shows that KLF11 does not alter the neurite formation upon NGF treatment whereas KLF10 increases neurite outgrowth. Transcript profiling demonstrates that KLF11 modulates distinct neurotransmitter receptors, including dopamine receptors. ChIP assays show that the dopamine receptor D2 (DRD2) is a direct target of KLF11, working via the epigenetic regulator HP1. Drugs that modulate the dopamine system such as antidepressants are often prescribed to pancreatic cancer patients and their effect on tolerating pancreatic pain may be a result of an effect on this pathway.
The Effects of Preoperative Epidural Catheter (PEC) Placement on Outcomes in Patients Undergoing Pancreatectomy
N. Shah,1,2 K. Rosborough,3 B. Bonaventura,3 J. Zhang,4 M. Bloomston,1 C. Schmidt,1 W. S. Melvin,1,2 E. C. Ellison,1 P. Muscarella.11Division of Gastrointestinal Surgery & 2Center for Minimally Invasive Surgery, Department of Surgery; 3Department of Anesthesia; 4Center for Biostatistics, Ohio State University Medical Center, Columbus, OH.
Introduction: The purpose of this study was to investigate the effects of preoperative epidural catheter (PEC) placement on operative factors and postoperative outcomes in patients undergoing pancreatectomy.
Methods: After obtaining Institutional Review Board (IRB) approval, we retrospectively reviewed the charts of 426 patients who underwent pancreatectomy at our institution from 1997 through 2007. Operative data and postoperative outcomes were compared using the Chi-square test and Student's t-test.
Results: Four hundred and twenty-six patients with a mean age of 62.6 ± 12.5 years underwent pancreatectomy. Patients were divided in two groups: Group 1 - PEC placed (n = 202, 47.4%) and Group 2 - PEC not placed (n = 224, 52.6%). PEC placement was associated with decreased episodes of hypertension, operative time (299.1 vs. 316.6 minutes; p=0.02), length of SICU stay (2.5 vs. 4.4 days; p=0.02), length of hospitalization (12.4 vs. 15.3 days; p=0.006), and increased episodes of intraoperative hypotension. There were no significant differences in postoperative morbidity or mortality.
Conclusion(s): The operative time, length of SICU stay and length of hospitalization was significantly less with PEC placement. Although PEC placement was associated with increased episodes of intraoperative hypotension, this did not result in increased adverse patient outcomes. Prospective, randomized clinical trials are required to confirm these findings.
Alcohol Promotes Pancreatic Mitochondria Depolarization by Sensitizing the Permeability Transition Pore to Ca2+
N. Shalbueva, O. A. Mareninova, S. J. Pandol, A. S. Gukovskaya. VAGLAHS, University of California Los Angeles & Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA.
Introduction: We showed that loss of mitochondrial membrane potential ΔΨm, due to opening of the permeability transition pore (PTP), mediates pancreatic acinar cell death. Here we investigate ethanol's effects on ΔΨm and the role of PTP regulation by Ca2+ in these effects, by using mice deficient in cyclophilin D (CypD), a key component of PTP.
Methods: Changes in ΔΨm elicited by 50-100 mM ethanol alone or combined with 10 pM CCK-8 were measured with TMRM dye in mouse acinar cells.
Results: Ethanol decreased ΔΨm and potentiated CCK-induced mitochondrial depolarization. These effects are mediated through PTP as they were absent in CypD null acinar cells. Using permeabilized cells in which free Ca2+ concentrations were clamped with Ca/EGTA buffers, we tested the hypothesis that alcohol promotes depolarization by increasing PTP's sensitivity to Ca2+. Ca2+ dose-dependently depolarized acinar cell mitochondria, which was manifest in control cells at above 0.6 μM Ca2+ and resulted in complete loss of ΔΨm at 2 μM Ca2+. In ethanol treated cells, this dose-dependence curve shifted towards lower Ca2+, with depolarization already evident at 0.3 μM Ca2+ and complete loss of ΔΨm at 1.3 μM Ca2+. PTP inactivation in CypD null cells prevented Ca2+-induced depolarization, with and without ethanol.
Conclusion: Ethanol depolarizes pancreatic mitochondria and further, potentiates CCK-induced depolarization. Ethanol's effects are mediated by increased sensitivity of PTP to Ca2+. Mitochondrial depolarization, mediated through ethanol's effect on PTP and leading to acinar cell necrosis, represents one mechanism underlying ethanol's toxicity to pancreas.
Mist1 Suppresses Kras-Induced Acinar-to-Ductal Metaplasia Through the Mitogen-Activated Protein Kinase Pathway
G. Shi, D. DiRenzo, S. F. Konieczny. Department of Biological Sciences and the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the US. Several mouse models have been generated to recapitulate the development of PDAC, as well as PDAC precursor pancreatic intraepithelial neoplasia (PanIN), by expressing a KrasG12D oncogene from the endogenous Kras promoter. Previous studies in our laboratory have established that PanIN lesions can arise from differentiated acinar cells through acinar-to-ductal metaplasia (ADM). However, the regulatory mechanism involved in KrasG12D-induced ADM has not been well characterized. To better understand the signaling networks that regulate ADM, primary pancreatic acinar cells were maintained in vitro in a collagen-based 3D culture system, where acinar cells undergo an ADM process and form ductal cysts upon expression of KrasG12D. Utilizing a series of pharmacological inhibitors, we have found that activation of the MAPK pathway is required for ADM, whereas the Kras-dependent PI3K/Akt pathway is dispensable in this context. The transition from acinar to ductal phenotypes is also affected by the status of acinar cell differentiation. Loss of Mist1, a transcription factor that regulates acinar cell differentiation, accelerated PanIN formation in mouse models, and a similar acceleration of KrasG12D-induced ADM in the absence of Mist1 was observed in 3D acinar cell culture. Interestingly, sustained expression of Mist1 drastically reduced the numbers of ductal cysts in vitro and ADM in vivo, possibly through inhibition of MAPK activity. Future studies will identify the molecular mechanism(s) through which Mist1 represses ADM, which may lead to novel strategies to prevent the development of advanced PDAC disease.
Collagen Promotes Snail-driven Invasion through Increased TGF-β Signaling in Human Pancreatic Cancer Cells
M. A. Shields,1 S. Dangi-Garimella,1 S. Krantz,2 D. J. Bentrem,2,3,4 H. G. Munshi.1,3,41Division of Hematology/Oncology, Department of Medicine, and 2Division of Surgical Oncology, Department of Surgery, Feinberg School of Medicine, Northwestern University, 3The Jesse Brown VA Medical Center, and 4The Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL 60611.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by pronounced fibrotic reaction composed primarily of type I collagen. Collagen has been shown to promote invasion and metastases, but the underlying mechanisms remain poorly understood. Since epithelial-mesenchymal transition (EMT) is also associated with cancer invasion, we examined the extent to which collagen modulated the expression of Snail, a well-known regulator of EMT. Relative to cells grown on tissue culture plastic, PDAC cells grown in collagen induced Snail. Inhibiting the activity or expression of the TGF-β type I receptor abrogated collagen-induced Snail. Downstream of the receptor, Smad3 and Smad4 were critical for the induction of Snail by collagen. In contrast, Smad2 or ERK1/2 was dispensable for collagen-mediated Snail expression. Over-expression of Snail in PDAC cells resulted in a membrane type 1-matrix metalloproteinase (MT1-MMP)-dependent increase in invasion through 3D collagen. Mechanistically, Snail increased the expression of MT1-MMP through activation of ERK-MAPK signaling. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that collagen-rich milieu can increase the expression of Snail, and suggests that the fibrotic reaction actively contributes to PDAC progression.
Impact of the Histone Deacetylase Inhibitor 4-Phenylbutyrate on Apoptotic Tumor Cell Clearance by Macrophages in Pancreatic Carcinoma
L. Shmorgun, D. Dovzhanskiy, K. Felix, T. Hackert, W. Hartwig, T. Welsch, M. W. Büchler, J. Werner. Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Germany.
Background: Histone deacetylases (HDACs) inhibitors have been found to have potent anticancer activities, partly induced by tumor cell apoptosis. Clearance of apoptotic tumor cells is an important mechanism of antitumor immune response. Aim of the study was to assess the impact of 4-phenylbutyrate (4-PB) and its immunological effect on macrophage clearance of apoptotic pancreatic ductal adenocarcinoma (PDAC) cells.
Methods: A co-culture system of human macrophages and PDAC cell lines (T3M4, PANC-1 and AsPC-1) was used to assess phagocytic activity, cytokine expression, and apoptosis by fluorescence-activated cell sorting, western blot analysis and confocal microscopy.
Results: Apoptotic PDAC cells were phagocytosed by human macrophages. 4-PB treatment resulted in a dose-dependent increase of tumor cell apoptosis and a significantly increased phagocytosis of T3M4- and PANC-1 cells of 11.2 and 11.3% (P<0.05), respectively, compared to untreated cells. Likewise, 4-PB stimulated expression of the apoptotic marker p21 in those cells by 60 and 40%, respectively. Phagocytic activity semiquantified by AnnexinA1 protein expression was not significantly altered. 4-PB treatment induced the expression of the inflammatory cytokines IL-6 and IL-10 in human macrophages from PDAC patients.
Conclusion: 4-PB treatment activated tumor cell apoptosis in PDAC cells resulting in unspecific tumor cell phagocytosis by macrophages. The latter was characterized by a pro-inflammatory cytokine response, which might lead to inhibition of tumor cell growth through immunomodulation.
Advanced Pancreatic Cancer Treated With Neoadjuvant Therapy and Surgical Resection: A SEER-Medicare Analysis
J. K. Smith, S. C. Ng, J. P. Simons, S. A. Shah, T. P. McDade, J. F. Tseng. Surgical Outcomes Analysis & Research, University of Massachusetts Medical School, Worcester, MA.
Background: Pancreatic cancer is often diagnosed at late stages when surgical resection is no longer possible. However, the introduction of neoadjuvant therapy has given rise to potential downstaging of advanced disease and increased opportunities for resection. This study examined the use of neoadjuvant therapy in the U.S. for patients with advanced (stage III or IV) pancreatic cancer.
Methods: The Surveillance, Epidemiology, and End Results (SEER)-Medicare linked databases (1991-2007) were used to identify all Medicare-eligible patients ≥ 65 years with a diagnosis of pancreatic cancer. Treatment patterns for patients with advanced cancer were examined using univariate and survival analyses.
Results: Overall, 23,836 pancreatic cancer patients were identified, 905 (4.3%) stage III, and the majority, 14,323 (67.8%) stage IV. Of those with stage III or IV, treatments included: chemotherapy (4320, 28.4%), radiation (576, 3.8%), chemoradiation (1609, 10.6%), resection (189, 1.2%), resection with adjuvant therapy (136, 0.9%), and neoadjuvant therapy with resection for a minority of patients (42; 0.3%). The majority underwent no treatment (8356, 54.9%). Survival analyses demonstrated that patients who underwent neoadjuvant therapy had longer median survival than those who underwent resection alone or even resection with adjuvant therapy (13 months vs. 4 and 9 months, respectively; p<0.0001).
Conclusions: Traditionally, stage III or IV pancreatic cancer is considered unresectable; however, these findings suggest that there may be a highly selected group of patients who may gain a survival benefit from neoadjuvant therapy followed by resection.
Pancreatic Fluid Oxidized Fatty Acids (OxFA) are Elevated in Non-Calcific Chronic Pancreatitis (CP)
T. Stevens, M. P. Berk, Y. M. Chung, R. Zhang, A. Costanzo, M. P. Bronner, A. E. Feldstein. Cleveland Clinic, Cleveland, OH, USA.
Introduction: Oxidative stress plays a central role in the pathogenesis of CP. A specialized liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) assay was developed to quantify a profile of OxFAs, even at low concentrations. This method has not been studied in pancreatic fluid.
Aim: Compare the OxFA profile in pancreatic fluid from controls and pts with CP using HPLC-MS.
Methods: Pts undergoing EUS and endoscopic pancreatic function test (ePFT) for evaluation of CP were included. Pts with other inflammatory diseases or taking antioxidants were excluded. Two groups were studied: 1. Symptomatic controls [abdominal pain, no risk factors (e.g. acute pancreatitis, heavy alcohol), "normal" EUS (Rosemont Class), and normal ePFT (peak bicarb ≥80 mM)] vs. 2. Non-calcific CP [abdominal pain, previous acute pancreatitis, and either EUS "suggestive" or "indeterminate" with abnl ePFT]. A duodenal fluid sample was obtained endoscopically in the first 10 min after secretin. Samples were preserved with antioxidant and argon gas overlay, and frozen at -80°C. Samples were analyzed for hydroxy-eicosatetranoic acids (HETEs 5, 8, 9, 11, 12 and 15) and hydroxy-octadecadienoic acids (HODEs 9 and 13) using LC-ESI-MS/MS. Statistical analyses utilized student's t-test.
Results: 18 patients included (9 controls; 9 CP). Levels of all OxFAs were higher in CP compared to controls (7 out of 10 statistically-significant). The most differentially-expressed product was 9-HODE, with a mean (SD) concentration of 1703 fmol/μl (891) in CP vs. 715 (359) in controls (p=0.007).
Conclusions: Pancreatic fluid OxFA levels are elevated in non-calcific CP and may represent a useful biomarker for diagnosing early CP.
Factors Associated With Survival after Surgery for Recurrent Pancreatic Cancer
O. Strobel, W. Hartwig, T. Hackert, U. Hinz, S. Fritz, L. Schneider, M. Büchler, J. Werner. Dept. of Surgery, University of Heidelberg, Heidelberg, Germany.
Background: Local recurrence of pancreatic cancer occurs in up to 80% of pats. within 2 years after a potentially curative resection. Around 15% of these pats. have isolated local recurrence without systemic progression. In spite of localized disease these pats. usually only receive palliative chemotherapy.
Aims: To evaluate the outcome of surgery in locally recurrent pancreatic cancer and to assess factors associated with survival.
Methods: Local recurrence after pancreatic resections was diagnosed early by rigorous follow-up examination every three months after resection. From 10/01 to 10/09 112 pats. underwent exploration for suspected isolated local recurrence. Perioperative outcome and survival were analyzed.
Results: 104 pats. had a histologically proven recurrence. 61 (59%) pats. had an isolated local recurrence, in 43 (41%) pats. surgical exploration revealed metastases. Resection could be performed in 45 (74%) pats. with isolated local recurrence, 16 (26%) recurrent tumors were irresectable. Morbidity and mortality were 24% and 1.9% in resections and 9% and 0% in explorations w/o resection. Median survival in isolated local recurrence was 17 months vs. 9.1 months in metastatic disease (p<0.0001). In isolated local recurrence median survival was significantly longer after resection (21.6 months) compared to exploration w/o resection (10.7 months, p=0.0089). Presence of metastases, the status of resection, the use of intraoperative radiotherapy, and preoperative tumor markers CA19-9 and CEA were associated with survival. Pat. age and disease-free interval were not associated with survival.
Conclusion: Resection for recurrent pancreatic cancer is safe and resection may prolong survival in isolated local recurrence. In irresectable local recurrences intraoperative radiotherapy may be beneficial. CA19-9 and CEA-levels may be useful for patient selection.
Results of Surgery after Neoadjuvant Therapy for Primarily Irresectable Pancreatic Cancer in 257 Patients
O. Strobel, V. Berens, W. Hartwig, T. Hackert, U. Hinz, S. Fritz, L. Schneider, M. Büchler, J. Werner. Dept. of Surgery, University of Heidelberg, Heidelberg, Germany.
Background: In pancreatic cancer (PanCan) complete macroscopic resection is the only potentially curative therapy. Many patients present with locally advanced tumors that are primarily irresectable.
Aim: To assess the results of surgery after neoadjuvant therapy for primarily irresectable PanCan.
Methods: From a prospective database all consecutive patients undergoing surgery from 10/01 to 12/09 after neoadjuvant therapy for primarily irresectable PanCan were identified. Main criteria for irresectability were involvement of the celiac axis and superior mesenteric artery. Resection rates, perioperative results and survival were analyzed.
Results: Of 257 pats. 199 (77.4%) had received chemoradiation, 58 (22.6%) chemotherapy only. 120 (46.7%) pats. underwent resection, 137 (53.3%) exploration only. 47 (39.2%) multivisceral and 45 (37.5%) vascular resections (11 arterial reconstructions) were performed. In the resection group, there were 6 (5%) ypT0 tumors, 29 (24.2%) R0-, 57 (47.5%) R1-, 14 (11.7%) R2- and 14 (11.7%) Rx-resections. Overall morbidity and mortality were 30.7% and 5.0% and associated with multivisceral/vascular resections. 38 patients are still alive with a median follow-up of 11 months. Median survival was significantly longer after resection (12.7 months) than after explorations (8.8 months, p<0.0001). Median survival was 24.6 months after R0-, 12.2 months after R1- and 8.9 months after R2-resection (p=0.0340).
Conclusions: In primarily irresectable PanCan R0/R1-resections can be achieved in up to 40% of patients that undergo surgery after neoadjuvant therapy and result in survival rates similar to that observed for primarily resectable PanCan.
Putative Pancreatic Cancer Stem Cell Marker DCAMKL-1: Role in EMT
S. M. Sureban,1,3 R. May,1,3 S. A. Lightfoot,2,3 J. H. Wyche,4 S. Anant,1 C. W. Houchen.1,3Departments of 1Medicine and 2Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 3Veterans Affairs Medical Center, Oklahoma City, OK; 4Howard University, Washington, DC.
Background and Aim: The stem cell origin of pancreatic cancer has generated great interest in the identification of cancer stem cells (CSCs). Epithelial-mesenchymal transition (EMT) plays a key role in cancer invasion/metastasis. Recently, a link between EMT and the gain of epithelial "stem cell" properties has been described. Here, we sought to determine the expression patterns and involvement of the stem cell/CSC marker DCAMKL-1 in pancreatic cancer.
Methods: Immunohistochemistry was performed (DCAMKL-1, 14-3-3s, Vimentin, Snail and Slug) on human pancreatic tumor samples and uninvolved tissue. Total RNA isolated following siRNA-mediated knockdown of DCAMKL-1 was subjected to real-time RT-PCR for mRNA (DCAMKL-1, c-Myc, KRAS, Notch-1, ZEB1, ZEB2, Snail, Slug and Twist) and microRNA (pri-miR-200a, pri-miR-144 and pri-let-7a) analysis in AsPC-1 cells. Luciferase reporter assay was performed to measure let-7a and miR-144 expression in AsPC-1 cells.
Results: We found increased expression of DCAMKL-1 that co-localized with Vimentin, 14-3-3s, Snail and Slug in human pancreatic adenocarcinomas. Furthermore, siRNA-mediated knockdown of DCAMKL-1 in AsPC-1 cells resulted in down-regulation of ZEB1, ZEB2, Snail, Slug and Twist following induction of miR-200a (EMT inhibitor). Finally, knockdown of DCAMKL-1 also resulted in down-regulation of the proto-oncogenes c-Myc and KRAS via let-7a, and Notch-1 via miR-144 microRNA dependent mechanisms.
Conclusion: These findings illustrate a direct regulatory link between pancreatic CSC marker DCAMKL-1 and EMT, and suggest an important target for stem-cell-directed therapy in pancreatic cancer.
Pancreatic Cancer Microenvironment: Tumor-Host Interactions
D. Swartz-Basile,1 D. Basile,2 J. Friedrich,2 P. White,1 K. Ziegler,1 S. Wang,1 H. Pitt,1 N. Zyromski.11Department of Surgery; 2Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN.
Background: The importance of the pancreatic cancer stroma is increasingly recognized. However, little is known about the origin of these stromal cells. Recent advanced color-coded imaging technology allows visualization of the tumor microenvironment. These imaging models may provide greater understanding of the pancreatic cancer stroma cellular origin. We hypothesized that visualizing the pancreatic tumor microenvironment with color-coded fluorescence imaging can distinguish pancreatic cancer cells from host stromal cells and thereby determine tumor cell lineage.
Methods: Five C57Bl/6J and 5 Red Fluorescent protein-expressing (RFP) B6.Cg-Tg (CAG-mRFP1)1F1Hadj/J female mice (5 wks) were studied. At 8 wks of age all mice were inoculated with Green Fluorescent protein (GFP)-expressing murine pancreatic cancer (PAN 02) cells (2.5 × 105 cells) in the right flank. After 5 weeks of growth, tumors were evaluated by immunofluorescent staining of GFP and RFP and visualized using confocal microscopy.
Results: Immunofluorescent imaging of GFP tumors from RFP immunocompetent mice demonstrated interspersed GFP and RFP cells. RFP labeled cells generally had stromal phenotype (i.e. fibroblast, vascular endothelium). No overlapping signal was seen, suggesting that stromal cells are recruited from the host into the tumor.
Conclusions: These data suggest that pancreatic cancer stromal cells are recruited predominantly from the host. Improved understanding of stromal cell recruitment mechanisms may identify novel therapeutic targets for pancreatic cancer.
Selective, High-Affinity Inhibitors of Human Chymotrypsin C (CTRC)
A. Szabó,1 K. Zboray,2 D. Héja,2 D. Szakács,2 G. Pál,2 M. Sahin-Tóth.11Department of Molecular and Cell Biology, Boston University, Boston, MA; 2Department of Biochemistry, Eotvos University, Budapest, Hungary.
Background and Aims: The digestive enzyme chymotrypsin C (CTRC) proteolytically regulates activation and degradation of human cationic trypsinogen. Mutations in CTRC are risk factors for chronic pancreatitis. The aim of the present work was to develop selective peptide-inhibitors against CTRC.
Methods: A chymotrypsin inhibitor from the desert locust Schistocerca gregaria served as the parent molecule for an inhibitor library displayed on M13 phage. Amino acids of the reactive loop of the inhibitor are numbered as P4-P3-P2-P1-P1'-P2'-P3'-P4'. The P1 amino-acid of the inhibitor interacts with the primary specificity pocket of CTRC. Positions P4, P2, P1, P1', P2' and P4' of the reactive loop were randomized and phages binding to CTRC were selected. A consensus reactive sequence was deduced after DNA-sequencing 25 phage clones. The newly identified inhibitors were recombinantly expressed, purified and tested against human chymotrypsin B1, B2, and C; elastase 2A, 3A and 3B; and chymotrypsin-like enzyme 1.
Results: The best CTRC inhibitor exhibited a KD of 60 pM and a selectivity of 300-200,000-fold over other pancreatic proteases. A Leu at the P1 position and acidic residues (Asp or Glu) at the P4' position were important for high affinity binding, whereas an Asp residue at the P2' position increased selectivity.
Conclusions: Novel inhibitors against CTRC will be useful reagents to study the role of CTRC in cellular and animal models of pancreatitis.
Chymotrypsin C is Required for the Full Activation of Human Procarboxypeptidases A1 and A2
R. Szmola,1,3 M. Bence,1 A. Carpentieri,1,2 J. Samuelson,1 C. E. Costello,2 M. Sahin-Tóth.11Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA; 2Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, Boston, MA; 32nd Department of Medicine, Semmelweis University, Budapest, Hungary.
Background and Aims: Procarboxypeptidases A1 and A2 (pro-CPA1/A2) are major digestive zymogens in the human pancreatic juice with an unclear activation mechanism. Activation of pro-CPA1/A2 has been suggested to occur through tryptic cleavage of the activation peptide followed by limited proteolysis of the inhibitory prosegment by CPA1/A2 itself. However, in vitro studies indicate that the activation peptide is highly resistant against CPA1/A2 cleavage, suggesting the involvement of other proteases in the activation process. The aim of the present study was to investigate the possible role of chymotrypsin C (CTRC), a known regulator of trypsin activity, in the activation of procarboxypeptidases.
Methods: Human procarboxypeptidase and chymotrypsinogen isoforms were expressed in HEK 293T cells via transient transfection. Proteases were purified with ion-exchange and inhibitor affinity chromatography. Activation of procarboxypeptidases was followed by enzymatic assays, SDS-PAGE, and mass spectrometry analysis.
Results: Tryptic activation of pro-CPA isoforms resulted in partial activity only (10-15%), however, after addition of CTRC rapid and full activation of the zymogens was observed. CTRC selectively cleaved the evolutionarily conserved Leu96-Leu97 peptide bond in the activation peptide followed by multiple cleavages resulting in complete degradation of the activation peptide. Importantly, chymotrypsins B1 and B2, elastase 3B and chymotrypsin-like protease 1 were ineffective in promoting activation of the two pro-CPA isoforms.
Conclusions: Our results clearly demonstrate that cleavage by CTRC is required for full activation of human pro-CPA1 and pro-CPA2. The findings extend the role of CTRC as a key regulator of protease activity in the human pancreas.
Gene Expression Profiling Reveals Long Intronic Non-Coding RNAs Differentially Expressed in Pancreatic Cancer and Metastasis
A. C. Tahira,1 M. S. Kubrusly,2 M. F. Faria,1 S. Verjovski-Almeida,1 E. M. Reis,1 M. C. C. Machado.21Department of Biochemistry, Instituto de Química, 2Department of Gastroenterology, School of Medicine (LIM-37), University of Sao Paulo, Sao Paulo, SP, Brazil.
Aim: To investigate the expression of noncoding RNAs (ncRNAs) in clinical samples from pancreatic cancer patients.
Introduction: Pancreatic Adenocarcinoma (PA) is known by its aggressiveness and poor prognosis. Therefore, new molecular markers for detection and prognosis are needed. Long ncRNAs expressed from intronic regions play different roles in regulation of gene expression, but its relevance in pancreatic cancer has not been investigated.
Methods: Spotted cDNA microarrays interrogating ncRNAs were used to profile RNA samples from 15 primary tumors (T), 8 chronic pancreatitis (CP), 9 non-tumor pancreatic tissue (N) and 6 biopsies from distant metastasis (M).
Results: We identified 142 transcripts differentially expressed in T relative to N and CP samples (FDR<10%) comprising 37 ncRNAs. Comparison between M to T (FDR<5%) showed a signature of 143 transcripts, including 45 ncRNAs, revealing transcripts possibly associated with the invasive phenotype. Gene enrichment analysis indicated that the metastasis signature is enriched in ncRNAs transcribed from intronic regions of genes from the MAPK pathway and involved in apoptosis, being 9 transcripts in total. Increased expression in metastatic samples of intronic ncRNAs mapping to the PPP3CB and MAP3K14 loci was confirmed by qPCR.
Conclusion: Our work revealed a gene expression profile correlated to metastasis and pancreatic cancer that is enriched in long intronic ncRNAs. Further investigation is warranted to determine the functional role played by these transcripts during transformation and progression of pancreatic tumors.
Down-Regulation of Mcl-1 by Baicalein Induces Apoptosis through the Mitochondrial Pathway in Human Pancreatic Cancer Cells
H. Takahashi,1 M. C. Chen,1 D. M. Harris,2 H. Pham,2 H. A. Reber,1 O. J. Hines,1 V. L. W. Go,2 G. Eibl.11Department of Surgery and 2Medicine, UCLA Center for Excellence in Pancreatic Diseases, David Geffen School of Medicine at UCLA, Los Angeles, California.
Introduction: We previously reported that baicalein, derived from ScutellariaBaicalensis, decreased proliferation and stimulated apoptosis in pancreatic cancer (PaCa) cell lines. However, the precise molecular mechanism of baicalein-induced apoptosis on PaCa cells is still poorly understood.
Aim: Our aim was to delineate the cellular mechanisms for the pro-apoptotic effects of baicalein in PaCa cells.
Methods and Results: We first confirmed that baicalein induced apoptosis in four PaCa cell lines (BxPC-3, HPAF-II, MIA PaCa-2, and Panc-1) by cell death ELISA and Hoechst staining. BxPC-3 cells were most sensitive for baicalein treatment. Treatment with baicalein (5-15 μM) resulted in mitochondrial cytochrome c release, cleavage of caspase-3/-7 and PARP in BxPC-3 and MIA PaCa-2 cells. Furthermore, the pan-caspase inhibitor zVAD-fmk reversed baicalein-induced cleavage of PARP and apoptosis by 75 % (BxPC-3) and 90 % (MIA PaCa-2), indicating a caspase-dependent apoptosis by baicalein. Baicalein decreased mRNA and protein expression of the anti-apoptotic Bcl-2 family protein Mcl-1in a dose-dependent manner, but did not alter Bcl-xL protein expression, as shown by real-time PCR and Western blot. To delineate whether Mcl-1 is essential for PaCa cell survival, Mcl-1 expression was knock-down by siRNA that resulted in marked cleavage of PARP and induction of apoptosis.
Conclusion: These results demonstrate that the pro-apoptotic effect of baicalein is mediated through a mitochondrial pathway and at least partially through a reduction of the pro-survival function of Mcl-1.
New Surgical Procedure for Groove Pancreatitis
Y. Takeyama, T. Yasuda, H. Ishikawa, T. Nakai, M. Yamasaki, K. Kamei, Y. Nakata, M. Takemoto. Department of Surgery, Kinki University Faculty of Medicine, Osaka, Japan.
Introduction and Aim: In groove pancreatitis, pylorus-preserving pancreatoduodenectomy is usually indicated when symptoms do not improved, or when malignancy cannot be denied. However, when malignancy can be denied this procedure may be too invasive. Thus, we formulated a less-invasive procedure to treat this condition and applied clinically.
Method: Upper part of the duodenum and the groove area of the head of the pancreas are resected. The anal cut end of the duodenum is set between the minor and the major papillas. The main pancreatic duct should be completely preserved. To avoid the injury of the main pancreatic duct, the drainage tube in the main pancreatic duct is inserted preoperatively or intraoperatively. Gastrojejunostomy is performed in Roux-en-Y fashion. The tube inserted into the main pancreatic duct is kept and drained externally.
Results: This new procedure was indicated in two cases. The both cases were classified as a pure form of groove pancreatitis which means the lesion is restricted to the groove area without involvement of the main pancreatic duct. The common bile duct cold be preserved in one case, but it was resected and reconstructed in the other case. A minor leakage of the pancreatic juice in one case could be treated conservatively. In the long-term observation, the pancreatic function and morphology have been maintained.
Discussion & Conclusion: The new less-invasive operative procedure has been successfully applied to tow cases with groove pancreatitis. The target case of this procedure should be limited to a pure form of groove pancreatitis, and malignancy must be strictly denied preoperatively.
The Role of Cyclooxygenase-2 Gene Polymorphisms in Pancreatic Carcinogenesis
R. Talar-Wojnarowska,1 A. Gasiorowska,1 M. Olakowski,2 A. Lekstan,2 P. Lampe,2 B. Smolarz,3 H. Romanowicz-Makowska,3 A. Kulig,3 E. Malecka-Panas.11Dep of Digestive Tract Diseases, Medical University of Lodz; 2Dep of Digestive Tract Surgery, Silesian Medical University, Katowice; 3Lab of Molecular Genetics, Institute of Polish Mother's Memorial Hospital, Lodz, Poland.
Background: Cyclooxygenase-2 (COX-2) is a key enzyme involved in many biologic processes, such as cell proliferation, invasion, metastases and angiogenesis, which are all relevant to cancer development and progression. The purpose of this study was to evaluate the clinical significance of -765G/C and -1195G/A COX-2 gene polymorphisms in patients with pancreatic cancer.
Methods: The study included 201 patients: 85 with pancreatic cancer (PC) and 116 healthy controls. -765G/C and -1195G/A COX-2 genes polymorphisms have been studied in DNA isolated from blood samples. The associations of the analysed genotypes and clinical data at diagnosis have been evaluated.
Results: We found an increased frequency of the homozygous -1195AA COX-2 genotype in patients with PC (53,7%) compared with control group (21%) (p<0,01). In contrast, the distribution of genotype and allele frequencies of the -765G/C COX-2 polymorphism in the PC patients did not differ from those in control groups. The correlation between presence of homozygous -1195AA COX-2 genotype and tumor size > 3 cm has been observed (p<0,05). Analysed polymorphisms were unrelated to the patients sex and age as well as with the presence of regional or distant metastases.
Conclusion: These preliminary results indicate that -1195G/A COX-2 polymorphism may play important role in pancreatic carcinogenesis, however further studies are needed to investigate its possible association with PC prognosis.
All Patients With Acute Pancreatitis and Persistent Organ Failure Do Not Require Organ Failure Specific Interventions
R. Talukdar, S. S. Vege, M. Clemens. Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic College of Medicine, Rochester, MN, USA.
Background and aim: Patients with persistent organ failure (OF) (> 48hrs) in acute pancreatitis (AP) is associated with high morbidity and mortality. All guidelines recommend that these patients be treated in the ICU with OF-specific interventions. The aim of this study was to evaluate if OF-specific intervention was necessary for all patients with AP and persistent OF.
Methods: We studied 32 (11.7%) patients with persistent OF from a prospective database of 274 patients admitted with AP over 2 years. We defined OF according to the Atlanta Criteria and OF-specific interventions as hemofiltration/dialysis, BiPAP/CPAP/mechanical ventilation, and pressor support/intra-aortic balloon pump. Non-OF specific measures included fluid and oxygen therapy for renal, circulatory and respiratory failures respectively. Outcome variables were OF-specific intervention and ICU care requirement for different OFs.
Results: Of the 32 (11.7%) patients with persistent OF, 17 (53.1%) developed OF during hospital stay. Nine patients (28.1%) had single OF, 17 (53.1%) had two OF and 6 (18.8%) had three OF. 26 (81.3%) had respiratory failure, 24 (75%) patients had renal failure, and 13 (40.6%) had circulatory failure.
All patients with respiratory and circulatory failure required OF-specific treatment. Among the patients with renal failure, 15/24 (62.5%) could be managed with intravenous fluid therapy only. Overall, 5/32 (20.8%) patients did not require any OF-specific interventions. 29 (90.6%) of all patients with OF required ICU care.
Conclusion: This study shows that nearly 21% of patients with AP with persistent OF do not require any OF-specific intervention and nearly 10% of such patients can be treated outside the ICU.
Admission Systemic Inflammatory Response Syndrome (SIRS) Score Predicts the Development of Primary Intra-Abdominal Infection in Patients With Acute Pancreatitis
R. Talukdar, S. S. Vege, M. Clemens. Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic College of Medicine, Rochester, MN, USA.
Background and aim: Presence of organ failure in acute pancreatitis (AP) is known to be associated with infected necrosis and high mortality. There are no known predictors of intra abdominal infection in AP. The aim of this study is to assess the capability of simple baseline parameters to predict intra-abdominal infection.
Methods: We studied 274 patients with AP admitted to Mayo Clinic hospitals over a 2 year period and identified the patients who had microbiologically confirmed infections of pancreatic necrosis and peripancreatic fluid collections. We defined primary intra-abdominal infection as any infection that developed prior to any abdominal intervention. We recorded admission hematocrit, BMI, serum BUN/creatinine and SIRS score. We used univariate and multivariate analysis and expressed results as odds ratio(OR) [95% confidence intervals (CI)].
Results: Overall, 44(16.1%) patients had infections. 26(9.5%) had intra-abdominal infection, of whom 20(79.9%) had primary intra-abdominal infection. Among patients with primary intra-abdominal infection, 8(40%) had gram positive, 1(5%) gram negative and 11 (55%) mixed bacterial infection while 5(25%) had additional fungal infection. On univariate analysis, BUN≥25 mg/dL and SIRS≥2 were found to significantly predict primary intra-abdominal infection with OR (95% CI) of 2.68(0.96 -6.96) and 3.59(1.42-9.51)[2-tailed p = 0.048 and 0.007 respectively]. On multivariate analysis, only SIRS ≥2 was predictive with an adjusted OR (95% CI) of 3.13(1.21-8.49)[p=0.018].
Conclusions: SIRS is a simple severity assessment tool that can predict the development of primary intra-abdominal infection in AP early in the disease course.
Does Stopping Drinking Alcohol Even After the Onset of Overt Exocrine and Endocrine Pancreatic Insufficiency Improve the Prognosis of Patients With Alcohol-Induced Chronic Pancreatitis?
Y. Tando,1 M. Yanagimachi,1 Y. Matsuhashi,1 H. Tanaka,1 E. Sato,1 A. Matsumoto,1 S. Chikazawa,1 A. Kon,1 T. Suda,1 T. Nakamura.2Department of 1Internal Medicine III and 2Health Science, Hirosaki University School of Medicine.
Introduction and Aim: Excessive alcohol consumption is the major etiological cause in chronic pancreatitis. Natural course in patients with alcohol-induced chronic pancreatitis (ACP) was previously reported by Ammann or Lankisch. They indicated that alcohol abstinence brings a beneficial effect on mortality, invalidity, and endocrine pancreatic function. However, stopping drinking is not easy for patients and many patients continue alcohol intake despite doctor's advice. The aims of this study are to: (1) estimate drinking habit in patients with ACP and (2) make clear whether stopping drinking alcohol even in "burned-out" ACP improve the prognosis.
Patients and Methods: The records of 28 patients with ACP (10 continued and 18 discontinued alcohol intake) were reviewed focusing on drinking habit, clinical features and mortality. They visited our out-patient clinic on "burned-out" stage.
Results: All patients were free of pancreatic pain. Four patients with continued drinking and one patient with discontinued drinking needed increasing both pancreatic enzyme supplements and insulin. Frequencies of hypoglycemia (in particular, unawareness hypoglycemia in night) and fecal incontinence (not steatorrhea) were increased in ACP continued drinking. Extrapancreatic malignancy, infection and cardio-vascular disease were observed in ACP continued drinking.
Conclusion: Stopping drinking alcohol is necessary even after the onset of pain relief and overt pancreatic insufficiency in all patients with ACP.
Plasma Metabonomic Change of Acute Pancreatitis in Rats
W. F. Tang, J. Li, H. L. Gong, M. H. Wan, Y. X. Liu, Q. Xia. Department of Integrative Medicine, West China Hospital, Sichuan University, Chengdu, PR China.
Aim: To explore the plasma metabonomic changes of acute pancreatitis in rats.
Method: Rats were randomly divided into sham operation group (J), and acute pancreatitis group (Y, sodium taurocholate). 1HNMR was applied for metabolomic detection and analysis with 600MHz NMR spectrometer detection technology. The LED on the CPMG result-analysis and comprehensive use of PCA were used after OSC filtering noise PLS-DA analysis, to screen the differences in metabonimics.
Results: The component analysis and partial least squares discriminant analysis indicated the metabonomic difference between the two groups. Many different metabolites between the two groups were identified after analyzing the score maps, load charts and the original map, including lactic acid (lactate, d 1.3,1.34,4.1), Val (valine, d 0.98,1.02), succinic (amber acid, d 2.38), 3 - hydroxybutyric acid (3-HB, d 1.18), high density lipoprotein (HDL, d 0.86) and unsaturated fatty acids (UFA, d 2.78, 5.3),which were higher than those in the sham group. The metabolites of glycerol (Glycerol, d 3.58, 3.66), choline (choline, d 3.22); trimethylamine oxide (TMAO, d 3.26), glucose (glucose, d 3-4), glycine (glycine, d 3.54), (VLDL / LDL, d 1.3), (HDL / LDL, d 0.9), very low density lipoprotein (VLDL, d 1.34) and phosphatidylcholine (Ptd, d 2.78) were lower than those in the sham group.
Conclusion: There are significant metabonomic changes in rat plasma with acute pancreatitis, which might be part of the potential biological markers of the disease and therapeutic targets.
Multiple Organ Failure Following Acute Necrotizing Pancreatitis With Hemorrhage, and Ruptured Pseudocyst
D. Thirabanjasak. Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Aim: To describe the multiple organ failure following acute necrotizing pancreatitis accompanying with extensive hemorrhage and perforation of pseudocyst.
Background: Acute pancreatitis is a group of inflammation happening in the pancreas ranging in severity from edema and fat necrosis to parenchymal necrosis with severe hemorrhage. Acute pancreatitis is the serious condition with high mortality and morbidity. Either alcoholic or biliary-induced can lead to acute pancreatitis in several degrees. Besides, pseudocyst usually arise follow the episode of acute pancreatitis. Rupture, hemorrhage, or infection of pseudocyst are life-threatening condition and multiple organs failure, and all involved clinicians should aware of.
Methods: In the author's file, there is a complete autopsy case of patient expired from fatal complication of acute necrotizing pancreatitis with parenchymaal hemorrhage and rupture of pre-existing pseudocyst at the head of pancreas.
Results: A 46-year-old male with clinically acute abdomen was present to the doctor. History of long-term alcohol abuse was noted. Multiple organ failure developed during the hospital course, leading to expire. Autopy disclosed acute necrotizing pancreatitis with extensive parenchymal hemorrhage and spotty fat necrosis. Ruptured pseudocyst was found at the head of pancreas, 2cm. Hemoperitoneum (2000 ml) and acute fibrinous peritonitis were detected. Multiple organs involvement led to acute tubular necrosis of renal parenchyma, pulmonary congestion and edema, passive congestion of liver and passive congestion of spleen.
Discussion & Conclusion: Necrotizing acute pancreatitis is defined as necrosis of the exocrine and endocrine counterpart, including peripancreatic adipose tissue. Several causes are known predominantly biliary lithiasis and alcoholism. Severe consequences due to the release of pancreatic enzymes include activation of inflammation mediators which can lead to multiple organ failure. The anatomic changes following acute pancreatitis suggest autodigestion of the pancreatic substance by inappropriately activated pancreatic enzymes, especially trypsin. Digestion of leakage enzyme will cause damage to adipocytes and damage to elastic fibers of blood vessels leading to tissue hemorrhage. Besides, the pre-existing pseudocyst at the head of pancreas that caused from earlier episode of inflammation could lead to anatomical deviation of ductal system. On-top obstruction and new episode of acute pancreatitis is like a serious "wax and wane" condition and can end up with serious complication, the multiple organ failure syndrome.
A Randomized, Double-Blind, Dose-Response Control, Crossover Study of 2 Doses of EUR-1008 (ZENPEP) in Chronic Pancreatitis (CP) Patients With Exocrine Pancreatic Insufficiency (EPI)
P. Toskes,1 A. Secci,2 D. Broussard,2 R. Thieroff-Ekerdt.21University of Florida College of Medicine, Gainesville, FL; 2Eurand Pharmaceuticals, Inc., Yardley, PA.
Background: EPI is a serious condition associated with a number of diseases, including CF, CP, and pancreatic cancer, as well as GI surgery. Pancreatic enzyme products (PEPs) are used to treat symptoms of EPI (malabsorption, malnutrition, and steatorrhea).
Aim: This study evaluated the efficacy and safety of 2 doses of EUR-1008 [Zenpep® (pancrelipase) Delayed-Release Capsules] in adults with CP with EPI.
Methods: The effect of ZENPEP on the coefficient of fat absorption (CFA) was investigated in a randomized, double-blind, dose-response control, crossover study with placebo run-in (9-11 days) and 2 treatment periods (7-9 days) with a high dose (7×20,000 U lipase/day) and low dose (7×5000 U lipase/day) of ZENPEP.
Results: 76 eligible CP patients (fecal elastase <100 μg/g stool) were randomized. Mean CFA was significantly higher with low- (88.9%) and high-dose (89.9%) ZENPEP versus placebo run-in (81.7%; P<0.001) (n=72) without a difference between doses (P=0.228, primary endpoint). Post-hoc analysis revealed that the treatment effect increased with more severe EPI. ZENPEP high dose had greater increases in CFA than low dose in patients with severe steatorrhea (CFA <65%). CNA, body weight, and BMI also increased significantly with both doses compared to placebo run-in. Percentage of days with clinical symptoms of EPI decreased with both doses. ZENPEP was safe and well-tolerated, with GI complaints being the most frequently reported adverse events.
Conclusions: Our findings suggest that CP patients with EPI benefit from the low-dose ZENPEP while the high dose used in this study might be needed for patients with more severe EPI.
The Greater Severity of Pancreatitis in CFTR Deficient Mice is Independent of Acinar Cell Effects
S. Trapp,1 P. Pallagi,3 J. Mayerle,1 G. Biczo,3 A. Dummer,1 Z. Rakonczay,3 M. Sendler,1 F.-U. Weiss,1 W. Halangk,2 P. Hegyi,3 M. M. Lerch.11Department of Medicine A, Ernst-Moritz-Arndt-University Greifswald, Germany; 2Division of Experimental Surgery, Otto-von-Guericke-University Magdeburg, Germany; 3University of Szeged, First Department of Medicine, Pancreatic Research Group.
Introduction: The incidence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is 17-26% in patients with non-alcoholic chronic pancreatitis. It remains unknown how CFTR mutations may cause or contribute to pancreatitis.
Methods: We investigated the course of acute pancreatitis in wild-type and transgenic CFTRtm1HGU mice which were generated by an insertion of a stop-cassette into exon 9 and retain only 20% of residual CFTR-function (Pathobiology 2002; 70: 89-97). Exocrine pancreatic function was determined by measuring stool chymotrypsin activity. Pancreatitis was induced by supramaximal caerulein stimulation and severity determined by serum-enzyme and myeloperoxidase measurements and lung and pancreatic histology. To study CCK effects in living isolated acini, intracellular trypsinogen and pro-elastase activation was determined. To investigate the pancreatic ductal function, intact pancreatic ducts were isolated and fluid and HCO3- secretion were measuered by video swelling technique and microfluorometry.
Results: CFTRtm1HGU mice develop no spontaneous phenotype and have no exocrine pancreatic insufficiency, clinically a precondition for developing pancreatitis. Pancreatitis in CFTR-transgenic animals animals was more severe as indicated by higher serum amylase and lipase activities as well as greater intrapancreatic trypsinogen activation. Myeloperoxidase activity in the pancreas was significantly increased in CFTRtm1HGU animals as early as 1h after onset. In isolated acini upon supramaximal CCK stimulation, however, intracellular trypsin and elastase activities were identical between wild type and CFTRtm1HGU mice. Co-incubation of activated leukocytes from CFTRtm1HGU mice with wild type animals and vice versa did not result in changes in premature intracellular zymogen activation. Induction of pancreatitis resulted in impaired apical HCO3- transport. Fluid secretion was also diminished in untreated CFTR transgenic animals and after induction of pancreatitis.
Conclusion: CFTR deficiency leads to more severe course of experimental pancreatitis in animals with intact pancreatic function. This increased disease severity, particularly after 8h, is not due to alterations that affect acinar cells directly or their CFTR expression and is due to either an impairment of the systemic inflammatory response or a diminished fluid secretion which highlights the importance of duct cells in the pathogenesis of pancreatitis.
The Mek/Erk Pathway Promotes Notch-Dependent Signaling in Human Pancreatic Cancer Cells
I. Tremblay, Marie-Josée Boucher. Gastroenterology Unit, Dept. of Medicine, Fac. of Medicine and Health Sciences, University of Sherbrooke, Canada.
Background: Recent studies suggested that the ability of Ras to transform cells depends on cooperation with the Notch signaling pathway. However, the molecular and cellular mechanisms involved in this cooperation remain unknown.
Aim: To evaluate the impact of the Mek/Erk pathway, downstream of Ras, on Notch signaling.
Methods: The MIA PaCa-2 and BxPC-3 cell lines were used. Mek/Erk activity was down-regulated by treatment with the specific inhibitor U0126. Transfections or retroviral infections of a constitutive active form of KRas or Mek (MekCA) were used to increase Mek/Erk activity. Notch-dependent transcription was measured by luciferase assays using a CSL-luciferase or hes1-luciferase reporter genes.
Results:1- Transient transfection of an activated KRas or a MekCA increased Notch-dependent transcription. In parallel, treatment with U0126 reduced the basal and completely blocked the KRas- and MekCA-induced Notch-dependent transcription. 2- Western blot analysis revealed higher molecular weight forms of NIC (active Notch) when cells were transfected/infected with MekCA as compared to control MekWT-transfected/infected cells. U0126 treatment prevented the MekCA-induced decreased in mobility shift of NIC suggesting that NIC is subjected to Mek-dependent post-translational modifications in vivo. 3- Immunoprecipitation followed by in vitro phosphatase assay revealed that these MekCA-induced higher molecular weight forms of NIC corresponded to phosphorylated forms. 4- By in vitro kinase assays, we demonstrated that NIC worked as an Erk substrate.
Conclusion: Our results provide the novel discovery that Mek/Erk signaling promotes NIC phosphorylation which correlates with increased Notch-dependent transcription.
Claudin-4 (CLDN4) is Associated With Neoplastic Progression of Intraductal Papillary Mucinous Neoplasms (IPMNs) of the Pancreas
K. Tsutsumi, N. Sato, H. Fujita, K. Ohuchida, T. Ohtsuka, S. Takahata, K. Mizumoto, M. Tanaka. Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Background: Claudin-4 (CLDN4), encoding a protein for tight junction formation and function, is highly overexpressed in pancreatic ductal adenocarcinoma and is also associated with invasive phenotype of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. However, the expression pattern of CDLN4 during neoplastic progression of IPMNs remains unknown.
Methods: Using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), we analyzed CLDN4 mRNA in formalin-fixed paraffin-embedded (FFPE) tissues from 80 patients with IPMNs of different histological grades and papillary subtypes and 6 pancreatic juice samples (3 adenomas & 3 invasive carcinomas) obtained by endoscopic retrograde pancreatography for preoperative cytology.
Results: The CLDN4 expression was significantly higher in high grade IPMNs (borderline & carcinoma) than in low grade IPMNs (adenoma) (P < 0.0001). In addition, CLDN4 mRNA levels were significantly higher in intestinal type IPMNs than in non-intestinal type IPMNs based on papillary subclassification (P < 0.0001). Furthermore, the CLDN4 mRNA expression levels were significantly higher in pancreatic juice samples from patients with invasive carcinoma than in those from patients with adenoma (P = 0.0495).
Conclusion: Our findings suggest that CLDN4 may play an important role in the neoplastic progression of IPMNs and represents a novel diagnostic and therapeutic target.
L1 Cell Adhesion Molecule (L1CAM) Expression at the Cancer Invasive Front is a Novel Prognostic Marker of Pancreatic Ductal Adenocarcinoma
S. Tsutsumi,1,2 S. Morohashi,1,2 Y. Kudo,1 H. Akasaka,1,2 H. Ogasawara,1,2 M. Ono,1 K. Takasugi,1 K. Ishido,2 K. Hakamada,2 H. Kijima.1Departments of 1Pathology and 2Surgery, Hirosaki University School of Medicine, Aomori, Japan.
Background and Objectives: Pancreatic ductal adenocarcinoma (PDAC) is one of the most extremely aggressive cancers with a poor prognosis after curative resection. L1 cell adhesion molecule (L1CAM) is a 200-220 kDa type I transmembrane glycoprotein of the immunoglobulin superfamily, which has been shown affect the prognosis of several cancers. No clinicopathological significance of L1CAM expression has been examined at the invasive front of PDAC. In this study, we examined the relationship between L1CAM expression and clinicopathological features in PDAC by immunohistochemistry.
Methods: One hundred seven surgically resected specimens of PDAC were immunohistochemically examined using a monoclonal antibody against L1CAM.
Results: Positive expression of L1CAM was found in 23 of 107 cases with PDAC. In most cases (21/23), L1CAM expression was localized at the invasive front of the tumor tissue. Positive expression of L1CAM was significantly correlated with the histological grade, lymph node involvement, and distant metastasis. In univariate analysis, a positive expression of L1CAM was associated with short overall survival (P = 0.0002), and this was significant in multivariate analysis (P=0.009).
Conclusions: L1CAM could play an important role in the invasive process in vivo, and is thought to be a good indicator of prognosis in PDAC.
TNF-α-Induced NF-κB Activation in AR42J Cells Requires Both Serine and Tyrosine Phosphorylation of IκBα
E. Twait, D. Williard, I. Samuel. VAMC & UI CCOM, Iowa City, IA.
Nuclear factor-kappa B (NF-κB) is a transcription factor necessary for induction of many proinflammatory cytokines. Inhibitor of κB (IκB) is composed of the subunits IκBα and IκBβ, which maintain NF-κB in its inactive form in the cytosol. The classical pathway mediating NF-κB activation involves serine phosphorylation of IκBα (residues 32 and 36) in response to stimulation by agonists such as TNF-α. Our current findings provide evidence that not only is TNF-α-stimulated NF-κB activation in rat exocrine pancreatic AR42J cells mediated by this classical pathway, but also by an alternative oxidative stress related pathway involving tyrosine phosphorylation (residue 42) of IκBα. AR42J cells infected with an adenovirus containing a NF-κB responsive luciferase reporter showed a strong increase in NF-κB activation when stimulated with TNF-α, as measured by luciferase activity. Electrophoretic Mobility Shift Assay (EMSA) data using AR42J cell nuclear extracts further confirmed this activation by showing increased NF-κB DNA:protein interaction in the nucleus in response to TNF-α stimulation, while supershift analysis showed this interaction was NF-κB p65 subunit specific. By expressing two dominant-negative forms of IκBα (DN.IκBα), we were able to investigate NF-κB p65 activation in relation to both agonist stimulation (S32A/S36A mutation) and oxidative stress (Y42F mutation). Control empty vector (EV) infected cells showed strong NF-κB activation in response to TNF-α stimulation, which was completely prevented by expression of either the S32A/S36A or the Y42F form of DN.IκBα (p65 immunoblots and ELISA using nuclear extracts). These findings indicate that TNF-α-induced NF-κB activation in AR42J cells requires the phosphorylation of both the serine 32/36 and the tyrosine 42 activation sites of IκBα.
BMP Regulates Autophagy in Acute Pancreatitis (AP)
M. Tyler,1 Y. Cao,1 W. Yang,1 J. Aronson,2 V. Popov,2 C. Chao,2 M. R. Hellmich,2 T. C. Ko.1,21Department of Surgery, UTHSC-Houston; 2UTMB, Galveston, TX.
Introduction: Autophagy is a homeostatic mechanism in which a cell 'recycles' cellular material in response to dynamic changes in its energy pools. Recently, it has been shown that dysregulated autophagy contributes to AP. Autophagy is controlled by sequential expression of proteins such Beclin-1 and LC3-II. Beclin-1 is expressed during phagophore formation which precedes LC3-II expression during autophagosome formation. Previously, we have shown that BMP signaling is activated in cerulein (CR)-induced AP, and treatment with noggin, a BMP antagonist, attenuates CR-induced AP (Yang W et al. APA Annual meeting 2009). The purpose of this study is to test the hypothesis that BMP regulates authophagy in CR-induced AP.
Methods: C57BL/6 mice were randomized into two groups (n=7/group): (1) 9 hourly injections of CR (50 μg/kg, ip); (2) pretreatment with noggin, (0.5 μg/kg, ip) followed by CR (50 μg/kg, 9 hourly ip injections). Mice were euthanized 1 hr after last CR injection. Pancreas tissue was harvested to examine for the presence of autophagic vacuolization by electron microscopy and for autophagy markers by immunoblotting.
Results: Noggin pretreatment attenuated CR-induced autophagic vacuolization, and lowered LC3-II levels by 72.5% compared to CR treatment alone (p<0.05). Interestingly, in the presence of noggin, CR resulted in a 2.5-fold increase in Beclin-1 levels compared to CR treatment alone (p<0.05).
Conclusion and Discussion: These results suggest that noggin blocks autophagy at the transition from phagophore to autophagosome which is associated with attenuated AP. Targeting BMP signaling pathway may be a novel strategy to block disregulated autophagy seen in AP.
Analysis of ICOS and IL-10 Positive Regulatory T cells in Patients With Autoimmune Pancreatitis
K. Uchida, T. Kusuda, T. Ikeura, Y. Sakaguchi, K. Yoshida, T. Fukui, M. Shimatani, M. Matsushita, M. Takaoka, A. Nishio, K. Okazaki. Department of Gastroenterology and Hepatology, Kansai Medical University, Moriguchi, Japan.
Background & Aim: Autoimmune pancreatitis (AIP) have several immunologic abnormalities, including increased levels of serum IgG4 and infiltration of IgG4-positive plasmacytes. However, the role of IgG4 is unclear. Recently ICOS is paid attention to the key molecule of IL-10 secretion from Tregs. We analyzed the Tregs to clarify the role of IgG4 production in AIP.
Subjects and Methods: We obtained pancreatic tissue from 6 AIP and 6 cases of chronic pancreatitis. Hepatic tissue was sectioned from 16 IgG4-SC patients and 26 patients with PSC. We studied infiltrating IgG4-positive cells and Tregs in the pancreas and liver by immunohistochemistry. ICOS positive and IL-10 producing Tregs were analyzed from peripheral blood by flow cytometry.
Results: Tregs were increased in the pancreas with AIP (24.6±18.0 cells/HPF) compared with controls (5.1±4.3 cells/HPF). The numbers of IgG4-positive plasmacytes were significantly higher in AIP (18.6±10.3 cells/HPF) than in controls (1.1±0.7 cells/HPF). However, there were no significant differences in CD3+, CD4+, or CD79+ cells between the AIP and controls. In the liver, IgG4-positive plasmacytes were significantly higher in IgG4-SC (16.6±7.7 cells/HPF) than in PSC (4.0±0.7 cells/HPF). Tregs in the patients with IgG4-SC (5.4±1.5 cells/HPF) were also significantly increased compared with PSC (2.0±0.3 cells/HPF). In the patients with IgG4-SC, the numbers of infiltrated Tregs were significantly positively correlated with IgG4-positive plasma cells (R=0.75). ICOS+Tregs (3.45±1.58%) were significantly increased in comparison with healthy control (1.57±0.69%), alcoholic chronic pancreatitis (1.71±0.98%), idiopathic chronic pancreatitis (1.80±0.86%). Furthermore, IL10+ICOS+Tregs (3.81±1.52%) in LPSP were significantly increased compared with healthy control (1.38±0.64%). IL-10 producing Tregs and IgG4 also positively correlated in untreated AIP, (R=0.53).
Conclusion: In AIP, Tregs may affect the switching of B cells to IgG4-producing plasmacytes.
Race Does Not Substantially Affect Outcomes in Patients With Pancreatic Cancer
N. P. Valsangkar,1 D. M. Bush,2 J. S. Michaelson,1,2 C. Fernandez-del Castillo,1 A. L. Warshaw,1 S. P. Thayer.11Department of Surgery; 2Laboratory for Quantitative Medicine, Mass General Hospital, Boston, MA.
Background: It has been postulated that race and ethnicity play a major role in outcomes of patients with pancreatic cancer. Prior studies have attributed this difference to differences in health care access and quality of care received.
Methods: A large population-based database (SEER) of 64,200 patients from 1980 to 2005 was reviewed. One-, three- year and median survivals were calculated across two groups: African-American (AA) and Caucasian (C) patients. Univariate and multivariate analyses were done with Kaplan-Meier curves and the Cox proportional hazards model.
Results: There were no significant differences between the two groups in: age at presentation, tumor size, stage or primary site of tumor (C, n=58623; AA, n= 7645). There was no statistical difference in one-, three-year and median survivals (C= 40%, 10%, 9mo vs. AA= 39%, 8%, 9mo). A statistically significant difference in one-year survival between the two groups was identified for pancreatic head cancers (C 46% vs. AA 34%) that did not extend to tail cancers (C 34% vs. AA 33%), but this difference was not significant at three years. There was also a lower one-year survival for AA with grade II cancers (C 40% vs. AA 36%, P< 0.05).
Conclusions: This analysis suggests that AA and C patients present with similar demographics and extent of disease. Overall, there were no statistically significant differences in survival, although some differences were found in small subsets of patients. Despite previous reports, race and ethnicity does not appear to play a major role in determining survival. This finding may be due to the aggressive biology coupled with ineffective treatments that ultimately define the survival in PDAC.
Importance of Apical BK Channels on Pancreatic Duct Bicarbonate Secretion
V. Venglovecz,1,5 P. Hegyi,2 Z. Rakonczay,2 L. Tiszlavicz,3 M. Grunet,4 A. Nardi,4 M. A. Gray.51Department of Pharmacology and Pharmacotherapy; 2First Department of Medicine; 3Department of Pathology, University of Szeged; 4NeuroSearch A/S, Ballerup, Denmark and National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark; 5Institute for Cell and Molecular Biosciences, Newcastle upon Tyne, UK.
Introduction: We have previously shown that luminal administration of chenodeoxycholate (CDC) at a low concentration (0.1 mM) stimulated HCO3- secretion in intact pancreatic ducts via an elevation of intracellular calcium concentration. Our aim was to study the mechanism underlying the CDC-stimulated HCO3- secretion.
Methods: We used patch clamp and microfluorimetry to measure whole cell currents and rates of HCO3- secretion, respectively, from isolated guinea pig pancreatic duct epithelial cells (PDEC). Expression of ion channels was investigated by immunohistochemistry of intact pancreatic tissue.
Results: CDC (0.1 mM) reversibly increased whole cell K+ currents in PDEC. Activated K+ currents were inhibited by Ba2+ (2 mM), iberiotoxin (100 nM), and suppressed by strong intracellular Ca2+ buffering. Luminally applied iberiotoxin also blocked CDC-stimulated HCO3- secretion from microperfused ducts. Moreover, the specific large-conductance Ca2+-activated K+ channel (BK) activator, NS11021 induced a similar increase in HCO3- secretion to CDC. Immunohistochemical analysis showed strong BK channel protein expression on the apical membrane of PDEC.
Conclusion: We show for the first time that BK channels are expressed at the apical membrane of guinea pig PDECs and play a crucial role in regulating HCO3- secretion. We speculate that BK channels represent a novel therapeutic target to develop new therapies for acute pancreatitis.
Protective Effect of Dexamethasone Against Acute Respiratory Distress Syndrome in Patients With Severe Acute Pancreatitis
M. H. Wan, J. Li, W. F. Tang, H. L. GONG, P. Xue, L. Zhu, G. Y. Cheng, Q. Xia. Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, China.
Introduction: In various experimental pancreatitis models, dexamethasone (Dx) has been shown to inhibit inflammatory mediators and ameliorate systematic inflammatory response syndrome (SIRS), including acute respiratory distress syndrome (ARDS). However, its effect on ARDS in patients with severe acute pancreatitis (SAP) is not well addressed. We therefore performed a prospective study to investigate the effect of Dx on ARDS in patients with SAP, based on routine Chinese purgative herbs treatment.
Methods: In total 83 patients admitted to our hospital diagnosed SAP were enrolled in this study. The diagnosis of acute pancreatitis was based on "Atlanta" criteria, and SAP was defined by Balthazar CT severity index (≥ 5) complicated with SIRS. They were randomly allocated to control group (n=38), received standard treatment including Chinese purgative herbs, and Dx group (n=43), received additional 1mg/kg/d Dx treatment for three days. Complications, mortality rate, operational rate and days of hospitalization were compared between two groups.
Results: Dx treatment significantly reduced ARDS rate (20% vs 42.9%, P < 0.05) and the length of hospitalization (32 ± 13.2 d vs 40 ± 17.5 d, P < 0.05) compared to those in control group. Other parameters including the mortality rate were not significant different between the two groups.
Conclusion: Our study demonstrated that Dx can decrease the risk of developing ARDS in patients with SAP and shorten their length of hospitalization.
Da-Cheng-Qi Decoction Reduces Intra-abdominal Hypertension and Acute Lung Injury in Experimental Acute Pancreatitis Model in Rats
M. H. Wan, J. li, L. Zhu, G. Y. Cheng, W. F. Tang, Q. Xia. Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, China.
Introduction: Intra-abdominal hypertension (IAH) and acute lung injury (ALI) are the most common complications of acute necrotising pancreatitis (ANP). We investigated the effects of Da-cheng-qi Decoction (DCQD) on IAH and ALI in experimental acute pancreatitis in rats.
Methods: Male SD rats were randomly divided into three groups with ten rats each: (1) Rats with sham-operation (SO); (2) rats with ANP; (3) rats with ANP plus DCQD treatment. ANP was induced by retrograde infusion of 5% taurocholic acid into pancreatic duct. Two hours after operation, normal saline 10 ml/kg was orally adminstered to both SO and ANP groups, whereas DCQD 10 ml/kg was adminstered to the treatment group. Twenty-four hours after operation, aterial blood, pancreas and lung were collected for biomarkers and histopathology. Intra-abdominal pressure and intestinal propulsion rate were also measured. Moreover, survival rate for all groups was calculated.
Results: DCQD treatment significantly reduced intra-abdominal pressure and simultanously improved intestinal propulsion rate of ANP rats than those treated by saline (P <0.05). Both the pancreas and lung wet to dry weight ratio, myeloperoxidase activity and histopathology were significantly lower in DCQD group compared to ANP group (P <0.05). The oxygenation index in the DCQT group was significantly higher than ANP group (P >0.05), while no difference of PCO2 was found between two groups. For all the three groups, only two rats died in the ANP group.
Conclusion: We demonstrated that DCQD treatment can effectively relieve IAH and ALI in experimental pancreatitis, as a result this might improve the survival rate of rats with ANP.
Extended Pancreaticoduodenectomy With Vascular Resection and Reconstruction for Pancreatic Cancer
H. Z. Wang, G. Chen, L. D. Zhang, J. Ding, T. b. Xu, L. Cai, P. Bie. Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University Chongqing, P. R. China.
Introduction: Vascular resection and reconstruction performed at the time of pancreaticoduodenectomy (PD) for pancreatic carcinoma remains controversial. Vascular resection for locally advanced pancreatic carcinoma has an advantage in en block local resection. There are potential cases in which good outcomes can be achieved by vascular resection and reconstruction. The aim of this study was to evaluate whether patients with pancreatic carcinoma can benefit from vascular resection and reconstruction combined with pancreaticoduodenectomy.
Methods: From January, 2007 to November, 2009, 101 patients with pancreatic carcinoma underwent PD at Southwest Hospital, Third Military Medical University. Among them, vascular resection (VR) and reconstruction were performed in 42 patients, including SMV resection/reconstruction in 33 cases and portal vein resection/reconstruction in 9 cases. Data of patients was retrospectively collected and analyzed.
Results: There was no surgical mortality. No significant difference was observed in incidence of postoperative complications. Average survival time was 9.34 months in the group that required VR and 17.43 months in the group underwent standard PD (P<0.01). One year survival rate was 20.1% in VR group and 61.5% in PD group. Total recurrence rate was 59.5% in VR group and 37.3% in PD group respectively. A Cox proportional hazards model was constructed to analyze the effects of potential prognostic factors (vascular invasion, age, gender, tumor size, T stage, N status, margin status) on survival. Major vascular invasion had no impact on survival duration. However, lymph node invasion (LNI) had great negative impact on patient survival. There was no significant difference in the average survival time between the patients with/without LNI in VR and PD groups respectively. On the contrary, in each group, the survival time of patients without LNI was much longer than that of patients with LNI (P>0.05). There were 12 (28.6%) and 8 (13.6%) patients with LNI in VR and PD group respectively.
Conclusions: LNI is the most important risk factor on patient long-term survival after PD. Vascular invasion doesn't necessarily mean distant metastasis. The poor survival rate/time and high recurrence rate of the VR group are partly due to more patients with LNI in this group.
ATDC and KrasG12D Cooperate to Promote Pancreatic Adenocarcinoma in Mice
L. Wang,1 J. E. Wilkinson,2 H. Yang,1 J. Wu,1 M. Pasca Di Magliano,1,3 D. M. Simeone.1,4Departments of 1Surgery, 2Pathology, and 3Cell and Developmental Biology and 4Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI.
Pancreatic cancer is a deadly disease characterized by late diagnosis and therapeutic resistance. We recently showed that ATDC is overexpressed in 90% of pancreatic adenocarcinomas and acts as an oncogene by activating the Wnt signaling pathway.
To further explore the role of ATDC in pancreatic tumorigenesis, we generated transgenic mice with global ATDC expression driven by a CMV enhancer/β-actin promoter (CAG-ATDC). We verified the transgene expression in the mouse pancreas by qRT-PCR, Western blot and IHC analysis. The pancreas of CAG-ATDC mice presented with some mild acinar cell atypia starting at 6 months of age. However, we did not observe the development of PanIN lesions or carcinoma, even in older animals; therefore, ATDC expression is not sufficient to initiate pancreatic tumorigenesis. Next, we determined whether ATDC cooperates with oncogenic Kras in driving tumorigenesis. We generated CAG-ATDC;p48-Cre;LSL-KRASG12D (ATDC/Kras) transgenic mice and harvested animals at various time points (up to 3 months of age to date). Three month old CAG-ATDC mice showed no pancreatic abnormalities. At 3 months of age, PanIN1 lesions constitute 10% of the pancreatic parenchyma in p48-Cre;LSL-KRASG12D mice (n= 4), and no higher grade lesions are observed. In contrast, in age matched ATDC/Kras mice (n=6), up to 90% of the pancreatic parenchyma is formed by PanIN lesions, classified as PanIN1 (60%), PanIN2 (30%) and PanIN3, or carcinoma in situ (10%). Therefore, ATDC cooperates with Kras in driving PanIN formation and progression. We are currently aging a cohort of mice to determine the effect of ATDC overexpression on the onset of invasive and metastatic adenocarcinoma.
In conclusion, ATDC cooperates with oncogenic Kras to accelerate PanIN progression up to carcinoma in situ. ATDC/Kras mice may provide a useful tool to better understand the molecular events important in pancreatic cancer progression.
Isoflavone Enhanced the Efficacy of Gemcitabine In Vivo in Pancreatic Cancer
Z. Wang,1 A. Aboukameel,2 S. Banerjee,1 A. S. Asfar,2 R. Mohammad,2 F. H. Sarkar.11Department of Pathology; 2Department of Internal Medicine, Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI, USA.
Background: Pancreatic cancer (PC) remains one of the most aggressive cancers with a very poor prognosis. Previously in vitro studies have strongly supported that it has better killing of cells by gemcitabine when cells are pretreated with isoflavone. However, such studies have not been done to date in vivo for assessing survival benefit. Therefore, we investigated the combination effects of isoflavone with gemictibine on survival using an animal model.
Methods: The therapeutic utility of isoflavone and gemcitabine combination in SCID mice bearing subcutaneously implanted MIA PaCa-2 cells was investigated. 1 mg isoflavone/day/mouse was given by gavage while gemcitabine dose was 15 mg/kg administered intravenously.
Results: Our results showed, for the first time, the efficacy of combination of isoflavone and gemcitabine in the inhibition of pancreatic tumor growth in the model with improved survival. Isoflavone and gemcitabine alone caused 60% and 70% reduction in tumor weight, respectively, compared to control tumors. However, the combination of isoflavone and gemcitabine treatment showed significant decrease (p < 0.01) tumor weight compared to untreated control, isoflavone alone or gemcitabine alone treated group. Moreover, we found that isoflavone and gemcitabine increased the survival of animals significantly compared to control groups, respectively.
Conclusion: These results provide strong in vivo evidence in support of our hypothesis that isoflavone could be a novel agent in combination with a conventional therapeutic agent, which warrants further human investigation.
Eps8 is Subjected to Chaperone-Mediated Autophagy at Lysosomes in Pancreatic Cancer Cells
T. Welsch,1 A. Younsi,1 A. Disanza,2 A. M. Cuervo,3 G. Scita,2 J. Schmidt.11Department of Surgery, University of Heidelberg, Germany; 2FIRC Institute of Molecular Oncology, Milan, Italy; 3Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, USA.
Aim: Eps8 controls actin dynamics directly through its barbed end capping and actin-bundling activity, and has been associated with promotion of various solid malignancies. Here, we report a novel association of Eps8 with the late endosomal/lysosomal compartment, which is independent from actin polymerisation and specifically occurs in cancer cells.
Results: Endogenous Eps8 localized to large vesicular lysosomal structures in metastatic pancreatic cancer cell lines, such as AsPC-1 and Capan-1 that display high Eps8 levels. Additionally, ectopic expression of Eps8 increased the size of lysosomes. Structure-function analysis revealed that the region encompassing the amino acids 184-535 of Eps8 was sufficient to mediate lysosomal recruitment. Notably, this fragment harbors two KFERQ-like motifs required for chaperone-mediated autophagy (CMA). Furthermore, Eps8 co-immunoprecipitated with Hsc70 and LAMP-2, which are key elements for the CMA degradative pathway. Consistently, in vitro, a significant fraction of Eps8 bound to (11.9±5.1%) and was incorporated into (5.3±6.5%) lysosomes. Additionally, Eps8 binding to lysosomes was competed by other known CMA-substrates. Fluorescence recovery after photobleaching revealed that Eps8 recruitment to the lysosomal membrane was highly dynamic.
Conclusion: These results indicate that Eps8 in certain pancreatic cancer cells specifically localizes to lysosomes, and is directed to CMA. These results open a new field for the investigation of how Eps8 is regulated and contributes to tumor promotion in pancreatic cancers, probably effecting lysosomal degradation.
Evaluation of the ISGPS Definition of Delayed Gastric Emptying after Pancreatoduodenectomy in a High-volume Centre
T. Welsch, M. Borm, L. Degrate, M. W. Büchler, M. N. Wente. Department of General, Visceral and Transplantation Surgery, University Hospital Heidelberg, Heidelberg, Germany.
Background: Delayed gastric emptying (DGE) is a common complication after pancreatoduodenectomy (PD). The ISGPS (International Study Group of Pancreatic Surgery) definition of DGE has not been evaluated and validated in a high-volume centre.
Methods: Complete datasets including assessment of gastric emptying were identified from a database of patients undergoing PD between 2001 and 2008. Factors associated with DGE (grades A, B, and C) were assessed by uni- and multivariable analyses.
Results: DGE occurred in 340 of 764 patients (44.5%). Median hospital stays were significantly prolonged in patients with DGE (no DGE: 11, DGE A: 13, B: 21, C: 40 days, respectively). DGE was associated with prolonged (≥2 days) intensive care unit (ICU) admission (no DGE 9.4%, DGE A: 20.6%, B: 28.6%, C: 61.8%, respectively). Factors independently influencing DGE A were female sex, preoperative heart failure, and major complications (grade III-IV). Validation of the DGE definition revealed that DGE A and B were associated with interventional treatment in 20.1% and 44.4% of patients.
Conclusion: The identified risk factors for DGE are not amenable to perioperative improvement. The ISGPS DGE definition is feasible and applicable in patients with an uneventful postoperative course. Major postoperative complications and ICU treatment, however, limit its usefulness.
Effect of Underlying Disease Type on Efficacy of CREON® in a Randomized Trial of Patients With Exocrine Pancreatic Insufficiency (EPI) Due to Chronic Pancreatitis (CP) or Pancreatic Surgery (PS)
D. C. Whitcomb,1 E. Malecka-Panas,2 N. Gubergrits,3 S. Caras,4 Y. Shen,4 S. Sander-Struckmeier.51University of Pittsburgh, Pittsburgh, PA; 2Medical University of Lodz, Lodz, Poland; 3Donetsk National Medical University, Donetsk, Ukraine; 4Abbott, Marietta, GA; 5Abbott, Hannover, Germany.
Introduction: Efficacy of CREON® (pancrelipase [pancreatin] delayed-release capsules) has been shown in patients with EPI due to CP or PS in a randomized, controlled trial. Our aim was to determine any differences according to disease type (CP vs PS).
Methods: A sub-analysis of data from a double-blind, randomized, multicenter, placebo-controlled, parallel-group trial (NCT00414908) enrolling patients ≥18 years old with confirmed EPI due to CP or PS. After a 5-day placebo run-in period (baseline), subjects were randomized to CREON® (72,000 lipase units/meal) or placebo for 7 days. Exploratory sub-analysis according to disease type (CP vs PS) was carried out for the primary efficacy measure: change in coefficient of fat absorption (CFA) from baseline to end of double-blind period.
Results: Of 54 patients randomized, 40 had CP (16 received CREON® and 24 placebo) and 14 had PS (9 received CREON® and 5 placebo). There were no clinically meaningful differences according to disease type in CFA values at study end on CREON® (mean ± SD 87.9 ± 4.3% for CP and 83.3 ± 7.7% for PS); the greater mean ± SD changes in patients with PS (39.5 ± 23.4%) compared with those with CP (28.8 ± 14.5%) were most likely due to lower baseline values in the subgroup of patients with PS receiving CREON® (43.8 ± 24.1% vs 59.1 ± 14.5%).
Conclusion: In this study the beneficial effects of CREON® treatment were similar in patients with CP and PS, improving CFA to >80%.
Supported by Abbott, Marietta, GA
Significant Weight Gain After Long-Term Treatment With CREON® in Patients With Exocrine Pancreatic Insufficiency (EPI) Due to Chronic Pancreatitis (CP) or Pancreatic Surgery (PS), Despite Previous Oral Enzyme Therapy
D. C. Whitcomb,1 E. Malecka-Panas,2 G. A. Lehman,3 G. Vasileva,4 N. Gubergrits,5 S. Caras,6 Y. Shen,6 S. Sander-Struckmeier.71University of Pittsburgh, Pittsburgh, PA; 2Medical University of Lodz, Lodz, Poland; 3Indiana University Medical Center, Indianapolis, IN; 4MHAT Rousse AD, Rousse, Bulgaria; 5Donetsk National Medical University, Donetsk, Ukraine; 6Abbott, Marietta; GA, 7Abbott, Hannover, Germany.
Background: Pancreatic enzyme replacement therapy is vital to prevent severe maldigestion and undesired weight loss in patients with EPI due to CP or PS. Our objective was to assess long-term efficacy and safety of CREON® in this population.
Methods: A 6-month, open-label extension of a double-blind, placebo-controlled study enrolling patients ≥18 years old with confirmed EPI due to CP or PS who were previously taking oral enzymes. Patients received individualized CREON® doses as directed by investigators.
Results: 48 of 51 patients completed the open-label phase (one withdrew due to an unrelated treatment-emergent adverse event [TEAE] of burns; two were lost to follow-up). Mean age was 50.9 years, 70.6% were male, 76.5% had CP, and 23.5% had PS. The mean ± SD dose was 186,960 ± 74,640 lipase units/day. From baseline to study end subjects achieved a mean 2.7 ± 3.4 kg increase in body weight (p<0.0001). Mean change in stool frequency/day was -1.0 ± 1.3 (p<0.001). Improvements in abdominal pain, flatulence, and stool consistency were observed. TEAEs were reported by 22 patients (43%); only 7.8% were considered treatment related.
Conclusion: CREON® significantly increased weight, significantly reduced stool frequency, and was well tolerated over 6 months in patients with CP and PS previously managed with standard treatments.
Supported by Abbott, Marietta, GA
High-Fat Diet Hastens Mortality from Pancreatic Adenocarcinoma in Kras/P53/Cre Mice
P. B. White, H. Wu, M. Yip-Schneider, C. M. Schmidt, K. M. Ziegler, H. A. Pitt, D. A. Swartz-Basile, N. J. Zyromski. Department of Surgery, Indiana University School of Medicine, Indianapolis, IN.
Background: Obesity accelerates the development and progression of pancreatic cancer. We have previously shown that pancreatic cancers grow more rapidly and are more lethal in congenitally obese and diet-induced obese mice. Therefore, we hypothesized that high-fat diet induced obesity would accelerate pancreatic cancer growth in Kras/P53/Cre mice that spontaneously develop pancreatic adenocarcinoma.
Methods: KrasG12D/+;LSL-Trp53R172H;Pdx-1-Cre mice were randomized to either a 10% fat Low Fat Diet (LFD; n=4) or a 60% fat High Fat Diet (HFD; n=4) at an average age of 119 days. Mice were monitored daily and weighed weekly. Animals were sacrificed at the time of impending death (development of ascites, failure to eat, increasing lethargy). Pancreatic cancers were dissected, weighed, and measured. Body weight was compared with Student's t-test; survival was analyzed by log-rank and Wilcoxon rank-sum tests.
Results: The average peak weight of LFD mice was 29.4±1.7g and HFD mice was 33.7±3.1g (p=0.27). All mice spontaneously developed orthotopic pancreatic cancers. Multiple pancreatic tumors were seen in 1 of 4 LFD mice and 3 of 4 HFD mice. Metastases developed in 3 of 4 mice in each group. Median survival (range) was 126.5 (112-150) days in the LFD group and 99.5 (33-118) days in the HFD group (p=0.057).
Conclusion: These data suggest that a high-fat diet leads to: 1) modest (13%) weight gain; 2) development of multiple pancreatic tumors; and 3) decreased survival in Kras/P53/Cre mice that spontaneously develop pancreatic adenocarcinoma.
Cost Minimization Analysis Comparing Diagnostic Strategies in Idiopathic Pancreatitis: Importance of Prospective Data
C. M. Wilcox,1 M. L. Kilgore.21Division of Gastroenterology and Hepatology, 2School of Public Health, University of Alabama at Birmingham, Birmingham, Alabama.
Aim: Evaluate costs of diagnostic strategies for idiopathic pancreatitis (IP).
Background: The ideal endoscopic strategy for the evaluation of IP remains unsettled as some favor initial ERCP while others EUS. However, prospective trials encompassing both modalities are limited. A prior cost analysis using data from the literature suggests that EUS was the best initial strategy.
Methods: A decision analysis model of patients with two attacks of idiopathic pancreatitis with gallbladder in situ was constructed using TREE-AGE software. We analyzed cost and overall diagnostic ability of three strategies: EUS, ERCP with manometry, bile aspiration and endoscopic therapy as appropriate and laparoscopic cholecystectomy. Prevalence of causes for the base case analysis was taken from prospectively collected data on 33 such patients at our institution who underwent endoscopic evaluation. Medicare reimbursement rates were used to calculate costs.
Results: The identified causes after EUS +/- ERCP included microlithiasis (24%), sphincter of Oddi dysfunction (24%), pancreas divisum (21%), idiopathic (18%) and chronic pancreatitis (12%). Using the base case analysis, initial ERCP was the least costly strategy ($3,866) followed by EUS ($4,151) and laparoscopic cholecystectomy ($7,137). These results were sensitive to the prevalence of microlithiasis.
Conclusion: This cost minimization study suggests that initial ERCP with manometry and endoscopic therapy is the least costly initial test for the diagnostic evaluation of patients with recurrent IP with gallbladder in situ. These findings demonstrate the importance of determining causes based upon data unique to one's practice.
IκBα Inhibition Subdues LPS-Stimulated TNF-α Production in Isolated Acinar Cells
D. Williard, E. Twait, I. Samuel. VAMC & UI CCOM, Iowa City, IA.
Chemical inhibition of nuclear factor-kappa B (NF-κB) has shown reduced cytokine production following agonist stimulation, but chemical inhibitors can have non-specific targets other than NF-κB. Specific inhibition of NF-κB can be achieved by gene modulation of IκB. With adenoviral vector-mediated delivery of dominant-negative IκBα (DN.IκBα), we inhibited NF-κB in isolated acinar cells to clarify whether specific inhibition of NF-κB subdues agonist-stimulated cytokine production. We infected isolated acinar cells with empty vector (EV), DN.IκBα.Y42F, or DN.IκBα.S32A/S36A at 5MOI for 48h and found that DN.IκBα knocked down LPS-stimulated NF-κB activation and TNF-α production (*p<0.05, ANOVA). First, we stimulated infected cells with 10µg/ml LPS for 2.5 minutes and measured NF-κB p65 subunit activation in the nuclear fraction by ELISA. LPS resulted in a 2.7-fold increase in NF-κB p65 activation over unstimulated EV controls*. Incubation with DN.IκBα.Y42F or DN.IκBα.S32A/S36A returned the LPS-stimulated NF-κB p65 activation levels to those of the unstimulated EV controls*. In the second part of our study, we stimulated identical experimental groups with 10µg/ml LPS for 6h and measured TNF-α in the medium by ELISA. LPS resulted in a 170% increase in TNF-α production in the EV infected group compared to the unstimulated EV control*. DN.IκBα.Y42F limited the LPS-stimulated TNF-α increase by 46%*, while DN.IκBα.S32A/S36A limited the increase by 58%*. For each experiment, cell viability (ATP assay) was not significantly different between groups. Conclusions: The S32/S36 and Y42 phosphorylation sites of IκBα are both important in agonist-stimulated NF-κB activation. Specific inhibition of the NF-κB/IκB complex substantially subdues LPS-stimulated cytokine production in acinar cells.
Intracellular Calcium Signaling in Pancreatic Stellate Cells: Role in Pancreatic Disease
J. H. Won,1 J. Zhang,1 B. Ji,2 C. D. Logsdon,2 D. I. Yule.11Department of Pharmacology and Physiology, University of Rochester Medical School, Rochester, NY, 2Department of Cancer Biology, M.D Anderson Cancer, University of Texas, Houston, TX.
We have investigated the intracellular Ca2+ signaling characteristics of quiescent and activated stellate cells both in culture, and in situ in a pancreatic lobule preparation by multi-photon (MP) microscopy. We demonstrate that the complement of phospholipase C -coupled cell surface receptors is markedly altered following activation of stellate cells into a myoepithelial phenotype. Specifically, only activated cells (i.e those cells with prominent □-smooth muscle actin expression) express protease activated receptors (PAR). PAR activation results in prominent nuclear Ca2+ signals. These nuclear Ca2+ signals are greatly reduced by transfection of cells with a nuclear targeted parvalbumin construct which acts as a Ca2+ "sponge" specifically in this compartment. PAR activation results in the proliferation of activated stellate cells and this proliferation is inhibited by buffering nuclear calcium. In lobules of pancreas isolated from wild-type mice, and monitored with MP microscopy, Ca2+ signals were evoked in stellate cells by high concentrations of ATP and bradykinin, but not PAR agonists; characteristics consistent with quiescent stellate cells. In transgenic animals engineered to express a mutant KRas gene in acinar cells, a proportion of the stellate cells responded to low [ATP] and trypsin by increasing intracellular Ca2+, indicating an activated phenotype. In contrast to wild type animals, when experimental pancreatitis was induced in the KRas animals there was a marked increase in the number of stellate cells. Our data is consistent with the hypothesis that trypsin released from damaged acinar cells following induction of experimental pancreatitis may lead to proliferation of activated stellate cells in the KRas animals and contribute to the "chronic" pancreatitis phenotype observed in this animal model.
Binucleation and Polyploidization in Human Exocrine Pancreas
J. M. Xanthopoulos,1 D. Jain,2 F. S. Gorelick,1,3 E. S. Swenson.11Section of Digestive Diseases, Department of Internal Medicine, 2Department of Pathology, Yale University School of Medicine, New Haven, CT; 3West Haven Veteran's Affairs Medical Center, West Haven, CT.
Objectives: Most mammalian somatic cells contain a 2N complement of DNA. However, binucleation and incomplete mitosis may give rise to polyploid cells containing 4N or higher multiples of the haploid genome. Examples include megakaryocytes and hepatocytes. While polyploidy is generally associated with maturation, the dynamic nature and functional significance of polyploidization in acinar cells of the exocrine pancreas are not understood. We previously demonstrated age-dependent polyploidization in acinar cells of the normal mouse pancreas. Here we examine whether human acinar cells are binucleate and/or polyploid.
Methods: Human male pancreas sections were obtained through the Department of Pathology at Yale New Haven Hospital, with approval of the institutional Human Investigation Committee. Genome number was quantitated using fluorescence in situ hybridization for the Y chromosome. Cell membrane boundaries were delineated by E-cadherin immunolabeling. Nuclei were quantitated by DAPI staining.
Results: Preliminary results indicate that 10 percent of adult human male pancreatic acinar cells are binucleate (2N+2N); 10 percent contained a single tetraploid nucleus (4N) and the remainder were diploid (2N). Adult male mouse pancreatic acinar cells were 8 percent binucleate; 48 percent polyploid (4N or higher) and 44 percent diploid.
Conclusion: Binucleation and polyploidization are seen in adult human acinar cells, but at levels lower than that seen in adult mice. Future studies will address age and disease-related changes in acinar cell binucleation and polyploidization.
Protective Effect of Chai-Qin-Cheng-Qi Decoction on Acute Necrotising Pancreatitis Induced By L-Arginine in Mice
P. Xue,1 T. Jing,1 L. Wen,1 Z. Y. Cheng,1 L. Huang,1 W. Huang,2 X. N. Yang,1 Q. Xia.11Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, China, 2Departments of Surgery and Physiology, Royal Liverpool Hospital, University of Liverpool, UK.
Introduction: We previously reported that Chai-Qin-Cheng-Qi Decoction (CQCQD) ameilorates severity of caerulein-induced acute pancreatitis. Here we investigated the effects of CQCQD on acute necrotising pancreatitis (ANP) induced by L-Arginine.
Methods: Male KM mice were randomly divided into three groups with ten mice each: (1) Mice with saline injection, control group; (2) mice with ANP; (3) mice with ANP plus CQCQD treatment. ANP was induced by intraperitoneal injection of 4 g/kg L-arginine twice at one h apart. Seventy-two hours after begin of experiment, CQCQD 4 ml/kg × 3 at every 2 h was orally adminstered to the treatment group, whereas mice in other groups received saline. Mice were sacrificed 6 h after starting treatment, samples were collected for biomarkers and histopathology.
Results: Compared to the ANP group, CQCQD treatment significantly attenuated histopathological score of both the pancreas and the lung, and increased lung heat shock protein 70 expression. Moreover, CQCQD treatment significantly reduced serum interleukin-6 level and elevated interleukin-10 level, respectively.
Conclusion: CQCQD is protective against acute necrotising pancreatitis on a L-arginine-induced pancreatitis model.
Effects of Chai-Qin-Cheng-Qi Decoction on Serum Cholecystokinin Concentration and Acinar Cell Cytosolic Calcium Overload in Acute Pancreatitis
P. Xue,1 T. Jing,1 L. Wen,1 L. Huang,1 Z. Y. Cheng,1 W. Huang,2 X. N. Yang,1 Q. Xia.11Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, China; 2Departments of Surgery and Physiology, Royal Liverpool Hospital, University of Liverpool, UK.
Introduction: Our previous work demonstrated that Chai-Qin-Cheng-Qi Decoction (CQCQD) up-regulates the expression of sarco/endoplasmic reticulum Ca2+-ATPase2 mRNA and reduces cytosolic Ca2+ [Ca2+]c overload of pancreatic acinar cells. Here we tested the effects of CQCQD on serum cholecystokinin-8 (CCK-8) concentration and acinar cell [Ca2+]c overload in an acute necrotising pancreatitis (ANP) model.
Methods: Male KM mice were randomly divided into four groups with six mice each: (1) Mice with saline injection; (2) mice with ANP; (3) mice with ANP plus CQCQD treatment; (4) or plus CCK-siRNA treatment. ANP was induced by intraperitoneal (ip) injection of 4 g/kg L-arginine twice at one h apart. Seventy-two hours after begin of experiment, CQCQD or siRNA was adminstered a treatment respectively. Mice were sacrificed 6 h after starting treatment, blood was collected for serum CCK-8 concentratioin and pancreas was for histopathology. Pancreatic acinar cell [Ca2+]c was determined by staining with Fluo-3-AM using confocal microscopy.
Results: Both CQCQD and CCK-siRNA treatment significantly reduced serum CCK-8 concentration, acinar cell [Ca2+]c fluorescence intensity and pancreatic histopathology. Serum CCK-8 concentration positively correlated with pancreatic acinar cell [Ca2+]c (r = 0.793, P = 0.021) and pancreatic histopathological score (r= 0.847, P = 0.000), respectively.
Conclusion: CQCQD is protective against ANP through reducing increased serum CCK-8 concentration and pancreatic acinar cell [Ca2+]c overload.
A Population-Based Study of the Natural History of Chronic Pancreatitis
D. Yadav,1 L. Timmons,2 S. T. Chari.21University of Pittsburgh, Pittsburgh, PA, 2Mayo Clinic, Rochester, MN.
Background: Current understanding of chronic pancreatitis (CP) epidemiology and natural history is largely based on data from specialized centers. We studied the natural history of CP in Olmsted County, MN.
Methods: We reviewed medical records of all Olmsted County residents receiving a diagnosis of CP between 1977 and 2006 to identify 106 patients who met published criteria for definite CP. After excluding 17 patients diagnosed only at autopsy, there were 89 clinical CP cases whosecharts were reviewed in detail.
Results: The median age of CP diagnosis was 56 yrs (range 7-87) and 56% were male. Their median follow up was 9.7 yrs. Etiology of CP was alcohol-related (52%), idiopathic (40%) or others (8%). Compared with non-alcoholic CP, alcoholic CP patients were more likely to be younger at diagnosis (median 52 vs. 64 yrs, p<0.05), male (61 vs. 51%) and be current smokers (72 vs. 29%, p<0.05). Alcoholic CP patients were also more likely to have ever had pain (87 vs. 65%, p<0.05), acute pancreatitis (65 vs. 44%, p<0.05), recurrent pancreatitis (42 vs. 23%, p<0.05), pseudocysts (41 vs. 27%, p<0.05), exocrine insufficiency (45 vs. 20%, p<0.05), developed new diabetes (33 vs. 23%) or needed hospitalizations for pancreatitis (p<0.05). In contrast to historical data of 40-50% surgery rate during the disease course, only 18% alcoholic CP patients required surgery for CP or its complications. Pancreatic cancer developed in 6.5% alcoholic and 0% non-alcoholic CP patients during follow up. Mortality was similar in alcoholic and non-alcoholic CP.
Conclusions: Alcoholic CP patients are more symptomatic than patients with non-alcoholic CP. Low surgery rates during disease course indicates a milder natural history of alcoholic CP at population level than reported from specialized centers.
Characterization of Nestin-positive Blood Vessels in Human Pancreatic Cancer
K. Yamahatsu,1,2 Y. Matsuda,1 T. Ishiwata,1 T. Yamamoto,1 T. Fujii,1 T. Aimoto,2 Y. Nakamura,2 M. Hiroi,2 E. Uchida,2 Z. Naito.11Department of Pathology, Integrative Oncological Pathology, Nippon Medical School, Tokyo, Japan, 2Surgery for Organ and Biological Regulation, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Background: Tumor angiogenesis is an important factor in the proliferation and metastasis of human cancer. An increased number of Nestin-positive vessels in colorectal cancers correlated with worse prognosis, and co-expression of Nestin and Ki-67 in blood vessels was significantly increased in castration-resistant prostate cancer cases and the metastatic lesions. In the present study, we examined the expression of Nestin- and CD34-positive blood vessels in pancreatic cancer tissues and the relationship between Nestin- or CD34-positive vessels and the proliferative activity.
Methods: Immunohistochemical staining of Nestin, CD34, and PCNA, using serial tissue sections was performed on 45 pancreatic ductal adenocarcinoma cases. The number and dimension of the Nestin- or CD34- positive vessels and the number of PCNA positive cells in vessels was measured by use of image-analyzing software.
Results: In normal pancreases, Nestin was localized in a few capillary endothelial cells, nerve fibers, and a few spindle stromal cells, and CD34 was localized in most of the vascular endothelial cells and a few spindle stromal cells. In pancreatic cancer tissues, Nestin and CD34 were co-localized in endothelial cells of small vessels, while large vessels expressed only CD34, but not Nestin. The dimension of Nestin-positive vessels was approximately 74% of CD-34 positive vessels. The number of Nestin-positive vessels was approximately 20% of CD34 positive vessels. PCNA was expressed in 10% of Nestin-positive vascular endothelial cells, while it was expressed in 8% of CD34-positive endothelial cells in pancreatic cancer tissues. Expression levels of Nestin- and CD34-positive vessels did not correlate with prognosis in pancreatic cancer cases.
Conclusion: Nestin was expressed in small and proliferating blood vessels in pancreatic cancer tissues, and it may be a novel tumor angiogenetic marker in the cancer.
Collagen-I Increases the Expression of Transgelin and Lumican (Regulators of Cell Migration) by Activated Pancreatic Stellate Cells
L. Yang,* P. A. Phillips,* Z. Xu, J. Youkhana, A. Vonlaufen, R. Pirola, J. Wilson, M. V. Apte. *Equal first authors. Pancreatic Research Group, University of New South Wales, Sydney, Australia.
Activated pancreatic stellate cells (PSCs) play a central role in pancreatic fibrosis, a feature of chronic pancreatitis and pancreatic cancer. The fibrous matrix produced by PSCs can, in turn, regulate PSC function. Using microarrays, we have previously demonstrated that 146 genes were dysregulated (fold change>2, p<0.001, q<0.25, n≥3) in PSCs cultured on collagen-I (mimics fibrotic pancreas) versus Matrigel™ (mimics normal pancreatic basement membrane). Interestingly, genes for transgelin and lumican (which code for proteins known to regulate cell migration) were upregulated in PSCs cultured on collagen-I compared to Matrigel.
Aim: To assess transgelin and lumican mRNA and protein expression in: i) PSCs cultured on Matrigel versus collagen-I; ii) quiescent PSCs vs PSCs cultured on plastic.
Method: Transgelin and lumican expression was assessed by real-time PCR and western blotting in quiescent rat PSCs (24 hours after isolation or grown on Matrigel™ and activated PSCs (cultured on collagen-I for 72h or on plastic); n=5 preparations).
Results: Transgelin and lumican mRNA levels were upregulated by i) 45.5 and 24.7 fold (*p<0.05) respectively in PSCs cultured on collagen-I vs Matrigel™. and ii) 20 and 4.2 fold (*p<0.05) respectively in plastic-activated PSCs vs quiescent cells. Transgelin protein expression was also increased (% of control [mean±SE]: 295.7±28.92,*p<0.05) in activated PSCs compared to quiescent PSCs.
Conclusions: Transgelin and lumican are significantly increased in PSCs cultured on fibrous ECM and are associated with PSC activation.
Implication: Characterisation of genes that may play a role in PSC transformation may allow the identification of specific therapeutic targets for the treatment of fibrosis.
Do Clinical Outcomes post-ERCP Predict Clinical Outcomes Post-Puestow Procedure for Patients With Chronic Pancreatitis?
B. Young,1 D. Simeone,2 R. Kwon.31Divison of Gastroenterology, University of Michigan, Ann Arbor, MI; 2Department of Surgery, University of Michigan, Ann Arbor, MI; 3Division of Gastroenterology, University of Michigan, Ann Arbor, MI.
Background: Options for pain management in chronic pancreatitis include duct decompression with pancreatic duct (PD) stents or by surgery, such as a Puestow procedure. It is unclear whether a pain response to ERCP with PD stenting predicts clinical outcomes of surgical decompression.
Methods: Patients who underwent Puestow procedures at our institution from 2005 - 2010 were identified. Only patients with ERCP preceding surgery were studied. Clinical success (CS) was defined as documented significant reduction in narcotic requirement or narcotic independence within 2 months after ERCP or Puestow. Wilcoxan Rank Sum and Fisher's exact tests were used for analysis.
Results: 19 narcotic dependent patients underwent Puestow. 17 had pre-operative ERCPs, 12 with the intention for therapy. 8 (75%) of the 12 had successful stent placement across obstructing stones or strictures, while stent placement was unsuccessful in 4 patients. Of the 8 successful stents cases, 5 (62.5%) achieved post-ERCP CS, and all 5 subsequently had post-Puestow CS. All 3 ERCP failures had clinical failures with surgery. There was no significant difference in age, duration of disease, follow-up, current diabetes or tobacco use, presence of PD stone or stricture or mean dilation of the main PD between those with and without post-ERCP CS.
Conclusion: Our data suggests clinical success following ERCP with PD stenting is associated with the success of a Puestow procedure. Larger prospective studies are needed confirm the predictive value of ERCP for selection of patients for surgical intervention.
Adeno-Associated Virus-Mediated In Vivo Expression of Dominant Negative ERK Improves Mortality in a Novel Murine Model of Gallstone Pancreatitis
Z. Yuan, D. Williard, E. Twait, D. Kempuraj, I. Samuel. Surgery, VAMC/UI, Iowa City, IA.
This is the first report of the use of the adeno-associated virus (AAV) for in vivo gene modulation in an exocrine pancreatic disease. AAV vectors have several advantages over adenoviral vectors for in vivo work primarily due to low immunogenicity. Using a novel murine model of ligation-induced acute pancreatitis, we show that ERK inhibition significantly improves mortality. First, we showed that 3e12 viral genome particles (VGP) of AAV8.GFP i.p. results in robust pancreatic expression of GFP in 1 wk that peaks by 2 wk and plateaus at 3 wk (immunoblot). Immunohistochemistry confirmed GFP expression in acinar cells and H&E stain showed absence of neutrophilic infiltration. GFP expression was also seen in the liver but not the lung. To determine the effect of MAP kinase inhibition on mortality, we injected mice with 3e12 VGP of either AAV8.DN.ERK or AAV8.DN.p38 alone, or AAV8.DN.ERK and AAV8.DN.p38 combined, and ligated the pancreatic duct after two weeks. Sham-operated controls had no mortality. Diseased-controls showed median mortality of 3 days and 100% mortality by 5 days. AAV8.DN.ERK pretreatment alone significantly diminished the mortality with a median mortality of 5 days and only 80% mortality by 6 days (p<0.05; Gehan Wilcoxon test). AAV8.DN.p38 pretreatment alone showed no significant difference in survival, although we confirmed DN.p38 expression in the pancreas by immunoblotting. Pretreatment with both AAV8.DN.ERK and AAV8.DN.p38 showed a trend towards even better survival (66% mortality by 7 days) than AAV8.DN.ERK alone. Conclusion: Using an original murine model and a novel gene modulation technique, we show that ERK plays an essential role in disease pathogenesis and mortality, while p38 may have a possible contributory role.
Enhanced Sensitivity of Pancreatic Detection by Measuring the CA 19-9 Antigen on Selected Protein Carriers
T. Yue,1,5 L. Li,2 M. A. Anderson,3 D. E. Brenner,3 D. M. Simeone,3 Z. Feng,2 R. E. Brand, 4 B. B. Haab.11Laboratory of Cancer Immunodiagnostics, Van Andel Institute, Grand Rapids, MI; 2Fred Hutchinson Cancer Research Center, Seattle, WA; 3University of Michigan Medical Center, Ann Arbor, MI; 4University of Pittsburgh Medical Center, Pittsburgh, PA; 5Cell and Molecular Biology Program, Michigan State University, East Lansing, MI.
The goal of this study was to develop a blood test that can detect a higher percentage of pancreatic cancer patients than the tumor marker CA 19-9. We tested the hypothesis that the measurement of the CA 19-9 antigen (an oligosaccharide found on multiple proteins) on individual proteins could contribute to improved performance over the standard CA 19-9 assay, which measures the CA 19-9 antigen on all proteins. Serum or plasma samples were incubated on microarrays containing antibodies against the mucin proteins MUC1, MUC5AC, and MUC16. After the proteins were captured by the immobilized antibodies, the levels of the CA 19-9 antigen on the captured proteins were measured by incubation of the CA 19-9 monoclonal antibody. Four sample sets from three different institutions were examined, with a total of 333 individual samples from patients with pancreatic adenocarcinoma or pancreatitis. The CA 19-9 marker distinguished cancer from benign disease with 84-87% sensitivity at 75% specificity. The measurements of CA 19-9 on individual protein carriers contributed complementary information to the standard CA 19-9 marker. Thresholds could be set to detect elevations in many of the patients that showed no CA 19-9 elevations, while not increasing the false positive rate. In all sample sets, the panel showed improved sensitivity (85%-100%) over total CA19-9 alone (79%-90%). Among all false negatives classified by total CA19-9, the additional three markers in the panel picked up from 25% to 100% of them. The consistent performance over independent sample sets supports the generality and reliability of the novel marker panel as a tool for improving the accuracy of diagnoses of pancreatic cancer.
An Automated Analyzer Provides Clinically Concordant Results to Manual Back Titration for Quantitation of Bicarbonate in Pancreatic Juice
N. Zhong, M. Topazian, S. T. Chari, A. K. Saenger, J. E. Clain, F. C. Gleeson, M. J. Levy, E. Rajan, F. T. Enders. Mayo Clinic, Rochester, MN.
Introduction: Secretin pancreatic function tests play an important role in diagnosis of chronic pancreatitis. Back titration is the gold standard method for measurement of bicarbonate concentration in pancreatic juice, but is time consuming and manually performed. Use of an autoanalyzer for this purpose is not validated.
Methods: Bicarbonate concentrations in secretin-stimulated pancreatic juice specimens were quantitated by manual back titration, a clinical chemistry autoanalyzer (autoanalyzer bicarbonate, Roche Cobas c501), and a blood gas analyzer (calculated bicarbonate, IL GEM3000). Kappa statistic analysis, Bland-Altman analysis and Lin concordance correlation coefficients were calculated. Specimens were diluted 2:1 for autoanalyzer assays.
Results: Ninety specimens from 31 subjects were included. Using a bicarbonate concentration of 80 mEq/L as a cut-off value, there was poor agreement between back titration and calculated bicarbonate (kappa = 0.188), however only one specimen showed discrepancy between back titration and autoanalyzer bicarbonate (kappa = 0.977). In specimens with bicarbonate concentrations < 100 mEq/L, the limits of agreement between the back titration and autoanalyzer bicarbonate was -9.0 to 8.3 mEq/L. The Lin concordance correlation coefficient between the two methods was 0.931 (P<0.001). The limits of agreement between previously frozen and fresh pancreatic juice autoanalyzer results were -8.2 to 8.1 mEq/L.
Conclusion: There is strong concordance between manual back titration and chemistry autoanalyzer methods for measurement of bicarbonate concentrations in pancreatic juice. Autoanalyzers may replace back titration for this purpose. Blood gas analyzers are unsuitable.
Are Chronic Pancreatitis and Acute Recurrent Pancreatitis Two Different Faces Of the Same Coin or Two Different Diseases?
R. A. Zuppardo,1 L. Frulloni,2 S. Bertolini,1 C. Calzolari,1 S. K. Singh,3 A. Bertelè,4 V. Corrente,4 G. Leandro,1 F. Di Mario,1 A. Franzè,4 G. M. Cavestro.11University of Parma, Italy, Dept of Clinical Science; 2University of Verona, Italy, Dept of Surgical and GI Sciences; 3Boston University School of Medicine, Medical Center, Section of Gastroenterology; 4Parma Medical Center, GI Unit, Italy.
Background: Clinical differences between chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP) are controversial.
Aim: To investigate the clinical phenotype of ARP and CP patients as two distinct group of patients.
PATIENTS 81 ARP and 162 CP patients were consecutively enrolled. We classified ARP on the basis of recurrence of acute pancreatitis in the absence of radiological findings of CP (ductal dilation/alteration; calcifications). Gender, age at diagnosis, alcohol intake, smoking habit, incidence of dyslipidemia, diabetes mellitus, colelithiasis and pancreas divisum were analyzed.
Results: ARP and CP are characterized by differences in clinical course and associated factors. ARP subjects had a significantly higher incidence of pancreas divisum (ARP=11,1%;CP=3,7%;p=0,02). With respect to mean alcohol intake, the percentage of drinkers in ARP was lower (ARP=27,1%;CP=40,1%;p=0,04). Smoking habit was significantly higher in CP (ARP=22,2%;CP=66%;p=0,006) even if the amount of cig/die was similar (ARP=23,7±14,2;CP=23,6±12,1). Moreover, ARP patients had a minor incidence of type II diabetes (p<0,0001) and dyslipidemia (ARP=3,7%;CP=14,8%;p=0,008).
Conclusions: CP and ARP patients are characterized by distinct endogenous and exogenous factors. We do believe ARP may progress to CP as final epiphenomenon: CP could be the final endpoint of different pancreatic inflammatory diseases much as cirrhosis is the common late manifestation of a variety of liver diseases.